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Recombinant expression vector, genetically engineered bacterium containing recombinant expression vector and application of genetically engineered bacterium

A technology for expressing vectors and genes, applied in the field of stilbene synthase and its coding gene and its application, and coumaroyl-CoA, can solve the problems of high cost, low content, and difficult separation and purification of by-products, so as to improve conversion rate and increase The effect of consumption

Pending Publication Date: 2021-06-01
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the way to obtain red pinetin is mainly based on plant extraction and chemical synthesis. Most red pinetin is extracted from the heart of pine trees, and its content is low, only 1-40mg / g pine wood. In addition, because many structural analogues are difficult to separate, Complicated and expensive extraction process
Chemical synthesis faces the problems of expensive reaction substrates, toxic by-products, difficulties in separation and purification of by-products, and environmental pollution.

Method used

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  • Recombinant expression vector, genetically engineered bacterium containing recombinant expression vector and application of genetically engineered bacterium
  • Recombinant expression vector, genetically engineered bacterium containing recombinant expression vector and application of genetically engineered bacterium
  • Recombinant expression vector, genetically engineered bacterium containing recombinant expression vector and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Optimization of coumaryl-CoA gene and construction of recombinant expression vector

[0040] The amino acid sequence of the original p-coumaryl-CoA (4-coumarate-CoA ligase family protein [Populustrichocarpa], GenBank: EEF00197.1) was screened from the annotation information of the NCBI database. On the premise of not changing the encoded amino acid sequence, according to the codon preference of Escherichia coli, the Ptr4CL4 gene was codon-optimized, and the whole gene sequence Ptr4CL4 of p-coumaroyl-CoA was synthesized by the whole gene synthesis method. The nucleotide sequence of the artificially synthesized p-coumaroyl-CoA gene Ptr4CL4 is shown in SEQ NO.4. Both ends of the synthesized Ptr4CL4 gene also have NdeI and XhoI restriction sites for connection with the expression vector. The connection result is as figure 1 shown. Using the Ptr4CL4 gene sequence as a template to amplify the stilbene synthase Ptr4CL4 gene, the primers used are:

[0041] Ptr4CL...

Embodiment 2

[0044] Example 2: Optimization of stilbene synthase gene and construction of recombinant expression vector

[0045] The amino acid sequence of the original stilbene synthase (stilbene synthase [Pinus pinea], GenBank: ALN42233.1) was screened from the annotation information of the NCBI database. Under the premise of not changing the encoded amino acid sequence, according to the codon preference of Escherichia coli, the gene encoding the stilbene synthase was codon optimized, and the whole gene synthesis method synthesized the whole gene sequence Ppsts of the stilbene synthase, and its nucleotide The sequence is shown in SEQ NO.4. Both ends of the synthesized Ppsts gene also have BamHI and HindIII restriction sites for connection with the expression vector. Adopting the nucleotide sequence of Ppsts gene is template amplification stilbene synthase Ppsts gene, used primer is:

[0046] PpSTS-up: 5'-CA GGATCC GATGGGCGCTGTTGACT-3'

[0047] PpSTS-dn: 5'-CGC AAGCTT TTATTGCAGCGGA...

Embodiment 3

[0049] Example 3: Construction of a strain for synthesizing saponin.

[0050] Ppsts (SEQ NO.4), Ptps (SEQ NO.5) and Psps synthesized by Chuzhou General Q361R The nucleotide sequence lengths of the gene (SEQNO.6) are 1182bp, 1179bp and 1194bp respectively, wherein, Ptps and Psps Q361R The gene is two other known stilbene synthase genes, the Genebank accession number of Ptps gene is KF998274.1, Psps Q361R The Genebank accession number of the gene is P48407.1, which were respectively inserted into the BamHI and HindIII restriction sites of pRSFDuet-1 (3829bp). The results of the double enzyme digestion verification experiment of the extracted plasmid were as follows: figure 2 shown. The pETDuet-Ptr4cl4 plasmid (the plasmid was entrusted to a commercial company to prepare) was used as a template to amplify the target gene Ptr4cl4, and the primers used were:

[0051] Ptr4CL4-up: 5'-A CATATG ATGAGTGTTGCCACCGTGG-3'

[0052] Ptr4CL4-dn: 5'-AGA CTCGAG TTAACTCATGGTGGTT-3'

[...

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Abstract

The invention discloses a recombinant expression vector. The recombinant expression vector comprises a specific gene of p-coumaroyl-CoA ligase and a specific gene of stilbene synthetase. When the recombinant expression transformant containing the recombinant expression vector is applied to the biosynthesis process of the denisolin, the inhibition of an intermediate metabolite cinnamyl-coenzyme A and the denisolin on cinnamic acid conversion can be effectively reduced, the consumption of a substrate cinnamic acid is increased, and the conversion efficiency from the cinnamic acid to the denisolin is greatly improved.

Description

technical field [0001] The invention relates to the fields of biotechnology and biocatalysis, in particular to p-coumaroyl-CoA, stilbene synthase, coding genes and applications thereof. Background technique [0002] Red pinesin, also known as silver pinesin, trans-3,5-dihydroxystilbene, etc., belongs to stilbene compounds. It is a low molecular weight secondary metabolite in plants, a plant polyphenol mainly present in pine heartwood, which protects plants from the erosion of bacteria and fungi. In recent years, many studies have confirmed that saponin has numerous biological activities, such as the prevention of cardiovascular disease, anti-oxidation, anti-cancer and treatment of arthritis and other biological activities. It has been reported that red pine can protect animal cells from oxidative stress, and this effect has been observed in human retinal cells. Adding a certain concentration of red pine can improve the survival rate after oxidative stress. It has also been...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/52C12N9/00C12N1/21C12P7/22C12R1/19
CPCC12N9/93C12N9/00C12N15/70C12P7/22C12Y602/01012
Inventor 欧阳嘉许倩张晨胡月莉郑兆娟
Owner NANJING FORESTRY UNIV
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