CN109486737A - A kind of recombination bacillus coli and its construction method of high yield L-Trp - Google Patents
A kind of recombination bacillus coli and its construction method of high yield L-Trp Download PDFInfo
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Abstract
The invention discloses the recombination bacillus colis and its construction method of a kind of high yield L-Trp, belong to genetic engineering technology field.The present invention is using Escherichia coli CICC 10303 as starting strain, and using CRISPR-Cas9 gene editing technology, replacement shikimate kinase encoding gene aroK promoter is strong promoter T7；The promoter for replacing prephenate dehydrogenase encoding gene pheA is weak promoter tac；Tryptophan transporter encoding gene mtr is knocked out, prevents fermentation process tryptophan transfer from transporting back intracellular.The Recombinant organism of accumulation L-Trp is finally obtained, yield reaches 36g/L, produces L-Trp for further metabolic engineering Escherichia coli and lays a good foundation.
Description
Technical field
The present invention relates to the recombination bacillus colis and its construction method of a kind of high yield L-Trp, belong to genetic engineering technology
Field.
Background technique
L-Trp is one of eight kinds of amino acid needed by human as a kind of very important aromatic amino acid.?
In organism, L-Trp can synthesize the important life such as serotonine, niacin, pigment, alkaloid, coenzyme, heteroauxin
Active substances play an important role to the growth and development of humans and animals, are widely used in food, medicine, feed etc..L- color
The production of propylhomoserin relies primarily on chemical synthesis and protein Hydrolyze method earliest, but there is material sources to have for these methods
The disadvantages of limit, period length, complex process, thus be gradually eliminated.Since low in cost, raw material sources are extensive, environmental pollution is small
The features such as, microbial method production L-Trp, which has been obtained, to be widely applied.
Currently, microbial method production L-Trp there are yield it is not high, complicated for operation the disadvantages of.2001, Wang Jian etc. was used
Dithyl sulfate (DES) is used as mutagens, has chosen one plant of tyrosine, phenylalanine two nutrient defect strain starting strain, passes through
After multiple mutagenic treatment, one plant of L-Trp production bacterial strain is selected, the 64h that continuously ferments in batches after, L-Trp accumulation
It can achieve 7.28g/L.2007, Chen Junfeng etc. utilized the separation method of multiple mutagenesis, was with one plant of corynebacterium glutamicum
Starting strain obtains the L. tryptophan Analogue resistant mutant of one plant of phenylalanine and tyrosine double auxotroph,
After shake flask fermentation 96h, L-Trp accumulation reaches 10.82g/L.
Escherichia coli (Escherichia coli) are applied to industrial fermentation and produce each amino acid.Therefore, it transports
It is the effective way for producing L-Trp with metabolic engineering means building recombination bacillus coli.Currently, being mediated using expression plasmid
Amino acid synthesis pathway and competition approach in key enzyme gene overexpression or reduction be to Escherichia coli carry out gene change
The main means made.However mediate gene overexpression that will introduce antibiotic resistance in Bacillus coli cells using expression plasmid
Gene simultaneously adds certain antibiotic during the growth process, causes doubt of the people to antibiotic usage.Therefore it provides a kind of peace
The method for efficiently carrying out genetic modification to Escherichia coli entirely, has amino acids production field important meaning.
Summary of the invention
The first purpose of the invention is to provide a kind of recombination bacillus colis for producing L-Trp, and shikimate kinase is substituted
Gene aroK promoter is T7 promoter, has knocked out tryptophan transporter encoding gene mtr and prephenate dehydrogenase volume is substituted
The promoter of code gene pheA is tac promoter.
In one embodiment of the invention, the nucleotide sequence of the tac promoter such as SEQ ID NO.1 institute
Show.
In one embodiment of the invention, the nucleotide sequence of shikimate kinase aroK gene such as SEQ ID NO.2
It is shown.
In one embodiment of the invention, the nucleotide sequence of tryptophan transporter encoding gene mtr such as SEQ ID
Shown in NO.3.
In one embodiment of the invention, the nucleotide sequence of prephenate dehydrogenase encoding gene pheA such as SEQ ID
Shown in NO.4.
It in one embodiment of the invention, is to carry out genome editor to Escherichia coli CICC 10303 to obtain.
In one embodiment of the invention, the genome editor is carried out using CRISPR-Cas9 technology.
A second object of the present invention is to provide the construction methods of above-mentioned recombination bacillus coli, and the method includes following
Step:
1) building T7 promoter replacement recombinant fragment, tac promoter replacement recombinant fragment and knockout mtr genetic recombination piece
Section: after the fusion of Escherichia coli shikimate kinase encoding gene aroK gene cluster initiation codon upstream and downstream homology arm sequence, draw
Enter T7 promoter, obtains recombinant fragment T7AROK；The initiation codon upstream and downstream of prephenate dehydrogenase encoding gene pheA is same
After the fusion of source arm sequence, promoter tac is introduced, segment TACPHE is obtained；Tryptophan transporter encoding gene mtr upstream and downstream is same
Source arm fusion, obtains segment MTRD.
2) construction recombination plasmid: by segment T7AROK, TACPHE, MTRD respectively with the linearized vector PCR containing sgRNA
Connection, respectively obtains recombinant plasmid of the recombinant plasmid containing T7AROK, the recombinant plasmid containing TACPHE, the recombinant plasmid containing MTRD；
3) it constructs recombination bacillus coli: Escherichia coli CICC 10303 will be converted containing the plasmid of cas9 albumen, obtained big
Enterobacteria CICC 10303-cas9；Then by the recombinant plasmid transformed Escherichia coli CICC 10303-cas9 containing T7AROK, obtain
To recombination bacillus coli CICC 10303-aroKT；By the recombinant plasmid transformed Escherichia coli CICC 10303- containing TACPHE
AroKT obtains recombination bacillus coli CICC 10303-pheAT；By the recombinant plasmid transformed Escherichia coli CICC containing MTRD
10303-pheAT obtains recombination bacillus coli CICC 10303-mtrD；After removing exogenous plasmid, recombination bacillus coli is obtained
CICC 10303-TRYP, the exogenous plasmid include the recombinant plasmid containing T7AROK, the recombinant plasmid containing TACPHE and containing MTRD
Recombinant plasmid.
In one embodiment of the invention, the plasmid containing cas9 albumen includes pCas9.
In one embodiment of the invention, the linearized vector containing sgRNA includes pTT7A, pTPH or pTMT.
In one embodiment of the invention, the nucleotide sequence of pTT7A is as shown in SEQ ID NO.5.
In one embodiment of the invention, the nucleotide sequence of pTPH is as shown in SEQ ID NO.6.
In one embodiment of the invention, the nucleotide sequence of pTMT is as shown in SEQ ID NO.7.
In one embodiment of the invention, the nucleotide sequence of pCas9 is as shown in SEQ ID NO.8.
Third object of the present invention is to provide application of the above-mentioned recombination bacillus coli in production L-Trp.
In one embodiment of the invention, the application includes that will cultivate 22- at 34-38 DEG C in plating medium
The single colonie of 26h is seeded to seed culture medium, cultivates 5-8h, then under the conditions of 34-38 DEG C, 180-220rpm with 20% inoculation
Measure inoculation fermentation culture medium, 34-38 DEG C, 10-14h is cultivated under the conditions of 180-220rpm, 0.1mM IPTG induces 45-50h.
Fourth object of the present invention is to provide above-mentioned recombination bacillus coli in feed, pharmacy, health care product or food work
Application in industry.
The present invention be using Escherichia coli CICC 10303 as starting strain, using CRISPR-Cas9 gene editing technology,
Replacement shikimate kinase encoding gene aroK promoter is strong promoter T7；Replacement prephenate dehydrogenase encoding gene pheA's opens
Mover is weak promoter tac；Tryptophan transporter encoding gene mtr is knocked out, prevents fermentation process tryptophan transfer from transporting back intracellular.Most
The Recombinant organism of accumulation L-Trp is obtained eventually, and yield reaches 36g/L, is further metabolic engineering large intestine
Bacillus production L-Trp is laid a good foundation.Recombination bacillus coli construction method provided by the invention is simple, is easy to use, has
Good application prospect.
Detailed description of the invention
Fig. 1: plasmid map, A:pTT7A；B:pTPH；C:pTMT.
The yield of Fig. 2: the recombination bacillus coli CICC 10303-TRYP tryptophan under different condition of culture.
Fig. 3: the promoter for only replacing prephenate dehydrogenase encoding gene pheA is tac promoter, is issued in 35 DEG C of conditions
Ferment recombinant bacterium produces L-Trp.
Fig. 4: the promoter for only replacing prephenate dehydrogenase encoding gene pheA is tac promoter, is issued in 35 DEG C of conditions
Ferment recombinant bacterium produces L-Trp.
Fig. 5: only knocking out tryptophan transporter encoding gene mtr, issues ferment recombinant bacterium production L- color in 35 DEG C of temperature conditions
Propylhomoserin.
Specific embodiment
Recombination bacillus coli seed culture and fermentation medium:
Plating medium (g/L): tryptone 10, yeast powder 5, sodium chloride 5, agar powder 15, pH is adjusted to 7.0.
Seed culture medium (g/L): glucose 30, yeast powder 15.2, dipotassium hydrogen phosphate 24, ammonium sulfate 5, potassium dihydrogen phosphate
9.6, epsom salt 1.
Fermentation medium (g/L): glucose 60, epsom salt 16, ammonium sulfate 24, yeast extract powder 10, citric acid three
Two water object 16 of sodium, dipotassium hydrogen phosphate 5.6.
Condition of culture: the plate picking single bacterium for cultivating for 24 hours at 37 DEG C is dropped down into seed culture medium, in 37 DEG C, 220rpm item
Cultivate 10h under part, 10% inoculum concentration inoculation fermentation culture medium, 35 DEG C, cultivate 42h under the conditions of 220rpm.
The measuring method of L-Trp:
1. sample treatment:
1mL fermentation liquid is taken, centrifugation removal thallus takes supernatant.After supernatant is suitably diluted with 5% trichloroacetic acid
12000rpm/min is centrifuged 10min, the membrane filtration that then via hole diameter is 0.22 μm.
2. analysis method: OPA boric acid pre-column derivatization, 13.768min eluting peak are tryptophan
3. chromatographic condition:
(1) chromatographic column: chromatographic column C18 (250 × 4.6) mm
(2) column temperature: 40 DEG C
(3) mobile phase A: weighing 3.01g anhydrous sodium acetate in beaker, adds deionized water dissolving and is settled to 1L, then
200 μ L triethylamines are added, pH is transferred to 7.20 ± 0.05 with 5% acetic acid；5mL tetrahydrofuran is added after suction filtration, mixes standby
With.Mobile phase B: 3.01g anhydrous sodium acetate is weighed in beaker；Add deionized water dissolving and is settled to 200mL；With 5% acetic acid
PH is transferred to 7.20 ± 0.05；It is spare after mixing after suction filtration, then to this solution addition 400mL acetonitrile and 400mL methanol.
(4) flow velocity: 1.0ml/min；
(5) UV detector: 338nm；
(6) column temperature: 40 DEG C；
In following embodiments, what is explained is all using conventional experimental methods of molecular biology more.
The building of 1 recombinant fragment of embodiment
According to genome of E.coli sequence information, design primer pT-aroK-1R and pT-aroK-2F, pT-aroK-1F,
PT-aroK-2R (is shown in Table 1), is expanded from 10303 genome of Escherichia coli CICC using above-mentioned four kinds of primers and is started with T7
AroK DNA homolog each 600bp of arm gene order of son obtains weight by 2 sections of amplified fragments of gained by fusion DNA vaccine technological incorporation
Group segment T7AROK；
According to genome of E.coli sequence information, design primer pT-pheA-1F, pT-pheA-1R, pT-pheA-2F and
PT-pheA-2R expands pheA gene start codon two sides using above-mentioned primer from 10303 genome of Escherichia coli CICC
Each 600bp of homology arm gene order obtains recombinant fragment by 2 sections of amplified fragments of gained by fusion DNA vaccine technological incorporation
TACPHE。
According to genome of E.coli sequence information, design primer pT-mtr-1F, pT-mtr-1R, pT-mtr-2F and pT-
Mtr-2R expands mtr gene two sides homology arm gene order using above-mentioned primer from 10303 genome of Escherichia coli CICC
Each 600bp obtains recombinant fragment MTRD by 2 sections of amplified fragments of gained by fusion DNA vaccine technological incorporation.
1 primer table of table
The building of 2 recombinant plasmid of embodiment
According to carrier pTarget sequence information, design primer pT-aroK-F, pT-aroK-R carries out PCR and is contained
The linearized vector pTT7A of sgRNA (sequence information is as shown in SEQ ID NO.5)；Design primer pT-pheA-F, pT-pheA-
R carries out PCR and obtains the linearized vector pTPH containing sgRNA (sequence information is as shown in SEQ ID NO.6)；Design primer pT-
Mtr-F, pT-mtr-R carry out PCR and obtain linearizing the pTMT (sequence information is as shown in SEQ ID NO.7) containing sgRNA,
PTT7A, pTPH, pTMT connect construction recombination plasmid with recombinant fragment T7AROK, TACPHE, MTRD respectively, and BamHI, BsgI are bis-
Digestion verification and sequencing, confirmation construction of recombinant plasmid success.PTT7A, pTPH, pTMT plasmid map are as shown in Figure 1.
The building of the recombination T7AROK segment Escherichia coli of embodiment 3
PCas9 plasmid containing cas9 albumen is converted into Escherichia coli CICC 10303.Turned using the choosing of Kana resistance screen
Change successful recombination bacillus coli CICC 10303-cas9, recombinant plasmid pTargetT7AROK is then converted into Escherichia coli
CICC 10303-cas9, screening confirmation T7 promoter successful integration add 0.05mM IPTG to induce 12h, removal weight at 30 DEG C
Group plasmid pTargetT7AROK selects primer pT-aroK-2F and pT-aroK-2R to select transformant and carries out bacterium colony PCR, occurs
600bp or so band after sequencing is correct, obtains the recombination bacillus coli CICC 10303- that aroK gene promoter is T7
aroKT。
The building of the recombination TACPHE segment Escherichia coli of embodiment 4
Recombinant plasmid pTargetTACPHE is converted into Escherichia coli CICC 10303-aroKT, selects primer pT-pheA-
2F and pT-pheA-2R selects transformant and carries out bacterium colony PCR, 600bp or so band occurs, obtains aroK gene after sequencing is correct
Promoter is the recombination bacillus coli CICC 10303-pheAT that T7, pheA gene promoter are tac.
The building of the recombination MTRD segment Escherichia coli of embodiment 5
By recombinant plasmid pTargetMTRD convert Escherichia coli CICC 10303-pheAT, select primer pT-mtr-1F and
PT-mtr-2R selects transformant and carries out bacterium colony PCR, 1200bp or so band occurs, sequencing confirmation mtr gene by successful knockout,
Add 0.05mM IPTG to induce 12h at 30 DEG C, remove obtain after recombinant plasmid pTargetMTRD aroK gene promoter be T7,
PheA gene promoter is the recombination bacillus coli CICC 10303-mtrD of tac and mtr gene delection；It is cultivated at 37 DEG C
12h obtains the recombination bacillus coli CICC 10303-TRYP of high yield L-Trp after removing plasmid pCas9.
6 ammonium sulfate of embodiment and soy peptone make nitrogen source fermentation production L-Trp
Recombination bacillus coli CICC 10303-TRYP is cultivated for 24 hours at 37 DEG C in plating medium, picking single bacterium drops down onto
Seed culture medium cultivates 10h under the conditions of 37 DEG C, 220rpm, with 10% inoculum concentration inoculation fermentation culture medium, in fermented and cultured
Ammonium sulfate 18g/L is added in base, adds soy peptone 6g/L.35 DEG C, cultivate 12h under the conditions of 220rpm, 0.1mM IPTG is lured
It leads 36h and no longer consumes sugar to thallus.It is measured using HPLC high performance liquid chromatography and contains L-Trp 22g/L in fermented liquid supernatant.
Embodiment 7 adds citric acid fermentation and produces L-Trp
Recombination bacillus coli CICC 10303-TRYP is cultivated for 24 hours at 37 DEG C in plating medium, picking single bacterium drops down onto
Seed culture medium cultivates 10h under the conditions of 37 DEG C, 220rpm, is seeded to fermentation medium with 10% inoculum concentration, trains in fermentation
It supports and removes trisodium citrate in base, add citric acid 3g/L.35 DEG C, 12h, 0.1mM IPTG induction are cultivated under the conditions of 220rpm
40h no longer consumes sugar to thallus.It is measured using HPLC high performance liquid chromatography and contains L-Trp 26g/L in fermented liquid supernatant.
8 37 DEG C of temperature conditions of embodiment issue ferment production L-Trp
Recombination bacillus coli CICC 10303-TRYP is cultivated for 24 hours at 37 DEG C in plating medium, picking single bacterium drops down onto
Seed culture medium cultivates 10h under the conditions of 37 DEG C, 220rpm, with 10% inoculum concentration inoculation fermentation culture medium, 37 DEG C,
12h is cultivated under the conditions of 220rpm, 0.1mM IPTG induction 48h to sugar exhausts.Fermentation liquid is measured using HPLC high performance liquid chromatography
Contain L-Trp 32g/L in supernatant.
9 35 DEG C of temperature conditions of embodiment issue ferment production L-Trp
Recombination bacillus coli CICC 10303-TRYP is cultivated for 24 hours at 37 DEG C in plating medium, picking single bacterium drops down onto
Seed culture medium cultivates 10h, 10% inoculum concentration inoculation fermentation culture medium, 35 DEG C, 220rpm under the conditions of 37 DEG C, 220rpm
Under the conditions of cultivate 12h, 0.1mM IPTG induces 42h.It is measured using HPLC high performance liquid chromatography and contains L- color in fermented liquid supernatant
Propylhomoserin 36g/L, as shown in Figure 2.
It is T7 promoter that comparative example 1, which only replaces shikimate kinase aroK promoter, issues ferment recombination in 35 DEG C of temperature conditions
Bacterium produces L-Trp
According to genome of E.coli sequence information, design primer pT-aroK-1R and pT-aroK-2F, pT-aroK-1F,
PT-aroK-2R expands the aroK with T7 promoter using above-mentioned four kinds of primers from 10303 genome of Escherichia coli CICC
2 sections of amplified fragments of gained are obtained recombinant fragment by fusion DNA vaccine technological incorporation by each 600bp of DNA homolog arm gene order
T7AROK；
According to carrier pTarget sequence information, design primer pT-aroK-F, pT-aroK-R, PCR is obtained containing sgRNA
Linearized vector pTT7A；Construction recombination plasmid, the verifying of BamHI, BsgI double digestion and survey are connect with recombinant fragment T7AROK
Sequence, confirmation construction of recombinant plasmid success.
PCas9 plasmid containing cas9 albumen is converted into Escherichia coli CICC 10303.Turned using the choosing of Kana resistance screen
Change successful recombination bacillus coli CICC 10303-cas9, recombinant plasmid pTargetT7AROK is then converted into Escherichia coli
CICC 10303-cas9, screening confirmation T7 promoter successful integration simultaneously remove recombinant plasmid pTargetT7AROK, select primer
PT-aroK-2F and pT-aroK-2R selects transformant and carries out bacterium colony PCR, 600bp or so band occurs and obtains after sequencing is correct
AroK gene promoter is the recombination bacillus coli CICC 10303-aroKT of T7；After removing plasmid pCas9, high yield L- color is obtained
The recombination bacillus coli CICC 10303-TRYP1 of propylhomoserin.
Recombination bacillus coli CICC 10303-TRYP1 is cultivated for 24 hours at 37 DEG C in plating medium, picking single colonie
To seed culture medium, 10h is cultivated under the conditions of 37 DEG C, 220rpm, 10% inoculum concentration inoculation fermentation culture medium,；35℃,
12h is cultivated under the conditions of 220rpm, 0.1mM IPTG induces 42h.Utilize L- in HPLC high performance liquid chromatography measurement fermented liquid supernatant
The content 12g/L of tryptophan, as shown in Figure 3.
The promoter that comparative example 2 only replaces prephenate dehydrogenase encoding gene pheA is tac promoter, in 35 DEG C of temperature strips
The recombinant bacterium that ferments under part produces L-Trp
According to genome of E.coli sequence information, design primer pT-pheA-1F, pT-pheA-1R, pT-pheA-2F and
PT-pheA-2R expands pheA gene start codon two sides using above-mentioned primer from 10303 genome of Escherichia coli CICC
2 sections of amplified fragments of gained are obtained recombinant fragment TACPHE by fusion DNA vaccine technological incorporation by each 600bp of homology arm gene order.
According to carrier pTarget sequence information, design primer pT-pheA-F and pT-pheA-R, PCR are obtained containing sgRNA
Linearized vector pTPH；Construction recombination plasmid, the verifying of BamHI, BsgI double digestion and sequencing are connect with recombinant fragment TACPHE,
Confirm construction of recombinant plasmid success.
PCas9 plasmid containing cas9 albumen is converted into Escherichia coli CICC 10303.Turned using the choosing of Kana resistance screen
Change successful recombination bacillus coli CICC 10303-cas9, recombinant plasmid pTargetTACPHE is then converted into Escherichia coli
CICC 10303-cas9, screening confirmation tac promoter successful integration simultaneously remove recombinant plasmid pTargetTACPHE, design primer
Bacterium colony PCR verifying is carried out, after sequencing result is correct, obtains the recombination bacillus coli CICC that pheA gene promoter is tac
10303-pheAT2；After removing plasmid pCas9, recombination bacillus coli CICC 10303-TRYP2 is obtained.
Recombination bacillus coli CICC 10303-TRYP2 is cultivated for 24 hours at 37 DEG C in plating medium, picking single colonie,
Cultivate 10h under the conditions of 37 DEG C, 220rpm, 10% inoculum concentration inoculation fermentation culture medium, 35 DEG C, cultivate under the conditions of 220rpm
12h, 0.1mM IPTG induce 42h.Utilize the content 20g/ of L-Trp in HPLC high performance liquid chromatography measurement fermented liquid supernatant
L, as shown in Figure 4.
Comparative example 3 only knocks out tryptophan transporter encoding gene mtr, issues ferment recombinant bacterium production L- in 35 DEG C of temperature conditions
Tryptophan
According to genome of E.coli sequence information, design primer pT-mtr-1F, pT-mtr-1R, pT-mtr-2F and pT-
Mtr-2R expands mtr gene two sides homology arm gene order using above-mentioned primer from 10303 genome of Escherichia coli CICC
2 sections of amplified fragments of gained are obtained recombinant fragment MTRD by fusion DNA vaccine technological incorporation by each 600bp.
According to carrier pTarget sequence information, design primer pT-mtr-F and pT-mtr-R, PCR are obtained containing sgRNA's
Linearized vector pTMT connect construction recombination plasmid, the verifying of BamHI, BsgI double digestion and sequencing, confirmation with recombinant fragment MTRD
Construction of recombinant plasmid success.
PCas9 plasmid containing cas9 albumen is converted into Escherichia coli CICC 10303.Turned using the choosing of Kana resistance screen
Change successful recombination bacillus coli CICC 10303-cas9, recombinant plasmid pTargetMTRD is then converted into Escherichia coli CICC
10303-cas9, screening confirmation mtr gene by successful knockout successful integration and remove recombinant plasmid pTargetMTRD, and design is drawn
Object selects transformant and carries out bacterium colony PCR verifying, after sequencing result is correct, obtains recombination large intestine bar of the mtr gene by successful knockout
After bacterium CICC 10303-mtrD removes plasmid pCas9, the recombination bacillus coli CICC 10303- of high yield L-Trp is obtained
TRYP3。
Recombination bacillus coli CICC 10303-TRYP3 is cultivated for 24 hours at 37 DEG C in plating medium, picking single colonie
To seed culture medium, 10h is cultivated under the conditions of 37 DEG C, 220rpm, 10% inoculum concentration inoculation fermentation culture medium, 35 DEG C,
12h is cultivated under the conditions of 220rpm, 0.1mM IPTG induces 42h.Utilize L- in HPLC high performance liquid chromatography measurement fermented liquid supernatant
The content 20g/L of tryptophan, as shown in Figure 5.
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 recombination bacillus coli and its construction method of high yield L-Trp
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 87
<212> DNA
<213>artificial synthesized
<400> 1
taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 60
tgtttaactt taagaaggag atatacc 87
<210> 2
<211> 978
<212> DNA
<213>artificial synthesized
<400> 2
acagccgtaa aagcggtaat gtttttacgc tgaacgtgtt tcatctattt gacgcgcgca 60
ggtatttagc atacaaggag taccgatttg agagttggtg ctcttcgctg cctgcgttcc 120
atgatgatga tttatcattc aggcggcatt ttgctgtctt ttttacgcta atcttacccg 180
gtgatttatc gccagagcgg tggtagcaag gcagcgcgct tgcagcgacc agatatgcag 240
agggatgggt gatttattca gttgccaaac ccgctggagt attgagataa ttttcagtct 300
gactctcgca atatcttatg aggtttcagt tcatgtcctg cggcgctctc tgagcgaagc 360
gggtttatca ttaacgaata gtcttagtag taccgaaaaa atggcagaga aacgcaatat 420
ctttctggtt gggcctatgg gtgccggaaa aagcactatt gggcgccagt tagctcaaca 480
actcaatatg gaattttacg attccgatca agagattgag aaacgaaccg gagctgatgt 540
gggctgggtt ttcgatttag aaggcgaaga aggcttccgc gatcgcgaag aaaaggtcat 600
caatgagttg accgagaaac agggtattgt gctggctact ggcggcggct ctgtgaaatc 660
ccgtgaaacg cgtaaccgtc tttccgctcg tggcgttgtc gtttatcttg aaacgaccat 720
cgaaaagcaa cttgcacgca cgcagcgtga taaaaaacgc ccgttgctgc acgttgaaac 780
accgccgcgt gaagttctgg aagcgttggc caatgaacgc aatccgctgt atgaagagat 840
tgccgacgtg accattcgta ctgatgatca aagcgctaaa gtggttgcaa accagattat 900
tcacatgctg gaaagcaact aattctggct ttatatacac tcgtctgcgg gtacagtaat 960
taaggtggat gtcgcgtt 978
<210> 3
<211> 1515
<212> DNA
<213>artificial synthesized
<400> 3
tcatcgctga acagcgaaca caatctgtaa aataatatat acagccccga tttttaccat 60
cggggctttt tttctgtctt ttgtactcgt gtactggtac agtgcaatgc ataacaacgc 120
agtcgcacta tttttcactg gagagaagcc ctcatggcaa cactaaccac cacccaaacg 180
tcaccgtcgc tgcttggcgg cgtggtgatt atcggcggca ccattattgg cgcagggatg 240
ttttctctgc cagtggtcat gtccggggcg tggtttttct ggtcaatggc ggcgctgatc 300
tttacctggt tctgtatgct gcattccggc ttgatgattc tggaagctaa cctgaattac 360
agaatcggtt cgagttttga caccatcacc aaagatttgc tgggcaaagg ctggaacgtg 420
gtcaacggca tttccattgc ctttgtgctc tatatcctga cctatgccta tatttctgcc 480
agtggttcga ttctgcatca caccttcgca gagatgtcac taaacgtccc ggcacgggcg 540
gcgggttttg gttttgcatt gctggtagcg tttgtggtgt ggttgagcac taaagccgtc 600
agtcgcatga cagcgattgt gctgggggcg aaagtcatta ccttcttcct cacctttggt 660
agcctgctgg ggcatgtgca gcctgcgaca ttgttcaacg tcgccgaaag caatgcgtct 720
tatgcaccgt atctgttgat gaccctgccg ttctgtctgg catcgtttgg ttatcacggt 780
aacgtgccaa gcctgatgaa gtattacggc aaagatccga aaaccatcgt gaaatgtctg 840
gtgtacggta cgctgatggc gctggcgctg tataccatct ggttgctggc gacgatgggt 900
aacatcccgc gtccggagtt tatcggtatt gcagagaagg gcggtaatat tgatgtgctg 960
gtacaggcgt taagcggcgt actgaacagc cgtagtctgg atctgctgct ggtcgtgttc 1020
tcaaactttg cggtagcgag ttcgttcctc ggcgtaacgc tgggtttgtt tgactatctg 1080
gcagatctgt ttggtttcga cgactcggct gtgggccgct tgaaaacggc attgctgacc 1140
tttgccccgc cagttgtggg ggggctgttg ttcccgaacg gattcctgta cgccattggt 1200
tatgctggtt tagcggctac catctgggcg gcaattgttc cggcgctgtt agcccgtgca 1260
tcgcgtaaac gctttggcag cccgaaattc cgcgtctggg gtggcaagcc gatgattgcg 1320
ctgattctgg tgtttggcgt cggcaacgca ctggtgcata ttttatcgag ctttaattta 1380
ctgccggtgt atcagtaatc agcggtgcct tatccgacat ttctgctgcc tacacaatgc 1440
ctgatgcgct tcgcttatca ggtctatgta ggacagcgtt gccagctcgg ataaggcttc 1500
ccgcgttaag acaca 1515
<210> 4
<211> 1301
<212> DNA
<213>artificial synthesized
<400> 4
atgggaggcg tttcgtcgtg tgaaacagaa tgcgaagacg aacaataagg cctcccaaat 60
cggggggcct tttttattga taacaaaaag gcaacactat gacatcggaa aacccgttac 120
tggcgctgcg agagaaaatc agcgcgctgg atgaaaaatt attagcgtta ctggcagaac 180
ggcgcgaact ggccgtcgag gtgggaaaag ccaaactgct ctcgcatcgc ccggtacgtg 240
atattgatcg tgaacgcgat ttgctggaaa gattaattac gctcggtaaa gcgcaccatc 300
tggacgccca ttacattact cgcctgttcc agctcatcat tgaagattcc gtattaactc 360
agcaggcttt gctccaacaa catctcaata aaattaatcc gcactcagca cgcatcgctt 420
ttctcggccc caaaggttct tattcccatc ttgcggcgcg ccagtatgct gcccgtcact 480
ttgagcaatt cattgaaagt ggctgcgcca aatttgccga tatttttaat caggtggaaa 540
ccggccaggc cgactatgcc gtcgtaccga ttgaaaatac cagctccggt gccataaacg 600
acgtttacga tctgctgcaa cataccagct tgtcgattgt tggcgagatg acgttaacta 660
tcgaccattg tttgttggtc tccggcacta ctgatttatc caccatcaat acggtctaca 720
gccatccgca gccattccag caatgcagca aattccttaa tcgttatccg cactggaaga 780
ttgaatatac cgaaagtacg tctgcggcaa tggaaaaggt tgcacaggca aaatcaccgc 840
atgttgctgc gttgggaagc gaagctggcg gcactttgta cggtttgcag gtactggagc 900
gtattgaagc aaatcagcga caaaacttca cccgatttgt ggtgttggcg cgtaaagcca 960
ttaacgtgtc tgatcaggtt ccggcgaaaa ccacgttgtt aatggcgacc gggcaacaag 1020
ccggtgcgct ggttgaagcg ttgctggtac tgcgcaacca caatctgatt atgacccgtc 1080
tggaatcacg cccgattcac ggtaatccat gggaagagat gttctatctg gatattcagg 1140
ccaatcttga atcagcggaa atgcaaaaag cattgaaaga gttaggggaa atcacccgtt 1200
caatgaaggt attgggctgt tacccaagtg agaacgtagt gcctgttgat ccaacctgat 1260
gaaaaggtgc cggatgatgt gaatcatccg gcactggatt a 1301
<210> 5
<211> 2118
<212> DNA
<213>artificial synthesized
<400> 5
tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatgctg 1920
gatccttgac agctagctca gtcctaggta taatactagt gcgtttctct gccatttttt 1980
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 2040
ggcaccgagt cggtgctttt tttgaattct ctagagtcga cctgcagaag cttagatcta 2100
ttaccctgtt atccctac 2118
<210> 6
<211> 2118
<212> DNA
<213>artificial synthesized
<400> 6
tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatgctg 1920
gatccttgac agctagctca gtcctaggta taatactagt aaaaggcaac actatgacat 1980
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 2040
ggcaccgagt cggtgctttt tttgaattct ctagagtcga cctgcagaag cttagatcta 2100
ttaccctgtt atccctac 2118
<210> 7
<211> 2118
<212> DNA
<213>artificial synthesized
<400> 7
tcgagttcat gtgcagctcc atcagcaaaa ggggatgata agtttatcac caccgactat 60
ttgcaacagt gccgttgatc gtgctatgat cgactgatgt catcagcggt ggagtgcaat 120
gtcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 180
atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 240
ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 300
gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 360
agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 420
cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 480
ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 540
gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 600
caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 660
gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 720
ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 780
tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 840
gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag 900
gtagtcggca aataagatgc cgctcgccag tcgattggct gagctcatga agttcctatt 960
ccgaagttcc gcgaacgcgt aaaggatcta ggtgaagatc ctttttgata atctcatgac 1020
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1080
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1140
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1200
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1260
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1380
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1440
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1500
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1560
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1620
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1740
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1800
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1860
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatgctg 1920
gatccttgac agctagctca gtcctaggta taatactagt ctttgccgta atacttcatc 1980
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 2040
ggcaccgagt cggtgctttt tttgaattct ctagagtcga cctgcagaag cttagatcta 2100
ttaccctgtt atccctac 2118
<210> 8
<211> 12545
<212> DNA
<213>artificial synthesized
<400> 8
gatctcaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta 60
ttttaacttg ctatttctag ctctaaaacc tggtaacagg attagcagat gtgtgaaatt 120
gttatccgct cacaattcca cacattatac gagccggatg attaattgtc aacagctcat 180
ttcagaatat ttgccagaac cgttatgatg tcggcgcaaa aaacattatc cagaacggga 240
gtgcgccttg agcgacacga attatgcagt gatttacgac ctgcacagcc ataccacagc 300
ttccgatggc tgcctgacgc cagaagcatt ggtgcaccgt gcagtcgatg ataagctgtc 360
aaaccagatc aattcgcgct aactcacatt aattgcgttg cgctcactgc ccgctttcca 420
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 480
tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat 540
tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 600
gcaggcgaaa atcctgtttg atggtggttg acggcgggat ataacatgag ctgtcttcgg 660
tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg 720
cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc 780
cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc 840
gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca 900
gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg 960
cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga 1020
tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca 1080
cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg 1140
cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca 1200
ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg 1260
gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca 1320
gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt 1380
cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag 1440
agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga 1500
attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg 1560
tgtcaacgta aatgcatgcc gcttcgcctt ccatgggtat ggacagtttt ccctttgata 1620
tgtaacggtg aacagttgtt ctacttttgt ttgttagtct tgatgcttca ctgatagata 1680
caagagccat aagaacctca gatccttccg tatttagcca gtatgttctc tagtgtggtt 1740
cgttgttttt gcgtgagcca tgagaacgaa ccattgagat catacttact ttgcatgtca 1800
ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg cagttaaagc atcgtgtagt 1860
gtttttctta gtccgttacg taggtaggaa tctgatgtaa tggttgttgg tattttgtca 1920
ccattcattt ttatctggtt gttctcaagt tcggttacga gatccatttg tctatctagt 1980
tcaacttgga aaatcaacgt atcagtcggg cggcctcgct tatcaaccac caatttcata 2040
ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa cccattggtt aagcctttta 2100
aactcatggt agttattttc aagcattaac atgaacttaa attcatcaag gctaatctct 2160
atatttgcct tgtgagtttt cttttgtgtt agttctttta ataaccactc ataaatcctc 2220
atagagtatt tgttttcaaa agacttaaca tgttccagat tatattttat gaattttttt 2280
aactggaaaa gataaggcaa tatctcttca ctaaaaacta attctaattt ttcgcttgag 2340
aacttggcat agtttgtcca ctggaaaatc tcaaagcctt taaccaaagg attcctgatt 2400
tccacagttc tcgtcatcag ctctctggtt gctttagcta atacaccata agcattttcc 2460
ctactgatgt tcatcatctg agcgtattgg ttataagtga acgataccgt ccgttctttc 2520
cttgtagggt tttcaatcgt ggggttgagt agtgccacac agcataaaat tagcttggtt 2580
tcatgctccg ttaagtcata gcgactaatc gctagttcat ttgctttgaa aacaactaat 2640
tcagacatac atctcaattg gtctaggtga ttttaatcac tataccaatt gagatgggct 2700
agtcaatgat aattactagt ccttttcctt tgagttgtgg gtatctgtaa attctgctag 2760
acctttgctg gaaaacttgt aaattctgct agaccctctg taaattccgc tagacctttg 2820
tgtgtttttt ttgtttatat tcaagtggtt ataatttata gaataaagaa agaataaaaa 2880
aagataaaaa gaatagatcc cagccctgtg tataactcac tactttagtc agttccgcag 2940
tattacaaaa ggatgtcgca aacgctgttt gctcctctac aaaacagacc ttaaaaccct 3000
aaaggcttaa gtagcaccct cgcaagctcg gttgcggccg caatcgggca aatcgctgaa 3060
tattcctttt gtctccgacc atcaggcacc tgagtcgctg tctttttcgt gacattcagt 3120
tcgctgcgct cacggctctg gcagtgaatg ggggtaaatg gcactacagg cgccttttat 3180
ggattcatgc aaggaaacta cccataatac aagaaaagcc cgtcacgggc ttctcagggc 3240
gttttatggc gggtctgcta tgtggtgcta tctgactttt tgctgttcag cagttcctgc 3300
cctctgattt tccagtctga ccacttcgga ttatcccgtg acaggtcatt cagactggct 3360
aatgcaccca gtaaggcagc ggtatcatca acggggtctg acgctcagtg gaacgaaaac 3420
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 3480
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 3540
tacgtttcca caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa 3600
actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 3660
atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 3720
cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 3780
tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat 3840
gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 3900
catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 3960
tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 4020
catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 4080
cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 4140
tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat 4200
tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 4260
atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 4320
aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat 4380
ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg 4440
atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttccc 4500
tgcagggttt gcagtcagag tagaatagaa gtatcaaaaa aagcaccgac tcggtgccac 4560
tttttcaagt tgataacgga ctagccttat tttaacttgc tatgctgttt tgaatggttc 4620
caacaagatt attttataac ttttataaca aataatcaag gagaaattca aagaaattta 4680
tcagccataa aacaatactt aatactatag aatgataaca aaataaacta ctttttaaaa 4740
gaattttgtg ttataatcta tttattatta agtattgggt aatatttttt gaagagatat 4800
tttgaaaaag aaaaattaaa gcatattaaa ctaatttcgg aggtcattaa aactattatt 4860
gaaatcatca aactcattat ggatttaatt taaacttttt attttaggag gcaaaaatgg 4920
ataagaaata ctcaataggc ttagatatcg gcacaaatag cgtcggatgg gcggtgatca 4980
ctgatgatta taaggttccg tctaaaaagt tcaaggttct gggaaataca gaccgccaca 5040
gtatcaaaaa aaatcttata ggggctcttt tatttgacag tggagagaca gcggaagcga 5100
ctcgtctcaa acggacagct cgtagaaggt atacacgtcg gaagaatcgt atttgttatc 5160
tacaggagat tttttcaaat gagatggcga aagtagatga tagtttcttt catcgacttg 5220
aagagtcttt tttggtggaa gaagacaaga agcatgaacg tcatcctatt tttggaaata 5280
tagtagatga agttgcttat catgagaaat atccaactat ctatcatctg cgaaaaaaat 5340
tggtagattc tactgataaa gcggatttgc gcttaatcta tttggcctta gcgcatatga 5400
ttaagtttcg tggtcatttt ttgattgagg gagatttaaa tcctgataat agtgatgtgg 5460
acaaactatt tatccagttg gtacaaacct acaatcaatt atttgaagaa aaccctatta 5520
acgcaagtgg agtagatgct aaagcgattc tttctgcacg attgagtaaa tcaagacgat 5580
tagaaaatct cattgctcag ctccccggtg agaagaaaaa tggcttattt gggaatctca 5640
ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg 5700
ctaaattaca gctttcaaaa gatacttacg atgatgattt agataattta ttggcgcaaa 5760
ttggagatca atatgctgat ttgtttttgg cagctaagaa tttatcagat gctattttac 5820
tttcagatat cctaagagta aatactgaaa taactaaggc tcccctatca gcttcaatga 5880
ttaaacgcta cgatgaacat catcaagact tgactctttt aaaagcttta gttcgacaac 5940
aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt 6000
atattgatgg gggagctagc caagaagaat tttataaatt tatcaaacca attttagaaa 6060
aaatggatgg tactgaggaa ttattggtga aactaaatcg tgaagatttg ctgcgcaagc 6120
aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta 6180
ttttgagaag acaagaagac ttttatccat ttttaaaaga caatcgtgag aagattgaaa 6240
aaatcttgac ttttcgaatt ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt 6300
ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg 6360
tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc 6420
ttccaaatga aaaagtacta ccaaaacata gtttgcttta tgagtatttt acggtttata 6480
acgaattgac aaaggtcaaa tatgttactg aaggaatgcg aaaaccagca tttctttcag 6540
gtgaacagaa gaaagccatt gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta 6600
agcaattaaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag 6660
gagttgaaga tagatttaat gcttcattag gtacctacca tgatttgcta aaaattatta 6720
aagataaaga ttttttggat aatgaagaaa atgaagatat cttagaggat attgttttaa 6780
cattgacctt atttgaagat agggagatga ttgaggaaag acttaaaaca tatgctcacc 6840
tctttgatga taaggtgatg aaacagctta aacgtcgccg ttatactggt tggggacgtt 6900
tgtctcgaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atattagatt 6960
ttttgaaatc agatggtttt gccaatcgca attttatgca gctgatccat gatgatagtt 7020
tgacatttaa agaagacatt caaaaagcac aagtgtctgg acaaggcgat agtttacatg 7080
aacatattgc aaatttagct ggtagccctg ctattaaaaa aggtatttta cagactgtaa 7140
aagttgttga tgaattggtc aaagtaatgg ggcggcataa gccagaaaat atcgttattg 7200
aaatggcacg tgaaaatcag acaactcaaa agggccagaa aaattcgcga gagcgtatga 7260
aacgaatcga agaaggtatc aaagaattag gaagtcagat tcttaaagag catcctgttg 7320
aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagaca 7380
tgtatgtgga ccaagaatta gatattaatc gtttaagtga ttatgatgtc gatcacattg 7440
ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcttaacg cgttctgata 7500
aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact 7560
attggagaca acttctaaac gccaagttaa tcactcaacg taagtttgat aatttaacga 7620
aagctgaacg tggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg 7680
ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta 7740
aatacgatga aaatgataaa cttattcgag aggttaaagt gattacctta aaatctaaat 7800
tagtttctga cttccgaaaa gatttccaat tctataaagt acgtgagatt aacaattacc 7860
atcatgccca tgatgcgtat ctaaatgccg tcgttggaac tgctttgatt aagaaatatc 7920
caaaacttga atcggagttt gtctatggtg attataaagt ttatgatgtt cgtaaaatga 7980
ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa atatttcttt tactctaata 8040
tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc 8100
taatcgaaac taatggggaa actggagaaa ttgtctggga taaagggcga gattttgcca 8160
cagtgcgcaa agtattgtcc atgccccaag tcaatattgt caagaaaaca gaagtacaga 8220
caggcggatt ctccaaggag tcaattttac caaaaagaaa ttcggacaag cttattgctc 8280
gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga tagtccaacg gtagcttatt 8340
cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag 8400
agttactagg gatcacaatt atggaaagaa gttcctttga aaaaaatccg attgactttt 8460
tagaagctaa aggatataag gaagttaaaa aagacttaat cattaaacta cctaaatata 8520
gtctttttga gttagaaaac ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa 8580
aaggaaatga gctggctctg ccaagcaaat atgtgaattt tttatattta gctagtcatt 8640
atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc 8700
ataagcatta tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt 8760
tagcagatgc caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa 8820
tacgtgaaca agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg 8880
ctgcttttaa atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag 8940
ttttagatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt 9000
tgagtcagct aggaggtgac tgaagtatat tttagatgaa gattatttct taatctagac 9060
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 9120
tttccccgaa aagtgccacc tgcatcgatt tattatgaca acttgacggc tacatcattc 9180
actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca ttttttaaat 9240
acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc 9300
atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc gcgccagctt 9360
aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa 9420
acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac 9480
tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt aatcgcttcc 9540
atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga atagcgccct 9600
tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct 9660
tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag ccattcatgc 9720
cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc ctccggatga 9780
cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca 9840
aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt gagaatataa 9900
cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc 9960
gttaaacccg ccaccagatg ggcattaaac gagtatcccg gcagcagggg atcattttgc 10020
gcttcagcca tacttttcat actcccgcca ttcagagaag aaaccaattg tccatattgc 10080
atcagacatt gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc 10140
ccgcttatta aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac 10200
aaaagtgtct ataatcacgg cagaaaagtc cacattgatt atttgcacgg cgtcacactt 10260
tgctatgcca tagcattttt atccataaga ttagcggatc ctacctgacg ctttttatcg 10320
caactctcta ctgtttctcc atacccgttt ttttgggaat tcgagctcta aggaggttat 10380
aaaaaatgga tattaatact gaaactgaga tcaagcaaaa gcattcacta accccctttc 10440
ctgttttcct aatcagcccg gcatttcgcg ggcgatattt tcacagctat ttcaggagtt 10500
cagccatgaa cgcttattac attcaggatc gtcttgaggc tcagagctgg gcgcgtcact 10560
accagcagct cgcccgtgaa gagaaagagg cagaactggc agacgacatg gaaaaaggcc 10620
tgccccagca cctgtttgaa tcgctatgca tcgatcattt gcaacgccac ggggccagca 10680
aaaaatccat tacccgtgcg tttgatgacg atgttgagtt tcaggagcgc atggcagaac 10740
acatccggta catggttgaa accattgctc accaccaggt tgatattgat tcagaggtat 10800
aaaacgaatg agtactgcac tcgcaacgct ggctgggaag ctggctgaac gtgtcggcat 10860
ggattctgtc gacccacagg aactgatcac cactcttcgc cagacggcat ttaaaggtga 10920
tgccagcgat gcgcagttca tcgcattact gatcgttgcc aaccagtacg gccttaatcc 10980
gtggacgaaa gaaatttacg cctttcctga taagcagaat ggcatcgttc cggtggtggg 11040
cgttgatggc tggtcccgca tcatcaatga aaaccagcag tttgatggca tggactttga 11100
gcaggacaat gaatcctgta catgccggat ttaccgcaag gaccgtaatc atccgatctg 11160
cgttaccgaa tggatggatg aatgccgccg cgaaccattc aaaactcgcg aaggcagaga 11220
aatcacgggg ccgtggcagt cgcatcccaa acggatgtta cgtcataaag ccatgattca 11280
gtgtgcccgt ctggccttcg gatttgctgg tatctatgac aaggatgaag ccgagcgcat 11340
tgtcgaaaat actgcataca ctgcagaacg tcagccggaa cgcgacatca ctccggttaa 11400
cgatgaaacc atgcaggaga ttaacactct gctgatcgcc ctggataaaa catgggatga 11460
cgacttattg ccgctctgtt cccagatatt tcgccgcgac attcgtgcat cgtcagaact 11520
gacacaggcc gaagcagtaa aagctcttgg attcctgaaa cagaaagccg cagagcagaa 11580
ggtggcagca tgacaccgga cattatcctg cagcgtaccg ggatcgatgt gagagctgtc 11640
gaacaggggg atgatgcgtg gcacaaatta cggctcggcg tcatcaccgc ttcagaagtt 11700
cacaacgtga tagcaaaacc ccgctccgga aagaagtggc ctgacatgaa aatgtcctac 11760
ttccacaccc tgcttgctga ggtttgcacc ggtgtggctc cggaagttaa cgctaaagca 11820
ctggcctggg gaaaacagta cgagaacgac gccagaaccc tgtttgaatt cacttccggc 11880
gtgaatgtta ctgaatcccc gatcatctat cgcgacgaaa gtatgcgtac cgcctgctct 11940
cccgatggtt tatgcagtga cggcaacggc cttgaactga aatgcccgtt tacctcccgg 12000
gatttcatga agttccggct cggtggtttc gaggccataa agtcagctta catggcccag 12060
gtgcagtaca gcatgtgggt gacgcgaaaa aatgcctggt actttgccaa ctatgacccg 12120
cgtatgaagc gtgaaggcct gcattatgtc gtgattgagc gggatgaaaa gtacatggcg 12180
agttttgacg agatcgtgcc ggagttcatc gaaaaaatgg acgaggcact ggctgaaatt 12240
ggttttgtat ttggggagca atggcgatga cgcatcctca cgataatatc cgggtaggcg 12300
caatcacttt cgtctactcc gttacaaagc gaggctgggt atttcccggc ctttctgtta 12360
tccgaaatcc actgaaagca cagcggctgg ctgaggagat aaataataaa cgaggggctg 12420
tatgcacaaa gcatcttctg ttgagttaag aacgagtatc gagatggcac atagccttgc 12480
tcaaattgga atcaggtttg tgccaatacc agtagaaaca gacgaagaat ccatgggtat 12540
ggaca 12545
<210> 9
<211> 60
<212> DNA
<213>artificial synthesized
<400> 9
tcggtgcttt ttttgaattc tttttcggta ctactaagac tattcgttaa tgataaaccc 60
<210> 10
<211> 32
<212> DNA
<213>artificial synthesized
<400> 10
gtcgttactc aaacgcaggt cgaggtcaaa ag 32
<210> 11
<211> 37
<212> DNA
<213>artificial synthesized
<400> 11
acctgcgttt gagtaacgac aatcctctcc ataacgc 37
<210> 12
<211> 74
<212> DNA
<213>artificial synthesized
<400> 12
taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatggcag 60
agaaacgcaa tatc 74
<210> 13
<211> 90
<212> DNA
<213>artificial synthesized
<400> 13
attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaaa 60
gcttagatct attaccctgt tatccctact 90
<210> 14
<211> 25
<212> DNA
<213>artificial synthesized
<400> 14
gaattcaaaa aaagcaccga ctcgg 25
<210> 15
<211> 56
<212> DNA
<213>artificial synthesized
<400> 15
tcggtgcttt ttttgaattc gtaaaattta tgacaatgaa cattaccagc aaacaa 56
<210> 16
<211> 67
<212> DNA
<213>artificial synthesized
<400> 16
gttttccgat gtcatgcggc cgcacctcct ttgtgaaaac acattatacg agccgatgat 60
taattgt 67
<210> 17
<211> 42
<212> DNA
<213>artificial synthesized
<400> 17
aggcaacact ttgacaatta atcatcggct cgtataatgt gt 42
<210> 18
<211> 45
<212> DNA
<213>artificial synthesized
<400> 18
agggtaatag atctaagctt taaatcagta gtgccggaga ccaac 45
<210> 19
<211> 32
<212> DNA
<213>artificial synthesized
<400> 19
aagcttagat ctattaccct gttatcccta ct 32
<210> 20
<211> 25
<212> DNA
<213>artificial synthesized
<400> 20
gaattcaaaa aaagcaccga ctcgg 25
<210> 21
<211> 41
<212> DNA
<213>artificial synthesized
<400> 21
tcggtgcttt ttttgaattc gttgaccacg ttccagcctt t 41
<210> 22
<211> 25
<212> DNA
<213>artificial synthesized
<400> 22
ctgatgcgct ggcggtggtc gtggt 25
<210> 23
<211> 29
<212> DNA
<213>artificial synthesized
<400> 23
gaccaccgcc agcgcatcag gcattgtgt 29
<210> 24
<211> 35
<212> DNA
<213>artificial synthesized
<400> 24
agggtaatag atctaagctt cgctgatggc gctgg 35
<210> 25
<211> 32
<212> DNA
<213>artificial synthesized
<400> 25
aagcttagat ctattaccct gttatcccta ct 32
<210> 26
<211> 25
<212> DNA
<213>artificial synthesized
<400> 26
gaattcaaaa aaagcaccga ctcgg 25
Claims (10)
1. a kind of recombination bacillus coli for producing L-Trp, which is characterized in that shikimate kinase gene aroK promoter is substituted
For T7 promoter, tryptophan transporter encoding gene mtr is knocked out and opening for prephenate dehydrogenase encoding gene pheA is substituted
Mover is tac promoter.
2. recombination bacillus coli as described in claim 1, which is characterized in that the nucleotide sequence of the tac promoter is such as
Shown in SEQ ID NO.1.
3. recombination bacillus coli as claimed in claim 1 or 2, which is characterized in that carried out to Escherichia coli CICC 10303
Genome editor obtains.
4. recombination bacillus coli as claimed in claim 3, which is characterized in that the genome editor is to utilize CRISPR-
What Cas9 technology carried out.
5. the construction method of any recombination bacillus coli of claim 1-4, which is characterized in that the method includes following
Step:
1) building T7 promoter replacement recombinant fragment, tac promoter replacement recombinant fragment and knockout mtr genetic recombination segment:
After the fusion of Escherichia coli shikimate kinase encoding gene aroK gene cluster initiation codon upstream and downstream homology arm sequence, T7 is introduced
Promoter obtains recombinant fragment T7AROK；By the initiation codon upstream and downstream homology arm of prephenate dehydrogenase encoding gene pheA
After sequence fusion, promoter tac is introduced, segment TACPHE is obtained；By tryptophan transporter encoding gene mtr upstream and downstream homology arm
Fusion, obtains segment MTRD.
2) construction recombination plasmid: segment T7AROK, TACPHE, MTRD are connect with the linearized vector containing sgRNA respectively, point
Recombinant plasmid of the recombinant plasmid containing T7AROK, the recombinant plasmid containing TACPHE, the recombinant plasmid containing MTRD are not obtained；
3) it constructs recombination bacillus coli: Escherichia coli CICC 10303 will be converted containing the plasmid of cas9 albumen, obtain large intestine bar
Bacterium CICC 10303-cas9；Then by the recombinant plasmid transformed Escherichia coli CICC 10303-cas9 containing T7AROK, weight is obtained
Group Escherichia coli CICC 10303-aroKT；By the recombinant plasmid transformed Escherichia coli CICC 10303-aroKT containing TACPHE,
Obtain recombination bacillus coli CICC 10303-pheAT；By the recombinant plasmid transformed Escherichia coli CICC 10303- containing MTRD
PheAT obtains recombination bacillus coli CICC 10303-mtrD；After removing exogenous plasmid, recombination bacillus coli CICC is obtained
10303-TRYP。
6. construction method as claimed in claim 5, which is characterized in that the plasmid containing cas9 albumen includes pCas9.
7. such as construction method described in claim 5 or 6, which is characterized in that the linearized vector containing sgRNA include pTT7A,
PTPH or pTMT.
8. application of any recombination bacillus coli of claim 1-4 in production L-Trp.
9. application as claimed in claim 8, which is characterized in that the application includes that will train at 34-38 DEG C in plating medium
The single colonie for supporting 22-26h is seeded to seed culture medium, cultivates 5-8h, then under the conditions of 34-38 DEG C, 180-220rpm with 20%
Inoculum concentration inoculation fermentation culture medium, 34-38 DEG C, 10-14h is cultivated under the conditions of 180-220rpm, 0.1mM IPTG induces 45-
50h。
10. application of any recombination bacillus coli of claim 1-4 in feed, pharmacy, health care product or food industry.
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