Engineering bacterium and application thereof in production of amorpha fruticosa-4,11-diene

A technology of engineering bacteria and amorpha, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc.

Inactive Publication Date: 2013-03-20
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Large-scale planting of Artemisia annua can increase the production of artemisinin, but it competes with food planting for limited land resources and is easily affected by external factors such as climate

Method used

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  • Engineering bacterium and application thereof in production of amorpha fruticosa-4,11-diene
  • Engineering bacterium and application thereof in production of amorpha fruticosa-4,11-diene
  • Engineering bacterium and application thereof in production of amorpha fruticosa-4,11-diene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, the preparation of DNA molecule

[0071] 1. Prepare Amorpha-4,11-diene synthase gene (ADS gene, according to the nucleotide sequence of the ADS gene in Artemisia annua, the DNA sequence was optimized according to the preference of Escherichia coli)

[0072] A double-stranded DNA molecule (1672bp) shown in Sequence 1 of the Sequence Listing was synthesized.

[0073] In Sequence 1 of the sequence listing, the 1st to 6th nucleotides from the 5' end are the restriction endonuclease XhoI restriction endonuclease recognition sequence, the 23rd to 1660th nucleotides are the coding region of the ADS gene, and the 1661st to 1660th nucleotides are the coding region of the ADS gene. Nucleotide 1666 is two consecutive stop codons, and nucleotides 1667 to 1672 are the restriction endonuclease BamHI recognition sequence.

[0074] 2. Preparation of HMG-CoA reductase gene (mvaE gene, biological source is Enterococcus faecalis)

[0075] A double-stranded DNA molecule (24...

Embodiment 2

[0101] Embodiment 2, construction of plasmid

[0102] 1. Construction of plasmid pUC57-ADS

[0103] 1. Digest the double-stranded DNA molecule shown in Sequence 1 of the Sequence Listing with restriction endonucleases Xho I and BamHI, and recover the digested product.

[0104] 2. Digest vector pUC57 with restriction endonucleases Xho I and BamHI to recover a vector backbone of about 2700 bp.

[0105] 3. Ligate the digested product of step 1 with the vector backbone of step 2 to obtain the recombinant plasmid pUC57ADS (see figure 1 ).

[0106] 2. Construction of plasmid pET3-E

[0107] 1. Digest the double-stranded DNA molecule shown in Sequence 2 of the Sequence Listing with restriction endonucleases Nde I and BamH I, and recover the digested product.

[0108] 2. Digest the vector pET3b with restriction endonucleases Nde I and BamH I to recover a vector backbone of about 4600 bp.

[0109] 3. Ligate the digested product of step 1 with the vector backbone of step 2 to obtai...

Embodiment 3

[0183] Embodiment 3, the construction of recombinant bacteria

[0184] 1. Construction of recombinant bacteria pET3-ES / DHGT7

[0185] The recombinant plasmid pET3-ES was introduced into Escherichia coli DHGT7 competent cells to obtain the recombinant bacteria, which was named recombinant bacteria pET3-ES / DHGT7 (produced mevalonic acid).

[0186] 2. Construction of recombinant bacteria pET3-ES / pAOC3-MD-ispA-ADS / DHGT7

[0187] The recombinant plasmid pET3-ES and the recombinant plasmid pAOC3-MD-ispA-ADS were jointly introduced into Escherichia coli DHGT7 competent cells to obtain recombinant bacteria, which were named recombinant bacteria pET3-ES / pAOC3-MD-ispA-ADS / DHGT7 (producing Amorpha-4,11-diene).

[0188] 3. Construction of recombinant bacteria DHGT7ES

[0189] 1. The plasmid pKDS (for the schematic diagram of the structure, see Figure 16 ) into Escherichia coli DHGT7 competent cells to obtain recombinant bacteria, which were named as recombinant bacteria pKDS / DHGT7. ...

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Abstract

The invention discloses an engineering bacterium and application thereof in production of amorpha fruticosa-4,11-diene. The invention provides an engineering bacterium which is a recombinant bacterium obtained by introducing the following genes, namely an amorpha fruticosa-4,11-diene enzyme gene, an HMG-CoA reductase gene, an HMG-CoA (hydroxy methylglutaryl coenzyme A reductase inhibitor) synthase gene, a mevalonate kinase gene, a phosphomevalonate kinase gene, a mevalonate diphosphate decarboxylase gene, an IPP isomerase gene and a geranyl transferase gene into escherichia coli. Both a mevalonic acid engineering bacterium and an amorpha fruticosa-4,11-diene engineering bacterium disclosed by the invention have high yield property, do not need antibiotic screening in a culture process, and are suitable for the requirements of modern commercial production. A heterogeneous metabolic pathway is integrated on an escherichia coli chromosome by adopting a genome engineering means to obtain the engineering bacterium, and artemisinin precursor amorpha fruticosa-4,11-diene can be efficiently and stably synthesized.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in producing amorpha-4,11-diene. Background technique [0002] Artemisinin, a sesquiterpene lactone peroxide, is an effective antimalarial monomer isolated and identified from the plant Artemisia annua L. by Chinese scientists in 1971. Artemisinin and its derivatives such as Artemether, Arteether, Artesunate, Dihydroartemisinin, etc. have good antimalarial properties. After artemisinin is combined with one or more other antimalarial drugs, a specific antimalarial drug—artemisinin-based combination therapies (ACTs) can be obtained. Compared with antimalarial drugs in the past, ACTs are a special case, and malaria has no resistance to them. In 2005, the World Health Organization (WHO) recommended ACTs as the first-line treatment for uncomplicated falciparum malaria. In 2010, WHO released the list of 5 ACTs drugs again, including artemether﹢lumefantrine (AL), artesunate﹢amodiaquine (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P5/00C12P7/42C12R1/19
Inventor 方宏清吴涛孙旭戴红梅李树龙
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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