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Recombinant bacterium for synthesizing 3-hydracrylic acid as well as construction method and application of recombinant bacterium

A technology of hydroxypropionic acid and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of high toxicity, carcinogenicity, high production cost, restricting industrial application, etc., and achieve the effect of low price and reduction of synthesis cost.

Pending Publication Date: 2022-04-22
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemical synthesis method has the disadvantages of high production cost and low yield; at the same time, most of the precursor substances used in chemical synthesis are highly toxic and carcinogenic, which seriously restricts industrial application
However, there is no report on the synthesis of 3-hydroxypropionic acid from malonate

Method used

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  • Recombinant bacterium for synthesizing 3-hydracrylic acid as well as construction method and application of recombinant bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Construction of recombinant bacteria that synthesize 3-hydroxypropionic acid.

[0049] 1. Construction of vector pACYC dute-matB-mcr.

[0050] In this embodiment, the gene matB (the protein number of matB in NCBI is WP_011155789.1) derived from the malonyl-CoA synthase of Rhodopseudomonas palustris and the gene matB derived from Chloroflexus aurantiacus (thermophilic light all green silk The gene mcr of the malonyl-CoA reductase of bacteria) (the protein number of mcr in NCBI is AAS20429.1) is synthesized through the whole gene, and matB is shown in the sequence SEQ ID No.1 after codon optimization; The synthesized sequence of mcr after codon optimization is shown in SEQ ID No.2; the plasmid containing MtaB and the plasmid of mcr were respectively obtained, and the vector was pACYCdute-1.

[0051] The MtaB plasmid was digested with BgIII, the mcr plasmid was digested with BamHI and BgIII, the pACYC dute-1 plasmid was digested with NcoI, EcorV and KpnI, the d...

Embodiment 2

[0062] Example 2. Introducing pACYC dute-matB-mcr and pBAD-madLM into E.coli BL21(DE3) to synthesize 3-HP.

[0063] 1) Introduce the vectors pACYC dute-matB-mcr and pBAD-madLM obtained in Example 1 into E.coli BL21(DE3) competent cells, and spread them on a medium containing 100 μg·mL -1 Ampicillin and 100 μg·mL -1 Chloramphenicol LB solid plate; place the coated plate in a constant temperature incubator at 37°C, and continue to culture until a single colony grows. Activate the single clone of the obtained engineering strain in LB culture to obtain the seed liquid;

[0064] 2) The activated strain was inoculated into a 250mL shake flask containing 50mL LB medium (containing 100μg·mL -1 Ampicillin and 100 μg·mL -1 Chloramphenicol), cultured with shaking at 37°C and 180rpm until OD 600 When it reaches about 0.6, the culture medium is obtained;

[0065] 3) Adjust the temperature to 30° C., add a final concentration of 0.5 mM IPTG and a final mass concentration of 0.02% arabi...

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Abstract

The invention discloses a recombinant bacterium for synthesizing 3-hydracrylic acid as well as a construction method and application thereof, and belongs to the technical field of genetic engineering. Aiming at biosynthesis of 3-hydracrylic acid, the invention provides a recombinant bacterium for biosynthesis of 3-hydracrylic acid by taking malonic acid as a substrate. The malonyl-coenzyme A gene is obtained by introducing a gene matB for coding malonyl-coenzyme A synthase, a gene mcr for coding malonyl-coenzyme A reductase, a gene madL for coding malonyl-coenzyme A transmembrane protein and a gene madM for coding malonyl-coenzyme A transmembrane protein into escherichia coli. The enzyme activity verifies that the recombinant bacterium prepared by the invention realizes the biosynthesis of the 3-hydracrylic acid, provides a new idea for the synthesis of the 3-hydracrylic acid, and is beneficial to the popularization and application of the 3-hydracrylic acid.

Description

technical field [0001] The invention relates to a recombinant bacterium for synthesizing 3-hydroxypropionic acid and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] 3-hydroxypropionic acid is a three-carbon platform compound that has emerged in recent years. The molecule has two functional groups—hydroxyl and carboxyl. It can be used as a starting material for the synthesis of coatings, adhesives, water treatment chemicals and personal Nursing products and other chemical products and daily necessities. 3-hydroxypropionic acid can also generate 1,3-propanediol through reduction, and 1,3-propanediol is also an important chemical raw material, which can be used as a protective agent, solvent, etc., and can also be used to synthesize polyurethane and polytrimethylene terephthalate Wait. The 3-hydroxypropionic acid polymer formed by the polymerization of 3-hydroxypropionic acid has the advantages of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0008C12Y102/01075C12N9/00C07K14/21C12N15/52C12N15/70C12P7/42C12N2800/22
Inventor 咸漠姜兴林刘炜
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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