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Construction method and application of a high-yielding phloroglucinol genetically engineered bacterium

A technology of genetically engineered bacteria and phloroglucinol is applied in the field of genetic engineering to achieve the effects of increasing production, improving cycle activity, and high industrial application value

Active Publication Date: 2018-05-04
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

There is no report on the effect of the genetic modification method of knocking out the regulatory factor iclR and overexpressing acs on the fermentation production of phloroglucinol, and there is no report on the genetic modification method of promoting the assimilation of acetic acid to increase the production of phloroglucinol by activating the glyoxylate cycle.

Method used

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  • Construction method and application of a high-yielding phloroglucinol genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of mutant strain E.coli(DE3)△iclR

[0059] In this example, Escherichia coli E.coliBL21(DE3) was used as the starting strain, and the iclR gene was knocked out by using the P1 phage transduction technology to construct the mutant strain ZG-2280( figure 1 ).

[0060] Those skilled in the art should understand that each step of the gene knockout experiment of Escherichia coli E.coliBL21(DE3) was carried out according to standard molecular cloning techniques.

[0061] 1 Preparation of donor bacteria lysate

[0062] 1) Cultivate the recipient strain E.coliBW25113iclR:Kan overnight;

[0063] 2) Add 4ml of heated and melted 0.4% agar medium to a sterile 10ml EP tube, add 400μL of overnight culture recipient bacteria, add 10μL of P1 phage stock solution, mix well, pour into non-antibiotic plate, and cultivate in a humid environment.

[0064] 3) After the appearance of irregular plaques, collect the proliferating phage lysate and add about 400 μL of chlo...

Embodiment 2

[0073] Example 2 Preparation of recombinant plasmid

[0074] For the construction process of the original plasmids pET-phlD-marA and pACYC-accADBC, please refer to the literature: Yujin Cao, Xinglin Jiang, Rubing Zhang, Mo Xian. Improved phloroglucinol production by metabolically engineered Escherichia coli[J]. 1552. In this example, on the basis of the original plasmid pET-phlD-marA, the acs gene was inserted, and the overexpression of acs was induced by the T7 promoter to obtain the recombinant plasmid pET-phlD-marA-acs.

[0075] 2.1 Preparation of recombinant plasmid pET-phlD-marA-acs

[0076] Using the Escherichia coli E.coli (DE3) genome as a template, design primers to amplify the acs gene. The primer sequences are as follows:

[0077] acs-5':5'-CTAGCCATGGCTAGCCAAATTCACAAACACACC-3'

[0078] acs-3':5'-CGGGATCCTTACGATGGCATCGCGATAGC-3'

[0079] The gene acs and the vector pET-phlD-marA were double-digested with NcoI and BamHI respectively, and after the fragments were re...

Embodiment 3

[0082] Example 3 Production of Phloroglucinol by Fermentation

[0083] 3.1 Shake flask fermentation experiment

[0084] 1) For the cultivation of primary seed liquid, inoculate the control strain ZG-1944 and the engineering strain ZG-2294 into 3 mL LB liquid medium respectively, add 50 μg / mL kanamycin and 50 μg / mL chloramphenicol, and incubate at 37°C Grow for 8-12h.

[0085] 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.

[0086] 3) Cell OD 600 When it reaches 0.6-0.8, 100μM / L IPTG can be added for induction.

[0087] 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 supernatan...

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Abstract

The invention discloses a construction method and application of a high-yielding phloroglucinol genetic engineering bacterium, belonging to the technical field of genetic engineering. The method provided by the present invention is to knock out the transcription control factor iclR gene of the starting strain to obtain a mutant strain, and then transfer the mutant strain to a competent state and then introduce polyketide synthase gene phlD, multiple resistance activator marA, and acetyl CoA carboxylation Enzyme gene Accase, acetyl-CoA synthetase gene acs recombinant plasmid to obtain recombinant cells. The present invention realizes for the first time the production of phloroglucinol by improving the glyoxylic acid cycle activity and promoting the assimilation of acetic acid in a metabolic transformation mode, and has high industrial application value.

Description

technical field [0001] The invention relates to a construction method and application of a high-yielding phloroglucinol genetically engineered bacterium, 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, includ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/22C12R1/19
Inventor 咸漠赵广刘敏
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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