Biological method capable of increasing resveratrol accumulation amount

A technology for resveratrol and the highest yield, applied in the field of bioengineering, can solve the problems of lack of cofactors and low enzyme activity, and achieve the effect of increasing production

Inactive Publication Date: 2019-04-12
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production methods of resveratrol mainly include solvent extraction, enzymatic extraction, supercritical CO 2 Extraction, microwave extraction, bio-fermentation extraction, etc., as a method that has emerged in recent years, bio-fermentation has the adva

Method used

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  • Biological method capable of increasing resveratrol accumulation amount
  • Biological method capable of increasing resveratrol accumulation amount
  • Biological method capable of increasing resveratrol accumulation amount

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Construction of two expression plasmids

[0052] 1.1 Primer design

[0053] According to the Rgtal, Pc4cl and Ahsts sequences reported by NCBI, the following 6 pairs of primers were designed:

[0054] Rgtal-F: 5′C CCATGG GCATGGCGCCTCGCCCGACTTCG 3′

[0055] Rgtal-R: 5′CGC GGATCC TGTGCCAGCATCTTCAGCAGAAC 3′

[0056] 4CL-STS-1F: 5′ CATATG GGCGATTGCGTGGCC 3′

[0057] 4CL-STS-1R: 5′GCCAGCGGCGATCTGCCGAAA GGAAGCGGA ATGGTTAGCGTGAGTGGTATC 3′

[0058] 4CL-STS-2F: 5′GATACCACTCACGCTAACCA TTCCGCTTCC TTTCGGCAGATCGCCGCTGGC 3′

[0059] 4CL-STS-2R: 5′ CTCGAG TTAAATGGCCATGCTAC 3′

[0060] 4CL-STS-1NF: 5′GTATAAGAAGGAGATATA CATATG GGCGATTGCGTGGCC 3′

[0061] 4CL-STS-2NR: 5′CGGTTTCTTTACCAGA CTCGAG TTAAATGGCCATGCTACGC 3′

[0062] AccBC-F: 5′ATAAGGAGATATA CCATGG GCGTGTCAGTCGAGACTAGGAAG 3′

[0063] AccBC-R: 5′GCCGAGCTCGAATTC GGATCC TGCTTGATCTCGAGGAGAAC 3′

[0064] PTDH-F: 5′ GCAGATCTCCAATTG GATATC GATGCTGCCGAAACTGGTCATC 3′

[0065] PTDH-R: 5′CTTTACCAGAC...

Embodiment 2

[0081] Example 2 Construction of fumB gene knockout strain

[0082] 2.1 Primer design

[0083] A total of 4 primers are needed to knock out fumB gene, primers S1 and S2 for homologous recombination and primers S3 and S4 for identifying defective strains.

[0084] S1: 5'ATGTCAAACAAACCCTTTATCTACCAGGCACCTTTCCCGAAGCGATTGTGTAGGCTGGAG

[0085] S2: 5'TTACTTAGTGCAGTTCGCGCACTGTTTGTTGACGATTTGCACGGCTGACATGGGAATTAG

[0086] S3: 5'CCTGCGCTATTCACAGAATG

[0087] S4: 5'ATTGCAGCAGATCCACGTAG

[0088] 2.2 Construction of gene-deficient strain ΔfumB

[0089] The homologous recombination helper plasmid pKD46 was transformed into the host strain E.coli BL21(DE3), induced by L-arabinose to prepare electroporation competent cells.

[0090]The plasmid pKD4 containing the kanamycin resistance gene kana was used as a template, and S1 and S2 were used as primers for PCR amplification. Add 2 μL of the resistant fragment pKD4-kana to electroporation competent cells on ice, mix well and place in a 1 m...

Embodiment 3

[0093] Example 3 Transformation of expression plasmids to synthesize resveratrol strains

[0094] The ΔfumB gene-deficient strain was made into a ΔfumB gene-deficient E. coli competent according to the standard procedure for E. coli competent production, and the successfully constructed pCDFduet-Rgtal-Pc4cl-Ahsts and pACYCduet-AccBC-PDTH recombinant plasmids were transformed into the gene-deficient strain ΔfumB, And spread a solid LB plate containing spectinomycin and chloramphenicol in one thousandth, and cultivate at 37°C until the transformants grow out. Among them, the gene-deficient strain ΔfumB is a strain without spectinomycin and chloramphenicol resistance, and cannot grow on solid LB plates containing spectinomycin and chloramphenicol. Therefore, pCDFduet-Rgtal-Pc4cl containing pCDFduet-Rgtal-Pc4cl was obtained by screening with two antibiotics. - Gene-deficient strains of the Ahsts and pACYCduet-AccBC-PTDH recombinant plasmids. Designated as pCDF-ΔfumB-Res

[0095]...

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Abstract

The invention discloses a gene-defective-type Escherichia coli strain containing a resveratrol synthase system and a malonyl coenzyme-A synthase system and a construction method thereof, as well as amethod for increasing resveratrol accumulation amount. A recombinant plasmid composed of the resveratrol synthase system and the malonyl coenzyme-A synthase system provided by the invention includes Rgtal, Pc4cl, Ahsts, accBC and PTDH sequences as well as two suitable vector fragments. The biological method capable of increasing resveratrol accumulation amount provided by the invention comprises the following steps: transforming the recombinant plasmids composed of the resveratrol synthase system and the malonyl coenzyme-A synthase system, namely pCDF-Rgtal-Pc4cl-Ahsts and pACYC-accBC-PTDH, into Escherichia coli strains with fumarinase gene deficiency (delta-fumB) so as to obtain engineering strains; and then, carrying out culturing with an optimized medium. Thus, yield of resveratrol withthe optimized medium can be up 22.62 mg/L, which is increased by 5.73 times compared with yield of original strains.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for high-yielding resveratrol by a resveratrol synthetase system and a fumaric acid gene B-deficient strain. Background technique [0002] Resveratrol (Res) is a class of polyphenolic compounds containing a stilbene structure, also known as stilbene triphenols, and its chemical formula is C 14 h 12 o 3 , colorless needle-like crystals, easily soluble in ether, methanol, ethanol and other organic solvents, first extracted from the root of Veratrum tomentosa in 1940. Resveratrol is found in many plants in nature, such as grapes, knotweed, peanuts, mulberry plants, etc. It is an anti-toxic substance produced by plants to protect themselves from being attacked by pathogens. Resveratrol has various physiological activities such as prevention and treatment of atherosclerosis, reduction of platelet aggregation, and anti-virus. It has been listed as a health...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/22C12R1/19
CPCC12N15/70C12P7/22
Inventor 许激扬卞筱泓王郑隆
Owner CHINA PHARM UNIV
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