CN110452865A - A kind of recombination bacillus coli producing tyrosol and its construction method and application - Google Patents

A kind of recombination bacillus coli producing tyrosol and its construction method and application Download PDF

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Publication number
CN110452865A
CN110452865A CN201910754497.9A CN201910754497A CN110452865A CN 110452865 A CN110452865 A CN 110452865A CN 201910754497 A CN201910754497 A CN 201910754497A CN 110452865 A CN110452865 A CN 110452865A
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aro10
gene
coli
recombination bacillus
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CN110452865B (en
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陈献忠
徐微
沈微
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01001—Pyruvate decarboxylase (4.1.1.1)

Abstract

The invention discloses a kind of recombination bacillus coli for producing tyrosol and its construction method and applications, belong to technical field of bioengineering.Saccharomyces cerevisiae Pyruvate Decarboxylase Gene ARO10* after Escherichia coli heterogenous expression codon optimization.The recombination bacillus coli is to integrate upper ARO10* gene while five sites in the site pykF are deleted in the site lacI of genome of E.coli, the site trpE, the site pabB, the site pabA, obtained the bacterial strain of the ARO10* gene containing multiple copies.On the basis of above-mentioned recombinant bacterium, the integration of ARO10* gene is carried out in multiple sites at random, discovery is inserted into ARO10* gene in the site yccX, can obtain the bacterial strain of high yield tyrosol.Inducer and antibiotic are not needed using the strain fermentation.Ferment 48h, and tyrosol yield can reach 28mM.

Description

A kind of recombination bacillus coli producing tyrosol and its construction method and application
Technical field
The present invention relates to a kind of recombination bacillus coli for producing tyrosol and its construction method and applications, belong to biotechnology Field.
Background technique
Tyrosol (tyrosol) is a kind of phenolic compound with pharmacological activity, is a kind of derivative of benzyl carbinol, is one The single phenol antioxidant of kind, there are many natural origins, such as olive oil and green tea.Tyrosol has many physiological active functions, such as Anti-oxidant, antifatigue, anti anoxia, resisting stress, cold resistance, calmness, cardiovascular disease, hypertension etc..Tyrosol is also used as wine The flavoring agent of class plays an important role in the mouthfeel for promoting inebriant, especially in pure mellow wine, beer and grape wine. In addition, tyrosol is the precursor substance of hydroxytyrosol, hydroxytyrosol (2- (3,4-dihydroxyphenyl) ethanol) is a kind of For the antioxidant of human health, for comparing tyrosol, its inoxidizability is stronger, meanwhile, it can also be synthesized very Heteropolymer.Studies have shown that it has many biological properties, the generation of the diseases such as angiocarpy, osteohalsiteresis can be prevented.So junket Alcohol is as the bioactive compound in the fine chemicals and pharmaceuticals industry of chemical industry, always by the concern of researcher.
Tyrosol synthetic method mainly includes plant extract, chemical synthesis and biosynthesis.Currently, industrially prepared junket Alcohol mainly passes through chemically synthesized method.This process is during subsequent extracted tyrosol, there are many drawbacks, It is difficult to obtain high-purity tyrosol.In having been reported, the yield of tyrosol is up to 10.6mM.Therefore it provides a kind of high yield tyrosol Method, to it, further application has important value.
Summary of the invention
It is in E.coli MG1655 genome the first purpose of the invention is to provide a kind of recombination bacillus coli The site lacI, the site trpE, the site pabB, the site pabA, the site pykF five sites while deleted five positions Upper saccharomyces cerevisiae Pyruvate Decarboxylase Gene ARO10* gene is integrated on each site of point, obtains Escherichia coli YMGR5A。
The Escherichia coli YMGR5A is preserved in China typical culture collection on May 24th, 2019 Center, deposit number are CCTCC NO:M2019390, and preservation address is Wuhan, China, Wuhan University.
In one embodiment of the invention, the nucleotide sequence of ARO10* gene is as shown in SEQ ID NO.1.
In one embodiment of the invention, the recombination bacillus coli also deletes the site yccX, while in the position Upper ARO10* gene is integrated on point, obtains Escherichia coli YMGR6A (E.coli MG1655 Δ feaB Δ pheA ΔtyrB ΔtyrR lacI∷ARO10*trpE∷ARO10*pabB∷ARO10*pabA∷ARO10*pykF∷ARO10* yccx∷ARO10*)。
The Escherichia coli YMGR6A is preserved in China typical culture collection on May 24th, 2019 Center, deposit number are CCTCC NO:M2019391, and preservation address is Wuhan, China, Wuhan University.
In one embodiment of the invention, said gene editor is homologous using CRISPR-cas9 technology or Red What recombination carried out.
A second object of the present invention is to provide a kind of methods for producing tyrosol, apply above-mentioned recombination bacillus coli and carry out Fermentation.
In one embodiment of the invention, fermenting and producing tyrosol is carried out with M9Y culture medium.
In one embodiment of the invention, the flat lining out of LB by bacterial strain in non-resistant, culture；Picking single colonie It is inoculated in LB liquid medium, carries out seed liquor culture, cultivate 8-10h.
In one embodiment of the invention, seed liquor is inoculated in LB liquid medium with 1-5% inoculation volume ratio In, it is placed in 35-39 DEG C, 200-220rpm shaking table culture 8-12h；All thallus are collected, supernatant are removed after microorganism collection, and have It is primary that physiological saline cleans thallus；Thallus after cleaning is transferred in M9Y fermentation medium, is subsequently placed in 28-30 DEG C, 200-220rpm shaker fermentation 40-60h.It is primary every 12h sampling.
In one embodiment of the invention, seed liquor is taken to be inoculated in LB liquid medium with 1-5% inoculation volume ratio In, control initial OD 600 is 0.05-0.06, is placed in 35-39 DEG C, 200-220rpm shaking table culture works as OD600Reach 0.25- When 0.30, it is inoculated in the fermentor equipped with M9Y culture medium that liquid amount is 40-45%, adds glucose and ferment in fermentation process Female powder.
Third object of the present invention is to provide a kind of methods for constructing above-mentioned recombination bacillus coli, are in E.coli The site lacI of MG1655 genome, the site trpE, the site pabB, the site pabA, the site pykF five sites deleted While upper saccharomyces cerevisiae Pyruvate Decarboxylase Gene ARO10* gene, ARO10* are integrated on each site in five sites The nucleotide sequence of gene is as shown in SEQ ID NO.1.
Fourth object of the present invention is to provide above-mentioned recombination bacillus coli answering in food, chemical industry or pharmaceutical field With.
Beneficial effects of the present invention:
The present invention construct a plant height produce tyrosol bacterial strain, be in the site lacI of genome of E.coli, the site trpE, The site pabB, the site pabA are integrated upper ARO10* gene, have been obtained containing more while five sites in the site pykF are deleted The bacterial strain of the ARO10* gene of a copy.On the basis of above-mentioned recombinant bacterium, ARO10* gene has been carried out in multiple sites at random Integration, discovery the site yccX be inserted into ARO10* gene, the bacterial strain of high yield tyrosol can be obtained.It is not needed using the strain fermentation Inducer and antibiotic.Ferment 48h, and tyrosol yield can reach 28mM.
Biomaterial preservation
One plant of Escherichia coli (Escherichia coli), classification naming be Escherichia coli YMGR5A, in It is preserved in China typical culture collection center on May 24th, 2019, deposit number is CCTCC NO:M2019390, preservation Location is Wuhan, China, Wuhan University.
One plant of Escherichia coli (Escherichia coli), classification naming be Escherichia coli YMGR6A, in It is preserved in China typical culture collection center on May 24th, 2019, deposit number is CCTCC NO:M2019391, preservation Location is Wuhan, China, Wuhan University.
Detailed description of the invention
Fig. 1: 9 plants of bacterial strain (YMGRA constructed by the present invention；YMGEA, YMGR2A；YMGB2A, YMGR3A；YMGA3A, YMGR4A；YMGF4A, YMGR5A) fermentation tyrosol yield result.
Fig. 2: YMGR5A ferment tank tyrosol yield result constructed by the present invention.
Specific embodiment
The heterogenous expression saccharomyces cerevisiae Pyruvate Decarboxylase Gene in Escherichia coli MG1655 of embodiment 1 produces tyrosol
(1) plasmid pKK223-3-ARO10* is constructed
ARO10* gene order is obtained after codon optimization by the chemical synthesis of Suzhou Hong Xun biotech firm, and is inserted into plasmid The site EcoR I and Hind III of pKK223-3 obtains recombinant plasmid pKK223-3-ARO10*.
(2) lacI ∷ ARO10* deletes expression frame construction
According to primers ARO10-L, LacIR (table 1) of pKK223-3 plasmid, related promoter and termination are obtained The expression segment of the tac-ARO10*-rrnB of son is inserted into pMD19-T simple plasmid, and recombinant plasmid 19Ts-tac- is obtained ARO10*-rrnB.Kana resistance fragments are arrived according to pKD13 design primer LacIL, PKDR for template amplification.Confronted with Xho I Grain 19Ts-tac-ARO10*-rrnB and Kana resistance fragments carry out digestion connection, obtain recombinant plasmid 19Ts-Kana-tac- ARO10*-rrnB.Using the plasmid 19Ts-Kana-tac-ARO10*-rrnB built as template, for lacIL, lacIR primer The deletion expression cassette of lacI ∷ ARO10* is obtained with PCR amplification is carried out.
1 primer of table
(3) YMGRA (E.coli MG1655 Δ feaB Δ pheA Δ tyrB Δ tyrR lacI ∷ ARO10*) bacterial strain Building
Using the method for Red homologous recombination, by YMGR/pKD46 (E.coli MG1655 Δ feaB Δ pheA Δ tyrB Δ tyrR/pKD46) it is prepared into electricity and turns competence, competence is added in the deletion expression cassette of above-mentioned lacI ∷ ARO10*, is turned Change.Picking transformant carries out bacterium colony PCR verifying with primer YLACIL, YLACIR, and bacterial strain YMGR/pKD46 is as control.It utilizes Plasmid pCP20 is transferred to bacterial strain, eliminates kalamycin resistance.Utilize high temperature (42 DEG C) elimination plasmids pKD46 and pCP20.Obtain bacterium Strain YMGRA.
Embodiment 2
YMGEA(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔtrpE lacI∷ARO10* TrpE), YMGR2A (E.coli MG1655 Δ feaB Δ pheA Δ tyrB Δ tyrR lacI ∷ ARO10*trpE ∷ ARO10*) strain construction
TrpE deletes box and trpE ∷ ARO10* deletes the building of expression cassette according to the gene order design primer of trpE 700trpE-U-L,ΔtrpE-U-R；ΔtrpE-D-L,700trpE-D-R.It is with E. coli MG1655 genome PCR amplification obtains segment DtrpEUP, DtrpEDown to template respectively, is primer with 500trpE-U-L, 500trpE-D-R, utilizes The method of nest-type PRC expands to obtain gene trpE deletion box.It is set according to the gene order of trpE and plasmid pKK223-ARO10* Count primer 700trpE-U-L, 700trpE-U-R；trpE-ARO10-L,trpE-ARO10-R；700trpE-D-L,700trpE- D-R.It is expanded respectively using E. coli MG1655 and plasmid pKK223-ARO10* genome as template, obtains segment trpEUP,trpEDown,ARO10.PTarget plasmid is subjected to digestion with Xba I, recycling obtains segment.With the one of Vazyme Cloning Kit is walked, four segments are attached, converts, correct plasmid is obtained, with 500trpE-U-L, 500trpE-D-R For primer, PCR is carried out, trpE ∷ ARO10* is obtained and deletes expression cassette.
YMGEA(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔtrpE lacI∷ARO10* TrpE), YMGR2A (E.coli MG1655 Δ feaB Δ pheA Δ tyrB Δ tyrR lacI ∷ ARO10*trpE ∷ ARO10*) the method that strain construction utilizes CRISPR-cas9, by YMGRA/pCas (E.coli MG1655 Δ feaB Δ pheA Δ tyrB Δ tyrR lacI ∷ ARO10*/pCas) it is prepared into electricity and turns competence, the plasmid sg-pTarget- of sgRNA will be had TrpE and above-mentioned trpE deletes box or competence, conversion is added in the deletion expression cassette of trpE ∷ ARO10*.Picking transformant, Bacterium colony PCR verifying is carried out with primer 700trpE-U-L, 700trpE-D-R, bacterial strain YMGRA/pCas is as control.Utilize IPTG It is induced, eliminates sg-pTarget-trpE plasmid, utilize (42 DEG C) elimination pCas plasmids of high temperature.Obtain bacterial strain YMGEA and YMGR2A。
Embodiment 3
YMGB2A(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔpabB lacI∷ARO10* trpE∷ARO10*),YMGR3A(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR lacI∷ARO10* TrpE ∷ ARO10*pabB ∷ ARO10*) strain construction
PabB deletes box and pabB ∷ ARO10* deletes the building of expression cassette and trpE deletes box and trpE ∷ ARO10* is deleted Except the building of expression cassette is similar, YMGR2A/pCas is prepared into electricity and turns competence, is converted, method is similar to Example 2. Obtain bacterial strain YMGB2A and YMGR3A.
Embodiment 4
YMGA3A(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔpabA lacI∷ARO10* trpE∷ARO10*pabB∷ARO10*),YMGR4A(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR LacI ∷ ARO10*trpE ∷ ARO10*pabB ∷ ARO10*pabA ∷ ARO10*) strain construction
PabA deletes box and pabA ∷ ARO10* deletes the building of expression cassette and trpE deletes box and trpE ∷ ARO10* is deleted Except the building of expression cassette is similar, YMGR3A/pCas is prepared into electricity and turns competence, is converted, method is similar to Example 2. Obtain bacterial strain YMGA3A and YMGR4A.
Embodiment 5
YMGF4A(E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔpykF lacI∷ARO10* trpE∷ARO10*pabB∷ARO10*pabA∷ARO10*),YMGR5A(E.coli MG1655 ΔfeaB ΔpheA Δ TyrB Δ tyrR lacI ∷ ARO10*trpE ∷ ARO10*pabB ∷ ARO10*pabA ∷ ARO10*pykF ∷ ARO10*) bacterial strain Building
PykF deletes box and pykF ∷ ARO10* deletes the building of expression cassette and trpE deletes box and trpE ∷ ARO10* is deleted Except the building of expression cassette is similar, YMGR4A/pCas is prepared into electricity and turns competence, is converted, method is similar to Example 2. Obtain bacterial strain YMGF4A and YMGR5A.
The fermentation of the synthesis tyrosol microorganism of embodiment 6
By bacterial strain in the flat lining out culture of LB of non-resistant, picking single colonie is inoculated in 20mL liquid LB, planted 8-10h is cultivated in sub- liquid culture.It takes 500 μ L of seed liquor to be inoculated in 50mL liquid LB and expands culture, be placed in 37 DEG C, 200r min-1Shaking table culture 10h.All thallus are collected, supernatant is removed after microorganism collection, and have physiological saline cleaning thallus primary.It will cleaning Later thallus is transferred in 50mL M9Y fermentation medium, is subsequently placed in 30 DEG C, 200rmin-1Shaker fermentation 48h.Every 12h sampling.Utilize the yield of high performance liquid chromatography (HPLC) detection tyrosol.The yield result of tyrosol as shown in Fig. 1 and table 2, The yield of the related gene and the appropriate copy number tyrosol for increasing ARO10* gene that knock out competition approach is incremented by step by step, works as knockout The yield of tyrosol reaches 10.84mM when pykF gene, and the yield of tyrosol reaches when knocking out pykF gene and integrating ARO10* gene 10.92mM, it is believed that it is little to continue growing ARO10* gene pairs tyrosol yield effect.
The tyrosol yield that the fermentation different strains of table 2 obtain
7 fermentation tank culture YMGR5A of embodiment produces tyrosol
Fermentation tank culture produces tyrosol, and YMGR5A is crossed on LB plate, cultivates；Picking single colonie is inoculated in 20mL liquid In body LB, seed liquor culture is carried out, cultivates 8-10h.It takes seed liquor to be inoculated in 50mL liquid LB and controls initial OD600It is 0.05, 37 DEG C are placed in, 200rmin-1Shaking table culture 5h expands culture, works as OD600When reaching 0.25, it is inoculated in 5L and is cultivated equipped with 2L M9Y The fermentor of base samples every 4h, adds appropriate glucose and yeast powder.Tyrosol is detected using high performance liquid chromatography (HPLC) Yield.The yield result of tyrosol as shown in Fig. 2, when ferment 48h when, the yield of tyrosol in the fermenter reaches 27.96mM.
Embodiment 8 detects the yield of tyrosol using high performance liquid chromatography (HPLC)
Chromatographic test strip part is specific as follows: Agela Innoval C18 chromatographic column (4.6 × 250mm, aperture are 5 μm)；Stream Dynamic is mutually 80% 0.1% formic acid and the methanol of water 20%；Flow velocity 1mLmin-1；10 μ L of sample volume；UV detector, detection Wavelength 276nm；Column temperature is 30 DEG C.
9 YMGR6A of embodiment (E.coli MG1655 Δ feaB Δ pheA Δ tyrB Δ tyrR lacI ∷ ARO10* TrpE ∷ ARO10*pabB ∷ ARO10*pabA ∷ ARO10*pykF ∷ ARO10*yccx ∷ ARO10*) strain construction
The building and the trpE ∷ ARO10* deletion building of expression cassette of yccx ∷ ARO10* deletion expression cassette are similar, will YMGR5A/pCas is prepared into electricity and turns competence, is converted, and method is similar to Example 2.Bacterial strain YMGR6A is obtained, tyrosol produces Amount reaches 11.74mM.
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 for producing tyrosol and its construction method and application
<160> 66
<170> PatentIn version 3.3
<210> 1
<211> 1908
<212> DNA
<213>artificial synthesized
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aaatctgttt tcggtgttcc gggtgacttc aacctgtctc tgctggaata cctgtactct 180
ccgtctgttg aatctgctgg tctgcgttgg gttggtacct gcaacgaact gaacgctgct 240
tacgctgctg acggttactc tcgttactct aacaaaatcg gttgcctgat caccacctac 300
ggtgttggtg aactgtctgc tctgaacggt atcgctggtt ctttcgctga aaacgttaaa 360
gttctgcaca tcgttggtgt tgctaaatct atcgactctc gttcttctaa cttctctgac 420
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ggttacacca tcgaacgtgc tatcatgggt ccgacccgtt cttacaacga cgttatgtct 1680
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aaacgttctg gtatcgaact gctggaagtt aaactgggtg aactggactt cccggaacag 1860
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<213>artificial synthesized
<400> 15
gcctttttgc gtttctacaa acggagactt tctgatggct gacattc 47
<210> 16
<211> 53
<212> DNA
<213>artificial synthesized
<400> 16
ggtaatagat ctaagcttct gcaggtcgac gctgaaaaca gctggtggct ttc 53
<210> 17
<211> 22
<212> DNA
<213>artificial synthesized
<400> 17
ccagaccgtg gaaatttcca cg 22
<210> 18
<211> 25
<212> DNA
<213>artificial synthesized
<400> 18
gagaatggat tccggatgga actgg 25
<210> 19
<211> 47
<212> DNA
<213>artificial synthesized
<400> 19
gaatgtcagc catcagaaag tctcccgggc tgggtatctg attgctt 47
<210> 20
<211> 47
<212> DNA
<213>artificial synthesized
<400> 20
aagcaatcag atacccagcc cgggagactt tctgatggct gacattc 47
<210> 21
<211> 60
<212> DNA
<213>artificial synthesized
<400> 21
gtcctaggta taatactagt taaccggggc tccgaaagta gttttagagc tagaaatagc 60
<210> 22
<211> 20
<212> DNA
<213>artificial synthesized
<400> 22
taaccggggc tccgaaagta 20
<210> 23
<211> 49
<212> DNA
<213>artificial synthesized
<400> 23
gagtcggtgc tttttttgaa ttctctagac cctggatttc attggtgcc 49
<210> 24
<211> 46
<212> DNA
<213>artificial synthesized
<400> 24
atttacgacc tgcacagcca tcagtcctga ctctactggc tatgtg 46
<210> 25
<211> 46
<212> DNA
<213>artificial synthesized
<400> 25
cacatagcca gtagagtcag gactgatggc tgtgcaggtc gtaaat 46
<210> 26
<211> 42
<212> DNA
<213>artificial synthesized
<400> 26
aggctacggt attccacgtc gtttgtagaa acgcaaaaag gc 42
<210> 27
<211> 42
<212> DNA
<213>artificial synthesized
<400> 27
gcctttttgc gtttctacaa acgacgtgga ataccgtagc ct 42
<210> 28
<211> 50
<212> DNA
<213>artificial synthesized
<400> 28
ggtaatagat ctaagcttct gcaggtcgac cacgaattat gcctgcggtc 50
<210> 29
<211> 22
<212> DNA
<213>artificial synthesized
<400> 29
gcctgctgta atagataaag cc 22
<210> 30
<211> 22
<212> DNA
<213>artificial synthesized
<400> 30
ggcgactggc ttaactattc ac 22
<210> 31
<211> 44
<212> DNA
<213>artificial synthesized
<400> 31
caggctacgg tattccacgt ccagtcctga ctctactggc tatg 44
<210> 32
<211> 44
<212> DNA
<213>artificial synthesized
<400> 32
catagccagt agagtcagga ctggacgtgg aataccgtag cctg 44
<210> 33
<211> 60
<212> DNA
<213>artificial synthesized
<400> 33
gtcctaggta taatactagt acgttattcg ccactatgcc gttttagagc tagaaatagc 60
<210> 34
<211> 20
<212> DNA
<213>artificial synthesized
<400> 34
acgttattcg ccactatgcc 20
<210> 35
<211> 53
<212> DNA
<213>artificial synthesized
<400> 35
gagtcggtgc tttttttgaa ttctctagag cctttagtca ctcttactgc cgc 53
<210> 36
<211> 43
<212> DNA
<213>artificial synthesized
<400> 36
atttacgacc tgcacagcca tggcggctcc ggtacaaaag aac 43
<210> 37
<211> 43
<212> DNA
<213>artificial synthesized
<400> 37
gttcttttgt accggagccg ccatggctgt gcaggtcgta aat 43
<210> 38
<211> 44
<212> DNA
<213>artificial synthesized
<400> 38
gatcaccctg ttacgcataa acgtttgtag aaacgcaaaa aggc 44
<210> 39
<211> 44
<212> DNA
<213>artificial synthesized
<400> 39
gcctttttgc gtttctacaa acgtttatgc gtaacagggt gatc 44
<210> 40
<211> 49
<212> DNA
<213>artificial synthesized
<400> 40
ggtaatagat ctaagcttct gcaggtcgac tggatcggct caaccacca 49
<210> 41
<211> 20
<212> DNA
<213>artificial synthesized
<400> 41
gaccattgag cttggtccgc 20
<210> 42
<211> 21
<212> DNA
<213>artificial synthesized
<400> 42
ccacccaccg aaacggtaaa c 21
<210> 43
<211> 44
<212> DNA
<213>artificial synthesized
<400> 43
gatcaccctg ttacgcataa acggcggctc cggtacaaaa gaac 44
<210> 44
<211> 44
<212> DNA
<213>artificial synthesized
<400> 44
gttcttttgt accggagccg ccgtttatgc gtaacagggt gatc 44
<210> 45
<211> 60
<212> DNA
<213>artificial synthesized
<400> 45
gtcctaggta taatactagt atggttgcgg taacgtatga gttttagagc tagaaatagc 60
<210> 46
<211> 20
<212> DNA
<213>artificial synthesized
<400> 46
atggttgcgg taacgtatga 20
<210> 47
<211> 52
<212> DNA
<213>artificial synthesized
<400> 47
gagtcggtgc tttttttgaa ttctctagag gctaatgctg tacgtaatac gc 52
<210> 48
<211> 42
<212> DNA
<213>artificial synthesized
<400> 48
atttacgacc tgcacagcca tgttgagaag gatgggagaa ac 42
<210> 49
<211> 42
<212> DNA
<213>artificial synthesized
<400> 49
gtttctccca tccttctcaa catggctgtg caggtcgtaa at 42
<210> 50
<211> 43
<212> DNA
<213>artificial synthesized
<400> 50
catcagggcg cttcgatata cgtttgtaga aacgcaaaaa ggc 43
<210> 51
<211> 43
<212> DNA
<213>artificial synthesized
<400> 51
gcctttttgc gtttctacaa acgtatatcg aagcgccctg atg 43
<210> 52
<211> 50
<212> DNA
<213>artificial synthesized
<400> 52
ggtaatagat ctaagcttct gcaggtcgac cagcaatgcg ccttcagtag 50
<210> 53
<211> 23
<212> DNA
<213>artificial synthesized
<400> 53
ctgcacattt ctcggtacag ttc 23
<210> 54
<211> 19
<212> DNA
<213>artificial synthesized
<400> 54
cgcacaatgt gcgccattt 19
<210> 55
<211> 42
<212> DNA
<213>artificial synthesized
<400> 55
gtttctccca tccttctcaa cgtatatcga agcgccctga tg 42
<210> 56
<211> 42
<212> DNA
<213>artificial synthesized
<400> 56
catcagggcg cttcgatata cgttgagaag gatgggagaa ac 42
<210> 57
<211> 60
<212> DNA
<213>artificial synthesized
<400> 57
gtcctaggta taatactagt gaaagtctgc ataattgcct gttttagagc tagaaatagc 60
<210> 58
<211> 20
<212> DNA
<213>artificial synthesized
<400> 58
gaaagtctgc ataattgcct 20
<210> 59
<211> 51
<212> DNA
<213>artificial synthesized
<400> 59
gagtcggtgc tttttttgaa ttctctagag tgtccgtgct gaatatccac c 51
<210> 60
<211> 45
<212> DNA
<213>artificial synthesized
<400> 60
atttacgacc tgcacagcca ttgctgctct ccttatcctt aatgg 45
<210> 61
<211> 45
<212> DNA
<213>artificial synthesized
<400> 61
ccattaagga taaggagagc agcaatggct gtgcaggtcg taaat 45
<210> 62
<211> 46
<212> DNA
<213>artificial synthesized
<400> 62
cctgccaaaa ccggtaaaat gtatgtttgt agaaacgcaa aaaggc 46
<210> 63
<211> 46
<212> DNA
<213>artificial synthesized
<400> 63
gcctttttgc gtttctacaa acatacattt taccggtttt ggcagg 46
<210> 64
<211> 50
<212> DNA
<213>artificial synthesized
<400> 64
ggtaatagat ctaagcttct gcaggtcgac ccacccgcaa agatatgtcg 50
<210> 65
<211> 22
<212> DNA
<213>artificial synthesized
<400> 65
gatattctgc cccagcactc ag 22
<210> 66
<211> 20
<212> DNA
<213>artificial synthesized
<400> 66
gtgccacggt tagcctgtat 20

Claims (10)

1. a kind of recombination bacillus coli, which is characterized in that be the site lacI in E.coli MG1655 genome, the site trpE, The site pabB, the site pabA, five sites in the site pykF are whole on each site in five sites while deleted Close saccharomyces cerevisiae Pyruvate Decarboxylase Gene ARO10* gene, the nucleotide sequence of ARO10* gene such as SEQ ID NO.1 institute Show.
2. recombination bacillus coli as described in claim 1, which is characterized in that the recombination bacillus coli also deletes yccX Point, while upper ARO10* gene is integrated on the site.
3. recombination bacillus coli as claimed in claim 1 or 2, which is characterized in that utilize CRISPR-cas9 technology or Red Homologous recombination carries out site deletion or gene integration.
4. a kind of method for producing tyrosol, which is characterized in that apply any recombination bacillus coli of claim 1-3 into Row fermentation.
5. method as claimed in claim 4, which is characterized in that using M9Y culture medium as fermentation medium.
6. method as claimed in claim 4, which is characterized in that culture that bacterial strain is crossed on LB plate；Picking single colonie connects Kind carries out seed liquor culture in LB liquid medium, cultivates 8-10h.
7. method as claimed in claim 6, which is characterized in that seed liquor is inoculated in liquid LB with 1-5% inoculation volume ratio In culture medium, it is placed in 35-39 DEG C, 200-220rpm shaking table culture 8-12h；All thallus are collected, are gone after microorganism collection Clearly, thallus is cleaned；Thallus after cleaning is transferred in M9Y fermentation medium, is subsequently placed in 28-30 DEG C, 200-220rpm Shaker fermentation 40-60h.
8. method as claimed in claim 6, which is characterized in that seed liquor is taken to be inoculated in liquid LB with 1-5% inoculation volume ratio In culture medium, initial OD is controlled600For 0.05-0.06, it is placed in 35-39 DEG C, 200-220rpm shaking table culture works as OD600Reach When 0.25-0.30, it is inoculated in the ferment tank 40-60h equipped with M9Y culture medium that liquid amount is 40-45%.
9. the method for constructing any recombination bacillus coli of claim 1-3, which is characterized in that be in E.coli The site lacI of MG1655 genome, the site trpE, the site pabB, the site pabA, the site pykF five sites deleted While upper saccharomyces cerevisiae Pyruvate Decarboxylase Gene ARO10* gene, ARO10* are integrated on each site in five sites The nucleotide sequence of gene is as shown in SEQ ID NO.1.
10. any recombination bacillus coli of claim 1-3 is in the application of food, chemical industry or pharmaceutical field.
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CN113493758A (en) * 2021-05-31 2021-10-12 江南大学 Tyrosol-producing recombinant escherichia coli capable of shortening fermentation period and application thereof
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CN108779470A (en) * 2015-12-17 2018-11-09 赢创德固赛(中国)投资有限公司 The box gene knocked out for homologous recombination in yeast cells
WO2021027175A1 (en) * 2019-08-15 2021-02-18 江南大学 Recombinant escherichia coli strain for producing tyrosol, construction method therefor and use thereof
US11286475B2 (en) 2019-08-15 2022-03-29 Jiangnan University Tyrosol-producing recombinant Escherichia coli and construction method and application thereof
CN112094829A (en) * 2020-09-22 2020-12-18 江南大学 Amino deoxy-chorismate synthetase mutant T426I with changed enzyme activity and application thereof
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
CN113493758A (en) * 2021-05-31 2021-10-12 江南大学 Tyrosol-producing recombinant escherichia coli capable of shortening fermentation period and application thereof
CN113493758B (en) * 2021-05-31 2022-08-02 江南大学 Tyrosol-producing recombinant escherichia coli capable of shortening fermentation period and application thereof
CN113897325A (en) * 2021-11-05 2022-01-07 江南大学 Recombinant escherichia coli for producing salidroside and construction method and application thereof
CN114736918A (en) * 2022-03-23 2022-07-12 江南大学 Recombinant escherichia coli for producing salidroside through integrated expression and application thereof
CN114736918B (en) * 2022-03-23 2023-08-25 江南大学 Recombinant escherichia coli for producing salidroside by integrated expression and application thereof

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