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Recombinant bacteria for synthesizing succinic acid from fixed amount of carbon dioxide, as well as construction method and application of recombinant bacteria

A technology of carbon dioxide and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of aggravating production costs in anaerobic environment, achieve broad development prospects and application potential, avoid excessive dependence, and improve utilization

Active Publication Date: 2017-07-21
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that an anaerobic environment is required to aggravate the production cost in the conventional synthesis process of succinic acid, the present invention firstly provides a recombinant bacterium for synthesizing succinic acid by fixing carbon dioxide. The role of energy saving and emission reduction

Method used

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  • Recombinant bacteria for synthesizing succinic acid from fixed amount of carbon dioxide, as well as construction method and application of recombinant bacteria
  • Recombinant bacteria for synthesizing succinic acid from fixed amount of carbon dioxide, as well as construction method and application of recombinant bacteria
  • Recombinant bacteria for synthesizing succinic acid from fixed amount of carbon dioxide, as well as construction method and application of recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of recombinant bacteria that fix carbon dioxide and synthesize succinic acid

[0054] 1)-2) Construction of vector pACYCDuet-accADBC-pcs

[0055] 1) Obtain the gene accADBC of acetyl-CoA carboxylase derived from Escherichia coli (the gene IDs of each subunit in NCBI are accA: 944895, accB: 947758, accC: 947761, accD: 946796), and use the gene of Escherichia coli The genome was used as a template and obtained by PCR amplification (primers: 5'-caggatccgatgagtctgaatttcctt-3' and 5'-CGGAATTCTTATTTTCCTGAAGACCGAGTTT-3'). The specific amplification procedure is as follows:

[0056]

[0057] After the PCR, 1% (wt / v) agarose gel electrophoresis was performed, and the target fragment with a size of about 4000 bp was recovered by using a recovery kit (OMEGA GelExtraction Kit).

[0058] The obtained accADBC gene fragment and plasmid pACYCDuet-1 were double digested with BamHI and XhoI restriction endonucleases at 37°C for 3.5 hours, and the digested produ...

Embodiment 2

[0091] Embodiment 2 fermentation produces succinic acid

[0092] The monoclonal engineered strain obtained in Example 1 is activated in LB culture, and the seed liquid after activation is inoculated into a 500mL block containing 100mL of the basic improved liquid medium by the volume ratio of seed liquid: basic improved liquid medium 1:100. Plate shake flasks (containing 100 μg·mL-1 ampicillin and 100 μg·mL-1 chloramphenicol) were shaken at 37°C and 180 rpm. OD 600 When reaching about 0.6, the temperature was adjusted to 30°C, and 0.05mM IPTG was added for induction. The pH was adjusted to about 7 with ammonia water every 12 hours, and the fermentation was terminated 48 hours after the initial induction.

[0093] Product detection:

[0094] After the fermentation, take the fermented liquid, centrifuge at 10000rpm for 2min, and filter the supernatant through a 0.22μm water phase filter membrane to a liquid phase autosampler bottle, and perform qualitative detection by liquid...

Embodiment 3

[0096] Embodiment 3 fermentation produces succinic acid

[0097] The single clone of the engineering strain obtained in Example 1 is activated in LB culture, and the seed liquid after activation is inoculated into the 500mL block containing the basic improved liquid medium of 100mL by the volume ratio of seed liquid: basic improved liquid medium 2:100 Shake flasks (containing 100 μg·mL-1 ampicillin and 100 μg·mL-1 chloramphenicol) were shaken at 37°C and 220 rpm. OD 600 When reaching about 0.8, the temperature was adjusted to 33°C, and 0.1 mM IPTG was added for induction. The pH was adjusted to about 7 with ammonia water every 12 hours, and the fermentation was terminated 24 hours after the initial induction.

[0098] After the fermentation, take the fermented liquid, centrifuge at 10000rpm for 2min, and filter the supernatant through a 0.22μm water phase filter membrane to a liquid phase autosampler bottle, and perform qualitative detection by liquid chromatography-triple q...

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Abstract

The invention provides recombinant bacteria for synthesizing succinic acid from a fixed amount of carbon dioxide, as well as a construction method and application of the recombinant bacteria, and belongs to the field of gene engineering and fermentation engineering. A recombinant bacteria host is escherichia coli, which can express a gene accADBC of acetyl-CoA carboxylase, a gene mcr of malonyl CoA reductase, a gene pcs of propionyl CoA synthase, a gene pcc of propionyl CoA carboxylase, a gene mcE of methylmalonyl-CoA isomerase, a gene mcM of methylmalonyl-CoA mutase and a gene sucCD of succinyl-coenzyme A synthase. According to the construction method, recombinant carriers pACYCDuet-accADBC-pcs and pETDuet-mcr-pcc-sucCD-mcEM are transformed into a host cell. The recombinant bacteria constructed through the construction method can adopt glucose serving as a unique carbon source to synthesize the succinic acid, and fermentation is performed for 48 hours, thus obtaining the succinic acid with the concentration of 6.06 g / L.

Description

technical field [0001] The present invention relates to a fixed CO 2 A recombinant bacterium producing succinic acid and its construction method and application belong to the technical field of genetic engineering. Background technique [0002] The current economic development consumes a large amount of fossil resources, and the excessive use of fossil resources has caused a large amount of greenhouse gas emissions. my country's CO 2 Its emission ranks first in the world, accounting for 1 / 3 of the global total. At the same time, my country is actively participating in the international action to deal with climate change, and regards green, low-carbon energy saving and emission reduction as one of the major strategies for national economic and social development. Utilization is imminent. [0003] Succinic acid (also known as succinic acid, succinic acid) is a common natural organic acid widely found in human body, animals, plants and microorganisms. As an important organic...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/46C12R1/19
CPCC12N9/0008C12N9/90C12N9/93C12N15/70C12P7/46C12Y102/01075C12Y501/99001C12Y504/99002C12Y602/01005C12Y604/01002C12Y604/01003Y02P20/582
Inventor 赵广刘修涛咸漠冯新军刘会洲
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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