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Escherichia coli genetically-engineered bacterium for producing protocatechuic acid and construction method and application of escherichia coli genetically-engineered bacterium

A technology of protocatechuic acid and Escherichia coli, which is applied in the field of protocatechuic acid recombinant Escherichia coli genetically engineered bacteria and its construction, can solve the problems of genetic instability and affecting the industrial application of products, and achieve genetic stability and reduce Production cost, cost reduction effect

Active Publication Date: 2019-06-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although the engineering strains of E. coli constructed in the above studies can produce high concentrations of protocatechuic acid, the related engineering strains contain recombinant plasmids that cause genetic instability, and at the same time, aromatic amino acids or PCA extractants need to be added to the medium to promote cell growth. , thus affecting the industrial application of the product

Method used

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  • Escherichia coli genetically-engineered bacterium for producing protocatechuic acid and construction method and application of escherichia coli genetically-engineered bacterium
  • Escherichia coli genetically-engineered bacterium for producing protocatechuic acid and construction method and application of escherichia coli genetically-engineered bacterium
  • Escherichia coli genetically-engineered bacterium for producing protocatechuic acid and construction method and application of escherichia coli genetically-engineered bacterium

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Embodiment 1

[0045] Embodiment 1, the construction of Escherichia coli genetically engineered bacteria WJ004

[0046] The Escherichia coli genetically engineered strain WJ004 was constructed by weakening the expression regulation of the 3-dehydrogenase shikimate dehydrogenase gene (aroE). A two-step homologous recombination method was used to insert a synthetic regulatory element upstream of the start codon of aroE. P1 (as shown in SEQ ID NO: 1), and replace the original start codon ATG with the rare start codon TTG. The specific construction steps are as follows:

[0047] With the plasmid pEASY-cat-sacB ( figure 2 ) as a template, using primers aroE1-up / aroE1-down to amplify the fragment aroE1 of the first step of homologous recombination. The primer sequences are:

[0048] aroE1-up: GATGCCCTGACGGGTGAACTGTTTCGACAGGGGTAACATA G TGACGGAAGATCACTTC

[0049] aroE1-down: CTGTGGGCTATCGGATTACCAAAAACAGCATAGGTTTCCA ATCAAAGGGAAAACTGTCC

[0050] Amplification system: 5×TransStart TM FastPf...

Embodiment 2

[0068] Embodiment 2, the construction of Escherichia coli genetic engineering bacterium WJ006

[0069] Escherichia coli genetically engineered strain WJ006 is obtained on the basis of Escherichia coli WJ004 through the mutation and expression regulation of the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene (aroF) construction, using two-step homologous recombination Method, replace the original aroF gene (such as SEQ ID NO:6) with the mutant gene aroF* (such as SEQ ID NO:5) that changes the 443rd base C to T, to remove the 3-deoxy - Tyrosine feedback inhibition of D-arabinoheptulose-7-phosphate synthase with insertion of synthesis regulatory element P2 (eg SEQ ID NO: 2) upstream of the start codon. The specific construction steps are as follows:

[0070] Using Escherichia coli DSM 1576 genomic DNA as a template, the aroF gene was amplified using primers aroF-F / aroF-R. The primer sequence is:

[0071] aroF-F: ATGCAAAAAGACGCGCTGAA

[0072] aroF-R: TTAAGCCACGCGAGCCGTCAG...

Embodiment 3

[0110] Embodiment 3, the construction of Escherichia coli genetic engineering bacterium WJ012

[0111] The Escherichia coli genetically engineered strain WJ012 was constructed on the basis of Escherichia coli WJ006 by regulating the expression of the transketolase gene (tktA). It inserted the synthesis regulation upstream of the tktA start codon through two homologous recombination methods. Element P4 (eg SEQ ID NO: 4). The specific construction steps are as follows:

[0112] With the plasmid pEASY-cat-sacB ( figure 2 ) as a template, using primers tktA1-up / tktA1-down to amplify the fragment tktA1 of the first step of homologous recombination. The primer sequences are:

[0113] tktA1-up: GCCCAAAACGCGCTGTCGTCAAGTCGTTAAGGGCGTGCCCTTCATCAT GTGACGGAAGATCACTTC

[0114] tktA1-down: CATGCTCAGCGCACGAATAGCATTGGCAAGCTCTTTACGTGAGGACAT ATCAAAGGGAAAACTGTCC

[0115] The amplification system is the same as in Example 1, and the amplified tktA1 product comprises the cat-sacB box ( ...

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Abstract

The method discloses an escherichia coli genetically-engineered bacterium for producing protocatechuic acid and a construction method and application of the escherichia coli genetically-engineered bacterium. The escherichia coli genetically-engineered bacterium expresses 3-dehydrogenase shikimate dehydratase (aroZ) in escherichia coli for producing 3-dehydroshikimate to produce the protocatechuicacid. The strain can utilize a glucose inorganic salt culture medium for fermentation to produce the protocatechuic acid, the cost of the culture medium is reduced, the production cost of the protocatechuic acid is reduced accordingly, and the recombinant strain does not contain a plasmid and is stable in heredity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to recombinant Escherichia coli genetically engineered bacteria for producing protocatechuic acid and its construction method and application. Background technique [0002] Protocatechuate (PCA) has partial effects such as antibacterial, antiasthma, expectorant, and detoxification, and has the effect of astringent and promoting wound healing. It can be used clinically to treat chronic bronchitis, burns, infantile pneumonia, bacillary dysentery, acute pyelonephritis, acute pancreatitis and certain ulcer diseases. It is also used in medicine, organic intermediates, materials and dye synthesis, as well as feed, food, environmental protection and other fields. PCA can be catalyzed by 3-dehydroshikimate dehydratase (3-dehydroshikimate dehydratase, aroZ) derived from Klebsiella pneumococcus (Klebsiellapneumoniae) on 3-dehydroshikimate (DHS) (Figure 1), At the same time, it is al...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/90C12P7/42C12R1/19
Inventor 王钦宏陈五九江小龙宋国田彭彦峰张媛媛
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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