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Improved resveratrol biological production method

A technology of resveratrol and construction method, which is applied in the direction of introducing foreign genetic material by using vectors, recombinant DNA technology, bacteria, etc., to achieve the effects of expanding production, increasing production, and promoting the secretion of resveratrol

Inactive Publication Date: 2018-01-12
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the method of embedding T7 RNA polymerase into high-yielding tyrosine engineering bacteria and combining the two aspects of promoting resveratrol efflux and enhancing malonyl-CoA supply to increase resveratrol production has not been reported in any literature

Method used

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  • Improved resveratrol biological production method
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  • Improved resveratrol biological production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Tyrosine deaminase TAL, 4-coumaroyl-CoA ligase 4CL, stilbene synthase STS, acetyl-CoA carboxylase subunit accBC, dtsR1 gene design

[0030] Tyrosine deaminase TAL of Phanerochaete chrysosporium origin is preferred;

[0031] 4-coumaroyl-coenzyme ligase 4CL derived from Arabidopsis thaliana;

[0032] Grape (Vitis vinifera) derived stilbene synthase STS;

[0033] The amino acid sequences of the acetyl-CoA carboxylase subunits AccBC and DtsR1 derived from Corynebacterium glutamicum (Corynebacterium glutamicum) are shown in sequence as SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, SEQ ID No.4, and SEQ ID No.5.

[0034] Use the JCAT online codon optimization software (http: / / www.jcat.de) combined with the OPTIMIZER codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ) to optimize the codon preference of E. coli , design full-length synTAL, syn4CL, synSTS, synaccBC-dtsR1 genes, the nucleotide sequences of the above genes are respectively as SEQ ID No.06, SEQ ID...

Embodiment 2

[0035] Embodiment 2: Chassis strain SyBE-002447 (DE3) construction

[0036] According to the Escherichia coli BL21 (DE3) genome, the T7 RNA polymerase insert fragment was designed, and the primers were designed as follows:

[0037] The 500bp homology arm primers upstream of the cloned ybhB gene are as follows:

[0038] F-ybhB: AGAAAGGAGGGTTCATGAAA (SEQ ID No. 13)

[0039] R-ybhB: GAGCGAAACGGGAAGGTAAA (SEQ ID No. 14)

[0040] Clone T7 RNA polymerase primers as follows:

[0041]F-T7 RNA: TTTACCTTCCCGTTTCGCTCGTATCAAGGTATTTTATGCG (SEQ ID No. 15)

[0042] R-T7 RNA: GAAGCAGCTCCAGCCTACTCACTCATTAGGCACCCC (SEQ ID No. 16)

[0043] The primers for cloning the chloramphenicol fragment are as follows:

[0044] F-Chl: GTAGGCTGGAGCTGCTTC (SEQ ID No. 17)

[0045] R-Chl: CTGCTTTTTTATACTAACTTGTAGGCTGGAGCTGCTTC (SEQ ID No. 18)

[0046] The 500bp homology arm primers downstream of the cloned ybhC gene are as follows:

[0047] F-ybhC: AAGTTAGTATAAAAAAGCAG (SEQ ID No. 19)

[0048] R-ybhC: ...

Embodiment 3

[0059] Embodiment 3: Construction of pSyBE-synTLS expression vector

[0060] Using the artificially synthesized synTAL gene as a template (the nucleotide sequence is shown in SEQ ID No.6), using the PCR method, the primers are designed as follows to obtain an artificially synthesized synTAL gene fragment with BamHI and HindIII at both ends, and the PCR program Referring to Example 2.

[0061] synTALup: CGC GATGCCGTCCCGCATCGACTA (SEQ ID No. 21)

[0062] synTAL down: CCC TTACGCCTTGATAGACTTAA (SEQ ID No. 22)

[0063] Wherein the double dashes are the restriction sites of BamHI and HindIII.

[0064] The expression vector backbone pSyBE-217101 was fully chemically synthesized, and its sequence is shown in SEQ ID No.11 in the sequence table. The purified and recovered synTAL gene PCR product and the extracted pSyBE-217101 vector were double digested with BamHI and HindIII. The digestion reaction system was: (1) 5 μL 10*FD buffer, 2.5 μL BamHI, 2.5 μL HindIII, 30 μL synTAL gen...

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Abstract

The invention discloses an improved resveratrol biological production method, and relates to an improved construction method of a recombinant bacterium for producing resveratrol. The construction method comprises the following steps: (1) carrying out complete synthesis to obtain genes: synTAL, syn4CL, and synSTS; (2) preparing a SyBE-002447(DE3) strain; (3) carrying out complete synthesis to obtain a gene: synaccBC-dtsR1; (4) cloning an efflux protein gene yddG; (5) carrying out total chemical synthesis to obtain SyBE-217101 and pSyBE-217102; (6) connecting synTAL, syn4CL, and synSTS with pSyBE-217101 to obtain a recombinant expression vector pSyBE-synTLS; and connecting synaccBC-dtsR1 and yddG with pSyBE-217102 to obtain a recombinant expression vector pSyBE-synADY; and (7) converting tworecombinant expression vectors into the SyBE-002447(DE3) strain, and carrying out monoclonal screening to obtain a recombinant bacterium SyBEREST. Through expressing the efflux protein, the secretionof resveratrol is promoted, the yield of resveratrol is obviously increased, and the cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an improved resveratrol-producing recombinant bacterium and a construction method thereof. Background technique [0002] Resveratrol (Resveratrol, Res), the molecular formula is C 14 h 12 o 3 , with a molecular weight of 228, easily soluble in chloroform, methanol, ethyl acetate, ethanol and other organic solvents. There are four main naturally occurring forms of resveratrol: trans-resveratrol, cis-resveratrol, trans-resveratrol glycosides, and cis-resveratrol glycosides. As a polyphenolic natural phytoalexin, resveratrol is widely found in more than 70 kinds of plants such as grapes, peanuts, and knotweed. It has gained widespread attention since the "French Paradox" was proposed in 1990. Studies have shown that it has physiological activities such as relaxing cardiovascular, anti-cancer, anti-oxidation, anti-inflammation, and prolonging animal life, so it can be used in h...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/22
Inventor 赵广荣赵莹
Owner TIANJIN UNIV
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