CN110358719A - A kind of engineered strain and its construction method of fermentation synthesis erythrothioneine - Google Patents
A kind of engineered strain and its construction method of fermentation synthesis erythrothioneine Download PDFInfo
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Abstract
The invention discloses the engineered strains and its construction method of a kind of fermentation synthesis erythrothioneine, belong to technical field of bioengineering.The present invention is host with Escherichia coli and bacillus subtilis, using three shuttle plasmid pEBS of bacillus subtilis, Escherichia coli and saccharomyces cerevisiae as expression vector, separate sources egtABCDE gene cluster is introduced in Escherichia coli and bacillus subtilis and carries out heterogenous expression.Realize biosynthesis erythrothioneine.Then expression is optimized to egtABCDE gene cluster, including codon optimization, promoter optimization and various combination expression, Optimal Medium and methionine, hercynine, histidine, the cysteine of addition simultaneously, by Optimal Expression and optimum culture condition, high-performance bio synthesis erythrothioneine is realized.The present invention is that microflora high-efficiency fermenting preparation erythrothioneine is laid a good foundation, and is suitable for industrialized production and application.
Description
Technical field
The present invention relates to the engineered strains and its construction method of a kind of fermentation synthesis erythrothioneine, belong to biotechnology
Field.
Background technique
Erythrothioneine is a kind of derivative class mercaptan compound of rare natural chiral histidine, has unique biology function
Energy and pharmacological activity.It not only has and very strong has antioxidant activity: can remove through free radical, Peroxynitrite, hypochlorous acid, huge legendary turtle
Close bivalent metal ion, activation antioxidant enzyme, inhibit superoxide dismutase etc., and have anti-inflammatory effect and protection thin
Born of the same parents effect, medicine, food, health care product and cosmetics and biotechnology field be with a wide range of applications with before market
Scape.
Erythrothioneine is isolated from ergot for the first time in 1909, in actinomyces, cyanobacteria and fission yeast, part
It can also be synthesized in the microorganisms such as gill fungus bacterium, streptomycete, mycobacteria, and other bacterial species are such as: bacillus subtilis, large intestine bar
Bacterium, proteus vulgaris and streptococcus etc. cannot synthesize.Erythrothioneine can not be synthesized by animal and plant biological, but can be planted
Object and animal absorb and accumulation, and is distributed widely in the cell and tissue of mammal.Erythrothioneine is in human physiology
Vital, people must obtain from dietary source, and in specific organization and cell such as liver, kidney, central nervous system
It is accumulated in red blood cell.At present erythrothioneine can be produced by chemical synthesis, extraction method and biofermentation synthetic method.Chemistry
Method synthesizes that left-handed erythrothioneine is very difficult, and the safety of product is difficult to ensure, synthesis material is expensive, and synthesis cost is high,
Not up to expected yield；There is also many difficulties when extraction method production erythrothioneine, as raw material sources are insufficient, ergot in raw material
The content of thioneine is still very low and extraction cost is high, has medicament residue etc., with the biological section such as molecular biology and metabolic engineering
Fast development, biofermentation synthetic method, which produces erythrothioneine, becomes most potential production method.
However, the method for producing erythrothioneine by building genetic engineering bacterium, effect is simultaneously paid no attention in existing report
Think.Heterogenous expression is such as carried out in Escherichia coli with the egt gene from mycobacterium smegmatis.Gene in gene cluster is split
It is connected in multiple plasmids, then plasmid is transformed into Escherichia coli, Optimal Expression recombinase improves erythrothioneine yield, to the greatest extent
Pipe is in this way, shaking flask yield only has 24mg/L (Osawa R, Kamide T, Satoh Y, et al.Heterologous and
High Production of Ergothioneine in Escherichia coli[J].Journal of
Agricultural&Food Chemistry,2017,66(5):1191-1196.)。
Therefore it provides a kind of method that can be realized high-performance bio synthesis erythrothioneine, for industrial production erythrothioneine
With important application value.
Summary of the invention
The first purpose of the invention is to provide a kind of engineered strain of fermentation synthesis erythrothioneine, be with Escherichia coli or
Bacillus subtilis is host, heterogenous expression egtABCDE gene cluster, or, express amino acid sequence albumen as shown in k or l
Matter, amino acid sequence such as e, f, g, h, i or j of the egtABCDE gene cluster are any shown.
In one embodiment of the invention, the Escherichia coli include Escherichia coli MG1655, bacillus coli DH 5 alpha,
Escherichia coli W3110 or e. coli bl21.
In one embodiment of the invention, the bacillus subtilis includes bacillus subtilis 168, withered grass gemma
Bacillus W600 or bacillus subtilis W800.
In one embodiment of the invention, egtABCDE gene cluster source is Mycobacterium avium
Subsp.paratuberculosis str.k10, Mycolicibacter icosiumassiliensis strain 8WA6,
Mycobacterium colombiense CECT 3035, Mycobacterium marinum E11, Mycobacterium
Sp.KMS or Mycobacteroides salmoniphilum.
In one embodiment of the invention, egtABCDE gene cluster nucleotide sequence such as SEQ ID NO.1,
Shown in SEQ ID NO.2, a, b, c or d are any.The nucleotide sequence of coded sequence k is as shown in SEQ ID NO.3.Coded sequence l
Nucleotide sequence as shown in SEQ ID NO.4.
In one embodiment of the invention, the expression vector of the engineered strain is bacillus subtilis, large intestine bar
(construction method is shown in Yang S, Liu Q, Zhang Y, et to three shuttle plasmid pEBS of bacterium and saccharomyces cerevisiae
al.Construction and Characterization of Broad-spectrum Promoters for
Synthetic Biology.[J].Acs Synthetic Biology,2017,7(1):287-291.)。
A second object of the present invention is to provide a kind of method for producing erythrothioneine, apply above-mentioned engineered strain into
Row fermentation.
In one embodiment of the invention, engineered strain is seeded to fermentation medium, in 35-38 DEG C of fermentation 40-
60h adds methionine, hercynine, histidine, cysteine, the first sulphur of addition when cultivating 8-12h as needed
Propylhomoserin final concentration 2-8g/L, the hercynine final concentration 10-15g/L of addition, the histidine final concentration 15-20g/L of addition,
The cysteine final concentration 15-20g/L of addition.
Used fermentation medium group is divided into (g/L): yeast powder 2.5, peptone 5.0, Na2HPO46.78 KH2PO4
3.0, NaCl 0.5, NH4Cl 1.0, MgSO4·7H2O 0.5, CaCl20.015, glucose 40；FeCl2·6H2O
0.013.5；MnCl2·4H2O, 0.001；ZnCl2, 0.0017；CuCl2·2H2O, 0.00043.
Third object of the present invention is to provide above-mentioned engineering bacterias in preparation erythrothioneine or containing the product of erythrothioneine
In application.
Beneficial effects of the present invention:
The present invention is host with Escherichia coli or bacillus subtilis, with bacillus subtilis, Escherichia coli and wine brewing ferment
Three female shuttle plasmid pEBS are expression vector, introduce separate sources egtABCDE gene in Escherichia coli and bacillus subtilis
Cluster carries out heterogenous expression.Realize biosynthesis erythrothioneine.Then expression, including password are optimized to egtABCDE gene cluster
Son optimization, promoter optimization and various combination expression, can obtain one plant of preferable Escherichia coli of production erythrothioneine and withered grass respectively
Bacillus recombinant bacterium realizes high-performance bio synthesis erythrothioneine by Optimal Expression and optimum culture condition.Energy of the present invention
So that Escherichia coli recombinant strain is cultivated 60h erythrothioneine accumulation in shaking flask and is up to 437.6mg/L, bacillus subtilis recombination
Bacterium cultivates 60h erythrothioneine accumulation in shaking flask and is up to 568.4mg/L, industrially has potential and is widely applied valence
Value.
Detailed description of the invention
The building of gene various combination Optimal Expression contained by Fig. 1: pEBS-egtABCDE and egtABCDE gene cluster is illustrated
Figure.
Fig. 2: the Escherichia coli recombinant strain of heterogenous expression separate sources egtABCDE gene cluster cultivates wheat after 60h in shaking flask
Angle thioneine accumulation.
Fig. 3: the bacillus subtilis recombinant bacterium of heterogenous expression separate sources egtABCDE gene cluster cultivates 60h in shaking flask
Erythrothioneine accumulation afterwards.
The Escherichia coli, bacillus subtilis of gene various combination Optimal Expression contained by Fig. 4: egtABCDE gene cluster recombinate
Bacterium cultivates erythrothioneine accumulation after 60h in shaking flask.
Specific embodiment
One, the nucleotide sequence information that embodiment is related to
(1) sequence a information is from Mycobacterium avium subsp.paratuberculosis
The egtABCDE gene cluster nucleotide sequence of str.k10, NCBI number: AE016958.1 (322465to 327878), sequence
(a) amino acid sequence encoded is sequence e；
(2) SEQ ID NO.1 sequence information is from Mycolicibacter icosiumassiliensis
The egtABCDE gene cluster nucleotide sequence of strain 8WA6, the amino acid sequence of SEQ ID NO.1 coding are sequence f；
(3) sequence b information is the egtABCDE gene from Mycobacterium colombiense CECT 3035
Cluster nucleotide sequence, NCBI number: the amino acid sequence of CP020821.1 (3701114to 3706623), sequence b coding is sequence
Arrange g；
(4) sequence c information is the egtABCDE gene cluster nucleotides sequence from Mycobacterium marinum E11
Column, NCBI number: the amino acid sequence of HG917972.2 (6005272to 6010682), sequence c coding is sequences h；
(5) sequence (d) information is the egtABCDE gene cluster nucleotide sequence from Mycobacterium sp.KMS,
NCBI number: the amino acid sequence of CP000518.1 (5206948to 5212322), sequence d coding is sequence i；
(6) SEQ ID NO.2 sequence information is Mycobacteroides salmoniphilum strain D16Q15's
EgtABCDE gene cluster nucleotide sequence, the amino acid sequence of SEQ ID NO.2 coding are sequence j；
(7) SEQ ID NO.3 sequence information is gene cluster BACDE nucleotide sequence, the amino acid of SEQ ID NO.3 coding
Sequence is sequence k；
(8) SEQ ID NO.4 sequence information is gene cluster BA-PBSX-CDE nucleotide sequence, SEQ ID NO.4 coding
Amino acid sequence is sequence l.
Two, the building of expression system
Respectively with Mycobacterium avium subsp.paratuberculosis str.k10 genome,
Mycolicibacter icosiumassiliensis strain 8WA6 genome, Mycobacterium colombiense
3035 genome of CECT, Mycobacterium marinum E11 genome, Mycobacterium sp.KMS genome and
Mycobacteroides salmoniphilum genome is that template is made by PCR amplification egtABCDE gene cluster DNA fragmentation
Primer be respectively egtABCDE (mp)-F/egtABCDE (mp)-R, egtABCDE (mi)-F/egtABCDE (mi)-R,
egtABCDE(mc)-F/egtABCDE(mc)-R、egtABCDE(mm)-F/egtABCDE(mm)-R、egtABCDE(mk)-F/
Resulting PCR product and pEBS (are used primer by egtABCDE (mk)-R, egtABCDE (ms)-F/egtABCDE (ms)-R
PEBS-F/pEBS-R amplification) skeleton group be filled with generate pEBS-egtABCDE (mp), pEBS-egtABCDE (mi), pEBS-
EgtABCDE (mc), pEBS-egtABCDE (mm), pEBS-egtABCDE (mk) and pEBS-egtABCDE (ms).With
Mycobacterium avium subsp.paratuberculosis str.k10 genome is template, is obtained by PCR amplification
To egtB (being expanded using primer B (1)-F/B (1)-R), egtA (being expanded using primer A (2)-F/A (2)-R), egtC (using drawing
Object C (3)-F/C (3)-R amplification), egtD (using primer D (4)-F/D (4)-R expand), egtE (use primer E (5)-F/E
(5)-R is expanded) DNA fragmentation of gene, resulting PCR product and pEBS (are expanded into) skeleton using primer pEBS-F/pEBS-R
Group, which is filled with, generates pEBS-BACDE.Using pEBS-BACDE as template, primer P is usedBSX-F/PBSX- R passes through PCR amplification promoter
pbsxResulting PCR product and pEBS-BACDE (are used primer AP by DNA fragmentationBSC-F/APBSC-R amplification) skeleton assembling
PSTOP1622 is to generate pEBS-BA-PBSX- CDE, as shown in Figure 1.
Three, the building of recombinant bacterium
1. respectively to Escherichia coli MG1655, bacillus coli DH 5 alpha, Escherichia coli W3110, e. coli bl21 is transferred to weight
Group plasmid pEBS-egtABCDE (mp), pEBS-egtABCDE (mi), pEBS-egtABCDE (mc), pEBS-egtABCDE
(mm), pEBS-egtABCDE (mk) and pEBS-egtABCDE (ms).Obtain Escherichia coli recombinant strain Mmp, Mmi, Mmc, Mmm,
Mmk、Mms、Dmp、Dmi、Dmc、Dmm、Dmk、Dms、Wmp、Wmi、Wmc、Wmm、Wmk、Wms、Bmp、Bmi、Bmc、Bmm、Bmk、
Bms。
2. being transferred to recombinant plasmid to bacillus subtilis 168, bacillus subtilis W600, bacillus subtilis W800 respectively
pEBS-egtABCDE(mp)、pEBS-egtABCDE(mi)、pEBS-egtABCDE(mc)、pEBS-egtABCDE(mm)、pEBS-
EgtABCDE (mk) and pEBS-egtABCDE (ms).Obtain bacillus subtilis recombinant bacterium 168mp, 168mi, 168mc,
168mm、168mk、168ms、600mp、600mi、600mc、600mm、600mk、600ms、800mp、800mi、800mc、
800mm、800mk、800ms。
3. bacillus coli DH 5 alpha is transferred to recombinant plasmid pEBS-BACDE and pEBS-BA- respectively to bacillus subtilis 168
PBSX- CDE obtains recombinant bacterium BS-BACDE, BS-BA-PBSX-CDE、EC-BACDE、EC-BA-PBSX-CDE。
Four, the processing of sample and HPLC analyze erythrothioneine yield
4mL fermentation liquid is taken, 10min is centrifuged at 12000r/min, supernatant analyzes erythrothioneine for HPLC, and precipitating is used
Ultrasonication is carried out after isometric 50% methanol suspension, then is centrifuged 10min at 12 000r/min, is collected supernatant and is used for
HPLC analyzes erythrothioneine HPLC, and erythrothioneine yield is the sum of erythrothioneine content intracellular and extracellular.
HPLC analysis condition: mobile phase is made of 0.1% formic acid/water (A) and 0.1% formic acid/second eyeball (B).Gradient program
To be terminated in 8.5 minutes 95%B at 12 minutes in 10 minutes 40%B in 2 minutes 95%B.Flow velocity is 0.5ml/
Min, 10 μ L of sample volume.Sample is analyzed using Dionex UPLC Ultimate 3000 (Sunnyvale, CA).In Atlantis
HILIC silicagel column (3.01 μm of partial size, diameter × length=2.1 × 100mm；Waters compound is separated on), and at 264nm
Detection.
Five, the primer information being related in embodiment
Primer information is as shown in table 1.
1 primer of table
The shaking flask culture and erythrothioneine of 1 24 plants of heterogenous expression egtABCDE gene cluster Escherichia coli recombinant strains of embodiment
Assay
Pick them separately 24 plants of heterogenous expression egtABCDE gene cluster E. coli recombinant stains and the control bacterium of above-mentioned building
(conversion pEBS empty plasmid) monoclonal is inoculated in 5mL LB culture medium, and the card of final concentration of 50 μ g/mL is added as needed, and that is mould
Element is placed in 37 DEG C of culture 10-12h of 200rpm, and then by 10% inoculum concentration switching in 250mL triangle shake bottle, culture medium is hair
Ferment culture medium, liquid amount 20mL are subsequently placed in 220rpm37 according to that need to have the kanamycins for adding final concentration of 50 μ g/mL
DEG C culture, the methionine of the methionine, hercynine, histidine, the cysteine that add when cultivating 10h, addition are whole
Concentration 2g/L, the hercynine final concentration 10g/L of addition, the histidine final concentration 15g/L of addition, the cysteine of addition
Final concentration 15g/L, final liquid amount are 25mL (insufficient then mend sterile deionized water).The processing of sample is carried out after culture 60h
And HPLC analyzes erythrothioneine yield.As a result as shown in Fig. 2, recombinant bacterium can realize heterologous synthesis erythrothioneine, wherein recombinating
The erythrothioneine yield of bacterium Mmp is higher, reaches 335.3mg/L.
The shaking flask culture of 2 18 plants of heterogenous expression egtABCDE gene cluster bacillus subtilis recombinant bacteriums of embodiment and ergot
Thioneine assay
Pick them separately 18 plants of heterogenous expression egtABCDE gene cluster bacillus subtilis recombinant bacterial strains of above-mentioned building and right
It is inoculated in 5mL LB culture medium according to bacterium (conversion pEBS empty plasmid) monoclonal, the card of final concentration of 50 μ g/mL is added as needed
That mycin is placed in 37 DEG C of culture 10-12h of 200rpm, then transfers by 10% inoculum concentration in 250mL triangle shake bottle, culture medium
For fermentation medium, liquid amount 20mL is subsequently placed according to that need to have the kanamycins for adding final concentration of 50 μ g/mL
37 DEG C of 220rpm cultures, methionine, hercynine, histidine, the cysteine added when cultivating 10h, the first of addition
Methyllanthionine final concentration 2g/L, the hercynine final concentration 10g/L of addition, the histidine final concentration 15g/L of addition, addition
Cysteine final concentration 15g/L, final liquid amount are 25mL (insufficient then mend sterile deionized water).Sample is carried out after culture 60h
The processing of product and HPLC analyze erythrothioneine yield.As a result as shown in figure 3, recombinant bacterium can realize heterologous synthesis erythrothioneine,
Wherein the erythrothioneine yield of recombinant bacterium 168mp is higher, reaches 412.2mg/L.
Escherichia coli, the bacillus subtilis of gene various combination Optimal Expression contained by 3 egtABCDE gene cluster of embodiment
The shaking flask culture of recombinant bacterium and erythrothioneine assay
Pick them separately Escherichia coli of gene various combination Optimal Expression contained by the egtABCDE gene cluster of above-mentioned building, withered
Careless bacillus recombinant bacterium and control bacterium (conversion pEBS empty plasmid) monoclonal are inoculated in 5mL LB culture medium, are added as needed
The kanamycins of final concentration of 50 μ g/mL is placed in 37 DEG C of culture 10-12h of 200rpm, then by 10% inoculum concentration switching in
250mL triangle shake bottle, culture medium are fermentation medium, liquid amount 20mL, according to need to have the card for adding final concentration of 50 μ g/mL
That mycin is subsequently placed in 220rpm, 37 DEG C of cultures, the methionine added when cultivating 10h, hercynine, histidine, half
The histidine of cystine, the methionine final concentration 8g/L of addition, the hercynine final concentration 15g/L of addition, addition is whole
Concentration 20g/L, the cysteine final concentration 20g/L of addition.Final liquid amount is 25mL (insufficient then mend sterile deionized water).
The processing of sample is carried out after cultivating 60h and HPLC analyzes erythrothioneine yield.As a result as shown in figure 4, recombinant bacterium can realize it is different
Source synthesizes erythrothioneine, and wherein the erythrothioneine yield of recombinant bacterium BS-BA-Pbsx-CDE is higher, reaches 568.4mg/L.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of engineered strain and its construction method of fermentation synthesis erythrothioneine
<130> 233
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 5315
<212> DNA
<213>artificial sequence
<400> 1
atgggattcg ccgcggcgac gcaattgtcc cgcggagatg aagcttgcct gggcggcccc 60
gagctggccg gtgccgccga cgccgccgac tacatcaccc aacgttgcct cactgatgcc 120
ccgcacggcc gggtgggttt ggaggtcgag gcgcactgct tcgacccgac cgacccgcac 180
cgccggccgg gctgggcgga gatcggtgac gtgctgcagt ccctgccggc cttgccgggc 240
ggcggtgtcg tcacggtcga acccggcggc gctgtggagc tctccggccc gccgctgacc 300
gacgcacttt ccgcgatcga agcgatggtg gccgaccagg cggtgttgcg ggcggctttc 360
gccgccgccg ggctgggtct ggtgcagctg ggcgccgacc cgctgcggcc cacggagcgg 420
gtcaaccccg gcgagcgcta cgcggcgatg gagcagttct tcgccgccag ccacaccggt 480
tcggccggtg cggcgatgat gacgtcgacg gcgtcggtcc agctcaatct ggatgccggt 540
ccgcgttccg gttgggcaga ccgagtccgg ttggctcacg cgctgggccc gacgatggtc 600
gccatcgccg cgaactctcc gctgctggcc ggggagttct ccggctggct ttccacccgg 660
cagcgggtgt ggagccgact ggactcggcg cgctgcggcc ccatcctcgg ccttgacggc 720
accgaccccg tcaccgactg ggcgcggtac gcgctcaaag cgcccgtcat gctggtgcac 780
acgcccgaag cgattccggt cagccgctac gtgccgttcg ccgcctgggc cgacggacgg 840
gtgctgctcg gcgatcgccg cgccggcatt aaagatcttg attaccacct gaccacactg 900
tttcctccgg tgcgcccgcg cggatggctg gagatccgct acctcgacag cgtcgacgac 960
accctctggc cggcactggt attcaccctg gccaccctgc tcgacgaccc gctcgccgcc 1020
gacagcgctg ccgatgccgt cgcacccgtc gctaccgcct gggacgtcgc cgcccggatc 1080
ggcctggccg acccgcggct gcatgcggcc gcccggcact gcattgggct ggtcgccgac 1140
cgggccccgg catccctgcg cgacgggatc gatcggctgg ccggcatggt cgaacgcggc 1200
cgctgcccgg ccgacgatgt caccgacgag gtgacccggg ccggcaccgc ggcgatggtg 1260
acccgaatgg cgcgccgcca gtcatgacca cgtccgaaat attggcccgc aatctgaccc 1320
gggcgcggca gcgcacgctg gcgctcgtcg atttcgacga cgccgagctg caccgccagt 1380
acgacccgct gatgagcccg ctggtgtggg acctggccca tatcggtcag caggaggagc 1440
tgtggctgct gcgcgacggg gacccggcgc gcccaggcat gctgtccgcg gatgtcgagg 1500
gcctctacga cgccttcacc cacccacggg ccagccgcgt cgccctaccg ttgttggcgc 1560
ccgaacaggc ccggaagtat tgcgccaccg tgcgttccaa ggttttcgac aagctggggt 1620
ccctgccggg tgacgcaggg gatcgcacgg acggattcgt gttcggcctg gtgctcagcc 1680
atgaacacca gcacgacgag accatgctgc aggcgctgaa cctacgtgcc ggcccgccgc 1740
tgctcggtgc cggtgaccgc ctgcccgccg ggcagcccga cctggcagga acctcggtgt 1800
tggtaccggg cggtgaattc atgctgggcg tcgacgcaac cgacgagccc ttctcgctgg 1860
acaacgagcg ccccgcgcac cgcgtcgatg tggcggcgtt ccggatcggc cgggtgccgg 1920
tgaccaaccg cgaatggcgg gagttcatcg acgacggcgg ctaccggcag cctcagtggt 1980
ggagcgaacg gggttggcgc cactgcgtgc aagccgggtt gacggcgccg gagttctggg 2040
gccccgacca tcgatctcga atccggttcg gccacgtcga ggcccttccc ggcgacgagc 2100
cggtgcagca cgtcagctac ttcgaggcgc aggcctacgc gacatgggcc ggggcccggc 2160
tgcccaccga gatcgaatgg gagaaggcct gcgcctggga tccggccgcc ggcgcgcgac 2220
gccgctaccc ctggggcggc gccgacccgt cggccgaagt ggctaacctc gacggtgtgg 2280
cgctgcgacc ggcgccggtc ggggcctacc cggacggggc atcggcctat ggcgcgcaac 2340
aacttctcgg cgatgtctgg gagtggacca gttcgcccct gcaaccgtgg ccgggctttt 2400
ctccgatgat ctaccaacgc tattcggagc cgttcttcgg tggcgactac cgggtgctgc 2460
gcggcgggtc gtgggcggtt gcccgggaga tcctgcggcc gagcttccgc aactgggacc 2520
acccgtaccg gcggcagatc tttgccggcg tccgactggc ctgggacgcc tgatgtgccg 2580
acacctgggc tggctgggca agccgcggtc attggcccag ttgatgctgg agccgccgca 2640
gggcctgctg gtgcagtcct atgccccgcg ccgacagcag cacgggttgc tcaacgccga 2700
cggttggggc gcagggtttt tcgatgcctc ccagggcgac gcgccgcgac ggtggcgcag 2760
tgcggcacca ctgtggagcg atgggtcgtt cgcctcggtc gccccggtac tcgccagcca 2820
ctgcgtggtg gccgcggtgc gttcggcgac tgtcgggatg cctatcgagg ccagcgccac 2880
cgcgccgttc actgacggca gttggctgct gtcgcacaac ggcgtggtga accgtgaggt 2940
tctgaccgcc atcgatcgtg ccgaatcggt ctgtgacagt gccgtcctgg ccgcagcgat 3000
cttcgcccgc ggggtggaat cgttgggcga ggtgatcgcc gagatcggtg cggccgatcc 3060
gcaagcccgg ctgaacatat tggccgccaa cggttcccgt atgttggcga cgacctgggg 3120
agacactttg tcgatgttgc acggtgccga tggtgtggtg ctggccagtg agccctacga 3180
cgacgacccg cgctgggtgg atattccaga caagcacctg gtggaaacgc acgatggcga 3240
ggtgtacgtc acggccctgg gtgacctccg atgacggtat ctattgccaa tcacctggcc 3300
gccgacgcgg ctcgatccgc actgcgggcc gacgtccgcc acgggctggc ccagcatccg 3360
aagtctttac cgcccaagtg gttctacgac gacactggca gtgagctgtt cgaccagatc 3420
acccggctgc ccgagtacta cccgacccgc gccgaggcgc agatcctgcg cacgcactcc 3480
gccgaggtgg cggcgctgtc gggcgcggac accctggtcg aactgggcag cggcacctcg 3540
gagaagaccc ggatgctgct ggacgcactg caggacgctg ggttgttgcg ccgattcatc 3600
ccctttgacg tcgacgccgg agtgctggag acggcggggg aggcgatcgc ccgggactat 3660
cccggcgtgc aagtgcaggc cgtctgcggc gatttcgagc accacctggc gcagatcccc 3720
gacggtggca gacgactgtt cgtgtttctg ggatcgacca ttggcaacct gacgccgggc 3780
ccgcgtgcgg acttcctggc cgacctcgcc gcggtcatgc acgacggtga cggcctgctg 3840
ttgggcaccg atctggtcaa ggacaccggc aggctggtgc gcgcctacga cgacgcggcc 3900
ggggtgaccg cggcgttcaa ccgcaacgtg ctcgcggtgg tcaaccgtga gcttgatgct 3960
gacttcgacc tggacgcgtt cgcccatgtg gcgcggtgga attcggcgga gcagcgcatg 4020
gagatgtggt tgcgcgcccg cagccgtcag cacgtcacag tgggggcctt ggacctggct 4080
gtcgacttcg cggccggaga ggagatgctg accgaggtgt cctgcaaatt ccgggccgag 4140
caggtggccg gcgagctggc cgccgcggga ctgcgccgcc gacggtggtg gaccgacgcg 4200
gcaggcgatt tcggcgtgtc gctcgccatc aaatgagcct cgccgacagc tggcgcgctg 4260
cgcgcccccc ggtcgcgggg ctgcacctgg acagcgcagc ctgctcacgg cagagccatg 4320
cggcgctaga tgccgcagcc tgccatgccc gtcacgaagc cgaggtcggt ggctacgttg 4380
ccgccgaggc ggccgcaccg gtgctcgacg ccggccgggc ggccttcgcc gtattgaccg 4440
gcatggccga tgccgacgtg gtgtttacta ccggctcgcg aaatgcactg gacctcctgc 4500
tgtcccgatg gccgggcgcg cagcgcaccc tggcctgcct gcccggtgaa tacggcccga 4560
atctggcggt gatggcggtg cacgggttca gccggcgggc gctgccggtg gaccacaccg 4620
gccgcttggt gctcgacgag gcggcgagcg cactcgccgc cgacccgccg agcctggtgc 4680
acctgacccc ggtcggcagt caccgcggcc tggtgcagcc cttggcgcag atggcggaca 4740
tctgccgcgc gttcgatgtc ccgatggtgg tcgacgcggc gcaggcactg ggccagatcg 4800
actgcgcggt gggcgccgcc gccgtctatt cctcatcgcg caagtggctt gccgggccgc 4860
gcggtgtcgg ggcgctggca gtacggccgg ccttggcgga actggcgtcc tcgggaccgg 4920
cggccggtga cgtgaatgtg gctgcccagg taggcttctc cgtggcgctg ggggaacatc 4980
tggcggccag gccggcagcg gttcgggccg cgctggccgg cgtgggtgcc cggagccggc 5040
gggtgctggc tcggctaccc ggctggcggg tggtcgaacc gctcgacgag ccgagcgcca 5100
tcaccacctt ggccccgccg gccggcatcg atccggtggt cgtgcgggcg tggctgatcg 5160
ccgagcacgg gatcgtcacc accgcagccg gagtcgaacg cgcgccgttg gagctgaccg 5220
tgccggtgct gcggatctca ccgcacgtcg acaccaccga agcagatttg gacgtgctcg 5280
ctgcggcgtt ggccgatgcg ccgcgcggtg gataa 5315
<210> 2
<211> 5260
<212> DNA
<213>artificial sequence
<400> 2
atggccacca cggcgtcggc ggctctcact gggcgaccgc tgtccggaca cgacgaggct 60
gccgagcata tcgctcggca gtctttcgcc gacgcacatg tcggcacggt cgggctggag 120
ttggaatctc ataccgtcga gctgacggct ccgcaccgcc gagtcagctg gcgtcgcctc 180
catgacgtcg ctgactcggt tcccgacctg cccggatcca gcgcgattac gttcgaaccg 240
ggcggtgccg tcgaactctc cggccctccg cgtgccgacg tgtggtccgc aatctccttg 300
atgcgggccg atcacgagat cctgacctcg acgtatcgtg acgccgggat tgcactcgcc 360
agcctgggca ccgacccgtt gcgcgcaccc gagcgcgtca atcccggggc ccgatatgcg 420
gcgatggccc gccatttcgg cgccgcagga tatggcgaaa ctgggctgca gatgatgacc 480
tgcaccgcgt cattacaggt caatctgcaa tccgggacgc cacgtcaatg gcgggacagg 540
ttcgcgctgg cgcaacggat cggtcctacg atggcggcgc tgtcggcatc ctcgccgatg 600
ctcgcgggtc gtcgtaccgg tcggcaaaac actcgacagt ggatctggga gaagctggat 660
ccgcggcgct gcgccccggt cgagatcggc gccgacccga tcgaatcctg ggccagatat 720
gccctgcgcg cgcccgtcat gctggtgcga aacaaggacg gcgccgaaga ggttgtcacc 780
catgtgccgt ttgaatcctg ggtcgacggg tccgtgcagc tagcggggcg cgcccccacc 840
accgaagacc tcgactatca cctgaccacg ctgttcccgc cggtgcgacc gcggcgctgg 900
ctggagctgc ggtatctgga tgccgcgccg gattggtggt ggcccgcgct ggccttcacc 960
gtggtggcgg cgctcgatca tccgcaggtc gccgacgttg ccgcggaggc ggttgaaccg 1020
cttggtgacg cctgggaggt ggcggcgcgg gtcggcctgg cggatccggc cctgcatgcg 1080
gccggccgcc ggttggtgtc ggccgcgtgc gcggtggcgc cgcccgaatt ggccgcggat 1140
atggccttcc ttgtggaacg tgtcgagcaa gggcgttgcc ctgcagatga tttcatcgat 1200
aacgtcaccg aatacggggt ggaacaggcc ttctctggag cggcccgatg atccgggaga 1260
aactggcgcg cgatcttgaa gcggcacgtg cgcgcacgtt gacgatcacc gaccacgacg 1320
atgccgagct gcatcgtcag tacgacccgt tgatgagtcc gttggtgtgg gacctcgcgc 1380
atatcggcca gcaggaagag ctatggctgc tgcgcggtgg agatccgcgc acgccgggga 1440
tgctgcccgg tgacatcgag tccctctacg acgccttccg acacacccgg gccagcaggg 1500
tgcaactccc gctgctctcg ccggcacagg cgcggtcctt ctgtcacgaa gtgcgggggc 1560
gggttttgga ccgactggat gcactgcccc tggacggttc tgcgcgttcg gacgagttca 1620
cctacgccat ggtgctcagc cacgagcatc aacatgacga aaccatgttg caggccttgt 1680
cgattcgccg cggtgccgca ctgttggagc gtgtcgaccc gttgccgccg ggacaccccg 1740
gtgtggcggg aacctcggtg ctggtgccgg agggtccgtt tgtgctgggc gtcgatgcga 1800
gcgacgagcc gttctcgctc gataacgagc ggcccgcaca tacggtggac ctcaagagct 1860
tccggatcgg cagggttccg gtgaccaacg ccgagtggtc ggcattcatc gccgacggcg 1920
gataccggcg cctggaattc tggaccgagg ccgggtggac acaccgatgc gcggaagatc 1980
tgacggcgcc caagttttgg aaccggggcg gaacactcac acggttcggt cgtgagctgg 2040
agatcgtgcc cgacgagccc gtgcagcatg tcagctttca cgaggcgcag gcctacgccg 2100
cctgggcggg tgcgcgatta cccaccgagg ccgaatggga aaaggcctgc gtctgggatc 2160
cggaagcctc tgcgcgacgg cggtttccgt ggggtgagga tgcacccggc cgtgaccggg 2220
ccaatctcgg cggaggtgcg ctgggccccg cgccggtcgg ggcctacccg gcgtcggcat 2280
cggcgtatgg cgctgagcag atgctcggtg atgtgtggga gtggacggca tccccgctgc 2340
ggccgtggcc cggttttacg ccgatgatct accagcagta cagcgagccg ttcttcgaag 2400
gttcgggggc cggtgactat cgcgtgctac gcggaggttc gtgggcggtg gcaccgtcga 2460
tcatgcggcc cagcttccgg aactgggatc atccgatccg tcggcaaatc ttcagcggcg 2520
ttcgtctggc ctgggacgtc tagcatgtgt cgtcatctgg gctggctggg cgagccgcgt 2580
aacctggcct cgctgatact tgaaccaccg catggccttc tggtgcagtc gtatgcgcca 2640
cggcgccaaa aacacggcct ggtgaatgcc gacgggtggg gggccggatt tttcgccgac 2700
ggaaaaccgc gcagatggcg tagcgcgcgg ccgctgtggg gtgatgcttc cttcgcctcg 2760
gtggccccgg tgctacgtag ccgatgcgtg gtggcggcgg tccgttccgc cagtgccgga 2820
atgcccatcg acgagtccgc ggccgcgccg ttcacggacg gaacatggct gttgtcgcac 2880
aacgggatag tcgatcgcgg tgcggtgggc gaggtttccg gggctgaatc ggtggtggac 2940
agtgcggtgc tggcggcgcg catattcgcg tcgggaccag agaacctggg ggaggtggtc 3000
cgccaggttg gggctgctga tccgggtgct cggctgaaca tactggcggc caatggaaat 3060
gagttgatcg gcacgacctg gggtgacacc ctgtcgattc tggaggccgc cgacggtgtg 3120
gtggtggcca gtgaaccgta cgacgacgac ccgtcctggg tcgacatccc ggaccggcac 3180
gtggtgcggg tacgcgatgg gaaggtagag ataagaggat tatgacgccg tcgtccccga 3240
tgaccctgga gaatcactta gcctctggtg cggcagcaga ggcgctacga cgtgatgtcc 3300
ggcagggcct gaccgcagaa gcgaaatcgc ttccccctaa atggttttac gacgaggtcg 3360
gtagcgacct gttcgacgag atcaccaggc tgccggagta ttacccgacg cggaccgagg 3420
ctgggttgtt gcgggcccat gcagccgata tcgcggcggc ctccggcgcc gacaccctgg 3480
tggagttggg cagcggcacc tccgagaaga cgcggatgct gctcgatgcg ttggctcccg 3540
gcaccttcat ccctttcgat gtggattccg gggtgctgcg cgcggcgggg gatgcgctcg 3600
tcgatgagta tccggggatg cgtgtccgcg ccgtctgtgg agacttcgag aaggacctcg 3660
ggcggattcc ccgggagggc cgccgtctgg tggcattcct gggatccacg atcgggaatc 3720
tgaccacgca gccgcgggcg cggttcctgg ccgatgtcgc agccacgctg caatctggcg 3780
aaatgctgtt actgggaacc gatttggtca aggataccga gcgtctggtg cgggcctacg 3840
acgacagcgc cggcgtgacc gctgccttca accgcaatgt gctggcggtg atcaatcgtg 3900
aactcgacgc ggatttctct ctcgaggctt ttgaacacgt cgccttgtgg aacggcgagc 3960
aggagcgcat ggagatgtgg ctgcggtcgg tgcgcgacca gcgggtaagg atcgacgctc 4020
tggatctcgc cgtcgaattc ggggtgggcg agatgatgct gaccgaggtg tcctgcaagt 4080
tccggcggga gggtgtcgcg cgcgagttgg ccgccgcggg cctggatcag acgcactggt 4140
ggacagacga ttcccatgat ttcggactat cgctggcagt gaagcggtga gcctgggcga 4200
gcagtggagg cagggcaggc caccggttct cggcgtgcat ttggactcgg ccgcctgctc 4260
acggcagagc atcgagacca ttcgtgcggt ggctcagcac gccgaacatg aggcgcagat 4320
cggcggttac gtggcgcagg aggccgcgac accggtcctc gacgccggtc gggccgcggt 4380
gcgccatgtg acgggtatgc ccgaggcgca ggtgcatttc accacgggcg cggccgatgc 4440
gttgcgcacg cttctgcagg cgtggcccgc cgataccggt cgggtaatcg cttgtttgcc 4500
aggagaattc ggacccaatc tgatgatcat gaaccacttc ggattcactc cggtgtggct 4560
gccggccgac ggggatggcc gggccgatct tgacgggatc gaggcgtttc tgcgccgcga 4620
gaaggtggat ctggtgcatt tgacggctgt cggcagccac cgcggcacgg tacagcccgc 4680
tgcggaggtg gtcgcatgcg cgcgggcggc cggggttccc gtcgtggttg atgctgccca 4740
ggcattggga catatcgact gcacctatgg cgccgacgcc atgtacgcac cgtcacgcaa 4800
gtggttggcc gggccccgtg gggtgggtgt gcttgccgtc aatccgtctc ttgcgcatct 4860
ggttccgcag tgggcgggcc acatcgaggc gcatgtcgcg ggctgggtgg ggctgtcggt 4920
cgcgctgggg cagcatctgg cggccggtgc cgcagaggtt caaggtgccc tggttgagcg 4980
cggaagatcg gcacgaaaga tcttggggga aatcgaggat tggcgggttg tggaatccgt 5040
ggacgagccc agtgccatca ccaccctgga gcccgccgac gacgtggacg tcgtcgcggt 5100
gcgggcgcgc ctcatcgcgg aacacgcgat cgtgacgacc gctgccgaga ccgtgcgcgc 5160
gccgttcgag atgaccaggc cggtgctgcg gatcagcccg cacgtcgacg gcaccgacga 5220
tgagttggag cagcttcggg ccgtgttgga ggcggtctaa 5260
<210> 3
<211> 5524
<212> DNA
<213>artificial sequence
<400> 3
gtgacgtcac cggaggcgat cgccgaccag ctggcgcgga cgcgggcgcg gacgctgcgg 60
ctggtcgact tcgacgacga cgagctgcgc cggcagtacg acccgttgat gagcccgctg 120
gtgtgggatc tcgcgcacat cggccagcag gaggagctgt ggctgttgcg gggcggcgac 180
cccgcccggc ccgggatgtt gccgccggct gtcgagggcc tctatgacgc gttcgtgcac 240
tcccgcgcca gccgggtcga cctgccgctg ctgtcgccgg agcaggcccg cgcctactgc 300
cgcacggtgc gcgcggcggt gctcgacacc ctggatgcgt tgcccgacga ccccgacgcc 360
gccttcgtgt acgcgatggt ggtcagccac gagaaccagc acgacgaaac catgctgcag 420
gcgctgaacc tgcgcagcgg cgcgccgctg ctgcgcgaca cgtcggtgct gccggccggc 480
cggcccgaac tggccggcac ctcggtgctg gtgcccggcg gcgagttcgt cctcggggtg 540
gacgccgccg acgaacccga gtcgctggac aacgaacgcc gggcgcacgt gctcgacctg 600
cccgcgttcc ggatcggcac ggttccggtg accaacggcg aatggcagca gttcgtcgcc 660
gacggcggct acgacgagcc gcggtggtgg tcgcggcgcg gctggcagca ccgccaggcc 720
gcgggcctga cggcgcccca gttctggcac cccgacgcgc gcacccgcac ccggttcggc 780
catgtcgaag acatcccggc cgacgaaccg gtgcagcacg tcagcttttt cgaggccgag 840
gcgtacgcgg cctgggccgg tgcccggctg cccaccgaga tggagtggga gaaggcctgc 900
gtgtgggatc cgtccaccgg cacccggcgc cgctacccgt ggggagcgac gccgccatcg 960
ccggccgtgg cgaacctggg cggcgccgcg ctgcgccccg cccccgtcgg cgcctacccg 1020
gccggcgcct cggcgtgcgg ggccgaacag atgctcggcg acgtgtggga gtggacgacg 1080
tccccgctgc ggccctggcc gggcttcgcg ccgatgctct acgaacgcta ttcgcagccg 1140
ttctttgatg gcgactaccg ggtgctgcgc ggcggctcgt gggcggtgga gccgggcatc 1200
ctgcggccca gcttccgcaa ctgggatcac ccgtaccggc gccagatctt cgccggtgtc 1260
cggttggcct gggacgtgcc tggcgcgcgg gaccaccgct gaaagagagg aatgtacaca 1320
tgacgttcgc cccgagcact gcggcggctt cgcggccggc cggtggcgac ccggcggccg 1380
agctgaggat cgccgagctg accgactccg cggccgtcgc gcagtacatc gcccggggct 1440
gtctggcgga cgctccactg ggccgggtgg gtctggaagt cgaagcccac tgccacgacc 1500
cgcaagaccc gtgccgccgt ccgggctggg acgagatcgc cgcggtgctc gacgcgctgc 1560
ccgcgctgcc cggcggcagc cgggtcacgg tggagccggg cggggccgtc gagctgtccg 1620
gcccgccggc cgacggcgtg ctggcggcca tcgacgccat gcgccgcgac caggccgtgc 1680
tgcggcccgc attcgccggg gccgggctgg gtctggtgtt tttgggcgcc gacccgctgc 1740
ggccgcccca gcggatcaac cccggcgccc gctaccgcgc catggaacgg ttcttcgcca 1800
ccagccgctc cggcgaggcg ggcgcggcaa tgatgacatc cacggcgtcg attcaggtca 1860
acgtggacgc cgggccgcgg gaaggctggg cggcgcgggt gcggctggca catgccttgg 1920
gccccaccat gattgcgatg acggccaact cgccgatgct ggacggcgat ttcaccggct 1980
gggtgtccag ccggcagcgg gtgtgggggc ggatggactc ggcgcgctgc ggcccagtcc 2040
tgggcgccag cggcgacgac ccgggcaccg actgggcgcg ctacgcgctc aaggccccgg 2100
tgatgttggt gcacaccccg gacgccgagg cggtcacgcg ctgggtgccg ttcgccgact 2160
gggtggacgg gcgggtgctg ctgggcggcc gccggcccac tgtcgccgac ctggagtacc 2220
acctgaccac gctgttcccg ccggtgcgcc cgcggcagtg gctggagatc cgctacctgg 2280
acagcgtgcc cgacaccctg tggcccgccg tggtgttcac cctggtcgcg ctgctcgacg 2340
acccggtcgc cgccggcctg gcggcccggg ccgtggaacc ggtggccacc gcctgggacg 2400
tcgcggcccg ggtggggctg cgggaccgac ggctgtacgc ggcggccaac gcctgcctgg 2460
cgatcgccgc cgagcgggcg ccggccgaac tcgccgagcc gatgcggcgg ctggtccgcc 2520
tcgtcgaaca gggccggtgt cccggcgacg acttcgccga ccaggtcatc gagcacggca 2580
tcgcggcgtc ggtttcccgg gccgcgcgcg gcccgcaagg ggggccgtga aagagaggaa 2640
tgtacacatg tgccggcatc tgggatggct gggggccgac gccacggtct cctcgctgat 2700
gctcgagccg ccgttcgggc tgcgggtgca gtcctacgcg ccgcgccggc agaaacacgg 2760
cctgctcaac gccgacggtt ggggcgcagg gtttttcgac gcggaggtgc cgcgccgctg 2820
gcgcagcccg gcgccgctgt ggggtgacgt gtccttcgag tccgtcgcgc cggcgctgca 2880
cagccactgc gtggtggccg cggtgcgctc ggcgacggtg ggcatgccga tcgagatcag 2940
cgccaccgca ccgttcaccg acggccggtg gctgctgtcg cacaacgggg tggtcgaccg 3000
gtctgtgctg ccggtgacct cgcgggctga atccgtttgc gacagcgcga tattggcggc 3060
agtcatcttg gagcggggcc tggacgcact cggcgaggtg atcgtcgaga tcggcgcggc 3120
cgacccggcg gcccggctta acatactggc cgccaacggt tctcggctgc tggccacggc 3180
ctggggggac accctgtcca tcctgcgccg cgccgacggt gtggtggtgg ccagtgaacc 3240
ctacgacgac gactccgact gggaggacgt gccggaccgc cacctggtcg atgtcacggc 3300
cgacggtgtc acgctgacac cgctggatca tccgaaggga ttttgaaaga gaggaatgta 3360
cacatgacgc tgtcgctgtc caaccacctc gccgccgact cggcccacca cgcgttgcgc 3420
cgcgacgtgc tcgacgggct gcggcgtaca ccgaagtcgt tgccgcccaa gtggttctac 3480
gattcggtgg gtagtgacct gttcgaccag atcacccgcc tgccggaata ctatccgacc 3540
cgcgccgagg ccgagatcct gcgcgcgcag gcgcccggca tcgcctcggc cagcgaggcc 3600
gacaccctgg tcgaattggg cagcggcacc tcggagaaga cccgggtgct gctcgacgcg 3660
ctgcgcgagc ggggcgcgct gcgcaggtac gtcccgttcg acgtcgacgc cggcatcctg 3720
tcggcatccg ccgccgccat ccagcgcgaa tacgccggca tcgaaatcca ggccgtctgc 3780
ggcgatttcg aggagcacct gaccgagatc cccgaaggcg ggcgacggct gttcgtgttt 3840
ctgggctcga cgatcggcaa cctgacgccc gggccgcgcg cgcagttcct ggccgcgctg 3900
tcggcccaga tgcggcccgg cgacagcctg ttgctgggca ccgacctggt caaggacacc 3960
gagcggctgg tgcgcgccta cgacgacgcc gccggggtga cggcggcgtt caaccgcaac 4020
gtgctcgcgg tgataaaccg ggaactggac gcggatttcg acgtcgacgc cttcgagcac 4080
gtggcccgct ggaacgcgga cgaggaacgc atcgagatgt ggctgcgcgc gaccgtgcgc 4140
cagcgggtgc gggtcggtgc gctcgggctg acggtggact tccgggcggg tgaggagatg 4200
ctcaccgagg tgtcgtgcaa gttccgcccg gagcgggtga gtgccgagct ggccgccgcc 4260
ggattgcgtc gcacccgttg gtggaccgac ggcgcggagg acttcgggtt gtcgttggcc 4320
gtcaagtgaa agagaggaat gtacacgtga gcggcggcac cccgctggcc gacacgtggc 4380
ggtcggcccg gccgccggcg gcgggtctgc acctcgacag cgcggcctgt tcccggcaga 4440
gcttcgcggt gatcgaggcc accgcccagc acgcccgcaa cgaggccgag ctcggcggct 4500
acgtggccgc cgaggcggcc gcaccggtgc tcgacgccgg gcggttcgca ttcaccgcgc 4560
tgaccggcat gaccgacgcc gaggcggtgt tcaccaccgg ctcgctgcac gccctggacc 4620
tgctgctggg cagctggccg gccgagcgcc gcaccctggc ctgcctgccc ggcgagtacg 4680
gccccaacct ggccgtgatg accgcccacg gattcgaccg ttggctgctg ccgaccctgg 4740
ccgacggccg ggtggcgctg gacgacgcgg cgctggccct gcagaccgac ccgccggacc 4800
tggtccacct gaccacgatt ggcagccaca gcggtgtggc gcaaccggtt tcgatgatcg 4860
cccggctctg ccgggagctg gggctgccgc tggtcgtcga cgcggcgcag gcgctgggtc 4920
aggtggactg cacggccggc gccgacgtga cctactcgtc gtcgcggaag tggatcgcgg 4980
ggccgcgcgg agtcgggatg ctcgcggtgc gccgggagct gatggaaaac ctgcacccga 5040
ggctggcggc gccggactgg gcgccgggct ccacggtggc tcagcaactc gagttcggtg 5100
aggccaatat cgctgcgcga gtggggtttt cgctggcgct gggcgaatac ctggggtacg 5160
ggccggccgc cgtctgcgcg cggctggccg agctgggcgg cctgagccgc ggcgtgctga 5220
ccgacgtgac cggctgggcg gtcgtcgaag acgccgaaga gcccagtgcc atcaccactt 5280
tggcgcccac cgacggcgca gacccgcggg cggtgcggga ctggttgctc gccgagcggc 5340
ggatcctgac caccttcgtc ggcgtgcagc gcgcgccgtt gcggctcacc gcgccggtgt 5400
tgcggatctc gccgcacctg gacaccaccg ccgaggacct ggagaccttc gccgaagcgc 5460
tcatcgccgc gacggcggcg acctcggcct gatcatcgcc gcgacggcgg cgacctcggc 5520
ctga 5524
<210> 4
<211> 5613
<212> DNA
<213>artificial sequence
<400> 4
gtgacgtcac cggaggcgat cgccgaccag ctggcgcgga cgcgggcgcg gacgctgcgg 60
ctggtcgact tcgacgacga cgagctgcgc cggcagtacg acccgttgat gagcccgctg 120
gtgtgggatc tcgcgcacat cggccagcag gaggagctgt ggctgttgcg gggcggcgac 180
cccgcccggc ccgggatgtt gccgccggct gtcgagggcc tctatgacgc gttcgtgcac 240
tcccgcgcca gccgggtcga cctgccgctg ctgtcgccgg agcaggcccg cgcctactgc 300
cgcacggtgc gcgcggcggt gctcgacacc ctggatgcgt tgcccgacga ccccgacgcc 360
gccttcgtgt acgcgatggt ggtcagccac gagaaccagc acgacgaaac catgctgcag 420
gcgctgaacc tgcgcagcgg cgcgccgctg ctgcgcgaca cgtcggtgct gccggccggc 480
cggcccgaac tggccggcac ctcggtgctg gtgcccggcg gcgagttcgt cctcggggtg 540
gacgccgccg acgaacccga gtcgctggac aacgaacgcc gggcgcacgt gctcgacctg 600
cccgcgttcc ggatcggcac ggttccggtg accaacggcg aatggcagca gttcgtcgcc 660
gacggcggct acgacgagcc gcggtggtgg tcgcggcgcg gctggcagca ccgccaggcc 720
gcgggcctga cggcgcccca gttctggcac cccgacgcgc gcacccgcac ccggttcggc 780
catgtcgaag acatcccggc cgacgaaccg gtgcagcacg tcagcttttt cgaggccgag 840
gcgtacgcgg cctgggccgg tgcccggctg cccaccgaga tggagtggga gaaggcctgc 900
gtgtgggatc cgtccaccgg cacccggcgc cgctacccgt ggggagcgac gccgccatcg 960
ccggccgtgg cgaacctggg cggcgccgcg ctgcgccccg cccccgtcgg cgcctacccg 1020
gccggcgcct cggcgtgcgg ggccgaacag atgctcggcg acgtgtggga gtggacgacg 1080
tccccgctgc ggccctggcc gggcttcgcg ccgatgctct acgaacgcta ttcgcagccg 1140
ttctttgatg gcgactaccg ggtgctgcgc ggcggctcgt gggcggtgga gccgggcatc 1200
ctgcggccca gcttccgcaa ctgggatcac ccgtaccggc gccagatctt cgccggtgtc 1260
cggttggcct gggacgtgcc tggcgcgcgg gaccaccgct gaaagagagg aatgtacaca 1320
tgacgttcgc cccgagcact gcggcggctt cgcggccggc cggtggcgac ccggcggccg 1380
agctgaggat cgccgagctg accgactccg cggccgtcgc gcagtacatc gcccggggct 1440
gtctggcgga cgctccactg ggccgggtgg gtctggaagt cgaagcccac tgccacgacc 1500
cgcaagaccc gtgccgccgt ccgggctggg acgagatcgc cgcggtgctc gacgcgctgc 1560
ccgcgctgcc cggcggcagc cgggtcacgg tggagccggg cggggccgtc gagctgtccg 1620
gcccgccggc cgacggcgtg ctggcggcca tcgacgccat gcgccgcgac caggccgtgc 1680
tgcggcccgc attcgccggg gccgggctgg gtctggtgtt tttgggcgcc gacccgctgc 1740
ggccgcccca gcggatcaac cccggcgccc gctaccgcgc catggaacgg ttcttcgcca 1800
ccagccgctc cggcgaggcg ggcgcggcaa tgatgacatc cacggcgtcg attcaggtca 1860
acgtggacgc cgggccgcgg gaaggctggg cggcgcgggt gcggctggca catgccttgg 1920
gccccaccat gattgcgatg acggccaact cgccgatgct ggacggcgat ttcaccggct 1980
gggtgtccag ccggcagcgg gtgtgggggc ggatggactc ggcgcgctgc ggcccagtcc 2040
tgggcgccag cggcgacgac ccgggcaccg actgggcgcg ctacgcgctc aaggccccgg 2100
tgatgttggt gcacaccccg gacgccgagg cggtcacgcg ctgggtgccg ttcgccgact 2160
gggtggacgg gcgggtgctg ctgggcggcc gccggcccac tgtcgccgac ctggagtacc 2220
acctgaccac gctgttcccg ccggtgcgcc cgcggcagtg gctggagatc cgctacctgg 2280
acagcgtgcc cgacaccctg tggcccgccg tggtgttcac cctggtcgcg ctgctcgacg 2340
acccggtcgc cgccggcctg gcggcccggg ccgtggaacc ggtggccacc gcctgggacg 2400
tcgcggcccg ggtggggctg cgggaccgac ggctgtacgc ggcggccaac gcctgcctgg 2460
cgatcgccgc cgagcgggcg ccggccgaac tcgccgagcc gatgcggcgg ctggtccgcc 2520
tcgtcgaaca gggccggtgt cccggcgacg acttcgccga ccaggtcatc gagcacggca 2580
tcgcggcgtc ggtttcccgg gccgcgcgcg gcccgcaagg ggggccgtga ggcgcgcccc 2640
tccttgacac tgaatttagc atgtgatata attaacttaa tattctaccc aagcttataa 2700
aagagcactg ttgggcgtga gtggaggcgc cggaaaaaag catcgaaaaa aaagagagga 2760
atgtacacat gtgccggcat ctgggatggc tgggggccga cgccacggtc tcctcgctga 2820
tgctcgagcc gccgttcggg ctgcgggtgc agtcctacgc gccgcgccgg cagaaacacg 2880
gcctgctcaa cgccgacggt tggggcgcag ggtttttcga cgcggaggtg ccgcgccgct 2940
ggcgcagccc ggcgccgctg tggggtgacg tgtccttcga gtccgtcgcg ccggcgctgc 3000
acagccactg cgtggtggcc gcggtgcgct cggcgacggt gggcatgccg atcgagatca 3060
gcgccaccgc accgttcacc gacggccggt ggctgctgtc gcacaacggg gtggtcgacc 3120
ggtctgtgct gccggtgacc tcgcgggctg aatccgtttg cgacagcgcg atattggcgg 3180
cagtcatctt ggagcggggc ctggacgcac tcggcgaggt gatcgtcgag atcggcgcgg 3240
ccgacccggc ggcccggctt aacatactgg ccgccaacgg ttctcggctg ctggccacgg 3300
cctgggggga caccctgtcc atcctgcgcc gcgccgacgg tgtggtggtg gccagtgaac 3360
cctacgacga cgactccgac tgggaggacg tgccggaccg ccacctggtc gatgtcacgg 3420
ccgacggtgt cacgctgaca ccgctggatc atccgaaggg attttgaaag agaggaatgt 3480
acacatgacg ctgtcgctgt ccaaccacct cgccgccgac tcggcccacc acgcgttgcg 3540
ccgcgacgtg ctcgacgggc tgcggcgtac accgaagtcg ttgccgccca agtggttcta 3600
cgattcggtg ggtagtgacc tgttcgacca gatcacccgc ctgccggaat actatccgac 3660
ccgcgccgag gccgagatcc tgcgcgcgca ggcgcccggc atcgcctcgg ccagcgaggc 3720
cgacaccctg gtcgaattgg gcagcggcac ctcggagaag acccgggtgc tgctcgacgc 3780
gctgcgcgag cggggcgcgc tgcgcaggta cgtcccgttc gacgtcgacg ccggcatcct 3840
gtcggcatcc gccgccgcca tccagcgcga atacgccggc atcgaaatcc aggccgtctg 3900
cggcgatttc gaggagcacc tgaccgagat ccccgaaggc gggcgacggc tgttcgtgtt 3960
tctgggctcg acgatcggca acctgacgcc cgggccgcgc gcgcagttcc tggccgcgct 4020
gtcggcccag atgcggcccg gcgacagcct gttgctgggc accgacctgg tcaaggacac 4080
cgagcggctg gtgcgcgcct acgacgacgc cgccggggtg acggcggcgt tcaaccgcaa 4140
cgtgctcgcg gtgataaacc gggaactgga cgcggatttc gacgtcgacg ccttcgagca 4200
cgtggcccgc tggaacgcgg acgaggaacg catcgagatg tggctgcgcg cgaccgtgcg 4260
ccagcgggtg cgggtcggtg cgctcgggct gacggtggac ttccgggcgg gtgaggagat 4320
gctcaccgag gtgtcgtgca agttccgccc ggagcgggtg agtgccgagc tggccgccgc 4380
cggattgcgt cgcacccgtt ggtggaccga cggcgcggag gacttcgggt tgtcgttggc 4440
cgtcaagtga aagagaggaa tgtacacgtg agcggcggca ccccgctggc cgacacgtgg 4500
cggtcggccc ggccgccggc ggcgggtctg cacctcgaca gcgcggcctg ttcccggcag 4560
agcttcgcgg tgatcgaggc caccgcccag cacgcccgca acgaggccga gctcggcggc 4620
tacgtggccg ccgaggcggc cgcaccggtg ctcgacgccg ggcggttcgc attcaccgcg 4680
ctgaccggca tgaccgacgc cgaggcggtg ttcaccaccg gctcgctgca cgccctggac 4740
ctgctgctgg gcagctggcc ggccgagcgc cgcaccctgg cctgcctgcc cggcgagtac 4800
ggccccaacc tggccgtgat gaccgcccac ggattcgacc gttggctgct gccgaccctg 4860
gccgacggcc gggtggcgct ggacgacgcg gcgctggccc tgcagaccga cccgccggac 4920
ctggtccacc tgaccacgat tggcagccac agcggtgtgg cgcaaccggt ttcgatgatc 4980
gcccggctct gccgggagct ggggctgccg ctggtcgtcg acgcggcgca ggcgctgggt 5040
caggtggact gcacggccgg cgccgacgtg acctactcgt cgtcgcggaa gtggatcgcg 5100
gggccgcgcg gagtcgggat gctcgcggtg cgccgggagc tgatggaaaa cctgcacccg 5160
aggctggcgg cgccggactg ggcgccgggc tccacggtgg ctcagcaact cgagttcggt 5220
gaggccaata tcgctgcgcg agtggggttt tcgctggcgc tgggcgaata cctggggtac 5280
gggccggccg ccgtctgcgc gcggctggcc gagctgggcg gcctgagccg cggcgtgctg 5340
accgacgtga ccggctgggc ggtcgtcgaa gacgccgaag agcccagtgc catcaccact 5400
ttggcgccca ccgacggcgc agacccgcgg gcggtgcggg actggttgct cgccgagcgg 5460
cggatcctga ccaccttcgt cggcgtgcag cgcgcgccgt tgcggctcac cgcgccggtg 5520
ttgcggatct cgccgcacct ggacaccacc gccgaggacc tggagacctt cgccgaagcg 5580
ctcatcgccg cgacggcggc gacctcggcc tga 5613
Claims (10)
1. a kind of engineered strain of fermentation synthesis erythrothioneine, which is characterized in that using Escherichia coli or bacillus subtilis as place
It is main, heterogenous expression egtABCDE gene cluster, or, express amino acid sequence protein as shown in k or l, the egtABCDE base
Because cluster amino acid sequence as e, f, g, h, i or j it is any shown in.
2. engineered strain according to claim 1, which is characterized in that the Escherichia coli include Escherichia coli MG1655,
Bacillus coli DH 5 alpha, Escherichia coli W3110 or e. coli bl21.
3. engineered strain according to claim 1, which is characterized in that the bacillus subtilis includes bacillus subtilis
168, bacillus subtilis W600 or bacillus subtilis W800.
4. engineered strain according to claim 1, which is characterized in that the egtABCDE gene cluster nucleotide sequence is such as
Shown in SEQ ID NO.1, SEQ ID NO.2, a, b, c or d are any.
5. engineered strain according to claim 1 to 4, which is characterized in that the expression vector of the engineered strain includes
pEBS。
6. engineered strain according to claim 1, which is characterized in that the nucleotide sequence of coded sequence k such as SEQ ID
Shown in NO.3.
7. engineered strain according to claim 1, which is characterized in that the nucleotide sequence of coded sequence l such as SEQ ID
Shown in NO.4.
8. a kind of method for producing erythrothioneine, which is characterized in that application engineered strain as claimed in claim 1 to 7 carries out
Fermentation.
9. method according to claim 8, which is characterized in that engineered strain is seeded to fermentation medium, is sent out at 35-38 DEG C
Ferment 40-60h adds methionine, hercynine, histidine, cysteine, the first sulphur ammonia of addition when cultivating 8-12h
Sour final concentration 2-8g/L, the hercynine final concentration 10-15g/L of addition, the histidine final concentration 15-20g/L of addition add
The cysteine final concentration 15-20g/L added.
10. application of the engineering bacteria as claimed in claim 1 to 7 in preparation erythrothioneine or product containing erythrothioneine.
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| CN115873886B (en) * | 2022-04-27 | 2023-11-10 | 武汉合生科技有限公司 | Method and carrier for biosynthesis of ergothioneine |
| CN118360229A (en) * | 2024-06-19 | 2024-07-19 | 山东君泰药业有限公司 | Recombinant bacillus subtilis, construction method thereof and production method of ergothioneine |
| CN118360229B (en) * | 2024-06-19 | 2024-09-10 | 山东君泰药业有限公司 | Recombinant bacillus subtilis, construction method thereof and production method of ergothioneine |
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