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Recombinant bacterium of synthesizing 3-hydracrylic acid, construction method and application of recombinant bacterium

A technology of hydroxypropionic acid and recombinant bacteria, applied in the field of genetic engineering, can solve problems such as hindering the production of 3-hydroxypropionic acid, and achieve the effects of increasing yield and improving tolerance

Active Publication Date: 2017-09-01
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the prior art, in the microbial fermentation process, with the synthesis and accumulation of 3-hydroxypropionic acid, when the concentration of 3-hydroxypropionic acid reaches a certain height, the growth of microorganisms will be inhibited, and the production of 3-hydroxypropionic acid will be hindered. To further improve the problem, the present invention firstly provides a recombinant bacterium for synthesizing 3-hydroxypropionic acid. The host bacterium of the recombinant bacterium is Escherichia coli, which expresses the gene clpP encoding the ATP-dependent protease hydrolysis subunit, encoding acetyl-CoA The gene accABCD for carboxylase and the gene mcr for malonyl-CoA reductase

Method used

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  • Recombinant bacterium of synthesizing 3-hydracrylic acid, construction method and application of recombinant bacterium
  • Recombinant bacterium of synthesizing 3-hydracrylic acid, construction method and application of recombinant bacterium
  • Recombinant bacterium of synthesizing 3-hydracrylic acid, construction method and application of recombinant bacterium

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Effect test

Embodiment 1

[0051] Construction of embodiment 1 recombinant strain

[0052] 1) Construction of vector pETDuet-clpP

[0053] In this example, the malonyl-CoA reductase gene mcr (the protein number of MCR in NCBI is AAS20429.1) derived from C. aurantiacus was obtained by PCR amplification using the C. aurantiacus genome as a template (Primers: 5'-CATGGTACCAGCGGAACAGGACGAC-3' and 5'-CCCTCGAGGAATTTACACGGTAATCGC-3'), the specific amplification procedure is as follows:

[0054]

[0055] After the PCR, 1% (wt / v) agarose gel electrophoresis was performed, and the target fragment with a size of about 3700 bp was recovered by using a recovery kit (OMEGA GelExtraction Kit).

[0056] The obtained mcr gene fragment and plasmid pETDuet-1 were double digested with KpnI and XhoI restriction endonucleases at 37°C for 3.5 hours, and the digested products were subjected to 1% (wt / v) agarose gel electrophoresis, and then Use the recovery kit (OMEGAGel Extraction Kit) to recover the digested product, con...

Embodiment 2

[0080] Embodiment 2 fermentation produces 3-hydroxypropionic acid

[0081] The monoclonal engineered strain obtained is activated in LB culture, and the activated seed liquid is inoculated into a 250mL shake flask containing 100mL of basic modified liquid medium according to the ratio of seed liquid: basic modified liquid medium volume ratio of 1:100 (Contains 100μg·mL -1 Ampicillin and 100 μg·mL -1 Chloramphenicol), cultured with shaking at 37°C and 180rpm. OD 600 When reaching about 0.6, the temperature was adjusted to 30°C, and 0.05mM IPTG was added for induction. Thereafter, 0.05mM IPTG, 100mg / mL ampicillin and 100μg·mL -1 Chloramphenicol, 48h after the initial induction of IPTG, the fermentation was terminated.

[0082] Take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 min at 4°C, take the supernatant, and detect the product concentration by high-performance liquid chromatography. Using an ultraviolet detector, the 3-HP yield is 6.7 g / L. Compared with...

Embodiment 3

[0084] The single clone of the obtained engineering strain was activated in LB culture, and the activated strain was inoculated into a 250mL shake flask containing 100mL basic modified liquid medium (containing 100μg·mL-1 ampicillin and 100 μg·mL-1 chloramphenicol), shaking at 37°C and 220 rpm. When OD600 reached about 0.8, the temperature was adjusted to 33°C, and 0.1mM IPTG was added for induction. Thereafter, 0.1 mM IPTG, 100 mg / mL ampicillin and 100 μg·mL-1 chloramphenicol were added every 12 h, and the fermentation was terminated 24 h after the initial induction of IPTG.

[0085] Take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 min at 4°C, take the supernatant, and detect the product concentration by high-performance liquid chromatography. Using an ultraviolet detector, the 3-HP yield is 3.5 g / L.

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Abstract

The invention provides a recombinant bacterium of synthesizing 3-hydracrylic acid, and a construction method and preparation of the recombinant bacterium and belongs to the fields of gene engineering and fermentation engineering. A host bacterium of the recombinant bacterium is escherichia coli, and the recombinant bacterium expresses and codes gene clpP of an ATP (adenosine triphosphate) dependent protease hydrolysis subunit, and codes a gene accABCD of acetylcoenzyme A carboxylase and a gene mcr of malony-coA reductase. A preparation method comprises the steps of obtaining and transforming recombinant vectors pETDuet-clpP-mcr, and pACYCDuet-accADBC into a host competent cell. The recombinant bacterium solves the problem that the increase of a concentration of 3-hydracrylic acid in a microbial fermentation process inhibits microbial growth to influence a yield; a fermentation level is increased by 116% compared with that of strains of the ATP dependent protease hydrolysis subunit without expression and coding; and the level is the highest known shake flask fermentation level.

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, as a platform compound, is one of the 12 high-value-added bio-based chemical products announced by the U.S. Department of Energy in the future. It has broad application prospects and high economic value. 3-Hydroxypropionic acid is a three-carbon organic compound with carboxyl and hydroxyl groups in its molecule. It is an isomer of lactic acid and can be used as a precursor for the synthesis of various organic compounds to produce a variety of commercially valuable compounds, such as Acrylic acid, 1,3-propanediol, malonic acid, propiolactone, etc., the market share of the above chemicals exceeds 1 billion US dollars per year. In addition, it is also a monomer that forms many macromolecular compounds and ester ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0008C12N9/50C12N9/93C12N15/70C12P7/42C12Y102/01075C12Y604/01002
Inventor 赵广李申冯新军
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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