Recombinant escherichia coli using glucose for producing hydroxytyrosol as well as recombination method and application

A technology for recombining Escherichia coli and hydroxytyrosol, applied in the field of bioengineering, can solve the problems of high cost, long reaction steps, pollute the environment and the like, and achieve the effect of increasing yield

Active Publication Date: 2015-07-29
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In summary, the plant extraction process of hydroxytyrosol is cumbersome, inefficient, and pollutes the environment; there are also chemical synthesis methods, but chemical synthesis methods have low raw material utilization, long reaction steps, difficult control conditions, and the presence of by-products, etc. Problem; biotransformation is safe and pollution-free, but the cost is high and it is difficult to produce on a large scale
Although HpaBC and other hydroxylases have been reported to convert tyrosol into hydroxytyrosol, there has been no report on the production of hydroxytyrosol by Escherichia coli using simple carbon sources such as glucose

Method used

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  • Recombinant escherichia coli using glucose for producing hydroxytyrosol as well as recombination method and application
  • Recombinant escherichia coli using glucose for producing hydroxytyrosol as well as recombination method and application
  • Recombinant escherichia coli using glucose for producing hydroxytyrosol as well as recombination method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025]Gene HpaBC and Escherichia coli expression vector pTrcHisB-ARO10-HpaBC acquisition method:

[0026] The extraction of genomic DNA can be accomplished using a bacterial genome extraction kit.

[0027] The PCR primer is HpaBC-5FPSac, the sequence of which is shown in SEQ ID No: 1, and the sequence of HpaBC-3RPKpn is shown in SEQ ID No: 2. Using Escherichia coli BW25113 genomic DNA as a template, the open reading frame of HpaBC gene was amplified by PCR.

[0028] The HpaBC gene was ligated into the vector pTrcHisB by double digestion with SacI and KpnI to obtain the intermediate plasmid pTrcHisB-HpaBC.

[0029] Then with primer trc-HpaBC-5FPKpn (SEQ ID No: 3) and primer HpaBC-3RPKpn (SEQ ID No: 2), with plasmid pTrcHisB-HpaBC as template, PCR amplification obtains the HpaBC gene fragment Trc with Trc promoter -HpaBC, KpnI single-enzyme digestion and connection into plasmid pTrcHisB-ARO10 to obtain the final expression vector pTrcHisB-ARO10-HpaBC. The construction method ...

Embodiment 2

[0032] Escherichia coli strain containing the expression vector pTrcHisB-ARO10-HpaBC

[0033] BMGA (pTrcHisB-ARO10-HpaBC&PbB3) acquisition method:

[0034] There is no special requirement for the type of Escherichia coli used to construct the Escherichia coli expression strain, and it can be various Escherichia coli commonly used in the art capable of expressing the target gene, for example, Escherichia coli can be MG1655, BL21 (DE3) or BMGA. In order to increase the output of hydroxytyrosol, the present invention adopts the high-yield strain BMGA of tyrosine, the construction method of the strain and the literature (Bai Y, Bi H, Zhuang Y, et al. Production of salidroside in metabolically engineered Escherichia coli[J]. Scientific reports, 2014, 4: 6640-6647.) The reports are consistent.

[0035] In addition to the plasmid pTrcHisB-ARO10-HpaBC, this application also uses another expression plasmid PbB3, which contains overexpressed genes related to tyrosine pathway optimizati...

Embodiment 3

[0039] Fermentation of Escherichia coli strain BMGA (pTrcHisB-ARO10-HpaBC&PbB3):

[0040] Strain BMGA (pTrcHisB-ARO10-HpaBC&PbB3) was cultured at 37° C. for 12 hours in 3 mL of LB liquid medium containing 100 mg / L ampicillin and 25 mg / L chloramphenicol to obtain seed liquid. Then the seed solution was transferred into 50ml of LB liquid medium with 100mg / L ampicillin and 25mg / L chloramphenicol according to the transfer amount (1ml) of 2% by volume, and cultured with shaking at 37°C until the OD600 reached 0.6. IPTG (isopropyl-β-D-thiogalactopyranoside) at a concentration of 0.1 mM was used for induction, and cultured at 30° C. for 16 hours. The cells were collected by centrifugation at 4000 rpm for 10 min at 4°C. Transfer to 50mL M9Y liquid medium, and continue culturing at 30°C for 48 hours. The fermentation broth of Escherichia coli strain BMGA (pTrcHisB-ARO10-HpaBC&PbB3) was obtained.

[0041] M9Y medium: glucose 20g / L, yeast extract 0.25g / L, Na 2 PO 4 7H2O 12.8g / L, KH ...

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Abstract

The invention discloses recombinant escherichia coli using glucose for producing hydroxytyrosol as well as a building method and application. The method comprises the following steps that SEQ ID NO.1 and 2 are used as primers; the escherichia coli BW25113 genome DNA (deoxyribonucleic acid) is used as a template; HpaBC gene PCR (polymerase chain reaction) augmentation is carried out; vectors pTrcHisB are connected; middle plasmids are obtained; the middle plasmids are used as templates; the SEQ ID NO.3 and 2 are used as primers; PCR augmentation is carried out, and plasmids pTrcHisB-ARO10 are connected; expression vectors pTrcHisB-ARO10-HpaBC are obtained; in addition, the vectors and the plasmids pBb3 are simultaneously transferred into escherichia coli strains BMGA to obtain the recombinant escherichia coli. The recombinant escherichia coli contains ARO10 genes from saccharomyces cerevisiae, and HpaBC genes from the escherichia coli endogenesis. The yield of the hydroxytyrosol can be obviously improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Escherichia coli that uses glucose to produce hydroxytyrosol, a recombinant method and an application. Background technique [0002] Hydroxytyrosol, Chinese name: 3,4-dihydroxyphenylethanol, English name: 3,4-Dihydroxyphenylethanol. Chinese aliases: 2-(3,4-dihydroxyphenyl)ethanol; hydroxytyrosol; 3,4-dihydroxyphenethyl alcohol. CAS number: 10597-60-1. Molecular formula: C 8 h 10 o 3 . Structural formula: Molecular weight: 154.1632. [0003] Hydroxytyrosol is a natural polyphenol compound, which mainly exists in various parts of olives in the form of esterified oleuropein, and free hydroxytyrosol can be obtained after oleuropein is hydrolyzed. Hydroxytyrosol exhibits a large number of beneficial properties, such as cancer chemoprevention, anti-atherosclerosis, inhibition of DNA oxidative damage, protection of skin photodamage, and anti-inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/22C12R1/19
Inventor 刘涛李晓林白艳芬毕慧萍庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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