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Recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as preparation method and application thereof

A terpenoid, Escherichia coli technology, applied in the field of recombinant Escherichia coli chassis cells, can solve the problem that the metabolic flux of the MEP pathway cannot be fully opened to produce terpenoid chassis cells, without synthetic biology and industrial microorganism technology, chassis cells cannot meet the requirements. Issues such as requirements for large-scale production of terpenoids

Active Publication Date: 2014-05-07
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although this series of modifications to the MEP pathway has achieved great success, the obtained chassis cells still cannot meet the requirements for large-scale production of terpenoids.
Moreover, there is still a lack of sufficient understanding of the regulatory mechanism and enzymatic characteristics of the endogenous MEP pathway in E. coli. Only relying on the enhancement of gene expression in certain rate-limiting steps cannot completely relieve the unknown regulation that may exist in the pathway, so it is impossible to fully open MEP. metabolic flux of pathways and enable construction of higher terpenoid-producing chassis cells
[0006] Therefore, there is no effective and reliable synthetic biology and industrial microorganism technology in this area to produce terpenoids on a large scale

Method used

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  • Recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as preparation method and application thereof
  • Recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as preparation method and application thereof
  • Recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as preparation method and application thereof

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Embodiment approach

[0125] As an embodiment of the present invention, the gene sequence of the present invention can be constructed by PCR.

[0126] Vectors and host cells

[0127] The present invention also provides a vector or a vector mixture, which contains the gene combination described in the present invention, and preferably also contains an expression control sequence operably linked to the gene sequence.

[0128] The term "operably linked" or "operably linked to" refers to the condition that certain parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence. Those skilled in the art can select a suitable expression vector according to the host cell. According to the cleavage map of the known empty expression vector, those skilled in the art can insert the gene sequence of the present invention into an appropriate restr...

Embodiment 1

[0146] Cloning of MEP pathway genes from different strains

[0147] Table 1

[0148]

[0149] In this embodiment, Table 1 is used as an example, and the above-mentioned genes are selected for cloning.

[0150] dxs, dxr, ispD, ispF, idi genes of Escherichia coli, dxs1, dxs2, dxr, ispD, ispF, idi genes of Streptomyces avermitilis, dxs, dxr, ispD, ispF, idi1, idi2 gene, Bacillus subtilis dxs, dxr, ispD, ispF, idi gene, Erwinia (Erwinia taxi ATCC55669, referred to as ATCC) dxs, dxr, ispD gene, and Staphylococcus aureus idi gene design primers, primers Designs were designed and synthesized using conventional methods.

[0151] Genomic DNA of Streptomyces avermitilis, Saccharopolyspora erythromycin, Bacillus subtilis, Erwinia ATCC and Staphylococcus aureus were extracted using a genome extraction kit (Axygen Company). Using the corresponding genomic DNA as a template, the above 26 genes were amplified by PCR.

[0152] The PCR product was purified using a conventional Axygen DN...

Embodiment 2

[0158] Expression of each gene module

[0159] Each plasmid containing each module gene was transformed into commercially available Escherichia coli BL21(DE3), and then a single colony was picked and cultured overnight. Inoculate 1% inoculum into a test tube containing 2ml LB medium (100ml / L ampicillin), incubate at 37°C for 2h, add 0.1mM IPTG, place at 28°C, 200rpm for 6h induction. The bacteria in the culture medium were collected and ddH 2 After O suspension, prepare samples for SDS-PAGE followed by protein electrophoresis.

[0160] expression results see Figure 2-Figure 6 . Figure 2-Figure 6 The expression results of dxs module, dxr module gene, ispD module gene, ispF module gene and idi module gene from different species of MEP precursor pathway, dxs2AV, dxs1AV, dxs SE, dxr SE, dxr ATCC, dxr BS and ispD SE, confirmed by sequencing performed by Shanghai Boshang Biotechnology Co., Ltd.

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Abstract

The invention relates to a recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as a preparation method and application thereof. Particularly, an escherichia coli endogenous 2-C-methyl-D-erythritol-4-phosphoric acid (MEP) precursor pathway is reconstructed; the reconstructed escherichia coli base cell is utilized to perform the efficient biological synthesis of the terpene chemical compounds; the reconstruction of the precursor pathway mainly comprises the steps of fully digging the MEP precursor pathway gene modules of the sources of other natural microorganisms, screening gene modules with excellent characteristics to be expressed in the escherichia coli, and at the same time, performing the downstream synthesis pathway for integrally assembling the colibacillus chemical compounds in the reconstructed base cell, wherein the colibacillus chemical compounds include sesquiterpene chemical compounds such as amorphadiene, diterpene chemical compounds such as shell alkene, tetraterpenes chemical compounds such as lycopene, polyterpene chemical compounds, other terpene alkaloid chemical compounds and the like. The escherichia coli base cell can remarkably facilitate the synthesis of the terpene chemical compounds.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and industrial biology. Specifically, the invention relates to a recombinant Escherichia coli chassis cell for efficiently synthesizing terpenoids and its preparation method and application. Background technique [0002] Synthetic biology is based on rational design, integrating and assembling standardized biological components to construct artificial life systems with excellent performance. Once synthetic biology was born, its ideas and designs have profoundly influenced the development of industrial microbial technology, enabling microbial technology to play a greater role in the development and production of drugs, biofuels, and fine chemicals. [0003] The Keasling laboratory at the University of California, Berkeley, put the acadiene synthase gene (ads), acadiene oxidase gene (cyp71av1) and cytochrome P450 reduction protein gene (cpr) from Artemisia annua into yeast cells Under the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P1/04C12P5/02C12P5/00C12R1/19
Inventor 王勇熊智强李诗渊汪建峰
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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