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Recombinant bacterium for producing Cembrenediol and application thereof

A technology of cerebrotrienol and cerebrotrienol, which is applied in the field of metabolic engineering to achieve the effect of efficient production and application promotion

Pending Publication Date: 2021-05-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no strain suitable for the efficient catalytic production of cembratriene monool

Method used

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  • Recombinant bacterium for producing Cembrenediol and application thereof
  • Recombinant bacterium for producing Cembrenediol and application thereof
  • Recombinant bacterium for producing Cembrenediol and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] In this example, the GGPP synthetase gene (ggpps) was excised with NdeI and XhoI restriction endonucleases and connected to pETDuet to obtain the recombinant plasmid pETDuet-ggpps, and the two recombinant plasmids were transformed into E.coli JM109 to obtain the recombinant strain E. coli JM109 pETDuet-ggpps. Cultivate the recombinant strain E.coli JM109 pETDuet-ggpps, collect the cells, and extract the recombinant plasmid pETDuet-ggpps. The cembratrienol synthase gene cbts was digested with NcoI and EcoRI restriction enzymes, and pETDuet-ggpps was digested with NcoI and EcoRI restriction enzymes at the same time, and the cbts gene was connected to the recombinant plasmid pETDuet-ggpps to construct the recombinant Plasmid pETDuet-ggpps-cbts was transformed into E.coli BL21(DE3) to construct recombinant strain Z13. Put the constructed recombinant strain Z13 on the LB solid medium containing the corresponding antibiotics, and carry out three-section lines. After a single...

Embodiment 2

[0025] In this example, the 1-deoxy-D-xylulose-5-phosphate synthase gene dxs was digested with NdeI and XhoI restriction endonucleases and the pACYCDuet vector to construct the recombinant plasmid pACYCDuet-dxs, and transform the recombinant plasmid into E .coliJM109 was screened and verified to obtain the recombinant strain Z11. Cut the isoprene pyrophosphate isomerase gene (idi) with NcoI and EcoRI restriction enzymes, and treat pACYCDuet-dxs with the same restriction enzymes at the same time, and use DNA ligase to connect idi to pACYCDuet- dxs, the recombinant plasmid pACYCDuet-dxs-idi was obtained. The recombinant plasmid pACYCDuet-dxs-idi was transformed into the recombinant strain Z13 in Example 1, and the recombinant strain Z16 was obtained through screening verification. Z16 was induced to ferment for 2 days. After the fermentation, the cells were collected by centrifuging at 5000 rpm for 10 min. After extraction and reconstitution, adopt GC-MS to measure the output ...

Embodiment 3

[0027]In this example, the 1-deoxy-D-xylulose-5-phosphate synthase gene dxs was digested with NdeI and XhoI restriction endonucleases and the pCDFDuet vector to construct the recombinant plasmid pCDFDuet-dxs, and transform the recombinant plasmid into E .coliJM109 was screened and verified to obtain the recombinant strain Z11. Cut the isoprene pyrophosphate isomerase gene (idi) with NcoI and EcoRI restriction enzymes, and treat pCDFDuet-dxs with the same restriction enzymes at the same time, and use DNA ligase to connect idi to pCDFDuet- dxs, obtain the recombinant plasmid pCDFDuet-dxs-idi, transform the recombinant plasmid into the recombinant strain Z13 in Example 1, and obtain the recombinant strain Z17 through screening verification. The GGPP synthetase gene (ggpps) was excised with NdeI and XhoI restriction endonucleases and connected to pRSFDuet to obtain the recombinant plasmid pRSFDuet-ggpps, and the two recombinant plasmids were transformed into E.coli JM109 to obtain...

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Abstract

The invention relates to a recombinant strain for producing Cembrenediol and application of the recombinant strain, wherein the recombinant strain capable of producing Cembrenediol from plants is constructed by heterologous expression of a GGPP synthase gene gpps and a Cembrenediol synthase gene cbts. The yield of Cembrenediol is improved by overexpressing key genes of carbon metabolism flux in a DXP (Dixanthone) pathway, namely a 1-deoxy-D-xylulose-5-phosphate synthase gene dxs and an isoprene pyrophosphate isomerase gene idi. The expression plasmid is further optimized to excessively express dxs and idi, and the yield of Cembrenediol is increased to 2.60 times of that of a control group. By adopting the high-yield Cembrenediol strain and the method disclosed by the invention, the Cembrenediol can be efficiently produced, and the application of the strain in the fields of agriculture, tobaccos and the like can be promoted.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and in particular relates to a recombinant bacterium producing cembratriene-ol and its application. Background technique [0002] In the context of the global insecticide market, chemically synthesized active compounds such as artificial pyrethrum derivatives and most notably neonicotinoids (e.g. imidacloprid, thiamethoxam and clothianidin; valued at US$ 1.89 billion), which can Fast-acting pest but non-target discriminant product. Neonicotinoids are the main class of insecticides that mediate the neurotoxic effects of all insects through irreversible binding to nicotinic acetylcholine receptors. Chemical pesticides have many disadvantages, such as: easy to cause environmental pollution, target populations gradually develop resistance, and they have negative effects on non-target populations. These compounds thus affect pests as well as beneficial insects such as bees and bumblebe...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/75C12P7/02C12R1/19C12R1/125
CPCC12N9/90C12N9/1085C12N9/1022C12Y503/03002C12Y205/01C12Y202/01007C12N15/70C12N15/75C12P7/02
Inventor 杨海泉陈献忠张坤杰董田田刘万隆沈微夏媛媛陈磊
Owner JIANGNAN UNIV
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