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Actinobacillus succinogenes genetically engineered bacterium and construction method and application thereof

A technology of genetically engineered bacteria and actinomycetes, applied in the field of metabolic engineering, can solve the problems of increased cost, easy death, weak acid resistance of Actinobacillus succinates, etc.

Inactive Publication Date: 2021-03-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the high yield of succinic acid has been successfully achieved by Actinobacillus succinogenes, during the fermentation process, the pH of the medium is continuously reduced with the continuous accumulation of succinic acid and other by-product acids, and the production of succinic acid Actinobacillus has weak acid resistance and is easy to die under acidic conditions, and most fermentations are carried out under neutral conditions, so a large amount of alkali is needed to maintain neutral pH conditions, and there is a possibility of bacterial contamination
Furthermore, approximately equivalent amounts of acid must be consumed to re-acidify succinic acid to succinic acid in downstream industrial treatment steps, which not only adds to the cost of the overall process, but also due to the discharge of large amounts of ion-dominated wastewater, also damage to the environment

Method used

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  • Actinobacillus succinogenes genetically engineered bacterium and construction method and application thereof
  • Actinobacillus succinogenes genetically engineered bacterium and construction method and application thereof
  • Actinobacillus succinogenes genetically engineered bacterium and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Overexpression strains gadB-WS058, gadB-gadC-WS058 and gadB △C11 - build of gadC-WS058

[0027] This embodiment specifically illustrates the overexpression strains gadB-WS058, gadB-gadC-WS058 and gadB △C11 The construction process of -gadC-WS058, the specific steps are as follows:

[0028] Use the Ezvp Column Bacterial Genomic DNA Extraction Kit (Shanghai Shenggong) to extract the genome of Lactobacillus brinneri preserved in the laboratory, and use it as a template to use gadB gene primers (F1 and F2) / gadB △C11 (F1 and F5) and gadC gene primers (F3 and F4) PCR amplified gadB and gadC / gadB △C11 Fragment where gadB / gadB △C11 and gadC gene fragments are about 1449bp and 1521bp respectively, and then use primers F1 and F4 to amplify gadB-gadC / gadB by fusion PCR △C11 - The gadC fragment, whose size is about 2979bp, obtains the Gad system gene expression cassette.

[0029] Primer sequence (SEQ ID NO.7~11):

[0030] F1: TTATCAATGAGGTGA TCTAGA ATGAGTGAAAAAAA...

Embodiment 2

[0036] Embodiment 2: Enzyme activity assay of Gad system in Actinobacillus succinate

[0037] This embodiment specifically illustrates whether there is enzyme activity after the Gad system is introduced into Actinobacillus succinogenes, and the specific determination method is as follows:

[0038] Take out the strains WS058, pLGZ922-WS058, gadB preserved in glycerol tubes from the refrigerator △C11 -gadC-WS058, gadB-gadC-WS058, melted at room temperature (take 750 μL) into TSB liquid (50ml / 100ml) medium, anaerobic culture at 37°C for 30h.

[0039] Centrifuge the cultured bacterial solution at 6000 rpm at 4°C for 10 minutes, collect the bacterial cells, wash with water once, add 10 mL of distilled water, suspend the bacterial cells, and obtain a sample solution.

[0040] 0.2mol·L -1 Sodium acetate-acetic acid buffer (pH 4.6) was used as the reaction buffer, and 60 mM sodium glutamate and 0.6 mM pyridoxal phosphate were added to prepare the substrate buffer. Mix 1 mL of the sub...

Embodiment 3

[0043] Embodiment 3: the mensuration of acid resistance

[0044] The present embodiment specifically illustrates the influence of Actinobacillus succinate into the Gad system on the acid resistance of the bacterial strain, and the specific assay method is as follows:

[0045] Take out the strains stored in glycerol tubes from the refrigerator, melt them at room temperature, and activate them by streaking. Pick a single colony and place them in TSB medium for anaerobic culture at 37°C for 24 hours; then absorb 30 mL of bacterial liquid and add them to 50 mL centrifuge tubes, centrifuge at 8000 rpm After 10 minutes, the supernatant was discarded, and 30 mL of sterile water was added to suspend the bacteria, and then 5 mL were drawn and added to TSB medium containing 45 mL of pH 4.6 with different concentrations of sodium glutamate, and cultured anaerobically at 37 °C for 1.5 h. Draw 1 mL, wash it with water once, and dilute it according to a certain ratio, then draw 5 μL and spo...

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Abstract

The invention discloses an actinobacillus succinogenes genetically engineered bacterium and a construction method and application thereof. According to the invention, glutamic acid decarboxylase and an amino acid transporter are overexpressed in actinobacillus succinogenes, and glutamic acid is catalyzed by the glutamic acid decarboxylase to generate gamma-aminobutyric acid, then the gamma-aminobutyric acid is transported out of cells through the amino acid transporter, and a proton is consumed in such process to neutralize the pressure of acid, so that the acid resistant capability of the actinobacillus succinogenes is improved, thereby reducing the use amount of a pH regulator and generation of byproduct acetic acid in the fermentation process.

Description

technical field [0001] The invention relates to a gene engineering bacterium of Actinobacillus succinogenes and its construction method and application, belonging to the technical field of metabolic engineering. Background technique [0002] Succinic acid (also known as succinic acid) is a C4 platform chemical, a natural organic acid widely found in animals, plants, humans and microorganisms. It can be widely used in food industry, chemical industry and pharmaceutical industry. In recent years, since succinic acid can be used as a C4 platform compound for the synthesis of a large number of chemicals and biodegradable plastics, it has been listed as one of the 12 potential chemicals by the US Department of Energy. [0003] Actinobacillus succinogenes is a Gram-negative bacterium with high succinic acid production isolated from the rumen of cattle. It can naturally produce succinic acid, and can use a broad-spectrum carbon source for the production of succinic acid. One of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/60C12N15/31C12P7/46C12R1/01
CPCC12N9/88C07K14/335C12N15/74C12P7/46C12Y401/01015
Inventor 郑璞陈春梅陈鹏程吴丹
Owner JIANGNAN UNIV
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