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Microorganism and use thereof

A technology of microorganisms and plasmids, applied in the field of bioengineering, can solve the problems of residual chemical reagents, low farnesene content, and inability to apply, and achieve the effects of overcoming difficulties in genetic manipulation, improving yield and efficiency, and short production cycles

Active Publication Date: 2019-11-19
SHENZHEN ACTION TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Farnesene is present in very low levels in plants and cannot be marketed as an extracted natural product
Although the chemical synthesis method has the advantages of easy to obtain and cheap raw materials, mild reaction conditions, fast reaction rate, and easy separation of the product from the reaction system, it is difficult to control the stereo configuration of the β-farnesene double bond and varying degrees of Chemical reagent residues, the quality, safety and scope of use of its products are restricted

Method used

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  • Microorganism and use thereof
  • Microorganism and use thereof

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Experimental program
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Effect test

Embodiment 1

[0017] The recombinant escherichia coli of the optimal collocation of embodiment 1MVA and MEP approach

[0018] Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are two general substrates for isoprenoid biosynthesis. Two isoprenoid pathways, the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway, are responsible for IPP and DMAPP synthesis in nature. The MEP pathway is more efficient than the MVA pathway in converting carbon sources (eg, glucose, glycerol) into isoprenoids. However, energy and reducing power will be consumed in this process, so additional energy molecule ATP and reducing equivalent NADPH provided from other ways are needed. At the same time, the MVA pathway, which overproduces isoprenoids, produces excess reducing equivalent NADH. How can the MVA pathway be used to provide the reducing equivalents required for the MEP pathway? By changing the flux of the MEP pathway and the MVA pathway, a more balanced metabolic pat...

Embodiment 2

[0025] The optimal matching recombinant yeast of embodiment 2MVA and MEP approach

[0026] At the same time, the inventor also constructed recombinant Saccharomyces cerevisiae based on this theoretical content. Plasmid pJ1-018-11 contains ERG10, ERG12, and tHMG1 in the MVA pathway of Saccharomyces cerevisiae. These genes were obtained by PCR, and the control gene expression was pGAL1 and pGAL7 respectively. , pGAL10 promoter. These fragments were connected sequentially by designing primers to form a 50bp overlapping sequence between two adjacent genes, with 1.5kb sequences homologous to the integration site on both sides of the target fragment, and these fragments were cloned in the DNA assembly method. The pRS423 vector contained a NotI restriction site between the fragment and the vector, and the positive clone was sequenced and named pJ1-018-11.

[0027] Plasmid pJ1-018-12 contains Erg13, ERG8, and MVD1 in the MVA pathway of Saccharomyces cerevisiae. These genes are obtain...

Embodiment 3

[0034] The optimal matching recombinant Streptomyces of embodiment 3MVA and MEP approach

[0035] According to the above conclusions, when the flux ratio of the MEP pathway to the MVA pathway is 4:3, farnesene can be produced more efficiently, so the inventors made the related proteins ERG10, ERG12, tHMG1, Erg13, ERG8 in the Saccharomyces cerevisiae MVA pathway , MVD1 and ERG20 were cloned on pIB139 according to the Gibson method, and the positive clone was sequenced and named pJ1-018-26. The plasmid controls gene expression by a low-strength ermE promoter, contains attP sites and Int integrase, and is resistant to abramycin.

[0036]Using PCR-targetting (Gust. B., 2003. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of thesesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A. 100 (4): 1541-1546.) Plasmid pSET152 (Qian Liu.,2016.Development of Streptomyces sp.FR-008as anemerging chassis.Synth Syst Biotechnol.1(3):207-214.) T...

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Abstract

The invention provides a microorganism. The microorganism comprises an MEP pathway and an MVA pathway in a flux ratio of 4:3. A relative balance of energy and reducing equivalent in the metabolic pathway of the microorganism is favorable for production of farnesene. The microorganism has a high yield of farnesene.

Description

technical field [0001] The present invention relates to the field of bioengineering, specifically, the present invention relates to microorganisms and uses thereof, more specifically, the present invention relates to microorganisms, a method for preparing farnesene and a method for preparing microorganisms. Background technique [0002] Farnesene was first isolated from apple trees and plays an important role in plant defense. Because it is an important chemical raw material, it has broad market value in emerging aviation fuel, rubber, natural vitamin E and other fields, and has received extensive attention in recent years. [0003] Farnesene is present in very low levels in plants and cannot be marketed as a natural product. Although the chemical synthesis method has the advantages of easy to obtain and cheap raw materials, mild reaction conditions, fast reaction rate, and easy separation of the product from the reaction system, it is difficult to control the stereo config...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/19C12N15/70C12N15/81C12N15/76C12P5/02C12R1/19C12R1/865C12R1/465
CPCC12N15/70C12N15/81C12N15/76C12P5/026
Inventor 叶紫玲
Owner SHENZHEN ACTION TECH CO LTD
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