CN110358719B - Engineering strain for fermentation synthesis of ergothioneine and construction method thereof - Google Patents
Engineering strain for fermentation synthesis of ergothioneine and construction method thereof Download PDFInfo
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Abstract
The invention discloses an engineering strain for fermentation synthesis of ergothioneine and a construction method thereof, belonging to the technical field of biological engineering. The invention takes escherichia coli and bacillus subtilis as hosts and three shuttle plasmids pEBS of the bacillus subtilis, the escherichia coli and saccharomyces cerevisiae as expression vectors, and introduces egtABCDE gene clusters from different sources into the escherichia coli and the bacillus subtilis to carry out heterologous expression. Realizes the biosynthesis of ergothioneine. And then carrying out optimized expression on the egtABCDE gene cluster, wherein the optimized expression comprises codon optimization, promoter optimization and different combination expressions, simultaneously optimizing a culture medium and methionine, histidine betaine, histidine and cysteine which are added, and realizing the high-efficiency biosynthesis of ergothioneine through optimized expression and optimized culture conditions. The invention lays a foundation for the efficient fermentation preparation of ergothioneine by a microbial system, and is suitable for industrial production and application.
Description
Technical Field
The invention relates to an engineering strain for fermentation synthesis of ergothioneine and a construction method thereof, belonging to the technical field of biological engineering.
Background
Ergothioneine is a rare natural chiral histidine derivative thiol compound and has unique biological function and pharmacological activity. It not only has strong antioxidant activity: can remove free radicals, peroxynitrite, hypochlorous acid, chelate divalent metal ions, activate antioxidant enzyme, inhibit superoxide dismutase and the like, has the effects of resisting inflammation and protecting cells, and has wide application prospect and market prospect in the fields of medicines, foods, health care products, cosmetics, biotechnology and the like.
Ergothioneine was first isolated from ergot in 1909 and is also synthesized in microorganisms such as actinomycetes, cyanobacteria and Schizosaccharomyces, partial mycoses, streptomycetes, mycobacteria, and other bacterial species such as: bacillus subtilis, Escherichia coli, Proteus vulgaris and Streptococcus cannot be synthesized. Ergothioneine is also not biosynthesized by animals and plants, but is absorbed and accumulated by plants and animals and is widely distributed in cells and tissues of mammals. Ergothioneine is vital in human physiology, and a human must acquire from dietary sources and accumulate in specific tissues and cells such as liver, kidney, central nervous system, and red blood cells. Ergothioneine can be produced by chemical synthesis, extraction and biological fermentation synthesis. The synthesis of the L-ergothioneine by a chemical method is very difficult, the safety of the product is difficult to ensure, the synthesis raw materials are expensive, the synthesis cost is high, and the expected yield is not reached; the extraction method for producing ergothioneine has many difficulties, such as insufficient raw material sources, low content of the ergothioneine in the raw material, high extraction cost, drug residue and the like, and the biological fermentation synthesis method for producing the ergothioneine becomes the most potential production method along with the rapid development of biological sciences such as molecular biology, metabolic engineering and the like.
However, the existing reports show that the method for producing ergothioneine by constructing genetically engineered bacteria has not ideal effect. For example, the egt gene from Mycobacterium smegmatis can be used for heterologous expression in E.coli. The genes in the gene cluster were resolved and ligated into a number of plasmids, which were then transformed into E.coli, and the expression of the recombinant enzyme was optimized to increase Ergothioneine Production, although shake flask Production was only 24mg/L (Osawa R, Kamid T, Satoh Y, et al. heterologous and High Production of Ergotheine in Escherichia coli [ J ]. Journal of Agricultural & Food Chemistry 2017,66(5): 1191-1196.).
Therefore, the method for efficiently biosynthesizing the ergothioneine is provided, and has important application value for industrial production of the ergothioneine.
Disclosure of Invention
The first purpose of the invention is to provide an engineering strain for fermenting and synthesizing ergothioneine, which takes escherichia coli or bacillus subtilis as a host and expresses an egtABCDE gene cluster in a heterologous way, or expresses a protein with an amino acid sequence shown as k or l, wherein the amino acid sequence of the egtABCDE gene cluster is shown as any one of e, f, g, h, i or j.
In one embodiment of the invention, the Escherichia coli comprises Escherichia coli MG1655, Escherichia coli DH5 α, Escherichia coli W3110 or Escherichia coli BL 21.
In one embodiment of the present invention, the bacillus subtilis comprises bacillus subtilis 168, bacillus subtilis W600 or bacillus subtilis W800.
In one embodiment of the invention, the egtABCDE gene cluster is derived from Mycobacterium subsp.paratuberculosis strain.k 10, Mycobacterium icosium assiliensis strain 8WA6, Mycobacterium colombiensis CECT 3035, Mycobacterium marinum E11, Mycobacterium sp.KMS or Mycobacterium salmoniphilium.
In one embodiment of the invention, the egtABCDE gene cluster nucleotide sequence is as shown in any one of SEQ ID NO.1, SEQ ID NO.2, a, b, c or d. The nucleotide sequence of the coding sequence k is shown in SEQ ID NO. 3. The nucleotide sequence of the coding sequence l is shown in SEQ ID NO. 4.
In one embodiment of the present invention, the expression vector of the engineered strain is a triple shuttle plasmid pEBS of Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae (see Yang S, Liu Q, Zhang Y, et al. construction and Characterization of Broad-spectrum microorganisms for Synthetic Biology [ J ]. Acs Synthetic Biology,2017,7(1):287 and 291.).
The second purpose of the invention is to provide a method for producing ergothioneine, which applies the engineering strain to ferment.
In one embodiment of the invention, the engineering strain is inoculated to a fermentation medium, fermented at 35-38 ℃ for 40-60h, and methionine, histidine betaine, histidine and cysteine are added as required when the engineering strain is cultured for 8-12h, the final concentration of the added methionine is 2-8g/L, the final concentration of the added histidine betaine is 10-15g/L, the final concentration of the added histidine is 15-20g/L, and the final concentration of the added cysteine is 15-20 g/L.
The components of the fermentation medium used are (g/L): 2.5 parts of yeast powder, 5.0 parts of peptone and Na2HPO4 6.78，KH2PO43.0，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。
The third purpose of the invention is to provide the application of the engineering bacteria in the preparation of ergothioneine or products containing the ergothioneine.
The invention has the beneficial effects that:
the invention takes escherichia coli or bacillus subtilis as a host, takes three shuttle plasmids pEBS of the bacillus subtilis, the escherichia coli and saccharomyces cerevisiae as expression vectors, and introduces egtABCDE gene clusters from different sources into the escherichia coli and the bacillus subtilis to carry out heterologous expression. Realizes the biosynthesis of ergothioneine. And then performing optimized expression on the egtABCDE gene cluster, wherein the optimized expression comprises codon optimization, promoter optimization and different combined expression, so that an escherichia coli and bacillus subtilis recombinant strain with better ergothioneine production can be obtained respectively, and the efficient biosynthesis of the ergothioneine is realized through optimized expression and optimized culture conditions. The invention can ensure that the ergothioneine accumulation amount of 60h of the escherichia coli recombinant bacterium cultured on the shake flask reaches 437.6mg/L, and the ergothioneine accumulation amount of 60h of the bacillus subtilis recombinant bacterium cultured on the shake flask reaches 568.4mg/L, thus having potential and wide application value in industry.
Drawings
FIG. 1: the pEBS-egtABCDE gene cluster and the construction scheme of different combinations of genes contained in the egtABCDE gene cluster for optimizing expression.
FIG. 2: the ergothioneine accumulation amount of the recombinant Escherichia coli which heterologously expresses egtABCDE gene clusters from different sources is 60 hours after the recombinant Escherichia coli is cultured on a shake flask.
FIG. 3: the ergothioneine accumulation amount of the recombinant bacillus subtilis heterologously expressing the egtABCDE gene cluster from different sources is 60 hours after the recombinant bacillus subtilis is cultured on a shake flask.
FIG. 4: the ergothioneine accumulation amount of escherichia coli and bacillus subtilis recombinant bacteria which are optimally expressed by different combinations of genes contained in the egtABCDE gene cluster is cultured on a shake flask for 60 hours.
Detailed Description
Nucleotide sequence information related to the first embodiment
(1) Sequence a information is the egtABCDE gene cluster nucleotide sequence derived from Mycobacterium subsp.paratuberculosis str.k10, NCBI accession number: AE016958.1(322465to 327878), the amino acid sequence encoded by sequence (a) being sequence e;
(2) the sequence information of SEQ ID NO.1 is an egtABCDE gene cluster nucleotide sequence derived from Mycolibacter icosiumassiliensis strain 8WA6, and the amino acid sequence coded by SEQ ID NO.1 is a sequence f;
(3) the sequence b information is an egtABCDE gene cluster nucleotide sequence derived from Mycobacterium colombiense CECT 3035, and the NCBI number: CP020821.1(3701114to 3706623), the amino acid sequence encoded by sequence b is sequence g;
(4) sequence c information is the egtABCDE gene cluster nucleotide sequence from Mycobacterium marinum E11, NCBI accession number: HG917972.2(6005272to 6010682), wherein the amino acid sequence encoded by the sequence c is the sequence h;
(5) sequence (d) information is the egtaccde gene cluster nucleotide sequence derived from Mycobacterium sp. CP000518.1(5206948to 5212322), the amino acid sequence encoded by sequence d is sequence i;
(6) the sequence information of SEQ ID NO.2 is the gene cluster nucleotide sequence of the egtABCDE gene of Mycobacterium salmoniphilium strain D16Q15, and the amino acid sequence coded by SEQ ID NO.2 is the sequence j;
(7) the sequence information of SEQ ID NO.3 is a gene cluster BACDE nucleotide sequence, and the amino acid sequence coded by SEQ ID NO.3 is a sequence k;
(8) the sequence information of SEQ ID NO.4 is a nucleotide sequence of a gene cluster BA-PBSX-CDE, and the amino acid sequence coded by the SEQ ID NO.4 is a sequence l.
Second, construction of expression System
Amplifying the DNA fragment of the egtABCDE gene cluster by PCR using as templates the Mycobacterium avium subsp.parturculosis strain 10 genome, Mycobacterium icobacter icosium assilytics strain 8WA6 genome, Mycobacterium colombiensis CECT 3035 genome, Mycobacterium marinum E11 genome, Mycobacterium sp.KMS genome and Mycobacterium salmonitum genome, respectively, using as primers egtABCDE (mp) -F/egtABCDE (mp) -R, egtABCDE (mi) -F/egtABCDE (mp) -R, egtABCDE (mc) -F/egtABCDE (mc) -R, egtABCDE (mm) -F/egtABCDE mm) -pE R, egtABCDE (mk) -F/egtABCDE (mk) -R, egtABCDE (mp) -F/egtABCDE (mp) -pEBS, assembling the resulting primers with PCR-pEBS to produce PCR products (pEBS), (mK) -F/etABBCDE) and PCR products (pEmBS) using the PCR products obtained by PCR, pEBS-egtABCDE (mc), pEBS-egtABCDE (mm), pEBS-egtABCDE (mk), and pEBS-egtABCDE (ms). With the genome of Mycobacterium subsp.paratuberculosis str.k10 as a template, DNA fragments of the genes egtB (amplified using primers B (1) -F/B (1) -R), egtA (amplified using primers A (2) -F/A (2) -R), egtC (amplified using primers C (3) -F/C (3) -R), egtD (amplified using primers D (4) -F/D (4) -R), and egtE (amplified using primers E (5) -F/E (5) -R) were obtained by PCR amplification, and the resulting PCR products were assembled with a pEBS (amplified using primers pEBS-F/pEBS-R) backbone to produce pEBS-BACDE. Using pEBS-BACDE as template and primer PBSX-F/PBSX-R amplification of promoter p by PCRbsxDNA fragment, and the PCR product obtained was ligated with pEBS-BACDE (using primer AP)BSC-F/APBSC-R amplification) backbone Assembly pSTOP1622 to generate pEBS-BA-PBSXCDE, as shown in figure 1.
Third, construction of recombinant bacteria
1. The recombinant plasmids pEBS-egtABCDE (mp), pEBS-egtABCDE (mi), pEBS-egtABCDE (mc), pEBS-egtABCDE (mm), pEBS-egtABCDE (mk) and pEBS-egtABCDE (ms) were transferred into E.coli MG1655, E.coli DH5 alpha, E.coli W3110 and E.coli BL21, respectively. The Escherichia coli recombinant bacteria Mmp, Mmi, Mmc, Mmm, Mmk, Mms, Dmp, Dmi, Dmm, Dmk, Dms, Wmp, Wmi, Wmc, Wmm, Wmk, Wms, Bmp, Bmi, Bmc, Bmm, Bmk and Bms are obtained.
2. The recombinant plasmids pEBS-egtABCDE (mp), pEBS-egtABCDE (mi), pEBS-egtABCDE (mc), pEBS-egtABCDE (mm), pEBS-egtABCDE (mk) and pEBS-egtABCDE (ms) were transferred into Bacillus subtilis 168, Bacillus subtilis W600 and Bacillus subtilis W800, respectively. The Bacillus subtilis recombinant bacteria 168mp, 168mi, 168mc, 168mm, 168mk, 168ms, 600mp, 600mi, 600mc, 600mm, 600mk, 600ms, 800mp, 800mi, 800mc, 800mm, 800mk and 800ms are obtained.
3. Respectively transferring the recombinant plasmids pEBS-BACDE and pEBS-BA-P into bacillus subtilis 168 and escherichia coli DH5 alphaBSXCDE to obtain recombinant bacteria BS-BACDE, BS-BA-PBSX-CDE、EC-BACDE、EC-BA-PBSX-CDE。
Fourthly, processing samples and analyzing the yield of ergothioneine by HPLC
Taking 4mL of fermentation liquor, centrifuging at 12000r/min for 10min, using the supernatant for HPLC analysis of ergothioneine, suspending the precipitate with equal volume of 50% methanol, performing ultrasonication, centrifuging at 12000r/min for 10min, collecting the supernatant for HPLC analysis of ergothioneine HPLC, wherein the yield of ergothioneine is the sum of intracellular and extracellular ergothioneine contents.
HPLC analysis conditions mobile phase consisted of 0.1% formic acid/water (A) and 0.1% formic acid/acetonitrile (B). The gradient program was terminated at 2 min at 95% B, 10min at 40% B, 8.5 min at 95% B, and 12 min. The flow rate was 0.5ml/min and the amount of sample was 10. mu.L. Samples were analyzed using a Dionex UPLC Ultimate 3000(Sunnyvale, CA). The compounds were isolated on an Atlantis HILIC silica gel column (particle size 3.01 μm, diameter. times. length 2.1X 100 mm; Waters) and detected at 264 nm.
Fifth, primer information referred to in the examples
The primer information is shown in Table 1.
TABLE 1 primers
Example 124 Shake flask culture and ergothioneine content determination of recombinant E.coli strains heterologously expressing the egtABCDE Gene Cluster
Respectively selecting 24 strains of the escherichia coli recombinant strain for heterologous expression of the egtABCDE gene cluster constructed above and a control bacterium (a transformed pEBS empty plasmid) to perform monoclonal inoculation on 5mL of LB culture medium, adding kanamycin with the final concentration of 50 mu g/mL according to needs, culturing at the temperature of 200rpm and 37 ℃ for 10-12h, then transferring to a 250mL triangular flask according to the inoculation amount of 10%, wherein the culture medium is a fermentation culture medium, the liquid loading amount is 20mL, adding kanamycin with the final concentration of 50 mu g/mL according to needs, culturing at the temperature of 220rpm and 37 ℃, adding methionine, histidine betaine, histidine and cysteine when culturing for 10h, adding methionine with the final concentration of 2g/L, adding histidine betaine with the final concentration of 10g/L, adding histidine with the final concentration of 15g/L, and adding cysteine with the final concentration of 15g/L, the final liquid loading was 25mL (sterile deionized water was supplemented if insufficient). After 60h of incubation, samples were processed and analyzed for ergothioneine production by HPLC. The result is shown in figure 2, the recombinant bacteria can realize the heterologous synthesis of ergothioneine, wherein the recombinant bacteria Mmp has higher ergothioneine yield which reaches 335.3 mg/L.
Example 218 Shake flask culture and ergothioneine content determination of recombinant strain of Bacillus subtilis for heterologous expression of egtABCDE Gene Cluster
Respectively selecting 18 strains of heterogeneously expressed egtABCDE gene cluster bacillus subtilis recombinant strains constructed above and a control bacterium (a transformed pEBS empty plasmid) to perform monoclonal inoculation on 5mL of LB culture medium, adding kanamycin with the final concentration of 50 mu g/mL according to needs, culturing at the temperature of 200rpm and 37 ℃ for 10-12h, then transferring to a 250mL triangular flask according to the inoculation amount of 10 percent, wherein the culture medium is a fermentation culture medium, the liquid loading amount is 20mL, adding kanamycin with the final concentration of 50 mu g/mL according to needs, culturing at the temperature of 220rpm and 37 ℃, adding methionine, histidine betaine, histidine and cysteine when culturing for 10h, the final concentration of the added methionine is 2g/L, the final concentration of the added histidine betaine is 10g/L, the final concentration of the added histidine is 15g/L, and the final concentration of the added cysteine is 15g/L, the final liquid loading was 25mL (sterile deionized water was supplemented if insufficient). After 60h of incubation, samples were processed and analyzed for ergothioneine production by HPLC. As shown in FIG. 3, the recombinant bacteria can realize the heterologous synthesis of ergothioneine, wherein the yield of the ergothioneine of the recombinant bacteria 168mp is high and reaches 412.2 mg/L.
Example 3 Shake flask culture and ergothioneine content determination of recombinant bacteria of Escherichia coli and Bacillus subtilis with optimized expression of different combinations of genes contained in the egtABCDE Gene Cluster
Respectively selecting Escherichia coli, Bacillus subtilis recombinant bacteria and control bacteria (converted pEBS empty plasmids) contained in the egtABCDE gene cluster which are constructed in the way, performing single cloning and inoculation on 5mL of LB culture medium, adding kanamycin with the final concentration of 50 mu g/mL according to needs, culturing at the temperature of 200rpm and 37 ℃ for 10-12h, then transferring to a 250mL triangular shake flask according to the inoculation amount of 10 percent, wherein the culture medium is a fermentation culture medium, the liquid loading amount is 20mL, adding kanamycin with the final concentration of 50 mu g/mL according to needs, culturing at the temperature of 220rpm and 37 ℃, adding methionine, histidine betaine, histidine and cysteine when culturing for 10h, adding methionine with the final concentration of 8g/L, adding histidine betaine with the final concentration of 15g/L and adding histidine with the final concentration of 20g/L, the final concentration of added cysteine was 20 g/L. The final liquid loading was 25mL (sterile deionized water was supplemented if insufficient). After 60h of incubation, samples were processed and analyzed for ergothioneine production by HPLC. The result is shown in FIG. 4, the recombinant bacterium can realize the heterologous synthesis of ergothioneine, wherein the recombinant bacterium BS-BA-Pbsx-CDE has higher ergothioneine yield which reaches 568.4 mg/L.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> engineering strain for fermentation synthesis of ergothioneine and construction method thereof
<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 (9)
1. An engineering strain for fermenting and synthesizing ergothioneine is characterized in that escherichia coli or bacillus subtilis is used as a host, an egtABCDE gene cluster is expressed in a heterologous mode, or a protein with an amino acid sequence shown as k or l is expressed, wherein the amino acid sequence of the egtABCDE gene cluster is shown as any one of e, f, g, h, i or j; the nucleotide of the coding sequence e is shown as the 322465-327878 site of GenBank number AE 016958.1; the nucleotide of the coding sequence f is shown in SEQ ID NO. 1; the nucleotide sequence of the coding sequence g is shown as the 3701114-3706623 position of GenBank number CP 020821.1; the nucleotide of the coding sequence h is shown as position 6005272-6010682 of GenBank (HG 917972.2); the nucleotide of the coding sequence i is shown in GenBank number CP000518.1 No. 5206948-5212322; the nucleotide of the coding sequence j is shown as SEQ ID NO. 2; the nucleotide of the coding sequence k is shown in SEQ ID NO. 3; the nucleotide of the coding sequence l is shown in SEQ ID NO. 4.
2. The engineered strain of claim 1, wherein the escherichia coli comprises escherichia coli MG1655, escherichia coli DH5 a, escherichia coli W3110, or escherichia coli BL 21.
3. The engineered strain of claim 1, wherein the bacillus subtilis comprises bacillus subtilis 168, bacillus subtilis W600 or bacillus subtilis W800.
4. The engineered strain of any one of claims 1-3, wherein the expression vector of the engineered strain comprises pEBS.
5. The engineered strain of claim 1, wherein the nucleotide sequence of coding sequence k is shown in SEQ ID No. 3.
6. The engineered strain of claim 1, wherein the nucleotide sequence of coding sequence i is shown in SEQ ID No. 4.
7. A method for producing ergothioneine, which comprises fermenting with the engineered strain of any one of claims 1 to 6.
8. The method as claimed in claim 7, wherein the engineered strain is inoculated into a fermentation medium, fermented at 35-38 ℃ for 40-60h, and during the culture for 8-12h, methionine, histidine betaine, histidine and cysteine are added, the final concentration of added methionine is 2-8g/L, the final concentration of added histidine betaine is 10-15g/L, the final concentration of added histidine is 15-20g/L, and the final concentration of added cysteine is 15-20 g/L.
9. Use of the engineered bacterium of any one of claims 1-6 for the preparation of ergothioneine or products containing ergothioneine.
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