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A kind of production method of phloroglucinol

A technology of phloroglucinol and its production method, applied in the field of bioengineering, can solve the problems of expensive raw materials of phloroglucinol, complex separation and purification process, environmental pollution of by-products, etc.

Active Publication Date: 2021-09-21
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The chemical synthesis of phloroglucinol has problems such as expensive raw materials, complicated separation and purification process, and serious environmental pollution by by-products.

Method used

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  • A kind of production method of phloroglucinol
  • A kind of production method of phloroglucinol
  • A kind of production method of phloroglucinol

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, the determination of acetic acid production phloroglucinol approach:

[0022] Acetate can be catalyzed by acetyl-CoA synthase (Acs or AckA and Pta) to generate malonyl-CoA, and malonyl-CoA can be catalyzed by phloroglucinol synthase (PhlD) to generate phloroglucinol. The target genes PhlD, Acs, AckA and Pta were synthesized according to the gene synthesis method. The synthetic gene fragment PhlD was connected with the backbone PETDuet-1 to construct the plasmid pET-phlD. The fragments Acs, AckA and Pta were ligated with the backbone pET-phlD by Gibson's method to construct plasmids pET-phlD-acs and pET-phlD-ackA-pta. The plasmids pETDuet-1, pET-phlD, pET-phlD-acs and pET-phlD-ackA-pta were transformed into Escherichia coli BL21(DE3) respectively to obtain strains BL21(DE3)(pETDuet-1), BL21(DE3) (pET-phlD), BL21(DE3)(pET-phlD-acs) and BL21(DE3)(pET-phlD-ackA-pta). After a single colony grows, it is inoculated into LB liquid medium, cultured at 37°C for 8...

Embodiment 2

[0026] Embodiment 2, optimization M9 fermentation medium condition

[0027] The plasmid pET-phlD-ackA-pta was transferred into Escherichia coli BL21 (DE3), inoculated in 5mL liquid LB medium after growing a single colony, and transferred to cells containing different concentrations of acetic acid (0g) with 1% inoculum after overnight culture. / L, 2g / L, 4g / L, 6g / L, 8g / L, 10g / L) M9 fermentation medium, cultured in shake flasks. Take the fermented liquid every 12 hours and check the output. Fermentation results such as image 3 shown. It can be seen from the figure that the yield of fermentation in the medium containing 2g / L acetic acid was the highest at 24h, which was 78.21mg / L. Therefore, 2g / L acetic acid is the optimum concentration for phloroglucinol production. On this basis, the optimized 2g / L acetic acid concentration was used to optimize the concentration of yeast extract in M9 medium. Escherichia coli BL21 (DE3) (pET-phlD-ackA-pta) was used as the target strain, an...

Embodiment 3

[0030] Embodiment 3, utilize CRISPRi to improve phloroglucinol output

[0031]Verify the blocking effect of dCas9. The target fragments egfp and egfp-sgRNA were obtained by PCR, and the target fragments were connected to the backbone pZE and pCS-dCas9 respectively by Gibson's method to construct plasmids pZE-pferB-egfp and pCS-dCas9-egfp-sgRNA. After the construction, the two plasmids were co-transformed into Escherichia coli BL21(DE3), and after a single colony grew, they were inserted into 5 mL LB liquid medium for overnight culture. Then transfer the seed solution to a new 5mL liquid LB medium with a 1% inoculum amount, add IPTG with a final concentration of 1mM and corresponding antibiotics and culture it on a shaker at 37°C, and detect the fluorescence and OD after 4 hours 600 Value, after this; = check once every two hours. The result is as Figure 5 As shown, after culturing for 4 hours, the fluorescence intensity / OD of the control group BL21(DE3)(pCS-dCas9+PferB-egf...

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Abstract

As a high-value compound precursor, phloroglucinol has important application prospects in the fields of medical treatment and materials. Due to the disadvantages of traditional chemical synthesis methods in terms of cost and environmental protection, the present invention uses cheap and environmentally friendly acetic acid as a carbon source to realize the biosynthesis of phloroglucinol. The present invention first constructs two synthesis pathways using acetic acid as carbon source, verifies the feasibility of the method through fermentation, and determines that the pathway for expressing PhlD, AckA and Pta is the production pathway of phloroglucinol. The concentration of acetic acid and yeast extract in the M9 fermentation medium were respectively optimized to increase the production of phloroglucinol to 228mg / L. The feasibility of competitive pathway inhibition using CRISPR / dCas9 was demonstrated using the green fluorescent protein eGFP. Subsequent fermentation was carried out, and the expression level of citrate synthase in the competition pathway was reduced by CRISPRi, and the production of phloroglucinol was increased to 284mg / L. The invention proposes a novel production method of phloroglucinol, which provides a new strategy for the industrial production of phloroglucinol.

Description

technical field [0001] The invention relates to a production method of phloroglucinol, which belongs to the technical field of bioengineering Background technique: [0002] Phenolic compounds and their derivatives have good antioxidant activity and have been extensively studied in physiology and pharmacology. Phloroglucinol is a trihydric phenolic compound, an important drug intermediate, and has good pharmacological activities in many aspects such as anti-virus, anti-tumor, anti-inflammation, and hemostasis. In terms of biological reagents, phloroglucinol can be used as raw materials such as dye coupling agent, tire tackifier and azo composite ink. At the same time, as a precursor for the synthesis of triaminotrinitrobenzene (TABA), a heat-resistant low-sensitivity explosive, it also has important application prospects in the fields of high-energy materials, medicinal chemistry and material science. [0003] The chemical synthesis of phloroglucinol has problems such as ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/22C12R1/19
CPCC12N9/1025C12P7/22C12Y203/03
Inventor 霍毅欣于盛竹陈振娅
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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