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Engineering strain for biologically producing ferulic acid and establishing method of engineering strain

A construction method and technology for engineering strains, applied in the directions of bacteria, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of difficult separation, long reaction time, etc., and achieve high production efficiency, high industrialization value, and by-products less effect

Active Publication Date: 2014-05-28
湖州百肽诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction time of this method is long, and the mixture of trans and cis ferulic acid is produced, which is not easy to separate. At the same time, the chemical synthesis method has certain problems in environmental pollution.

Method used

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  • Engineering strain for biologically producing ferulic acid and establishing method of engineering strain
  • Engineering strain for biologically producing ferulic acid and establishing method of engineering strain
  • Engineering strain for biologically producing ferulic acid and establishing method of engineering strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Construction and identification of recombinant strains

[0038] 1. Construction of strains containing recombinant plasmid pET24a-vaoA

[0039] 1) Plasmid construction

[0040] Plasmid pET24a(+): a product of Novagen, with a size of 5.31kb, containing a kanamycin resistance gene, a lactose repressor lac I gene, a T7 promoter, and multiple restriction endonuclease sites.

[0041] The DNA fragment of the vanillyl oxidase gene (vaoA, 1683bp) was digested with Nde I and Bam HI. The vector pET24a(+) was double digested with Nde I and Bam HI. Enzyme digestion system: 43 μL DNA, 5 μL buffer R, 1 μL Bam HI, 1 μL Nde I, incubated at 37°C for 3 hours.

[0042] The enzyme-digested DNA fragments were gel recovered, and the vaoA and carrier DNA fragments were ligated with T4 ligase. The ligation system was as follows: vaoA 7.5 μL, pET24a vector 1.5 μL, buffer 1 μL, T4 ligase 1 μL, incubated overnight at 16°C, and the ligated products were heat-shocked Transformed in...

Embodiment 2

[0056] Embodiment 2: the fermentation of recombinant bacterial strain

[0057] Mix 100mL of seed solution, the seed solution contains 1% peptone, 0.5% yeast extract, 1% sodium chloride, and the balance is purified water. After being sterilized in a 250mL Erlenmeyer flask, inoculate a single colony on the plate medium, and the shaker rotation speed is 200rpm / min. After culturing at 37°C for 16 hours, inoculate into a 500mL Erlenmeyer flask containing 200mL fermentation broth, which contains 1.2% peptone, 2.4% yeast extract, 0.4% glycerin, 0.23% potassium dihydrogen phosphate, 1.25% dipotassium hydrogen phosphate, The balance is purified water. Fermentation culture conditions: Inoculate according to 1% of the fermentation volume, culture at 37°C, shaker rotation speed is 200rpm / min, after 1.5 hours after inoculation, cool down to 25-28°C, add final concentration of 0.6mM IPTG, and cultivate for 8 hours.

Embodiment 3

[0058] Embodiment 3: catalysis eugenol generates ferulic acid

[0059] After the fermentation, collect the cells by centrifugation at 4000rpm / min at 4°C, suspend the cells with 100mL of phosphate buffer, transfer them to a 250mL transformation bottle, add 0.06mL of eugenol, the transformation conditions are 25°C, and the rotation speed of the shaker is 200rpm / min, and 0.06 mL of eugenol substrate was added in batches every hour. After 8-10 hours, the yield of ferulic acid and the complete conversion of eugenol were detected by high performance liquid chromatography (HPLC).

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Abstract

The invention discloses an establishing method of an engineering strain for biologically producing ferulic acid and application of the engineering strain in production of the ferulic acid. The engineering strain enables a vanillyl alcohol alcohol oxidase gene, a coniferyl alcohol dehydrogenase gene and a coniferyl aldehyde dehydrogenase gene to be connected in series and multiply strongly expressed in escherichia coli by using a T7 promoter, and then eugenol is catalyzed to generate the ferulic acid through enzymatic reaction. Through the bioconversion method of the ferulic acid, poisoning of substrate eugenol to a production strain is avoided; the characteristics of simple process and high conversion rate are achieved.

Description

technical field [0001] The invention belongs to the technical field of production of pharmaceutical raw materials, and in particular relates to a recombinant bacterial strain simultaneously expressing vanillin alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase and a construction method thereof, and an organism for producing ferulic acid using the bacterial strain conversion method. Background technique [0002] The chemical name of ferulic acid is 4-hydroxy-3-methoxycinnamic acid, which is one of the derivatives of cinnamic acid. Because ferulic acid can be used as a precursor for microbial fermentation to produce vanillin, as well as its anti-oxidation, anti-thrombotic, blood lipid-lowering, and immune-regulating properties, ferulic acid and its derivatives are widely used in Lowering blood fat, preventing and treating coronary heart disease and cancer, antibacterial and anti-inflammatory, anti-mutation, promoting blood circulation, elimi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42
Inventor 林凌王俊政陈剑付成根
Owner 湖州百肽诺生物科技有限公司
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