CN107916245A - A kind of application for the method and the recombination engineering for producing L tyrosine recombination engineerings - Google Patents

A kind of application for the method and the recombination engineering for producing L tyrosine recombination engineerings Download PDF

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CN107916245A
CN107916245A CN201711044289.7A CN201711044289A CN107916245A CN 107916245 A CN107916245 A CN 107916245A CN 201711044289 A CN201711044289 A CN 201711044289A CN 107916245 A CN107916245 A CN 107916245A
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seq
sequence
sybe
sense primer
fragment
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赵广荣
周亮
丁琦
王海燕
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Frontier Technology Research Institute of Tianjin University Co Ltd
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Frontier Technology Research Institute of Tianjin University Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/225—Tyrosine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Abstract

A kind of application for the method and the recombination engineering for producing L tyrosine recombination engineerings, using Escherichia coli SyBE 002447 as starting strain, integrates full chemistry synthesis module P respectivelylacAroG tyrA aroE and PtrcPps tkt glk, build the chassis bacterial strain SyBE 22002 of L tyrosine；Then full chemistry synthesis module P is usedtacsHpaBC ldh, which are integrated, is inserted into nupG downstream positions on chromosome, obtains bacterial strain SyBE 22011.The present invention solves the problems, such as that fermentation process needs to add antibiotic and inducer isopropylthio β D Thiogalactopyranosides, L tyrosine and danshensu can efficiently be produced, simplify downstream separation purge process, reduce industrial production cost, the pollution of antibiotic and derivant to environment is avoided, is increased economic efficiency.

Description

A kind of application for the method and the recombination engineering for producing l-tyrosine recombination engineering
Technical field
Biological technical field belonging to the present invention, the structure and its production L- of more particularly to a kind of l-tyrosine recombination engineering The method of tyrosine and danshensu.
Background technology
L-tyrosine is a kind of important nutritional necessary amino acid, and metabolism, growth and development to humans and animals play Important effect.Its frequently as, phenylketonuria patient nutritional supplement, and peptide hormone, antibiotic, L-3,4 dihydroxyphenylalanine, The preparing raw material of melanin, p-Coumaric Acid, 4-Vinyl phenol etc. and be widely used in food, feed, medicine and chemical industry Etc. industry.Based on this, recent years l-tyrosine and its synthesis of derivative attracted the sight of more and more researchers.Due to The ability of natural microbial dynamic accumulation l-tyrosine is very low, even if the recombination engineering of structure, it is still desirable to use antibiotic Plasmid and derivant is maintained to make gene overexpression, there are destruction and the potential hazard of pollution environment.
Danshensu is a kind of derivative of l-tyrosine, its entitled β-(3,4- dihydroxy phenyl) lactic acid of chemistry.It is newest Pharmacological research shows that danshensu can suppress blood platelet synthesis and aggregation, improves myocardial hypoxia tolerance, protection cardiac muscle, at the same time Increase coronary flow.These pharmacological actions are Salvia Miltiorrhiza Injection coronary heart disease, the main mechanism of myocardial ischemia.Therefore danshensu Have effects that in terms of cardiovascular and cerebrovascular disease it is extraordinary, such as：Antiatherosclerosis and reducing blood lipid, antithrombus formation, expansion Coronary artery, protects the effect such as damage of lipid peroxide that myocardial mitochondria triggers from oxygen radical.In addition there is research It was found that it in antibacterial anti-inflammatory, prevents the excessive healing of the surface of a wound, anti-cerebral ischemia damnification, element is antitumor, treatment psoriasis etc. There is preferable effect.Therefore danshensu possesses wide market and application prospect.Danshensu is mainly extracted from red sage root at present, But content is low, limited be subject to planting season and raw material, production capacity is low.Microbe synthesis has that cost is low, yield is high Advantage, this patent are exactly to construct l-tyrosine production recombination engineering and its derivative strain, realize l-tyrosine and danshensu Microbial fermentation production.
The content of the invention
The purpose of the present invention is for current l-tyrosine and danshensu production status, there is provided production l-tyrosine and Radix Salviae Miltiorrhizae The recombination engineering of element.
Second object of the present invention be to provide above-mentioned recombination engineering in fermenting and producing l-tyrosine and danshensu should With.
Technical scheme is summarized as follows：
A kind of recombination engineering for producing l-tyrosine, is built with following methods：
(1) using Escherichia coli SyBE-002447 genomes as template, using sequence shown in SEQ ID No.1 as sense primer, Using sequence shown in SEQ ID No.2 as anti-sense primer, clone obtains the fragment F1 of 500bp；Design simultaneously chemistry synthesis module Plac- AroG-tyrA-aroE, sequence is as shown in SEQ ID No.3, using the sequence as template, using sequence shown in SEQ ID No.4 to be upper Primer is swum, using sequence shown in SEQ ID No.5 as anti-sense primer, clone obtains the fragment F2 of 3500bp or so；With chemical synthesis Sequence C HL genes are template, its sequence is as shown in SEQ ID NO.6, using sequence shown in SEQ ID No.7 as sense primer, with Sequence shown in SEQ ID No.8 is anti-sense primer, and amplification obtains the F3 fragments of 1000bp；With Escherichia coli SyBE-002447 bases Because group is template, using sequence shown in SEQ ID No.9 as sense primer, using sequence shown in SEQ ID No.10 as anti-sense primer, Clone obtains the fragment F4 of 500bp.
(2) four recombinant fragments obtained in (1) are assembled into recombinant fragment F1-F2-F3- by the method for overlap-extension PCR F4.Using Escherichia coli SyBE-002447 bacterial strains as chassis cell, F1-F2-F3-F4 is integrated using the method for λ red homologous recombinations Then fragment loses chloramphenicol resistance gene from chromosome, obtains recombinant bacterial strain on the chromosome of SyBE-002447 bacterial strains SyBE-22001, for fermenting and producing l-tyrosine.
(3) using the bacterial strain SyBE-22001 genomes obtained in (2) as template, using sequence shown in SEQ ID No.11 to be upper Primer is swum, using sequence shown in SEQ ID No.12 as anti-sense primer, clone obtains the fragment F5 of 500bp；Design simultaneously chemical synthesis L-tyrosine synthesis module Ptrc- pps-tkt-glk, sequence is as shown in SEQ ID No.13, using the sequence as template, with SEQ Sequence shown in ID No.14 is sense primer, and using sequence shown in SEQ ID No.15 as anti-sense primer, clone obtains a 5700bp left sides Right fragment F6；With sequence SEQ ID No.6 (CHL genes) for template, using sequence shown in SEQ ID No.16 as sense primer, Using sequence shown in SEQ ID No.17 as anti-sense primer, amplification obtains the fragment F7 of 1000bp.With bacterial strain SyBE-22001 genes Group is template, using sequence shown in SEQ ID No.18 as sense primer, using sequence shown in SEQ ID No.19 as anti-sense primer, gram The grand fragment F8 for obtaining 500bp.
(4) the method assembling structure recombinant fragment F5-F6- that four recombinant fragments obtained in (3) are passed through overlap-extension PCR F7-F8.Integrated using the method for λ red homologous recombinations on the chromosome of fragment F5-F6-F7-F8 to SyBE-22001 bacterial strains, so Chloramphenicol resistance gene is lost from chromosome afterwards, obtains recombinant bacterial strain SyBE-22002, for fermenting and producing l-tyrosine.
(5) using bacterial strain SyBE-22002 genomes as template, using sequence shown in SEQ ID No.20 as sense primer, with Sequence shown in SEQ ID No.21 is anti-sense primer, and clone obtains the fragment F9 of 500bp or so.Synthesized with design and full chemistry Danshensu synthesis module sequence SEQ ID No.22 (Ptacs- hpaBC-ldh) be template, using sequence shown in SEQ ID No.23 as Sense primer, using sequence shown in SEQ ID No.24 as anti-sense primer, clone 3600bp or so fragment F10；With sequence SEQ ID No.6 (CHL genes) are template, using sequence shown in SEQ ID No.25 as sense primer, with sequence shown in SEQ ID No.26 Anti-sense primer is classified as, amplification obtains the F11 fragments of 1000bp or so.Using bacterial strain SyBE-22002 genomes as template, with SEQ Sequence shown in ID No.27 is sense primer, and using sequence shown in SEQ ID No.28 as anti-sense primer, clone obtains 500bp or so Fragment F12.
(6) the method assembling structure recombinant fragment F9-F10- that four recombinant fragments obtained in (5) are passed through overlap-extension PCR F11-F12, using the method for λ red homologous recombinations on the chromosome of SyBE-22002 bacterial strains, loses chloramphenicol resistance gene, The integration bacterial strain SyBE-22011 recombinated, the danshensu of upper tank fermentation synthesis.
The solution have the advantages that：The present invention overcomes the l-tyrosine by plasmid expression system and danshensu production The deficiency of bacterial strain, there is provided it is a kind of it is new produce l-tyrosine and the method for danshensu using microbial fermentation, utilizing works are big Enterobacteria realize from glucose production l-tyrosine and danshensu, fermentation process need to need not add any precursor, antibiotic and Derivant, is conducive to isolating and purifying for downstream, reduces production cost, increases economic efficiency, greatly accelerate l-tyrosine and Radix Salviae Miltiorrhizae The industrialized production of element.
Embodiment
Technical scheme is further illustrated with reference to specific embodiment.It is each to use plasmid specifying information such as Under：Plasmid pJET1.2 is purchased from Beijing Quanshijin Biotechnology Co., Ltd (western No. 66 Zhong Guan-cun in osculum road in Haidian District, Beijing City east Rise four floor of Technology Park B-3 buildings)；Plasmid pKD46 and pCP20 are purchased from Beijing Central Plains company (Oriental East Road, Chaoyang District, Beijing City 11 Number).
Escherichia coli used in the present invention are SyBE-002447, now common in China Committee for Culture Collection of Microorganisms The preservation of microorganism center, collection numbering of registering on the books is CGMCC No.7962.The preservation time is on July 22nd, 2013, ground Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode 100101.
Embodiment 1：The structure of l-tyrosine recombinant strain
Using SyBE-002447 as starting strain, pass through λ red homologous recombinations paa gene clusters on its genome respectively Position synthesis genetic fragment Plac- aroG-tyrA-aroE, P is integrated in the position of LacI genestrc- pps-tkt-glk fragments, Obtain final chassis bacterial strain SyBE-22002.Detailed process is as follows：
(1) P is usedlac- aroG-tyrA-aroE replaces paa gene clusters on SyBE-002447 chromosomes.With Escherichia coli SyBE-002447 genomes are template, design upstream homology arm primers F 1-F and F1-R, using sequence shown in SEQ ID No.1 as Sense primer, using sequence shown in SEQ ID No.2 as anti-sense primer；Downstream homology arm primers F 4-F and F4-R are designed, with SEQ Sequence shown in ID No.9 is sense primer, using sequence shown in SEQ ID No.10 as anti-sense primer；
5’-GAAGTCGCTGCACCTAAGGAACCGGAGAGGCGGTTTGCGTATTGGG-3’；SEQ ID No.1
5’-CGCCTTCTCCTGTAGCGTTATCCCGATAT-3’；SEQ ID No.2
5’-TAATATTTCGTCTCAATACAAATTC-3’；SEQ ID No.9
5’-AGGCCGTGATTAACGCAGCAGCG-3’；SEQ ID No.10
50 μ LPCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 1 μ L sense primers, 1 μ L anti-sense primers, 1 μ L genomic templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃10min；4℃+ ∞。
PCR amplification respectively obtains the downstream homology arm F4 of 500bp upstreams homology arm F1 and 500bp.
Design simultaneously full chemistry synthesis module Plac- aroG-tyrA-aroE, its sequence is as shown in SEQ ID No.3, with the sequence Template is classified as, upstream and downstream primer F2-F and F2-R are designed, using sequence is sense primer shown in SEQ ID No.4, with SEQ ID Sequence shown in No.5 is anti-sense primer；
5’-CCCAATACGCAAACCGCCTCTCC-3’；SEQ ID No.4
5’-TCCCAATACGCAAACCGCCTCTCC-3’；SEQ ID No.5
50 μ LPCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 1 μ L sense primers, 1 μ L anti-sense primers, 1 μ L plasmid templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 56 DEG C of 45s, 72 DEG C of 2min, 30 circulations；72℃10min；4℃+ ∞.PCR amplification obtains the F2 fragments of 3500bp or so；
Using full chemistry composition sequence CHL genes as template, its sequence designs upstream and downstream primer as shown in SEQ ID NO.6 F3-F and F3-R, using sequence shown in SEQ ID No.7 as sense primer, using sequence shown in SEQ ID No.8 as anti-sense primer；
5’-GGAGAGGCGGTTTGCGTATTGGGAGTGTAGGCTG GAGCTGCTTC-3’；SEQ ID No.7
5’-GTCTCGAATTTGTATTGAGACGAAATATTAATGGGAATTAGCCATGGTCC-3’；SEQ ID No.8
50 μ LPCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 1 μ L sense primers, 1 μ L anti-sense primers, 1 μ L plasmid templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃10min；4℃+ ∞.Amplification obtains the F3 fragments of 1000bp or so.
After four fragments that PCR is obtained carry out PCR purifying recycling, using above-mentioned F1 fragments and F2 fragments as template (two A fragment is according to molar ratio 1：1 adds), using sequence shown in SEQ ID No.1 as sequence shown in sense primer and SEQ ID No.5 For anti-sense primer, Overlap extension PCR is carried out；
50 μ LPCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 1 μ L sense primers, 1 μ L anti-sense primers, 1 μ L fragment templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 2min10s, 30 circulations；72℃10min；4 ℃+∞.Amplification obtains the F1-F2 fragments of 4000bp or so, this fragment is carried out PCR recycling；
Using F3 fragments and F4 fragments, as template, (two fragments are according to molar ratio 1：1 adds), with sequence shown in SEQ ID No.7 It is anti-sense primer to be classified as sequence shown in sense primer and SEQ ID No.10, carries out PCR reactions；
50 μ L PCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 1 μ L sense primers, 1 μ L anti-sense primers, 1 μ L fragment templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72℃10min；4℃+ ∞.Amplification obtains the F3-F4 fragments of 1500bp or so, this fragment is carried out PCR recycling.
Finally using two fragments of F1-F2 and F3-F4 as template, (two fragments are according to molar ratio 1：1 adds), with SEQ ID Sequence shown in No.1 is that sequence shown in sense primer and SEQ ID No.4 is that anti-sense primer carries out PCR reactions,
50 μ LPCR reaction systems are：5 × buffer of 10 μ L, 5 μ L dNTPs, 1 μ L sense primers, 1 μ L anti-sense primers, 1 μ L fragment templates, 1 μ L polymerases, surplus pure water polishing.
PCR parameters are as follows：95℃5min；95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 3min, 30 circulations；72℃10min；4℃+ ∞.Amplification obtains the F1-F2-F3-F4 fragments of 5500bp or so, this fragment is carried out PCR recycling.
(2) according to pJET1.2 cloned plasmids kit specifications, which is connected on pJET1.2 plasmids and is obtained PJET-F1-F2-F3-F4 plasmids, and convert competent escherichia coli cell.37 DEG C recovery after the completion of be coated with ampicillin and Chlorampenicol resistant LB tablets, when 37 DEG C of cultures 12 are small.Then using using sequence shown in SEQ ID No.1 as sense primer and SEQ ID Sequence shown in No.10 carries out bacterium colony PCR verifications, screening positive clone for anti-sense primer, and PCR the results show stripe sizes are 5500bp represents that purpose fragment is successfully connected on pJET1.2 carriers.And it will verify that obtained positive clone strain carries out sequencing and tests Card, is as a result sequence designed before.
(3) conventional method imported according to escherichia coli plasmid is transferred to pKD46 plasmids in bacterial strain SyBE-002447. In 5mL LB culture mediums, (LB culture medium prescriptions are 10g/LNaCl to the Escherichia coli that picking contains pKD46 plasmids, 10g/L albumen Peptone, 5g/L yeast extracts), 30 DEG C are incubated overnight.100uL is taken to be incubated overnight bacterium solution switching in containing 10mL LB culture mediums Test tube, while the ampicillin of final concentration of 50ug/mL is added, 30 DEG C, 220rpm cultures.When thalline culture to OD600 exists When between 0.3-0.4, add the L-arabinose of final concentration of 100mM as derivant, continue culture to OD600 be about 0.6, Prepare electroporated competent cell.The F1-F2-F3-F4 recombinant fragments for taking 7.5uL to prepare, it is electroporated to enter treating for preparation Electroporated competent cell, 2.5KV, 5-6ms, 37 degree of recovery 1-2h.The flat of the LB of chlorampenicol resistant is coated with after the completion of recovery Plate, is incubated overnight.Second day is sequence shown in sense primer and SEQ ID No.10 as downstream using sequence shown in SEQ ID No.1 Primer carries out bacterium colony PCR verifications, picks out positive colony, while using original strain as control, PCR reaction systems and parameter are set Put in same embodiment 1 (1), obtained PCR product is subjected to sequence verification, to ensure that gene is replaced successfully.Verify correct bacterium Strain is deposited bacterium and is preserved (glycerol stock store method mixes for 600uL glycerine and 600uL bacterium solutions, places -20 degree and carries out long term storage).
(4) the correct bacterial strain of verification in (3) is taken to lack its pKD46 plasmid carried, step is as follows：Glycerol stock 50uL is taken to connect Enter in 5mL LB culture mediums, 42 DEG C, after 220rmp cultures 4-6h, take 50uL to be transferred in new 5mL LB culture mediums at once, 42 DEG C, 220rmp culture 4-6h, this step is repeated 2 times, and is rule to the bacterium solution finally obtained in the LB solid plates of nonreactive, 42 DEG C of trainings 12h is supported, is rule respectively in ammonia benzyl and the LB solid plates of the mould resistance of chlorine to the monoclonal bacterial strain on tablet, after cultivating 12h, Dan Ke Grand bacterial strain can not survive on ammonia benzyl antibiotic tablet, but survive in the LB tablets of chlorampenicol resistant, it was demonstrated that pKD46 plasmids are Through successfully lacking, the monoclonal bacterial strain is preserved, store method is same as above.
(5) the mould resistant gene of chlorine (F3) lacked in pKD46 strain gene groups obtained in (4) is lacked.Step It is rapid as follows：The conventional method imported using escherichia coli plasmid, which imported into pcp20 plasmids, has integrated F1-F2-F3-F4 fragments In bacterial strain SyBE-002447, at 30 DEG C, recovery culture 1-2h, is coated on ammonia benzyl and the mould dual anti-LB of chlorine under conditions of 160rpm On solid plate, 12h is cultivated, obtains the purpose bacterial strain with pCP20 plasmids；Purpose bacterial strain of the picking with pCP20 plasmids exists 42 DEG C of secondary cultures 3 times (step is with the operation of missing pKD46 plasmid procedures herein) in nonreactive culture medium, then take the culture of third time Liquid is scoring to nonreactive LB solid plates, and 42 DEG C are incubated overnight；The single bacterium colony that next day picking is stayed overnight on tablet is distinguished respective score line and is arrived (since pCP20 be able to not can be replicated in temperature more than 37 DEG C and lost) on the tablet of the mould resistance of chlorine and nonreactive, survive in nonreactive and put down Plate and the single bacterium colony that can not be survived in the mould resistance of chlorine, further using sequence shown in SEQ ID No.1 as sense primer and SEQ ID Sequence shown in No.10 carries out bacterium colony PCR verifications for anti-sense primer, verifies whether the mould resistant gene of chlorine successfully lacks by bacterium colony PCR Lose, PCR the results shows stripe size represents successfully to lack the mould resistant gene of chlorine for 4500bp, and the bacterial strain obtained after lacking successfully is deposited Bacterium, store method are same as above, strain was named SyBE-22001.
(5) on the basis of bacterial strain SyBE-22001, by the method for λ red homologous recombinations fragment Ptrc-pps-tkt-glk It is incorporated on bacterial strain SyBE-22001 genomes.Build the experimental procedure of recombinant fragment F5-F6-F7-F8 and P in (1)lac- It is similar that aroG-tyrA-aroE integrates fragment (F1-F2-F3-F4) building process.Using Escherichia coli SyBE-22001 genomes as Template, designs upstream and downstream homology arm primers F 5-F and F5-R, using sequence is sense primer shown in SEQ ID No.11, with SEQ ID Sequence shown in No.12 is anti-sense primer；Design downstream homology arm primers F 8-F and F8-R, using sequence shown in SEQ ID No.18 as Sense primer, using sequence shown in SEQ ID No.19 as anti-sense primer；
5’-CGGCATTGGCTGGGTCGCGG-3’；SEQ ID No.11
5’-CGTTAATTACACTCAATCGAGTGCAGGTGAAACTGACCGATAAGCCG-3’；SEQ ID No.12
5’-CGCGGTAAGCTACCACAGTTGCATTTAC-3’；SEQ ID No.18
5’-CGTTACCACCATGCGCTTTGGTGAGTGGC-3’；SEQ ID No.19
Pass through PCR amplification 500bp upstreams homology arm F5 fragments and 500bp downstreams homology arm F8 fragments；
Design simultaneously full chemistry synthesis module Ptrc- pps-tkt-glk, its sequence is as shown in SEQ ID No.13, and design is up and down Primers F 6-F and F6-R are swum, using the sequence as template, using sequence is sense primer shown in SEQ ID No.14, with SEQ ID Sequence shown in No.15 is anti-sense primer, and amplification obtains the F6 fragments of 5700bp or so；
5’-CGGCTTATCGGTCAGTTTCACCTG-3’；SEQ ID No.14
5’-TGCAGCGACTGCACGGTGCACC-3’；SEQ ID No.15
Again using full chemistry composition sequence CHL genes as template, its sequence as shown in SEQ ID NO.6, draw by design upstream and downstream Thing F7-F and F7-R, using sequence shown in SEQ ID No.16 as sense primer, are drawn using sequence shown in SEQ ID No.17 as downstream Thing, amplification obtain the chlorampenicol resistant fragment F7 of 1000bp or so；
5’-GGTGCACCGTGCAGTCGCTGCAGTGTAGGCTG GAGCTGCTTC-3’；SEQ ID No.16
5’-GTACGTAAATGCAACTGTGGTAGCTTACCGCGATGGGAATTAGCCATGGTC-3’；SEQ ID No.17
Above-mentioned 4 PCR reaction systems and parameter setting are the same as (1) in embodiment 1 (wherein primer is different)
After obtaining 4 fragments, with DNA purification kits, PCR fragment is recycled.Finally the homologous F5 in upstream, purpose fragment F6, chlorampenicol resistant fragment F7 and downstream homology arm F8 are connected by the method for overlap-extension PCR, obtain the F5- of 7700bp F6-F7-F8 recombinant fragments.It is connected to according to method same in (1) on pJET1.2 plasmids, and will verifies positive gram obtained Grand bacterial strain carries out sequence verification, and bacterium preservation is deposited after correctly being cloned.
(6) pKD46 plasmids are transferred to the bacterial strain SyBE-22001 obtained in (4), then and (3) in take same experiment Operation, electric shock is transferred to F5-F6-F7-F8 recombinant fragments in the Escherichia coli of the plasmid containing pKD46 after being induced with L-arabinose, After obtaining correct clone strain, the pKD46 plasmids of its carrying are lacked；Further bacterium is obtained by importing pCP20 Plasmid eliminations The mould resistant gene of chlorine (F7) in pnca gene group.Using sequence shown in SEQ ID No.11 as sense primer and SEQ ID No.19 institutes It is anti-sense primer to show sequence, verifies whether the mould resistant gene of chlorine successfully lacks by bacterium colony PCR, PCR the results show stripe sizes Represent successfully to lack the mould resistant gene of chlorine, the optimization chassis bacterial strain for seamless integration of succeeding, strain was named for 6700bp SyBE-22002。
Fermentation medium composition contains for every liter of culture medium：K2HPO41.32mM、NH4Cl 50mM、MgCl20.523mM、 K2SO40.276mM、FeSO40.01mM、CaCl20.0005mM, NaCl 50mM, yeast extract 1g/L, glucose 5g/L.Adjust Section pH sterilizes 20min under 7.0 or so, 0.1Mpa pressure.Flow the glucose solution that the carbon source added is 100g/L, 105 DEG C of high pressures Sterilize 30min.
Cultivated in fermentation medium, without adding antibiotic and derivant, when shake flask fermentation 24 is small.SyBE-22001 Bacterial strain production l-tyrosine yield is 380mg/L.SyBE-22002 bacterial strains production l-tyrosine yield is 520mg/L.
2 danshensu of embodiment synthesizes the structure of bacterial strain
Devise the tac promoters (P of series connectiontacs) driving hpaBC and ldh genes coexpression, and Ptacs- HpaBC-ldh is incorporated on the chassis bacterial strain SyBE-22002 chromosomes obtained in embodiment 1, and integration site selects in the genome NupG downstreams.Comprise the following steps that：
(1) recombinant fragment F9-F10-F11-F12 is built
Recombinant fragment F9-F10-F11-F12 is built, fragment F1-F2-F3-F4 structures are integrated in its construction step and example 1 Process is similar.Using integration bacterial strain SyBE-22002 genomes as template, upstream homology arm primers F 9-F and F9-R are designed, with SEQ Sequence shown in ID No.20 is sense primer, using sequence shown in SEQ ID No.21 as anti-sense primer；Design downstream homology arm draws Thing F12-F and F12-R, using sequence shown in SEQ ID No.27 as sense primer, using sequence shown in SEQ ID No.28 as downstream Primer,
5’-GTTCATTCTGACCATCCCG-3’；SEQ ID No.20
5’-CAAATTCAGCCGATAGCGGAACGGGAAGGCACCCGTTTTTCTTTGCGTA-3’；SEQ ID No.21
5’-CGCCAGAAGGTGACCCGTT-3’；SEQ ID No.27
5’-TTGAGGATGACTCGCCGCT-3’；SEQ ID No.28
PCR amplification respectively obtains the downstream homology arm F12 of 500bp upstreams homology arm F9 and 500bp.
Design simultaneously full chemistry synthesis module Ptacs- hpaBC-ldh, its sequence is as shown in SEQ ID No.22, and design is up and down Swim primers F 10-F and F10-R, using sequence shown in SEQ ID No.23 as sense primer, using sequence shown in SEQ ID No.24 as Anti-sense primer；
5’-GCCTTCCCGTTCCGCTATC-3’；SEQ ID No.23
5’-CTGCGCTAGTAGACGAGTCC-3’；SEQ ID No.24
PCR amplification obtains the F10 fragments of 3600bp or so；
Again using full chemistry composition sequence CHL genes as template, its sequence as shown in SEQ ID NO.6, draw by design upstream and downstream Thing F11-F and F11-R, using sequence shown in SEQ ID No.25 as sense primer, using sequence shown in SEQ ID No.26 as downstream Primer；
5’-GCCAGCACATGGACTCGTCTACTAGCGCAGGTGTAGGCTGGAGCTGCTTC-3’；SEQ ID No.25
5’-TAAAAAAAACGGGTCACCTTCTGGCGATGGGAATTAGCCATGGTCC-3’；SEQ ID No.26
The chlorampenicol resistant fragment F11 of PCR amplification 1000bp or so；
Above-mentioned 4 PCR reaction systems are with parameter setting the same as embodiment 1 (wherein primer is different).
With DNA purification kits, four PCR fragments are separately recovered.Upstream homology arm F9, purpose piece degree F10, chloramphenicol Resistance fragments F11 and downstream homology arm F12 gets the F9-F10-F11-F12 weights of 5600bp continuously by the method for overlap-extension PCR Pack section.
Same method according to (1) in example 1 is connected on pJET1.2 plasmids, and will verify obtained positive colony Bacterial strain carries out sequence verification, and bacterium preservation is deposited after correctly being cloned.
(2) structure of integration bacterial strain
PKD46 plasmids are transferred in bacterial strain SyBE-22002, then same experimental implementation is taken with (1) in embodiment 1, Electric shock is transferred to F9-F10-F11-F12 weights in the Escherichia coli SyBE-22002 of the plasmid containing pKD46 after being induced with L-arabinose Pack section, after obtaining the clone for correctly integrating purpose fragment, lacks the pKD46 plasmids of its carrying；Further transduceed by electricity Enter the mould resistant gene of chlorine (F11) in pcp20 Plasmid eliminations acquisition strain gene group.Using sequence shown in SEQ ID No.20 to be upper It is anti-sense primer to swim sequence shown in primer and SEQ ID No.28, verifies whether the mould resistant gene of chlorine successfully lacks by bacterium colony PCR Lose, PCR the results shows stripe size represents successfully to lack the mould resistant gene of chlorine for 4600bp, and the bacterial strain obtained after lacking successfully is deposited Bacterium, integration bacterial strain are named as SyBE-22011.
(3) fermenting and producing danshensu
Fermenting and producing danshensu is carried out into SyBE-22011 using the integration bacterium that step (2) obtains, in the step (3) In, without adding antibiotic and derivant.
The glycerol stock of the integration bacterial strain SyBE-22011 of 50uL preservations is taken to be connected to the test tube of the LB culture mediums containing 5mL nonreactives In be incubated overnight, then the 100 μ L bacterium solutions being incubated overnight are transferred in the 250mL shaking flasks containing 100mL LB culture mediums, altogether The LB culture mediums of 4 bottles of 100mL are connect, at 37 DEG C, bacterium solution, is then linked into the fermentation tank of 5L and is mended by 220rpm culture 6-8h Material fermentation, fermentation tank contain 2.1L fermentation medium (fermentative medium formulas：Glucose 7.5g/L, Na2HPO46.8g/L, KH2PO48.5g/L,NH4CL 3g/L,NaCl 0.5g/L,CaCL2·2H2O 0.07g/L,MgSO4·7H2O 1g/L and ferment Female extract 5g/L).Fermentation process every 3h sampling analysis danshensus yield, remaining glucose sugar concentration and bacterial strain biomass, Flow feeding control glucose sugar concentration is consistently lower than 1g/L, and feed-batch culture based formulas is：Glucose 500g/L, yeast extract 60g/L。
The detection of content of Danshensu：Take 1mL zymotic fluids 12000r/min to centrifuge 3 minutes, supernatant is taken, with 0.22 μm of micropore HPLC detections are carried out after membrane filtration.Chromatographic condition is as follows：Chromatographic column：C18 (4.6mm × 250mm, 5um)；Mobile phase is 20% - 0.1% formic acid of the water of methanol -80%；Flow velocity 1mL/min；20 μ L of sample size；Column temperature room temperature；UV detector, Detection wavelength 281nm.Ferment 60 it is small when, bacterial strain SyBE-22011 finally synthesizes 6.2g/L danshensus.
Exemplary description has been done to the present invention above, it should explanation, in the situation for the core for not departing from the present invention Under, any simple deformation, modification or other skilled in the art can not spend the equivalent substitution of creative work equal Fall into protection scope of the present invention.
Sequence table：
SEQ ID NO.03:
CCCAATACGCAAACCGCCTCTCCACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAA GCGGTCCCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTC CCGACTGGAAAGCAATTGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTA CACTTTATGCTTCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAGATATCAAGCTTAC ATGCTCTAGAAAAGGAGAATATCATGAATTATCAGAACGACGATTTACGCATCAAAGAAATCAAAGAGTTACTTCCT CCTGTCGCATTGCTGGAAAAATTCCCCGCTACTGAAAATGCCGCGAATACGGTTGCCCATGCCCGAAAAGCGATCCA TAAGATCCTGAAAGGTAATGATGATCGCCTGTTGGTTGTGATTGGCCCATGCTCAATTCATGATCCTGTCGCGGCAA AAGAGTATGCCACTCGCTTGCTGGCGCTGCGTGAAGAGCTGAAAGATGAGCTGGAAATCGTAATGCGCGTCTATTTT GAAAAGCCGCGTACCACGGTGGGCTGGAAAGGGCTGATTAACGATCCGCATATGGATAATAGCTTCCAGATCAACGA CGGTCTGCGTATAGCCCGTAAATTGCTGCTTGATATTAACGACAGCGGTCTGCCAGCGGCAGGTGAGTTTCTCGATA TGATCACCCTACAATATCTCGCTGACCTGATGAGCTGGGGCGCAATTGGCGCACGTACCACCGAATCGCAGGTGCAC CGCGAACTGGCATCAGGGCTTTCTTGTCCGGTCGGCTTCAAAAATGGCACCGACGGTACGATTAAAGTGGCTATCGA TGCCATTAATGCCGCCGGTGCGCCGCACTGCTTCCTGTCCGTAACGAAATGGGGGCATTCGGCGATTGTGAATACCA GCGGTAACGGCGATTGCCATATCATTCTGCGCGGCGGTAAAGAGCCTAACTACAGCGCGAAGCACGTTGCTGAAGTG AAAGAAGGGCTGAACAAAGCAGGCCTGCCAGCACAGGTGATGATCGATTTCAGCCATGCTAACTCGTCCAAACAATT CAAAAAGCAGATGGATGTTTGTGCTGACGTTTGCCAGCAGATTGCCGGTGGCGAAAAGGCCATTATTGGCGTGATGG TGGAAAGCCATCTGGTGGAAGGCAATCAGAGCCTCGAGAGCGGGGAGCCGCTGGCCTACGGTAAGAGCATCACCGAT GCCTGCATCGGCTGGGAAGATACCGATGCTCTGTTACGTCAACTGGCGAATGCAGTAAAAGCGCGTCGCGGGTAAAA GCTTACATGCTCTAGAAAAGGAGAATATTATGGTTGCTGAATTGACCGCATTACGCGATCAAATTGATGAAGTCGAT AAAGCGCTGCTGAATTTATTAGCGAAGCGTCTGGAACTGGTTGCTGAAGTGGGCGAGGTGAAAAGCCGCTTTGGACT GCCTATTTATGTTCCGGAGCGCGAGGCATCTATTTTGGCCTCGCGTCGTGCAGAGGCGGAAGCTCTGGGTGTACCGC CAGATCTGATTGAGGATGTTTTGCGTCGGGTGATGCGTGAATCTTACTCCAGTGAAAACGACAAAGGATTTAAAACA CTTTGTCCGTCACTGCGTCCGGTGGTTATCGTCGGCGGTGGCGGTCAGATGGGACGCCTGTTCGAGAAGATGCTGAC CCTCTCGGGTTATCAGGTGCGGATTCTGGAGCAACATGACTGGGATCGAGCGGCTGATATTGTTGCCGATGCCGGAA TGGTGATTGTTAGTGTGCCAATCCACGTTACTGAGCAAGTTATTGGCAAATTACCGCCTTTACCGAAAGATTGTATT CTGGTCGATCTGGCATCAGTGAAAAATGGGCCATTACAGGCCATGCTGGTGGCGCATGATGGTCCGGTGCTGGGGCT ACACCCGATGTTCGGTCCGGACAGCGGTAGCCTGGCAAAGCAAGTTGTGGTCTGGTGTGATGGACGTAAACCGGAAG CATACCAATGGTTTCTGGAGCAAATTCAGGTCTGGGGCGCTCGGCTGCATCGTATTAGCGCCGTCGAGCACGATCAG AATATGGCGTTTATTCAGGCACTGCGCCACTTTGCTACTTTTGCTTACGGGCTGCACCTGGCAGAAGAAAATGTTCA GCTTGAGCAACTTCTGGCGCTCTCTTCGCCGATTTACCGCCTTGAGCTGGCGATGGTCGGGCGACTGTTTGCTCAGG ATCCGCAGCTTTATGCCGACATCATTATGTCGTCAGAGCGTAATCTGGCGTTAATCAAACGTTACTATAAGCGTTTC GGCGAGGCGATTGAGTTGCTGGAGCAGGGCGATAAGCAGGCGTTTATTGACAGTTTCCGCAAGGTGGAGCACTGGTT CGGCGATTACGTGCAGCGTTTTCAGAGTGAAAGCCGCGTGTTATTGCGTCAGGCGAATGACAATCGCCAGTAAAAGC TTACATGCTCTAGAAAAGGAGGAATATCATGGAAACCTATGCTGTTTTTGGTAATCCGATAGCCCACAGCAAATCGC CATTCATTCATCAGCAATTTGCTCAGCAACTGAATATTGAACATCCCTATGGGCGCGTGTTGGCACCCATCAATGAT TTCATCAACACACTGAACGCTTTCTTTAGTGCTGGTGGTAAAGGTGCGAATGTGACGGTGCCTTTTAAAGAAGAGGC TTTTGCCAGAGCGGATGAGCTTACTGAACGGGCAGCGTTGGCTGGTGCTGTTAATACCCTCATGCGGTTAGAAGATG GACGCCTGCTGGGTGACAATACCGATGGTGTAGGCTTGTTAAGCGATCTGGAACGTCTGTCTTTTATCCGCCCTGGT TTACGTATTCTGCTTATCGGCGCTGGTGGAGCATCTCGCGGCGTACTACTGCCACTCCTTTCCCTGGACTGTGCGGT GACAATAACTAATCGGACGGTATCCCGCGCGGAAGAGTTGGCTAAATTGTTTGCGCACACTGGCAGTATTCAGGCGT TGAGTATGGACGAACTGGAAGGTCATGAGTTTGATCTCATTATTAATGCAACATCCAGTGGCATCAGTGGTGATATT CCGGCGATCCCGTCATCGCTCATTCATCCAGGCATTTATTGCTATGACATGTTCTATCAGAAAGGAAAAACTCCTTT TCTGGCATGGTGTGAGCAGCGAGGCTCAAAGCGTAATGCTGATGGTTTAGGAATGCTGGTGGCACAGGCGGCTCATG CCTTTCTTCTCTGGCACGGTGTTCTGCCTGACGTAGAACCAGTTATAAAGCAATTGCAGGAGGAATTGTCCGCGTGA ACTAGTCCGGCTTATCGGTCAGTTTCACCTGATTTACGTAAAAACCCGCTTCGGCGGGTTTTTGCTTTTGGAGGGGC AGAAAGATGAATGACTGTCCACGACGCTATACCCAAAAGAAACGAGGAGAGGCGGTTTGCGTATTGGGA
SEQ ID NO.06:
GTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCA TTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGACATGG AAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCC CATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGAT TGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCT TGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTC ATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGTAATTCCG GATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTT AAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTC TTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTG AAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGC CGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTC TGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGA ACTAAGGAGGATATTCATATGGACCATGGCTAATTCCCAT
SEQ ID NO.013:
CTGCAGCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGT GCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACAT CATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGGAGAGGG AATTGTGAGCGGATAACAATTTCACACAGGAAACAGGAATTCCGGAGATGAAGATCTTATAAGAAGGAGATATACAT ATGTCCAACAATGGCTCGTCACCGCTGGTGCTTTGGTATAACCAACTCGGCATGAATGATGTAGACAGGGTTGGGGG CAAAAATGCCTCCCTGGGTGAAATGATTACTAATCTTTCCGGAATGGGTGTTTCCGTTCCGAATGGTTTCGCCACAA CCGCCGACGCGTTTAACCAGTTTCTGGACCAAAGCGGCGTAAACCAGCGCATTTATGAACTGCTGGATAAAACGGAT ATTGACGATGTTACTCAGCTTGCGAAAGCGGGCGCGCAAATCCGCCAGTGGATTATCGACACTCCCTTCCAGCCTGA GCTGGAAAACGCCATCCGCGAAGCCTATGCACAGCTTTCCGCCGATGACGAAAACGCCTCTTTTGCGGTGCGCTCCT CCGCCACCGCAGAAGATATGCCGGACGCTTCTTTTGCCGGTCAGCAGGAAACCTTCCTCAACGTTCAGGGTTTTGAC GCCGTTCTCGTGGCAGTGAAACATGTATTTGCTTCTCTGTTTAACGATCGCGCCATCTCTTATCGTGTGCACCAGGG TTACGATCACCGTGGTGTGGCGCTCTCCGCCGGTGTTCAACGGATGGTGCGCTCTGACCTCGCATCATCTGGCGTGA TGTTCTCCATTGATACCGAATCCGGCTTTGACCAGGTGGTGTTTATCACTTCCGCATGGGGCCTTGGTGAGATGGTC GTGCAGGGTGCGGTTAACCCGGATGAGTTTTACGTGCATAAACCGACACTGGCGGCGAATCGCCCGGCTATCGTGCG CCGCACCATGGGGTCGAAAAAAATCCGCATGGTTTACGCGCCGACCCAGGAGCACGGCAAGCAGGTTAAAATCGAAG ACGTACCGCAGGAACAGCGTGACATCTTCTCGCTGACCAACGAAGAAGTGCAGGAACTGGCAAAACAGGCCGTACAA ATTGAGAAACACTACGGTCGCCCGATGGATATTGAGTGGGCGAAAGATGGCCACACCGGTAAACTGTTCATTGTGCA GGCGCGTCCGGAAACCGTGCGCTCACGCGGTCAGGTCATGGAGCGTTATACGCTGCATTCACAGGGTAAGATTATCG CCGAAGGCCGTGCTATCGGTCATCGCATCGGTGCGGGTCCGGTGAAAGTCATCCATGACATCAGCGAAATGAACCGC ATCGAACCTGGCGACGTGCTGGTTACTGACATGACCGACCCGGACTGGGAACCGATCATGAAGAAAGCATCTGCCAT CGTCACCAACCGTGGCGGTCGTACCTGTCACGCGGCGATCATCGCTCGTGAACTGGGCATTCCGGCGGTAGTGGGCT GTGGAGATGCAACAGAACGGATGAAAGACGGTGAGAACGTCACTGTTTCTTGTGCCGAAGGTGATACCGGTTACGTC TATGCGGAGTTGCTGGAATTTAGCGTGAAAAGCTCCAGCGTAGAAACGATGCCGGATCTGCCGTTGAAAGTGATGAT GAACGTCGGTAACCCGGACCGTGCTTTCGACTTCGCCTGCCTACCGAACGAAGGCGTGGGCCTTGCGCGTCTGGAAT TTATCATCAACCGTATGATTGGCGTCCACCCACGCGCACTGCTTGAGTTTGACGATCAGGAACCGCAGTTGCAAAAC GAAATCCGCGAGATGATGAAAGGTTTTGATTCTCCGCGTGAATTTTACGTTGGTCGTCTGACTGAAGGGATCGCGAC GCTGGGTGCCGCGTTTTATCCGAAGCGCGTCATTGTCCGTCTCTCTGATTTTAAATCGAACGAATATGCCAACCTGG TCGGTGGTGAGCGTTACGAGCCAGATGAAGAGAACCCGATGCTCGGCTTCCGTGGCGCGGGCCGCTATGTTTCCGAC AGCTTCCGCGACTGTTTCGCGCTGGAGTGTGAAGCAGTGAAACGTGTGCGCAACGACATGGGACTGACCAACGTTGA GATCATGATCCCGTTCGTGCGTACCGTAGATCAGGCGAAAGCGGTGGTTGAAGAACTGGCGCGTCAGGGGCTGAAAC GTGGCGAGAACGGGCTGAAAATCATCATGATGTGTGAAATCCCGTCCAACGCCTTGCTGGCCGAGCAGTTCCTCGAA TATTTCGACGGCTTCTCAATTGGCTCAAACGATATGACGCAGCTGGCGCTCGGTCTGGACCGTGACTCCGGCGTGGT GTCTGAATTGTTCGATGAGCGCAACGATGCGGTGAAAGCACTGCTGTCGATGGCTATCCGTGCCGCGAAGAAACAGG GCAAATATGTCGGGATTTGCGGTCAGGGTCCGTCCGACCACGAAGACTTTGCCGCATGGTTGATGGAAGAGGGGATC GATAGCCTGTCTCTGAACCCGGACACCGTGGTGCAAACCTGGTTAAGCCTGGCTGAACTGAAGAAATAAAATAAATC CCCGGCGGCGTTGGATCTTATAAGAAGGAGATATACATATGTCCTCACGTAAAGAGCTTGCCAATGCTATTCGTGCG CTGAGCATGGACGCAGTACAGAAAGCCAAATCCGGTCACCCGGGTGCCCCTATGGGTATGGCTGACATTGCCGAAGT CCTGTGGCGTGATTTCCTGAAACACAACCCGCAGAATCCGTCCTGGGCTGACCGTGACCGCTTCGTGCTGTCCAACG GCCACGGCTCCATGCTGATCTACAGCCTGCTGCACCTCACCGGTTACGATCTGCCGATGGAAGAACTGAAAAACTTC CGTCAGCTGCACTCTAAAACTCCGGGTCACCCGGAAGTGGGTTACACCGCTGGTGTGGAAACCACCACCGGTCCGCT GGGTCAGGGTATTGCCAACGCAGTCGGTATGGCGATTGCAGAAAAAACGCTGGCGGCGCAGTTTAACCGTCCGGGCC ACGACATTGTCGACCACTACACCTACGCCTTCATGGGCGACGGCTGCATGATGGAAGGCATCTCCCACGAAGTTTGC TCTCTGGCGGGTACGCTGAAGCTGGGTAAACTGATTGCATTCTACGATGACAACGGTATTTCTATCGATGGTCACGT TGAAGGCTGGTTCACCGACGACACCGCAATGCGTTTCGAAGCTTACGGCTGGCACGTTATTCGCGACATCGACGGTC ATGACGCGGCATCTATCAAACGCGCAGTAGAAGAAGCGCGCGCAGTGACTGACAAACCTTCCCTGCTGATGTGCAAA ACCATCATCGGTTTCGGTTCCCCGAACAAAGCCGGTACCCACGACTCCCACGGTGCGCCGCTGGGCGACGCTGAAAT TGCCCTGACCCGCGAACAACTGGGCTGGAAATATGCGCCGTTCGAAATCCCGTCTGAAATCTATGCTCAGTGGGATG CGAAAGAAGCAGGCCAGGCGAAAGAATCCGCATGGAACGAGAAATTCGCTGCTTACGCGAAAGCTTATCCGCAGGAA GCCGCTGAATTTACCCGCCGTATGAAAGGCGAAATGCCGTCTGACTTCGACGCTAAAGCGAAAGAGTTCATCGCTAA ACTGCAGGCTAATCCGGCGAAAATCGCCAGCCGTAAAGCGTCTCAGAATGCTATCGAAGCGTTCGGTCCGCTGTTGC CGGAATTCCTCGGCGGTTCTGCTGACCTGGCGCCGTCTAACCTGACCCTGTGGTCTGGTTCTAAAGCAATCAACGAA GATGCTGCGGGTAACTACATCCACTACGGTGTTCGCGAGTTCGGTATGACCGCGATTGCTAACGGTATCTCCCTGCA CGGTGGCTTCCTGCCGTACACCTCCACCTTCCTGATGTTCGTGGAATACGCACGTAACGCCGTACGTATGGCTGCGC TGATGAAACAGCGTCAGGTGATGGTTTACACCCACGACTCCATCGGTCTGGGCGAAGACGGCCCGACTCACCAGCCG GTTGAGCAGGTCGCTTCTCTGCGCGTAACCCCGAACATGTCTACATGGCGTCCGTGTGACCAGGTTGAATCCGCGGT CGCGTGGAAATACGGTGTTGAGCGTCAGGACGGCCCGACCGCACTGATCCTCTCCCGTCAGAACCTGGCGCAGCAGG AACGAACTGAAGAGCAACTGGCAAACATCGCGCGCGGTGGTTATGTGCTGAAAGACTGCGCCGGTCAGCCGGAACTG ATTTTCATCGCTACCGGTTCAGAAGTTGAACTGGCTGTTGCTGCCTACGAAAAACTGACTGCCGAAGGCGTGAAAGC GCGCGTGGTGTCCATGCCGTCTACCGACGCATTTGACAAGCAGGATGCTGCTTACCGTGAATCCGTACTGCCGAAAG CGGTTACTGCACGCGTTGCTGTAGAAGCGGGTATTGCTGACTACTGGTACAAGTATGTTGGCCTGAACGGTGCTATC GTCGGTATGACCACCTTCGGTGAATCTGCTCCGGCAGAGCTGCTGTTTGAAGAGTTCGGCTTCACTGTTGATAACGT TGTTGCGAAAGCAAAAGAACTGCTGTAATTAGCATTTCGGATCTTATAAGAAGGAGATATACATATGACAAAGTATG CATTAGTCGGTGATGTGGGCGGCACCAACGCACGTCTTGCTCTGTGTGATATTGCCAGTGGTGAAATCTCGCAGGCT AAGACCTATTCAGGGCTTGATTACCCCAGCCTCGAAGCGGTCATTCGCGTTTATCTTGAAGAACATAAGGTCGAGGT GAAAGACGGCTGTATTGCCATCGCTTGCCCAATTACCGGTGACTGGGTGGCGATGACCAACCATACCTGGGCGTTCT CAATTGCCGAAATGAAAAAGAATCTCGGTTTTAGCCATCTGGAAATTATTAACGATTTTACCGCTGTATCGATGGCG ATCCCGATGCTGAAAAAAGAGCATCTGATTCAGTTTGGTGGCGCAGAACCGGTCGAAGGTAAGCCTATTGCGGTTTA CGGTGCCGGAACGGGGCTTGGGGTTGCGCATCTGGTCCATGTCGATAAGCGTTGGGTAAGCTTGCCAGGCGAAGGCG GTCACGTTGATTTTGCGCCGAATAGTGAAGAAGAGGCCATTATCCTCGAAATATTGCGTGCGGAAATTGGTCATGTT TCGGCGGAGCGCGTGCTTTCTGGCCCTGGGCTGGTGAATTTGTATCGCGCAATTGTGAAAGCTGACAACCGCCTGCC AGAAAATCTCAAGCCAAAAGATATTACCGAACGCGCGCTGGCTGACAGCTGCACCGATTGCCGCCGCGCATTGTCGC TGTTTTGCGTCATTATGGGCCGTTTTGGCGGCAATCTGGCGCTCAATCTCGGGACATTTGGCGGCGTGTTTATTGCG GGCGGTATCGTGCCGCGCTTCCTTGAGTTCTTCAAAGCCTCCGGTTTCCGTGCCGCATTTGAAGATAAAGGGCGCTT TAAAGAATATGTCCATGATATTCCGGTGTATCTCATCGTCCATGACAATCCGGGCCTTCTCGGTTCCGGTGCACATT TACGCCAGACCTTAGGTCACATTCTGTAAATCCTTCCTTTTATATCGGGGGTGCACCGTGCAGTCGCTGCA
SEQ ID NO.022:
GCCTTCCCGTTCCGCTATCGGGCCCTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGTTG ACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGGAGCTCTTGACAATTAATCATCGGCTCGTATAATGTGTG GAATTGTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCGGCTCGTATAAT GTGTGGAATTGTGCCCAAGCTTACATGCTCTAGAACATCCCGTCAAAGGAGCATCGACCATGAAACCAGAAGATTTC CGCGCCAGTACCCAACGTCCTTTCACCGGGGAAGAGTATCTGAAAAGCCTGCAGGATGGTCGCGAGATCTATATCTA TGGCGAGCGAGTGAAAGACGTCACCACTCATCCGGCATTTCGTAATGCGGCAGCGTCTGTTGCCCAGCTGTACGACG CACTGCACAAACCGGAGATGCAGGACTCTCTGTGTTGGAACACCGACACCGGCAGCGGCGGCTATACCCATAAATTC TTCCGCGTGGCGAAAAGTGCCGACGACCTGCGCCAGCAACGCGACGCCATCGCTGAGTGGTCACGCCTGAGCTATGG CTGGATGGGCCGTACCCCAGACTACAAAGCCGCTTTCGGTTGCGCACTGGGCGCGAATCCGGGCTTTTACGGTCAGT TCGAGCAGAACGCCCGTAACTGGTACACCCGTATTCAGGAAACTGGCCTCTACTTTAACCACGCGATTGTTAACCCA CCGATCGATCGTCATTTGCCGACCGATAAAGTGAAAGACGTTTACATCAAGCTGGAAAAAGAGACTGACGCCGGGAT TATCGTCAGCGGTGCGAAAGTGGTTGCCACCAACTCGGCGCTGACTCACTACAACATGATTGGCTTCGGCTCGGCAC AAGTGATGGGCGAAAACCCGGACTTCGCACTGATGTTCGTTGCGCCAATGGATGCCGATGGCGTGAAATTAATCTCC CGCGCCTCTTATGAGATGGTCGCGGGTGCTACCGGCTCGCCATACGACTACCCGCTCTCCAGCCGCTTCGATGAGAA CGATGCGATTCTGGTGATGGATAACGTGCTGATTCCATGGGAAAACGTGCTGATCTACCGCGATTTTGATCGCTGCC GTCGCTGGACGATGGAAGGCGGTTTTGCCCGTATGTATCCGCTGCAAGCCTGTGTGCGCCTGGCAGTGAAATTAGAC TTCATTACGGCACTGCTGAAAAAATCACTCGAATGTACCGGCACCCTGGAGTTCCGTGGTGTGCAGGCCGATCTCGG TGAAGTGGTAGCGTGGCGCAACACCTTCTGGGCATTGAGTGACTCGATGTGTTCAGAAGCAACGCCGTGGGTCAACG GGGCTTATTTACCGGATCATGCCGCACTGCAAACCTATCGCGTACTGGCACCAATGGCCTACGCGAAGATCAAAAAC ATTATCGAACGCAACGTTACCAGTGGCCTGATCTATCTCCCTTCCAGTGCCCGTGACCTGAATAATCCGCAGATCGA CCAGTATCTGGCGAAGTATGTGCGCGGTTCGAACGGTATGGATCACGTCCAGCGCATCAAGATCCTCAAACTGATGT GGGATGCTATTGGCAGCGAATTTGGTGGTCGTCACGAACTGTATGAAATCAACTACTCCGGTAGCCAGGATGAGATT CGCCTGCAGTGTCTGCGCCAGGCACAAAACTCCGGCAATATGGACAAGATGATGGCGATGGTTGATCGCTGCCTGTC GGAATACGACCAGGACGGCTGGACTGTGCCGCACCTGCACAACAACGACGATATCAACATGCTGGATAAGCTGCTGA AATAACGCAGCAGGAGGTTAAGATGCAATTAGATGAACAACGCCTGCGCTTTCGTGACGCGATGGCCAGCCTGTCGG CAGCGGTAAATATTATCACCACCGAGGGCGACGCCGGACAATGCGGGATTACGGCAACGGCCGTCTGCTCGGTCACG GATACACCACCGTCGCTGATGGTGTGCATTAACGCCAACAGTGCGATGAACCCGGTTTTTCAGGGCAACGGCAAGTT GTGCGTCAACGTCCTCAACCATGAGCAGGAACTGATGGCACGCCACTTCGCGGGCATGACAGGCATGGCGATGGAAG AGCGTTTTAGCCTCTCATGCTGGCAAAAAGGTCCGCTGGCGCAGCCGGTGCTAAAAGGTTCGCTGGCCAGTCTTGAA GGTGAGATCCGCGATGTGCAGGCAATTGGCACACATCTGGTGTATCTGGTGGAGATTAAAAACATCATCCTCAGTGC AGAAGGTCATGGACTTATCTACTTTAAACGCCGTTTCCATCCGGTGATGCTGGAAATGGAAGCTGCGATTTAAACTA GTCGGAAATGTTAACGGCCGCATAATCGAAATCGCGAATTCCCACAATACCAAAGGAGCATCTAACATGAAAATTAT TGCCTATGCTGTACGTGATGACGAACGTCCATTCTTCGATACTTGGATGAAAGAAAACCCAGATGTTGAAGTTAAAT TAGTTCCAGAATTACTTACTGAAGACAACGTTGACTTAGCTAAAGGCTTCGACGGTGCCGATGTAGCCCAACAAAAG GACTATACTGCTGAAGTATTGAACAAGTTAGCCGACGAAGGGGTTAAGAACATCTCTCTTCGTAACGTTGGTGTTGA TAACTTGGACGTTCCTACTGTTAAAGCACGTGGCTTAAACATTTCTAACGTACCTGCATACTCACCAAATGCGATTG CTGAATTATCAGTAACGCAATTGATGCAATTATTACGTCAAACCCCATTGTTCAACAAGAAGTTAGCTAAGCAAGAC TTCCGTTGGGCACCAGATATTGCCAAGGAATTAAACACCATGACTGTTGGTGTTATCGGTACTGGTCGGATTGGCCG TGCTGCCATCGATATTTTCAAAGGCTTCGGCGCTAAGGTTATCGGTTACGATGTTTACCGGAATGCTGAACTTGAAA AGGAAGGCATGTACGTTGACACCTTGGACGAATTATACGCCCAAGCTGATGTTATCACGTTACACGTTCCTGCATTG AAGGATAACTACCACATGTTGAATGCGGATGCCTTCAGCAAGATGAAAGATGGCGCCTACATCTTGAACTTTGCTCG TGGGACACTCATCGATTCAGAAGACTTGATCAAAGCCTTAGACAGTGGCAAAGTTGCCGGTGCCGCTCTTGATACGT ATGAATACGAAACTAAAATCTTCAACAAAGACCTTGAAGGTCAAACGATTGATGACAAGGTCTTCATGAACTTGTTC AACCGCGACAATGTTTTGATTACACCACATACGGCTTTCTACACTGAAACTGCCGTTCACAACATGGTGCACGTTTC AATGAACAGTAACAAACAATTCATCGAAACTGGTAAAGCTGACACACAAGTTAAGTTTGACTAATCTCGATTATCAC TGGGATCCGCGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGT CGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATACTCGAGGGACTCGT CTACTAGCGCAG

Claims (9)

  1. A kind of 1. method for the recombination engineering for producing l-tyrosine, it is characterised in that built with following methods：
    (1) using Escherichia coli SyBE-002447 as starting strain, using λ red homologous recombinations and resistance loss method, with complete Chemistry synthesis module Plac- aroG-tyrA-aroE integrates the paa gene clusters position replaced on chromosome, obtains bacterial strain SyBE- 22001；
    (2) the bacterial strain SyBE-22001 obtained in (1), using λ red homologous recombinations and resistance loss method, is closed with full chemistry Into module Ptrc- pps-tkt-glk integrates the position for replacing the lacI genes on chromosome, obtains bacterial strain SyBE-22002；
    (3) the bacterial strain SyBE-22002 obtained in (2), using λ red homologous recombinations and resistance loss method, is closed with full chemistry Into module Ptacs- hpaBC-ldh, which is integrated, is inserted into nupG downstream positions on chromosome, obtains bacterial strain SyBE-22011.
  2. 2. the method for the recombination engineering of production l-tyrosine according to claim 1, it is characterised in that the tool of step (1) Body method is：
    Using Escherichia coli SyBE-002447 genomes as template, using sequence is sense primer shown in SEQ ID No.1, with SEQ Sequence shown in ID No.2 is anti-sense primer, and clone obtains the fragment F1 of 500bp；
    Design simultaneously chemistry synthesis module Plac- aroG-tyrA-aroE, sequence is as shown in SEQ ID No.3, using the sequence as mould Plate, using sequence shown in SEQ ID No.4 as sense primer, using sequence shown in SEQ ID No.5, as anti-sense primer, clone obtains The fragment F2 of 3500bp or so；
    Using chemical synthesis sequence C HL genes as template, its sequence is as shown in SEQ ID NO.6, with sequence shown in SEQ ID No.7 For sense primer, using sequence shown in SEQ ID No.8 as anti-sense primer, amplification obtains the F3 fragments of 1000bp；
    Using Escherichia coli SyBE-002447 genomes as template, using sequence is sense primer shown in SEQ ID No.9, with SEQ Sequence shown in ID No.10 is anti-sense primer, and clone obtains the fragment F4 of 500bp；Four recombinant fragments of middle acquisition are passed through weight The method of folded extension is assembled into recombinant fragment F1-F2-F3-F4, using Escherichia coli SyBE-002447 bacterial strains as chassis cell, profit F1-F2-F3-F4 fragments are integrated to the chromosome of SyBE-002447 bacterial strains with the method for λ red homologous recombinations, then from dyeing Chloramphenicol resistance gene is lost on body, obtains recombinant bacterial strain SyBE-22001.
  3. 3. the method for the recombination engineering of production l-tyrosine according to claim 1, it is characterised in that the tool of step (2) Body method is：
    Using the bacterial strain SyBE-22001 genomes obtained in step (1) as template, using sequence shown in SEQ ID No.11 as upstream Primer, using sequence shown in SEQ ID No.12 as anti-sense primer, clone obtains the fragment F5 of 500bp；
    Design simultaneously chemical synthesis l-tyrosine synthesis module Ptrc- pps-tkt-glk, sequence is as shown in SEQ ID No.13, with this Sequence is template, using sequence shown in SEQ ID No.14 as sense primer, using sequence shown in SEQ ID No.15 as anti-sense primer, Clone obtains the fragment F6 of 5700bp or so；
    With sequence SEQ ID No.6 (CHL genes) for template, using sequence is sense primer shown in SEQ ID No.16, with SEQ Sequence shown in ID No.17 is anti-sense primer, and amplification obtains the fragment F7 of 1000bp；Using bacterial strain SyBE-22001 genomes as mould Plate, using sequence shown in SEQ ID No.18 as sense primer, using sequence shown in SEQ ID No.19, as anti-sense primer, clone obtains The fragment F8 of 500bp；
    Four recombinant fragments of acquisition are built recombinant fragment F5-F6-F7-F8 by the method assembling of overlap-extension PCR, utilize λ The method of red homologous recombinations is integrated on the chromosome of fragment F5-F6-F7-F8 to SyBE-22001 bacterial strains, then from chromosome Chloramphenicol resistance gene is lost, obtains recombinant bacterial strain SyBE-22002.
  4. 4. the method for the recombination engineering of production l-tyrosine according to claim 1, it is characterised in that the tool of step (3) Body method is：
    Using the bacterial strain SyBE-22002 genomes obtained in step (2) as template, using sequence shown in SEQ ID No.20 as upstream Primer, using sequence shown in SEQ ID No.21 as anti-sense primer, clone obtains the fragment F9 of 500bp or so；
    With the danshensu synthesis module sequence SEQ ID No.22 (P of design and full chemistry synthesistacs- hpaBC-ldh) it is template, Using sequence shown in SEQ ID No.23 as sense primer, as anti-sense primer, cloned using sequence shown in SEQ ID No.24 The fragment F10 of 3600bp or so；
    With sequence SEQ ID No.6 (CHL genes) for template, using sequence is sense primer shown in SEQ ID No.25, with SEQ Sequence shown in ID No.26 is anti-sense primer, and amplification obtains the F11 fragments of 1000bp or so；
    Using bacterial strain SyBE-22002 genomes as template, using sequence is sense primer shown in SEQ ID No.27, with SEQ ID Sequence shown in No.28 is anti-sense primer, and clone obtains the fragment F12 of 500bp or so；
    Four recombinant fragments of acquisition are passed through the method assembling structure recombinant fragment F9-F10-F11-F12 of overlap-extension PCR, utilization The method of λ red homologous recombinations loses chloramphenicol resistance gene, what is recombinated is whole on the chromosome of SyBE-22002 bacterial strains Close bacterial strain SyBE-22011.
  5. 5. the method for the recombination engineering of production l-tyrosine according to claim 2, it is characterised in that：Wherein
    The sequence of SEQ ID No.1 is：5’-GAAGTCGCTGCACCTAAGGAACCGGAGAGGCGGTTTGCGTATTGGG-3’；
    The sequence of SEQ ID No.2 is：5’-CGCCTTCTCCTGTAGCGTTATCCCGATAT-3’；
    The sequence of SEQ ID No.3 is：CCCAATACGCAAACCGCCTCTCCACAGCTGATTGCCCTTCACCGCCTGGCCCT GAGAGAGTTGCAGCAAGCGGTCCCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGC TGGCACGACAGGTTTCCCGACTGGAAAGCAATTGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAG GAGATATCAAGCTTACATGCTCTAGAAAAGGAGAATATCATGAATTATCAGAACGACGATTTACGCATCAAAGAAAT CAAAGAGTTACTTCCTCCTGTCGCATTGCTGGAAAAATTCCCCGCTACTGAAAATGCCGCGAATACGGTTGCCCATG CCCGAAAAGCGATCCATAAGATCCTGAAAGGTAATGATGATCGCCTGTTGGTTGTGATTGGCCCATGCTCAATTCAT GATCCTGTCGCGGCAAAAGAGTATGCCACTCGCTTGCTGGCGCTGCGTGAAGAGCTGAAAGATGAGCTGGAAATCGT AATGCGCGTCTATTTTGAAAAGCCGCGTACCACGGTGGGCTGGAAAGGGCTGATTAACGATCCGCATATGGATAATA GCTTCCAGATCAACGACGGTCTGCGTATAGCCCGTAAATTGCTGCTTGATATTAACGACAGCGGTCTGCCAGCGGCA GGTGAGTTTCTCGATATGATCACCCTACAATATCTCGCTGACCTGATGAGCTGGGGCGCAATTGGCGCACGTACCAC CGAATCGCAGGTGCACCGCGAACTGGCATCAGGGCTTTCTTGTCCGGTCGGCTTCAAAAATGGCACCGACGGTACGA TTAAAGTGGCTATCGATGCCATTAATGCCGCCGGTGCGCCGCACTGCTTCCTGTCCGTAACGAAATGGGGGCATTCG GCGATTGTGAATACCAGCGGTAACGGCGATTGCCATATCATTCTGCGCGGCGGTAAAGAGCCTAACTACAGCGCGAA GCACGTTGCTGAAGTGAAAGAAGGGCTGAACAAAGCAGGCCTGCCAGCACAGGTGATGATCGATTTCAGCCATGCTA ACTCGTCCAAACAATTCAAAAAGCAGATGGATGTTTGTGCTGACGTTTGCCAGCAGATTGCCGGTGGCGAAAAGGCC ATTATTGGCGTGATGGTGGAAAGCCATCTGGTGGAAGGCAATCAGAGCCTCGAGAGCGGGGAGCCGCTGGCCTACGG TAAGAGCATCACCGATGCCTGCATCGGCTGGGAAGATACCGATGCTCTGTTACGTCAACTGGCGAATGCAGTAAAAG CGCGTCGCGGGTAAAAGCTTACATGCTCTAGAAAAGGAGAATATTATGGTTGCTGAATTGACCGCATTACGCGATCA AATTGATGAAGTCGATAAAGCGCTGCTGAATTTATTAGCGAAGCGTCTGGAACTGGTTGCTGAAGTGGGCGAGGTGA AAAGCCGCTTTGGACTGCCTATTTATGTTCCGGAGCGCGAGGCATCTATTTTGGCCTCGCGTCGTGCAGAGGCGGAA GCTCTGGGTGTACCGCCAGATCTGATTGAGGATGTTTTGCGTCGGGTGATGCGTGAATCTTACTCCAGTGAAAACGA CAAAGGATTTAAAACACTTTGTCCGTCACTGCGTCCGGTGGTTATCGTCGGCGGTGGCGGTCAGATGGGACGCCTGT TCGAGAAGATGCTGACCCTCTCGGGTTATCAGGTGCGGATTCTGGAGCAACATGACTGGGATCGAGCGGCTGATATT GTTGCCGATGCCGGAATGGTGATTGTTAGTGTGCCAATCCACGTTACTGAGCAAGTTATTGGCAAATTACCGCCTTT ACCGAAAGATTGTATTCTGGTCGATCTGGCATCAGTGAAAAATGGGCCATTACAGGCCATGCTGGTGGCGCATGATG GTCCGGTGCTGGGGCTACACCCGATGTTCGGTCCGGACAGCGGTAGCCTGGCAAAGCAAGTTGTGGTCTGGTGTGAT GGACGTAAACCGGAAGCATACCAATGGTTTCTGGAGCAAATTCAGGTCTGGGGCGCTCGGCTGCATCGTATTAGCGC CGTCGAGCACGATCAGAATATGGCGTTTATTCAGGCACTGCGCCACTTTGCTACTTTTGCTTACGGGCTGCACCTGG CAGAAGAAAATGTTCAGCTTGAGCAACTTCTGGCGCTCTCTTCGCCGATTTACCGCCTTGAGCTGGCGATGGTCGGG CGACTGTTTGCTCAGGATCCGCAGCTTTATGCCGACATCATTATGTCGTCAGAGCGTAATCTGGCGTTAATCAAACG TTACTATAAGCGTTTCGGCGAGGCGATTGAGTTGCTGGAGCAGGGCGATAAGCAGGCGTTTATTGACAGTTTCCGCA AGGTGGAGCACTGGTTCGGCGATTACGTGCAGCGTTTTCAGAGTGAAAGCCGCGTGTTATTGCGTCAGGCGAATGAC AATCGCCAGTAAAAGCTTACATGCTCTAGAAAAGGAGGAATATCATGGAAACCTATGCTGTTTTTGGTAATCCGATA GCCCACAGCAAATCGCCATTCATTCATCAGCAATTTGCTCAGCAACTGAATATTGAACATCCCTATGGGCGCGTGTT GGCACCCATCAATGATTTCATCAACACACTGAACGCTTTCTTTAGTGCTGGTGGTAAAGGTGCGAATGTGACGGTGC CTTTTAAAGAAGAGGCTTTTGCCAGAGCGGATGAGCTTACTGAACGGGCAGCGTTGGCTGGTGCTGTTAATACCCTC ATGCGGTTAGAAGATGGACGCCTGCTGGGTGACAATACCGATGGTGTAGGCTTGTTAAGCGATCTGGAACGTCTGTC TTTTATCCGCCCTGGTTTACGTATTCTGCTTATCGGCGCTGGTGGAGCATCTCGCGGCGTACTACTGCCACTCCTTT CCCTGGACTGTGCGGTGACAATAACTAATCGGACGGTATCCCGCGCGGAAGAGTTGGCTAAATTGTTTGCGCACACT GGCAGTATTCAGGCGTTGAGTATGGACGAACTGGAAGGTCATGAGTTTGATCTCATTATTAATGCAACATCCAGTGG CATCAGTGGTGATATTCCGGCGATCCCGTCATCGCTCATTCATCCAGGCATTTATTGCTATGACATGTTCTATCAGA AAGGAAAAACTCCTTTTCTGGCATGGTGTGAGCAGCGAGGCTCAAAGCGTAATGCTGATGGTTTAGGAATGCTGGTG GCACAGGCGGCTCATGCCTTTCTTCTCTGGCACGGTGTTCTGCCTGACGTAGAACCAGTTATAAAGCAATTGCAGGA GGAATTGTCCGCGTGAACTAGTCCGGCTTATCGGTCAGTTTCACCTGATTTACGTAAAAACCCGCTTCGGCGGGTTT TTGCTTTTGGAGGGGCAGAAAGATGAATGACTGTCCACGACGCTATACCCAAAAGAAACGAGGAGAGGCGGTTTGCG TATTGGGA；
    The sequence of SEQ ID No.4 is：5’-CCCAATACGCAAACCGCCTCTCC-3’；
    The sequence of SEQ ID No.5 is：5’-TCCCAATACGCAAACCGCCTCTCC-3’；
    The sequence of SEQ ID No.6 is：GTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTT CGGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAA GCATCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTT GCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTG AAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACC GTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAA ACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATT GCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATT TTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAA ATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTA GCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTT GGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACAC CAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAG GAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGACCATGGCTAATTCCCAT；
    The sequence of SEQ ID No.7 is：5’-GGAGAGGCGGTTTGCGTATTGGGAGTGTAGGCTG GAGCTGCTTC-3’；
    The sequence of SEQ ID No.8 is：5’-GTCTCGAATTTGTATTGAGACGAAATATTAATGGGAATTAGCCATGGTCC -3’；
    The sequence of SEQ ID No.9 is：5’-TAATATTTCGTCTCAATACAAATTC-3’；
    The sequence of SEQ ID No.10 is：5’-AGGCCGTGATTAACGCAGCAGCG-3’.
  6. 6. the method for the recombination engineering of production l-tyrosine according to claim 3, it is characterised in that：Wherein
    The sequence of SEQ ID No.11 is：5’-CGGCATTGGCTGGGTCGCGG-3’；
    The sequence of SEQ ID No.12 is：5’-CGTTAATTACACTCAATCGAGTGCAGGTGAAACTGACCGATAAGCCG- 3’；
    The sequence of SEQ ID No.13 is：CTGCAGCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGA AGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAAT GTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCGTA TAATGTGTGGGAGAGGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGAATTCCGGAGATGAAGATCTTAT AAGAAGGAGATATACATATGTCCAACAATGGCTCGTCACCGCTGGTGCTTTGGTATAACCAACTCGGCATGAATGAT GTAGACAGGGTTGGGGGCAAAAATGCCTCCCTGGGTGAAATGATTACTAATCTTTCCGGAATGGGTGTTTCCGTTCC GAATGGTTTCGCCACAACCGCCGACGCGTTTAACCAGTTTCTGGACCAAAGCGGCGTAAACCAGCGCATTTATGAAC TGCTGGATAAAACGGATATTGACGATGTTACTCAGCTTGCGAAAGCGGGCGCGCAAATCCGCCAGTGGATTATCGAC ACTCCCTTCCAGCCTGAGCTGGAAAACGCCATCCGCGAAGCCTATGCACAGCTTTCCGCCGATGACGAAAACGCCTC TTTTGCGGTGCGCTCCTCCGCCACCGCAGAAGATATGCCGGACGCTTCTTTTGCCGGTCAGCAGGAAACCTTCCTCA ACGTTCAGGGTTTTGACGCCGTTCTCGTGGCAGTGAAACATGTATTTGCTTCTCTGTTTAACGATCGCGCCATCTCT TATCGTGTGCACCAGGGTTACGATCACCGTGGTGTGGCGCTCTCCGCCGGTGTTCAACGGATGGTGCGCTCTGACCT CGCATCATCTGGCGTGATGTTCTCCATTGATACCGAATCCGGCTTTGACCAGGTGGTGTTTATCACTTCCGCATGGG GCCTTGGTGAGATGGTCGTGCAGGGTGCGGTTAACCCGGATGAGTTTTACGTGCATAAACCGACACTGGCGGCGAAT CGCCCGGCTATCGTGCGCCGCACCATGGGGTCGAAAAAAATCCGCATGGTTTACGCGCCGACCCAGGAGCACGGCAA GCAGGTTAAAATCGAAGACGTACCGCAGGAACAGCGTGACATCTTCTCGCTGACCAACGAAGAAGTGCAGGAACTGG CAAAACAGGCCGTACAAATTGAGAAACACTACGGTCGCCCGATGGATATTGAGTGGGCGAAAGATGGCCACACCGGT AAACTGTTCATTGTGCAGGCGCGTCCGGAAACCGTGCGCTCACGCGGTCAGGTCATGGAGCGTTATACGCTGCATTC ACAGGGTAAGATTATCGCCGAAGGCCGTGCTATCGGTCATCGCATCGGTGCGGGTCCGGTGAAAGTCATCCATGACA TCAGCGAAATGAACCGCATCGAACCTGGCGACGTGCTGGTTACTGACATGACCGACCCGGACTGGGAACCGATCATG AAGAAAGCATCTGCCATCGTCACCAACCGTGGCGGTCGTACCTGTCACGCGGCGATCATCGCTCGTGAACTGGGCAT TCCGGCGGTAGTGGGCTGTGGAGATGCAACAGAACGGATGAAAGACGGTGAGAACGTCACTGTTTCTTGTGCCGAAG GTGATACCGGTTACGTCTATGCGGAGTTGCTGGAATTTAGCGTGAAAAGCTCCAGCGTAGAAACGATGCCGGATCTG CCGTTGAAAGTGATGATGAACGTCGGTAACCCGGACCGTGCTTTCGACTTCGCCTGCCTACCGAACGAAGGCGTGGG CCTTGCGCGTCTGGAATTTATCATCAACCGTATGATTGGCGTCCACCCACGCGCACTGCTTGAGTTTGACGATCAGG AACCGCAGTTGCAAAACGAAATCCGCGAGATGATGAAAGGTTTTGATTCTCCGCGTGAATTTTACGTTGGTCGTCTG ACTGAAGGGATCGCGACGCTGGGTGCCGCGTTTTATCCGAAGCGCGTCATTGTCCGTCTCTCTGATTTTAAATCGAA CGAATATGCCAACCTGGTCGGTGGTGAGCGTTACGAGCCAGATGAAGAGAACCCGATGCTCGGCTTCCGTGGCGCGG GCCGCTATGTTTCCGACAGCTTCCGCGACTGTTTCGCGCTGGAGTGTGAAGCAGTGAAACGTGTGCGCAACGACATG GGACTGACCAACGTTGAGATCATGATCCCGTTCGTGCGTACCGTAGATCAGGCGAAAGCGGTGGTTGAAGAACTGGC GCGTCAGGGGCTGAAACGTGGCGAGAACGGGCTGAAAATCATCATGATGTGTGAAATCCCGTCCAACGCCTTGCTGG CCGAGCAGTTCCTCGAATATTTCGACGGCTTCTCAATTGGCTCAAACGATATGACGCAGCTGGCGCTCGGTCTGGAC CGTGACTCCGGCGTGGTGTCTGAATTGTTCGATGAGCGCAACGATGCGGTGAAAGCACTGCTGTCGATGGCTATCCG TGCCGCGAAGAAACAGGGCAAATATGTCGGGATTTGCGGTCAGGGTCCGTCCGACCACGAAGACTTTGCCGCATGGT TGATGGAAGAGGGGATCGATAGCCTGTCTCTGAACCCGGACACCGTGGTGCAAACCTGGTTAAGCCTGGCTGAACTG AAGAAATAAAATAAATCCCCGGCGGCGTTGGATCTTATAAGAAGGAGATATACATATGTCCTCACGTAAAGAGCTTG CCAATGCTATTCGTGCGCTGAGCATGGACGCAGTACAGAAAGCCAAATCCGGTCACCCGGGTGCCCCTATGGGTATG GCTGACATTGCCGAAGTCCTGTGGCGTGATTTCCTGAAACACAACCCGCAGAATCCGTCCTGGGCTGACCGTGACCG CTTCGTGCTGTCCAACGGCCACGGCTCCATGCTGATCTACAGCCTGCTGCACCTCACCGGTTACGATCTGCCGATGG AAGAACTGAAAAACTTCCGTCAGCTGCACTCTAAAACTCCGGGTCACCCGGAAGTGGGTTACACCGCTGGTGTGGAA ACCACCACCGGTCCGCTGGGTCAGGGTATTGCCAACGCAGTCGGTATGGCGATTGCAGAAAAAACGCTGGCGGCGCA GTTTAACCGTCCGGGCCACGACATTGTCGACCACTACACCTACGCCTTCATGGGCGACGGCTGCATGATGGAAGGCA TCTCCCACGAAGTTTGCTCTCTGGCGGGTACGCTGAAGCTGGGTAAACTGATTGCATTCTACGATGACAACGGTATT TCTATCGATGGTCACGTTGAAGGCTGGTTCACCGACGACACCGCAATGCGTTTCGAAGCTTACGGCTGGCACGTTAT TCGCGACATCGACGGTCATGACGCGGCATCTATCAAACGCGCAGTAGAAGAAGCGCGCGCAGTGACTGACAAACCTT CCCTGCTGATGTGCAAAACCATCATCGGTTTCGGTTCCCCGAACAAAGCCGGTACCCACGACTCCCACGGTGCGCCG CTGGGCGACGCTGAAATTGCCCTGACCCGCGAACAACTGGGCTGGAAATATGCGCCGTTCGAAATCCCGTCTGAAAT CTATGCTCAGTGGGATGCGAAAGAAGCAGGCCAGGCGAAAGAATCCGCATGGAACGAGAAATTCGCTGCTTACGCGA AAGCTTATCCGCAGGAAGCCGCTGAATTTACCCGCCGTATGAAAGGCGAAATGCCGTCTGACTTCGACGCTAAAGCG AAAGAGTTCATCGCTAAACTGCAGGCTAATCCGGCGAAAATCGCCAGCCGTAAAGCGTCTCAGAATGCTATCGAAGC GTTCGGTCCGCTGTTGCCGGAATTCCTCGGCGGTTCTGCTGACCTGGCGCCGTCTAACCTGACCCTGTGGTCTGGTT CTAAAGCAATCAACGAAGATGCTGCGGGTAACTACATCCACTACGGTGTTCGCGAGTTCGGTATGACCGCGATTGCT AACGGTATCTCCCTGCACGGTGGCTTCCTGCCGTACACCTCCACCTTCCTGATGTTCGTGGAATACGCACGTAACGC CGTACGTATGGCTGCGCTGATGAAACAGCGTCAGGTGATGGTTTACACCCACGACTCCATCGGTCTGGGCGAAGACG GCCCGACTCACCAGCCGGTTGAGCAGGTCGCTTCTCTGCGCGTAACCCCGAACATGTCTACATGGCGTCCGTGTGAC CAGGTTGAATCCGCGGTCGCGTGGAAATACGGTGTTGAGCGTCAGGACGGCCCGACCGCACTGATCCTCTCCCGTCA GAACCTGGCGCAGCAGGAACGAACTGAAGAGCAACTGGCAAACATCGCGCGCGGTGGTTATGTGCTGAAAGACTGCG CCGGTCAGCCGGAACTGATTTTCATCGCTACCGGTTCAGAAGTTGAACTGGCTGTTGCTGCCTACGAAAAACTGACT GCCGAAGGCGTGAAAGCGCGCGTGGTGTCCATGCCGTCTACCGACGCATTTGACAAGCAGGATGCTGCTTACCGTGA ATCCGTACTGCCGAAAGCGGTTACTGCACGCGTTGCTGTAGAAGCGGGTATTGCTGACTACTGGTACAAGTATGTTG GCCTGAACGGTGCTATCGTCGGTATGACCACCTTCGGTGAATCTGCTCCGGCAGAGCTGCTGTTTGAAGAGTTCGGC TTCACTGTTGATAACGTTGTTGCGAAAGCAAAAGAACTGCTGTAATTAGCATTTCGGATCTTATAAGAAGGAGATAT ACATATGACAAAGTATGCATTAGTCGGTGATGTGGGCGGCACCAACGCACGTCTTGCTCTGTGTGATATTGCCAGTG GTGAAATCTCGCAGGCTAAGACCTATTCAGGGCTTGATTACCCCAGCCTCGAAGCGGTCATTCGCGTTTATCTTGAA GAACATAAGGTCGAGGTGAAAGACGGCTGTATTGCCATCGCTTGCCCAATTACCGGTGACTGGGTGGCGATGACCAA CCATACCTGGGCGTTCTCAATTGCCGAAATGAAAAAGAATCTCGGTTTTAGCCATCTGGAAATTATTAACGATTTTA CCGCTGTATCGATGGCGATCCCGATGCTGAAAAAAGAGCATCTGATTCAGTTTGGTGGCGCAGAACCGGTCGAAGGT AAGCCTATTGCGGTTTACGGTGCCGGAACGGGGCTTGGGGTTGCGCATCTGGTCCATGTCGATAAGCGTTGGGTAAG CTTGCCAGGCGAAGGCGGTCACGTTGATTTTGCGCCGAATAGTGAAGAAGAGGCCATTATCCTCGAAATATTGCGTG CGGAAATTGGTCATGTTTCGGCGGAGCGCGTGCTTTCTGGCCCTGGGCTGGTGAATTTGTATCGCGCAATTGTGAAA GCTGACAACCGCCTGCCAGAAAATCTCAAGCCAAAAGATATTACCGAACGCGCGCTGGCTGACAGCTGCACCGATTG CCGCCGCGCATTGTCGCTGTTTTGCGTCATTATGGGCCGTTTTGGCGGCAATCTGGCGCTCAATCTCGGGACATTTG GCGGCGTGTTTATTGCGGGCGGTATCGTGCCGCGCTTCCTTGAGTTCTTCAAAGCCTCCGGTTTCCGTGCCGCATTT GAAGATAAAGGGCGCTTTAAAGAATATGTCCATGATATTCCGGTGTATCTCATCGTCCATGACAATCCGGGCCTTCT CGGTTCCGGTGCACATTTACGCCAGACCTTAGGTCACATTCTGTAAATCCTTCCTTTTATATCGGGGGTGCACCGTG CAGTCGCTGCA；
    The sequence of SEQ ID No.14 is：5’-CGGCTTATCGGTCAGTTTCACCTG-3’；
    The sequence of SEQ ID No.15 is：5’-TGCAGCGACTGCACGGTGCACC-3’；
    The sequence of SEQ ID No.16 is：5’-GGTGCACCGTGCAGTCGCTGCAGTGTAGGCTG GAGCTGCTTC-3’；
    The sequence of SEQ ID No.17 is：5’-GTACGTAAATGCAACTGTGGTAGCTTACCGCGATGGGAATTAGCCATGG TC-3’；
    The sequence of SEQ ID No.18 is：5’-CGCGGTAAGCTACCACAGTTGCATTTAC-3’；
    The sequence of SEQ ID No.19 is：5’-CGTTACCACCATGCGCTTTGGTGAGTGGC-3’.
  7. 7. the method for the recombination engineering of production l-tyrosine according to claim 4, it is characterised in that：Wherein
    The sequence of SEQ ID No.20 is：5’-GTTCATTCTGACCATCCCG-3’；
    The sequence of SEQ ID No.21 is：5’-CAAATTCAGCCGATAGCGGAACGGGAAGGCACCCGTTTTTCTTTGCGTA -3’；
    The sequence of SEQ ID No.22 is：GCCTTCCCGTTCCGCTATCGGGCCCTTGACAATTAATCATCGGCTCGTATAA TGTGTGGAATTGTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGGAGCTCTTGACAATTAATCATC GGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAA TCATCGGCTCGTATAATGTGTGGAATTGTGCCCAAGCTTACATGCTCTAGAACATCCCGTCAAAGGAGCATCGACCA TGAAACCAGAAGATTTCCGCGCCAGTACCCAACGTCCTTTCACCGGGGAAGAGTATCTGAAAAGCCTGCAGGATGGT CGCGAGATCTATATCTATGGCGAGCGAGTGAAAGACGTCACCACTCATCCGGCATTTCGTAATGCGGCAGCGTCTGT TGCCCAGCTGTACGACGCACTGCACAAACCGGAGATGCAGGACTCTCTGTGTTGGAACACCGACACCGGCAGCGGCG GCTATACCCATAAATTCTTCCGCGTGGCGAAAAGTGCCGACGACCTGCGCCAGCAACGCGACGCCATCGCTGAGTGG TCACGCCTGAGCTATGGCTGGATGGGCCGTACCCCAGACTACAAAGCCGCTTTCGGTTGCGCACTGGGCGCGAATCC GGGCTTTTACGGTCAGTTCGAGCAGAACGCCCGTAACTGGTACACCCGTATTCAGGAAACTGGCCTCTACTTTAACC ACGCGATTGTTAACCCACCGATCGATCGTCATTTGCCGACCGATAAAGTGAAAGACGTTTACATCAAGCTGGAAAAA GAGACTGACGCCGGGATTATCGTCAGCGGTGCGAAAGTGGTTGCCACCAACTCGGCGCTGACTCACTACAACATGAT TGGCTTCGGCTCGGCACAAGTGATGGGCGAAAACCCGGACTTCGCACTGATGTTCGTTGCGCCAATGGATGCCGATG GCGTGAAATTAATCTCCCGCGCCTCTTATGAGATGGTCGCGGGTGCTACCGGCTCGCCATACGACTACCCGCTCTCC AGCCGCTTCGATGAGAACGATGCGATTCTGGTGATGGATAACGTGCTGATTCCATGGGAAAACGTGCTGATCTACCG CGATTTTGATCGCTGCCGTCGCTGGACGATGGAAGGCGGTTTTGCCCGTATGTATCCGCTGCAAGCCTGTGTGCGCC TGGCAGTGAAATTAGACTTCATTACGGCACTGCTGAAAAAATCACTCGAATGTACCGGCACCCTGGAGTTCCGTGGT GTGCAGGCCGATCTCGGTGAAGTGGTAGCGTGGCGCAACACCTTCTGGGCATTGAGTGACTCGATGTGTTCAGAAGC AACGCCGTGGGTCAACGGGGCTTATTTACCGGATCATGCCGCACTGCAAACCTATCGCGTACTGGCACCAATGGCCT ACGCGAAGATCAAAAACATTATCGAACGCAACGTTACCAGTGGCCTGATCTATCTCCCTTCCAGTGCCCGTGACCTG AATAATCCGCAGATCGACCAGTATCTGGCGAAGTATGTGCGCGGTTCGAACGGTATGGATCACGTCCAGCGCATCAA GATCCTCAAACTGATGTGGGATGCTATTGGCAGCGAATTTGGTGGTCGTCACGAACTGTATGAAATCAACTACTCCG GTAGCCAGGATGAGATTCGCCTGCAGTGTCTGCGCCAGGCACAAAACTCCGGCAATATGGACAAGATGATGGCGATG GTTGATCGCTGCCTGTCGGAATACGACCAGGACGGCTGGACTGTGCCGCACCTGCACAACAACGACGATATCAACAT GCTGGATAAGCTGCTGAAATAACGCAGCAGGAGGTTAAGATGCAATTAGATGAACAACGCCTGCGCTTTCGTGACGC GATGGCCAGCCTGTCGGCAGCGGTAAATATTATCACCACCGAGGGCGACGCCGGACAATGCGGGATTACGGCAACGG CCGTCTGCTCGGTCACGGATACACCACCGTCGCTGATGGTGTGCATTAACGCCAACAGTGCGATGAACCCGGTTTTT CAGGGCAACGGCAAGTTGTGCGTCAACGTCCTCAACCATGAGCAGGAACTGATGGCACGCCACTTCGCGGGCATGAC AGGCATGGCGATGGAAGAGCGTTTTAGCCTCTCATGCTGGCAAAAAGGTCCGCTGGCGCAGCCGGTGCTAAAAGGTT CGCTGGCCAGTCTTGAAGGTGAGATCCGCGATGTGCAGGCAATTGGCACACATCTGGTGTATCTGGTGGAGATTAAA AACATCATCCTCAGTGCAGAAGGTCATGGACTTATCTACTTTAAACGCCGTTTCCATCCGGTGATGCTGGAAATGGA AGCTGCGATTTAAACTAGTCGGAAATGTTAACGGCCGCATAATCGAAATCGCGAATTCCCACAATACCAAAGGAGCA TCTAACATGAAAATTATTGCCTATGCTGTACGTGATGACGAACGTCCATTCTTCGATACTTGGATGAAAGAAAACCC AGATGTTGAAGTTAAATTAGTTCCAGAATTACTTACTGAAGACAACGTTGACTTAGCTAAAGGCTTCGACGGTGCCG ATGTAGCCCAACAAAAGGACTATACTGCTGAAGTATTGAACAAGTTAGCCGACGAAGGGGTTAAGAACATCTCTCTT CGTAACGTTGGTGTTGATAACTTGGACGTTCCTACTGTTAAAGCACGTGGCTTAAACATTTCTAACGTACCTGCATA CTCACCAAATGCGATTGCTGAATTATCAGTAACGCAATTGATGCAATTATTACGTCAAACCCCATTGTTCAACAAGA AGTTAGCTAAGCAAGACTTCCGTTGGGCACCAGATATTGCCAAGGAATTAAACACCATGACTGTTGGTGTTATCGGT ACTGGTCGGATTGGCCGTGCTGCCATCGATATTTTCAAAGGCTTCGGCGCTAAGGTTATCGGTTACGATGTTTACCG GAATGCTGAACTTGAAAAGGAAGGCATGTACGTTGACACCTTGGACGAATTATACGCCCAAGCTGATGTTATCACGT TACACGTTCCTGCATTGAAGGATAACTACCACATGTTGAATGCGGATGCCTTCAGCAAGATGAAAGATGGCGCCTAC ATCTTGAACTTTGCTCGTGGGACACTCATCGATTCAGAAGACTTGATCAAAGCCTTAGACAGTGGCAAAGTTGCCGG TGCCGCTCTTGATACGTATGAATACGAAACTAAAATCTTCAACAAAGACCTTGAAGGTCAAACGATTGATGACAAGG TCTTCATGAACTTGTTCAACCGCGACAATGTTTTGATTACACCACATACGGCTTTCTACACTGAAACTGCCGTTCAC AACATGGTGCACGTTTCAATGAACAGTAACAAACAATTCATCGAAACTGGTAAAGCTGACACACAAGTTAAGTTTGA CTAATCTCGATTATCACTGGGATCCGCGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCG TTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTT ATACTCGAGGGACTCGTCTACTAGCGCAG；
    The sequence of SEQ ID No.23 is：5’-GCCTTCCCGTTCCGCTATC-3’；
    The sequence of SEQ ID No.24 is：5’-CTGCGCTAGTAGACGAGTCC-3’；
    The sequence of SEQ ID No.25 is：5’-GCCAGCACATGGACTCGTCTACTAGCGCAGGTGTAGGCTGGAGCTGCTT C-3’；
    The sequence of SEQ ID No.26 is：5’-TAAAAAAAACGGGTCACCTTCTGGCGATGGGAATTAGCCATGGTCC- 3’；
    The sequence of SEQ ID No.27 is：5’-CGCCAGAAGGTGACCCGTT-3’；
    The sequence of SEQ ID No.28 is：5’-TTGAGGATGACTCGCCGCT-3’.
  8. 8. the application of the recombination engineering of l-tyrosine according to claim 1, it is characterised in that for fermenting and producing L- Tyrosine, step are：
    SyBE-22001, SyBE-22002 bacterial strain are taken, is respectively connected to the LB culture mediums of 5mL, 37 DEG C, 220rmp cultures 12h lives Change；
    Then at 37 DEG C, 220rpm expands culture and prepares seed, seed liquor is inoculated into 10% ratio the M9 culture mediums of improvement In, without adding any antibiotic and derivant, feed supplement glucose ferments.
  9. 9. the application of the recombination engineering of l-tyrosine according to claim 1, it is characterised in that red for fermenting and producing Ginseng element, step are：
    SyBE-22011 bacterial strains are taken to be linked into the LB culture mediums of 5mL, 37 DEG C, 220rmp cultures 12h is activated；
    Then at 37 DEG C, 220rpm culture 6-8h, expand culture 400mL, take 400mL bacterium solutions to be linked into fermentation tank, without adding Add any antibiotic and derivant, feed supplement glucose ferments.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949650A (en) * 2018-04-19 2018-12-07 江南大学 A kind of production method and engineering bacteria of danshensu
CN108949657A (en) * 2018-04-19 2018-12-07 江南大学 A kind of engineering bacteria and its application in danshensu and α-ketoglutaric acid coproduction
CN108949655A (en) * 2018-04-19 2018-12-07 江南大学 A kind of engineering bacteria and its application in danshensu and pyruvic acid coproduction
CN108949648A (en) * 2018-04-19 2018-12-07 江南大学 A kind of engineering bacteria and its with the application of cheap substrates production danshensu
WO2019200873A1 (en) * 2018-04-19 2019-10-24 江南大学 Recombinant escherichia coli and method for producing tanshinol by using same
CN112813013A (en) * 2021-02-06 2021-05-18 江南大学 Recombinant escherichia coli for producing hydroxytyrosol and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013033652A1 (en) * 2011-09-02 2013-03-07 The Regents Of The University Of California Metabolic engineering of the shikimate pathway
CN103667371A (en) * 2013-11-11 2014-03-26 天津大学 Biological production method of tanshinol

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013033652A1 (en) * 2011-09-02 2013-03-07 The Regents Of The University Of California Metabolic engineering of the shikimate pathway
CN103667371A (en) * 2013-11-11 2014-03-26 天津大学 Biological production method of tanshinol

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIANG ZHOU等: "Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A", 《MICROBIAL CELL FACTORIES》 *
YUAN-FENG YAN等: "Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway", 《METABOLIC ENGINEERING》 *
杨安钢等: "《生物化学与分子生物学实验技术》", 30 June 2008 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949650A (en) * 2018-04-19 2018-12-07 江南大学 A kind of production method and engineering bacteria of danshensu
CN108949657A (en) * 2018-04-19 2018-12-07 江南大学 A kind of engineering bacteria and its application in danshensu and α-ketoglutaric acid coproduction
CN108949655A (en) * 2018-04-19 2018-12-07 江南大学 A kind of engineering bacteria and its application in danshensu and pyruvic acid coproduction
CN108949648A (en) * 2018-04-19 2018-12-07 江南大学 A kind of engineering bacteria and its with the application of cheap substrates production danshensu
WO2019200873A1 (en) * 2018-04-19 2019-10-24 江南大学 Recombinant escherichia coli and method for producing tanshinol by using same
CN108949655B (en) * 2018-04-19 2019-10-25 江南大学 A kind of engineering bacteria and its application in danshensu and pyruvic acid coproduction
CN108949648B (en) * 2018-04-19 2019-10-25 江南大学 A kind of engineering bacteria and its with the application of cheap substrates production danshensu
CN108949657B (en) * 2018-04-19 2019-10-25 江南大学 A kind of engineering bacteria and its application in danshensu and α-ketoglutaric acid coproduction
CN108949650B (en) * 2018-04-19 2019-10-25 江南大学 A kind of production method and engineering bacteria of danshensu
US10829790B2 (en) 2018-04-19 2020-11-10 Jiangnan University Recombinant E. coli and method of producing Danshensu by using same
CN112813013A (en) * 2021-02-06 2021-05-18 江南大学 Recombinant escherichia coli for producing hydroxytyrosol and application thereof
CN112813013B (en) * 2021-02-06 2023-04-28 江南大学 Recombinant escherichia coli for producing hydroxytyrosol and application thereof

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