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A kind of biological preparation method of caffeic acid

An amino acid and tryptophan technology, applied in the field of microorganisms, can solve problems such as affecting food safety and affecting the application prospect of caffeic acid, and achieve the effects of high conversion rate and high yield

Active Publication Date: 2022-02-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Escherichia coli has defects such as endotoxins that affect food safety, which affects the application prospect of the above method in the industrial production of caffeic acid

Method used

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  • A kind of biological preparation method of caffeic acid
  • A kind of biological preparation method of caffeic acid
  • A kind of biological preparation method of caffeic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of recombinant Candida glycerologenus Cg-1 producing caffeic acid

[0033] Construction of recombinant bacteria: using Candida glycerinogenes CCTCC M 93018 as the starting strain, using CRISPR-Cas9 technology to knock out and knock in genes, by knocking out L-tryptophan shown in SEQ ID NO.7 Synthetic gene trp1 and the L-phenylalanine synthetic gene pheA shown in SEQ ID NO.8 knock out the competing pathway L-tryptophan and L-phenylalanine in the starting strain for L-tyrosine synthesis; Overexpression of the 3-deoxy-D-arabinoheptonate-7-phosphate synthase gene aro4 shown in SEQ ID NO.9 and the bifunctional choroidal acid synthase and flavin reductase genes shown in SEQ ID NO.10 aro7 releases the feedback inhibition effect of L-tyrosine, and transforms into the production host Cg-1.

Embodiment 2

[0035] Gene mutation: According to the software analysis of the protein structure of the three genes of tyrosine ammonia lyase FjTAL, 4-hydroxyphenylacetic acid-3-monooxygenase HpaB and NADPH flavin oxidoreductase HpaC, Leu151 of FjTAL and Ala195 of HpaB were found Form a hydrogen bond with the substrate, and analyze the possible combination with the substrate and the important role of glycosidic bond breaking. Therefore, saturation mutations were performed on the coding genes of the above three enzymes to increase the conversion rate. After fusion expression of the mutant genes, it was found that only by mutating the leucine Leu at position 151 of FjTAL to glycine Gly, and the alanine Ala at position 195 of HpaB to serine Ser, the production of caffeic acid was significantly improved. The gene loses its activity or the yield decreases, and the mutated fusion gene is expressed in the production host Cg-1 to obtain the bacterial strain Cg-2. Compared with the unmutated engineeri...

Embodiment 3

[0036] Example 3 Construction of a linearized plasmid expressing a fusion gene

[0037]Linearized vector preparation: The fusion gene FjTAL-HpaB-HpaC was obtained by tandem encoding mutant enzyme genes FjTAL, HpaB and HpaC using the connecting peptide GSG, digested with restriction endonucleases BamHI and NotI, and connected to the same enzyme A recombinant expression vector was constructed on the excised pURGAPU plasmid (SEQ ID NO.11), and SacI was used to linearize the recombinant expression vector.

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Abstract

The invention discloses a biological preparation method of caffeic acid, which belongs to the technical field of microorganisms. The present invention selects high osmotic pressure Candida glycerol producing yeast CCTCC M93018 to construct engineering bacteria, knocks out L-tryptophan synthesis gene trp1 and L-phenylalanine synthesis gene pheA, and overexpresses 3-deoxy-D-arabinan Keto-7-phosphate synthase gene aro4 and bifunctional choroidate synthase and flavin reductase gene aro7, and tyrosine ammonia lyase, and 4-hydroxyphenylacetic acid-3-monooxygenase Mutation, in the yeast to achieve de novo synthesis of caffeic acid with glucose as carbon source, caffeic acid production reached 87.6mg / L. The preparation method of the invention has the characteristics of convenient operation, high conversion efficiency, low production cost, wide industrial application prospect and the like.

Description

technical field [0001] The invention relates to a biological preparation method of caffeic acid, which belongs to the technical field of microorganisms. Background technique [0002] Caffeic acid, also known as 3,4-dihydroxycinnamic acid, is a natural phenolic compound widely present in plants. It has attracted much attention due to its proven anticancer, antibacterial, antiviral, and antioxidant activities. In addition, among its derivatives, chlorogenic acid (CGA) and caffeic acid phenethyl ester (CAPE) are widely used in medicine, food and other life science fields. [0003] So far, commercially available caffeic acid is usually extracted from plants, however, the complicated isolation process as well as the low content of caffeic acid in plants and the associated growth rate of plants seriously hinder the efficient extraction of caffeic acid from plants. Caffeic acid can also be produced chemically, but this is energy intensive, environmentally unfriendly and expensive...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/42C12R1/72
CPCC12N9/1085C12Y205/01054C12Y105/0103C12Y402/0102C12Y402/01051C12Y504/99005C12Y403/01025C12Y114/14009C12Y105/01036C12N9/0028C12N9/0071C12N9/88C12N9/90C12N15/815C12P7/42
Inventor 诸葛斌王希晖陆信曜宗红赵翠
Owner JIANGNAN UNIV
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