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Genetic engineering bacterium for high-yield phloroglucinol as well as construction method and application of genetic engineering bacterium

A technology of genetically engineered bacteria and phloroglucinol, which is applied in the field of genetic engineering, can solve the problem that the output and yield of phloroglucinol cannot meet the needs of industrial production, and achieve the effect of improving synthesis ability and yield

Active Publication Date: 2015-03-04
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Overexpression of genes such as phlD and ACCase can increase the production of phloroglucinol, but the current production and yield of phloroglucinol still cannot meet the needs of industrial production
CsrB is an important global regulatory factor in Escherichia coli. There is no report on the effect of this regulatory factor on the fermentation production of phloroglucinol, and there is no report on the genetic modification method to improve the synthesis ability of phloroglucinol through global regulation of carbon metabolism.

Method used

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  • Genetic engineering bacterium for high-yield phloroglucinol as well as construction method and application of genetic engineering bacterium
  • Genetic engineering bacterium for high-yield phloroglucinol as well as construction method and application of genetic engineering bacterium
  • Genetic engineering bacterium for high-yield phloroglucinol as well as construction method and application of genetic engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the construction of genetically engineered bacteria

[0063] Using Escherichia coli E.coliBL21(DE3) as the starting strain, the T7 promoter sequence was inserted in front of CsrB, and the overexpression of CsrB gene was induced by IPTG to construct the engineering strain ZG1563 ( figure 1 ), the related plasmids pET-phlD-mar and pACYC-accADBC for the production of phloroglucinol were introduced into ZG1563 and the control strain E.coliBL21(DE3) to detect phloroglucinol fermentation at shake flask and fermenter levels.

[0064] Those skilled in the art should understand that, the above-mentioned Escherichia coli E.coliBL21 (DE3) gene knockout experiment, each step is carried out according to the standard molecular cloning technology.

[0065] 1.1 Construction of homology arm

[0066] With the Escherichia coli E.coliBL21 (DE3) wild strain CsrB gene as a part of the downstream homology arm, the about 700bp of the CsrB upstream sequence is used as the upstrea...

Embodiment 2

[0086] Embodiment 2. Fermentation experiments of engineering strains

[0087] 2.1 Shake flask fermentation experiment

[0088] 1) For the cultivation of primary seed liquid, inoculate wild control strain E.coliBL21(DE3) and engineering strain ZG1563 containing pET-phlD-mar and pACYC-accADBC recombinant plasmids into 3mL LB liquid medium respectively, and add 50μg / mL kanamycin and 50 μg / mL chloramphenicol, grow at 37°C for 8-12h.

[0089] 2) Transfer the primary seed solution to a 250mL fermentation shaker flask with 1% inoculation amount, containing 50mL fermentation medium, and add 200g / L MgSO when transferring the seed solution 4 .7H 2 O 100 μL, 500 g / L glucose 2 mL, 1000 × trace elements 50 μL, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol double resistance, set 3 parallel controls for each strain, culture at 37°C, 180rpm.

[0090] 3) Cell OD 600 When it reaches 0.6-1.0, 100μM / L IPTG can be added for induction.

[0091] 4) After induction by IPTG, continue to culture a...

Embodiment 3

[0104] Embodiment 3. Fermentation experiments of engineering strains

[0105] 3.1 Shake flask fermentation experiment

[0106] 1) For the cultivation of the primary seed liquid, the inoculation method is according to Example 2.1.

[0107] 2) Transfer the primary seed solution to a 250mL fermentation shaker flask with 3% inoculum size, containing 50mL fermentation medium, and add 200g / L MgSO when transferring the seed solution 4 .7H 2 O 100 μL, 500 g / L glucose 2 mL, 1000 × trace elements 50 μL, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol double resistance, set 3 parallel controls for each strain, culture at 37°C, 180rpm.

[0108] 3) Cell OD 600 When it reaches 0.6-1.0, 100μM / L IPTG can be added for induction.

[0109] 4) After induction by IPTG, continue to culture at 37°C and 180rpm for 24 hours, then collect the bacterial liquid, centrifuge to get the supernatant, and measure the content of phloroglucinol.

[0110] 3.2 Fed-batch fermentation production of phloroglucinol...

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Abstract

The invention discloses a genetic engineering bacterium for high-yield phloroglucinol as well as a construction method and an application of the genetic engineering bacterium and belongs to the technical field of genetic engineering. The genetic engineering bacterium over-expresses carbon storage and regulation factor gene CsrB shown by SEQ ID NO.1 and co-expresses a polyketide synthase gene phlD, a multiple resistance activating factor gene marA and acetyl CoA carboxylase gene ACCase. The invention also provides the construction method and the application of the genetic engineering bacterium for the high-yield phloroglucinol. With the adoption of the genetic engineering bacterium, the yield of the phloroglucinol is firstly increased by 115.6 percent by adopting a mode of globally regulating carbon metabolism after post-transcriptional level of the genetic engineering bacterium, and the genetic engineering bacterium has a high industrial application value.

Description

technical field [0001] The invention relates to a genetically engineered bacterium with high phloroglucinol production and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Phloroglucinol is an important fine chemical product and an intermediate in the synthesis of flavonoids and isoflavones. Phloroglucinol itself can be used as an excellent antispasmodic drug for smooth muscle and is widely used in clinical practice. Phloroglucinol can also inhibit the activity of peroxidase, has anti-inflammatory and anti-oxidative effects, it can catalyze H 2 o 2 Decomposed into molecular oxygen and water, it is an important antioxidant enzyme. In addition, phloroglucinol is also an anti-curing agent, stabilizer, fuel coupling agent, tire tackifier, etc. with superior performance, and has a broad market demand. At present, the industrial production methods of phloroglucinol are mainly chemical synthesis metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/22
CPCC07K14/245C12N9/1029C12N9/93C12P7/22C12Y604/01002
Inventor 咸漠赵广刘敏曹玉锦刘长水
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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