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Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain

A technology of Escherichia coli and engineering strains, which is applied in the fields of metabolic engineering and microbial fermentation, and can solve the problems of long fermentation period, slow growth of Escherichia coli, and low carbon source conversion rate.

Inactive Publication Date: 2013-08-14
SHANDONG UNIV
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Problems solved by technology

The engineered strain has a faster carbon source metabolism rate, but the microaerobic growth is relatively slow, and the fermentation cycle is long, which is not conducive to the turnover of equipment
(3) Under anaerobic fermentation conditions, it has the highest conversion rate of succinic acid (the theoretical conversion rate is 71.4% higher than that under oxygen conditions), but the growth of E. coli under anaerobic conditions is relatively slow, and the fermentation cycle is long, which is not conducive to the turnover of equipment
(4) The two-stage strategy of aerobic growth-anaerobic fermentation is commonly used at present. Although this method uncouples the process of biomass accumulation and product production, the production capacity of succinic acid is usually limited by aerobic growth. The physiological state of cells at the time of transfer to anaerobic fermentation; and there is no accumulation of succinic acid during aerobic growth, which not only reduces the total conversion rate of carbon source, but also reduces the overall volumetric productivity of succinic acid during the entire fermentation process
Such an Escherichia coli succinic acid production strain and a full-stage fermentation strategy will be able to solve the problems of low conversion rate of carbon source in aerobic succinic acid fermentation, long micro-aerobic and anaerobic succinic acid fermentation cycle and low equipment turnover rate, And the shortcomings of unstable succinic acid production capacity and low overall productivity in the two-stage strategy of aerobic growth-anaerobic fermentation
[0005] After searching, there is no report on Escherichia coli engineering bacteria that produce succinic acid by aerobic-microaerobic-anaerobic full-stage fermentation

Method used

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  • Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain
  • Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain
  • Escherichia coli engineered strain and application in succinic acid production through aerobic-microaerobic-anaerobic full-stage fermentation of Escherichia coli engineered strain

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Experimental program
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Embodiment 1

[0066] The construction of embodiment 1 engineering strain

[0067] (1). Knockout of ptsG gene:

[0068] Bacteria: Escherichia coli MG1655

[0069] Described LB substratum is: peptone 10g / L, yeast powder 5g / L, NaCl10g / L, ampicillin 100mg / L, chloramphenicol 50mg / L.

[0070] The ampicillin-resistant plate is an LB solid medium containing 100 mg / L ampicillin and 1.5% agar powder.

[0071] The chloramphenicol-resistant plate is the LB solid medium containing 50 mg / L chloramphenicol and 1.5% agar powder.

[0072] The SOB medium is: peptone 2g / L, yeast powder 0.5g / L, NaCl0.0585g / L, KCl0.0186g / L, MgCl 2 0.203g / L.

[0073] a Cloning of homologous recombination fragments

[0074] The target gene was knocked out using the Red recombination system. According to the sequence number of the genome published by Genbank,

[0075] NC_000913.2 Escherichia coli MG1655 ptsG gene sequence with gene ID 945651 and the plasmid pKD3 sequence with GenBank ID AY048742.1 designed primers:

[0076...

Embodiment 2

[0398] Example 2. Engineering strain E.coli QZ1111 produces succinic acid under aerobic conditions

[0399] We carried out aerobic succinic acid fermentation of engineering strain E.coli QZ1111 in inorganic salt medium with glucose as the sole carbon source.

[0400] Strain: E.coli QZ1111 [The strain genotype is: MG1655(ΔptsGΔpoxBΔptaΔiclRΔsdhA)]

[0401] (Note, the unmodified original strain MG1655 does not produce succinic acid)

[0402] Seed medium: LB medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L).

[0403] Fermentation medium: AM1 medium (composed of (g / L): (NH 4 ) 2 HPO 4 12H 2 O, 2.63; NH 4 h 2 PO 4 , 0.87; KCl, 0.15; MgSO 4 ·7H 2 O, 0.37; trace elements (1000×: FeCl 3 ·6H 2 O, 2.4; CoCl 2 ·6H 2 O, 0.3; CuCl 2 2H 2 O, 0.15; ZnCl 2 , 0.3; Na 2 MoO 4 ·H 2 O, 0.3; H 3 BO 3 , 0.075; MnCl 2 4H 2 O, 0.5; HCl, 120mM); the balance is water).

[0404] Culture method: pick the single clone in the plate into a 300ml Erlenmeyer flask containing 50ml ...

Embodiment 3

[0407] Example 3. Engineering strain E.coli QMJ03 produces succinic acid under aerobic-microaerobic conditions

[0408] We carried out aerobic-microaerobic two-stage succinic acid fermentation on engineering strain E.coli QMJ03 in inorganic salt medium with glucose as the sole carbon source.

[0409] Strain: E.coli QZ1111 [The strain genotype is: MG1655(ΔptsGΔpoxBΔptaΔiclRΔsdhA)]

[0410] E.coli QMJ03[The strain genotype is: MG1655(ΔptsGΔpoxBΔptaΔiclRΔsdhAΔarcA)]

[0411] Seed medium: LB medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L).

[0412] Fermentation medium: AM1 medium (same as above).

[0413] Culture method: pick the single clone in the plate into a 300ml Erlenmeyer flask containing 50ml LB medium. Place them at 37°C for 12 hours, then transfer 5% of the inoculum into a 500ml Erlenmeyer flask containing 80ml of LB medium supplemented with 40g / L glucose, and place them at 37°C for 10 hours at a speed of 250 rpm. Then transfer 10% of the inoculum into a 1,200m...

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Abstract

The invention discloses an Escherichia coli engineered strain and application in succinic acid production through the aerobic-microaerobic-anaerobic full-stage fermentation of the Escherichia coli engineered strain. The name of the engineered strain is Escherichia coli YL106. The genotype of the strain is a formula shown in a drawing. The strain is collected by China Center for Type Culture Collection (CCTCC) on April 17th, 2013 and has the collection number of CCTCC NO: M2013149. According to the engineered strain disclosed by the invention, succinic acid is produced under the condition of aerobic-microaerobic-anaerobic full-stage fermentation, a large amount of succinic acid can be accumulated efficiently, and the total productivity (2.13 g / (L.h)) of the engineered strain is the highest among literature reports related to the succinic acid production using Escherichia coli currently, so that the engineered strain disclosed by the invention and an aerobic-microaerobic-anaerobic full-stage succinic acid fermentation process have considerable industrial application values and prospects.

Description

technical field [0001] The invention relates to an engineering strain of Escherichia coli and its application in aerobic-microaerobic-anaerobic full-stage fermentation to produce succinic acid, which belongs to the field of metabolic engineering and microbial fermentation. Background technique [0002] Succinic acid is a natural two-valent four-carbon carboxylic acid, an intermediate product in the tricarboxylic acid (TCA) cycle of organisms, and one of the end products of fermentation of many anaerobic and facultative anaerobic microorganisms one. As an important organic synthesis intermediate, succinic acid is widely used in the fields of food, medicine, spices, pesticides, dyes, paints and plastics, and is used to produce many chemical products such as adipic acid, 1,4- Butanediol, tetrahydrofuran, γ-butyrolactone, n-methylpyrrolidone and 2-pyrrolidone, etc., so there is a broad demand market. And with the continuous expansion of some new application fields, the demand ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/46C12R1/19
Inventor 祁庆生梁泉峰李宜奎
Owner SHANDONG UNIV
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