Recombinant escherichia coli for producing tyrosol and construction method and application thereof

A technology for recombining Escherichia coli and seeds, applied in the field of bioengineering, can solve problems such as difficulty in obtaining high-purity tyrosol

Active Publication Date: 2019-11-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process method has many disadvantages in the subsequent extraction of tyrosol, and it is difficult to obtain high-purity tyrosol

Method used

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  • Recombinant escherichia coli for producing tyrosol and construction method and application thereof
  • Recombinant escherichia coli for producing tyrosol and construction method and application thereof
  • Recombinant escherichia coli for producing tyrosol and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Production of Tyrosol by Heterologous Expression of Saccharomyces cerevisiae Pyruvate Decarboxylase Gene in Escherichia coli MG1655

[0025] (1) Construction of plasmid pKK223-3-ARO10*

[0026] The ARO10* gene sequence obtained after codon optimization was chemically synthesized by Suzhou Hongxun Biological Co., Ltd., and inserted into the EcoR I and Hind III sites of the plasmid pKK223-3 to obtain the recombinant plasmid pKK223-3-ARO10*.

[0027] (2) Construction of lacI::ARO10* deletion expression cassette

[0028] According to the sequence of the pKK223-3 plasmid, primers ARO10-L and LacIR (Table 1) were designed to obtain the expression fragment of tac-ARO10*-rrnB with promoter and terminator, and inserted into the pMD19-T simple plasmid to obtain the recombinant plasmid 19Ts- tac-ARO10*-rrnB. According to pKD13, primers LacIL and PKDR were designed to amplify Kana-resistant fragments as templates. The plasmid 19Ts-tac-ARO10*-rrnB was ligated with the K...

Embodiment 2

[0035] YMGEA (E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR ΔtrpE lacI::ARO10*trpE), YMGR2A (E.coli MG1655 ΔfeaB ΔpheA ΔtyrB ΔtyrR lacI::ARO10*trpE::ARO10*) strain construction

[0036]Construction of trpE deletion cassette and trpE::ARO10* deletion expression cassette Primers 700trpE-U-L, ΔtrpE-U-R; ΔtrpE-D-L, 700trpE-D-R were designed according to the gene sequence of trpE. Using Escherichia coli E.coli MG1655 genome as a template, the fragments DtrpEUP and DtrpEDown were amplified by PCR respectively. With 500trpE-U-L and 500trpE-D-R as primers, the gene trpE deletion cassette was amplified by nested PCR. According to the gene sequence of trpE and plasmid pKK223-ARO10*, primers 700trpE-U-L, 700trpE-U-R; trpE-ARO10-L, trpE-ARO10-R; 700trpE-D-L, 700trpE-D-R were designed. Using Escherichia coli E.coli MG1655 and the genome of plasmid pKK223-ARO10* as templates, the fragments trpEUP, trpEDown, and ARO10 were respectively amplified. The pTarget plasmid was digested with Xba I, and the...

Embodiment 3

[0039] 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*)

[0040] The construction of the pabB deletion cassette and the pabB::ARO10* deletion cassette is similar to the construction of the trpE deletion cassette and the trpE::ARO10* deletion cassette. The YMGR2A / pCas is prepared to be electrotransferable and transformed, and the method is similar to Example 2. Strains YMGB2A and YMGR3A were obtained.

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Abstract

The invention discloses recombinant escherichia coli for producing tyrosol and a construction method and application thereof and belongs to the technical field of bioengineering. The escherichia coliheterologously expresses a saccharomyces cerevisiae pyruvate decarboxylase gene ARO10* after codon optimization. According to the recombinant escherichia coli, the ARO10* gene is integrated while a lacI locus, a trpE locus, a pabB locus, a pabA locus and a pykF locus of an escherichia coli genome are deleted, and a strain containing multiple copies of the ARO10* gene is obtained. On the basis of the recombinant strain, the ARO10* gene is randomly integrated at the multiple loci, and the ARO10* gene is inserted at a yccX locus so that the strain with high tyrosol yield can be obtained. No inducer nor antibiotic is required for fermentation by using this strain. After 48 hours of fermentation, the yield of tyrosol can reach 28 mM.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli producing tyrosol and a construction method and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Tyrosol is a pharmacologically active phenolic compound, a derivative of phenylethyl alcohol, and a monophenolic antioxidant that has a variety of natural sources, such as olive oil and green tea. Tyrosol has many physiologically active functions, such as anti-oxidation, anti-fatigue, anti-hypoxia, anti-stress, anti-cold, sedation, cardiovascular disease, hypertension, etc. Tyrosol can also be used as a flavoring agent in alcohol, which plays an important role in enhancing the taste of alcoholic beverages, especially in sake, beer and wine. In addition, tyrosol is the precursor of hydroxytyrosol, and hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol) is an antioxidant beneficial to human health. Compared with tyrosol, its antioxidant Stronger, at the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/22C12R1/19
CPCC12N9/88C12N15/70C12P7/22C12Y401/01001
Inventor 陈献忠徐微沈微
Owner JIANGNAN UNIV
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