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Recombinant escherichia coli for efficiently producing succinic acid and construction method thereof

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of bioengineering, can solve the problems of cofactor metabolic imbalance, metabolic imbalance, product yield and low production intensity, and achieve the effect of increasing production and reducing the accumulation of by-products

Active Publication Date: 2021-01-29
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production efficiency of Escherichia coli fermentation is low, and there are usually by-products such as lactic acid, formic acid, acetic acid, ethanol, etc. in the fermentation broth. Excessive osmotic pressure and glucose absorption and utilization speed lead to metabolic imbalance, low product yield and low production intensity; in order to obtain high-performance production strains, it is usually necessary to use traditional breeding methods, various omics analysis and molecular biological transformation. combined method

Method used

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  • Recombinant escherichia coli for efficiently producing succinic acid and construction method thereof
  • Recombinant escherichia coli for efficiently producing succinic acid and construction method thereof
  • Recombinant escherichia coli for efficiently producing succinic acid and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Knockout of the gene encoding pyruvate formate lyase

[0047] (1) In order to knock out the gene encoding pyruvate formate lyase to reduce the amount of acid added by the by-product, a genome editing fragment was constructed with the help of Red homologous recombination technology. The gene editing fragments include upstream and downstream homology arm regions, and resistance screening cassettes. Using the plasmid pKD4 as a template, design sequences such as SEQ ID NO.6 / SEQ ID NO.7 primer pair pflB-focA-S / pflB-focA-A to amplify the resistance screening gene Kan to obtain the pflB-focA knockout frame fragment.

[0048] (2) Transform the pKD46 plasmid into the expression host E.coli FMME-N-5 competent cells, and obtain the recombinant strain E.coli FMME-N-5-pKD46 by colony PCR screening, and then knock out the obtained pflB-focA The frame fragment was transferred into competent cells of the recombinant strain E.coli FMME-N-5-pKD46 by electroporation, and posit...

Embodiment 2

[0052] Example 2: Knockout of lactate dehydrogenase expression gene

[0053] (1) The construction of E.coli FMME-N-5 (ΔfocA-pflB) is the same as in Example 1

[0054] (2) In order to further reduce the amount of by-product lactic acid to knock out the gene encoding lactate dehydrogenase, a genome editing fragment was constructed with the help of Red homologous recombination technology. The gene editing fragments include upstream and downstream homology arm regions, and resistance screening cassettes. Using the plasmid pKD4 as a template, design sequences such as SEQ ID NO.8 / SEQ ID NO.9 primer pair ldhA-S / ldhA-A amplification resistance screening gene Kan to obtain the ldhA knockout frame fragment.

[0055] (3) Transform the pKD46 plasmid into the expression host E.coli FMME-N-5-ΔfocA-pflB competent cells, obtain the recombinant strain E.coli FMME-N-5-ΔfocA-pflB-pKD46 by colony PCR screening, and then The obtained ldhA knockout frame fragment was transferred into competent ce...

Embodiment 3

[0059] Example 3: Knockout of Phosphotransacetylase Expression Gene

[0060] (1) The construction of E.coli FMME-N-5 (ΔfocA-pflB-ΔldhA) is the same as in Example 2

[0061] (2) In order to further reduce the amount of by-product acetic acid and ensure cell growth, the gene encoding the phosphoacetyl group was knocked out, and the genome editing fragment was constructed with the help of Red homologous recombination technology. The gene editing fragments include upstream and downstream homology arm regions, and resistance screening cassettes. Using the plasmid pKD4 as a template, design sequences such as SEQ ID NO.10 / SEQ ID NO.11 primer pair pta-S / pta-A amplification resistance screening gene Kan to obtain the pta knockout frame fragment.

[0062] The pKD46 plasmid was transformed into the expression host E.coli FMME-N-5-ΔfocA-pflB-ΔldhA competent cells, and the recombinant strain E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-pKD46 was obtained by colony PCR screening. Then, the obtained p...

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Abstract

The invention discloses recombinant escherichia coli for efficiently producing succinic acid and a construction method thereof, and belongs to the technical field of bioengineering. The method comprises the following steps: knocking out a by-product encoding gene of a succinic acid producing strain E.coli FMME-N-2 to obtain a strain E.coli FMME-N-5(delta foc A-pflB-delta ldhA-delta pta-ackA); performing overexpression from actinobacillus succinogenes phosphoenolpyruvate carboxykinase pck and bacillus stutzeri phosphite dehydrogenase ptxD; introducing the constructed plasmid pTrcHisA-pck-ptxD into an expression host E.coli FMME-N-5(delta foc A-pflB-delta ldhA-delta pta-ackA), and screening through an ampicillin resistance flat plate to obtain an engineering bacterium E.coli FMME-N-5(delta foc A-pflB-delta ldhA-delta pta-ackA)-pck-ptx-D for efficiently producing succinic acid. The engineering strain adopts a two-stage fermentation strategy on a 7.5 L fermentation tank, the fermentation time is 96h, the succinic acid yield reaches 137g / L, the succinic acid yield reaches 1g / g glucose, the production strength is 1.43 g / L / h, byproducts lactic acid and formic acid are not accumulated, acetic acid is 1-2g / L, and the engineering strain has certain industrial production potential.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli capable of efficiently producing succinic acid and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Succinic acid, scientific name succinic acid, is an important C4 platform compound. As a starting material for the synthesis of general chemicals, succinic acid is widely used in food, chemistry, medicine and other fields. Succinic acid is listed as the first among the 12 most potential bulk bio-based chemicals by the US Department of Energy. [0003] The traditional production method of succinic acid is chemical synthesis, mainly including paraffin oxidation, cyanidation hydrolysis of methyl chloroacetate and catalytic hydrogenation of vanadium pentoxide. However, due to the reduction of petroleum resources and the increasingly serious environmental pollution, The disadvantages of chemical synthesis methods are becoming more and more ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/54C12N15/53C12N15/60C12P7/46C12R1/19
CPCC12N15/52C12N9/1029C12N9/0006C12N9/88C12N9/0004C12N15/70C12P7/46C12Y203/01054C12Y101/01027C12Y401/01032C12Y120/01001C12Y203/01008C12N1/205C12N9/1025C12R2001/19C12P1/04
Inventor 刘立明唐文秀沈忱罗秋玲陈修来刘佳高聪宋伟
Owner JIANGNAN UNIV
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