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Gene modification method for increasing yield of phloroglucinol and application of same

A technology of phloroglucinol and gene, 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, achieve high industrial application value, and increase output

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

AI Technical Summary

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
arcA 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 increase the production of phloroglucinol through global regulation of carbon metabolism.

Method used

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  • Gene modification method for increasing yield of phloroglucinol and application of same
  • Gene modification method for increasing yield of phloroglucinol and application of same

Examples

Experimental program
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Effect test

Embodiment 1

[0060] The construction of embodiment 1 bacterial strain

[0061] Taking Escherichia coli E.coliBL21 (DE3) as the starting strain, the arcA gene in the genome was knocked out, and the engineering strain ZG1949 was constructed (the construction process is as follows: figure 1 shown), the related plasmids pET-phlD-mar and pACYC-accADBC for the production of phloroglucinol were introduced into ZG1949 and the control strain E.coliBL21(DE3) to detect phloroglucinol fermentation at shake flask and fermenter levels.

[0062] 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.

[0063] 1.1 Construction of homology arm

[0064] Using the upstream and downstream of the arcA gene of the Escherichia coli E.coliBL21 (DE3) wild strain (including the partial sequence of the arcA gene) about 600bp base fragments as templates, primers we...

Embodiment 2

[0084] Embodiment 2. Fermentation experiments of engineering strains

[0085] 2.1 Shake flask fermentation experiment

[0086] 1) For the cultivation of primary seed liquid, inoculate wild control strains E.coliBL21(DE3) and ZG1949 containing pET-phlD-marA and pACYC-accADBC recombinant plasmids into 3mL LB liquid medium respectively, and add 50μg / mL card Namycin and 50 μg / mL chloramphenicol were grown at 37°C for 8-12h.

[0087] 2) Transfer the primary seed solution to a 250mL fermentation shaker flask with 1% inoculation amount, containing 50mL fermentation medium, and add 200g / L MgSO4.7H2O 100μL, 500g / L glucose 2mL, 1000× 50 μL of trace elements, 50 μg / mL kanamycin, 50 μg / mL chloramphenicol double resistance, set 3 parallel controls for each strain, and culture at 37°C and 180rpm.

[0088] 3) When the OD600 of the cells reaches 0.6-1.0, 100 μM / L IPTG can be added for induction.

[0089] 4) After induction by IPTG, continue to culture at 37°C and 180rpm for 24 hours, then ...

Embodiment 3

[0102] Embodiment 3. Fermentation experiments of engineering strains

[0103] 3.1 Shake flask fermentation experiment

[0104] 1) For the cultivation of the primary seed liquid, the inoculation method is according to Example 2.1.

[0105] 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.

[0106] 3) When the OD600 of the cells reaches 0.6-1.0, 100 μM / L IPTG can be added for induction.

[0107] 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.

[0108] 3.2 Fed-batch fermentation production of phloro...

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Abstract

The invention discloses a gene modification method for increasing the yield of phloroglucinol and application of the same, and belongs to the technical field of genetic engineering. The gene modification method comprises the following steps: obtaining a mutant strain by virtue of a mode of knocking out or inserting a global regulating factor arcA gene of an original strain, converting the state of the mutant strain to a competent state, introducing recombinant plasmids containing a polyketide synthase gene phlD, a multiple resistance activating factor marA and acetyl CoA carboxylase gene ACCase, and obtaining a recombinant cell. The invention also provides a method of producing the phloroglucinol by utilizing the recombinant cell. According to the gene modification method and application disclosed by the invention, the yield of the phloroglucinol after fermentation is firstly increased by 2.06 times by adopting a mode of globally regulating carbon metabolism after post-transcriptional level, and the gene modification method and application have high industrial application value.

Description

technical field [0001] The invention relates to a genetic modification method and application for increasing the output of phloroglucinol, 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 methods, including trinitrotol...

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 咸漠赵广刘敏张汝兵
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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