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Recombinant bacteria for increasing yield of succinic acid and construction method thereof

A technology of recombinant bacteria and Escherichia coli, applied in the biological field, can solve problems such as energy loss, low affinity, and slow speed

Active Publication Date: 2013-06-05
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] PPC and PCK exhibit different reactivity properties in catalyzing the carboxylation of PEP: PPC enzymes on HCO 3 - High affinity (Km: 0.1μM, Kai et al., 1999), fast catalytic speed (Wohl and Markus, 1972), can quickly carboxylate PEP into OAA, providing precursors for the synthesis of succinic acid, but this This process is also accompanied by energy loss (release of Pi); in contrast, although PCK enzyme 3 - The affinity of PEP is low (Km: 13μM, Matte et al., 1996), the speed of PEP carboxylation is slow, and the catalytic activity (specific activity) is 28μmol / min / g (Krebs and Bridger, 1980), but in this process , along with the production of ATP, which will facilitate the growth of the strain under anaerobic conditions

Method used

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  • Recombinant bacteria for increasing yield of succinic acid and construction method thereof
  • Recombinant bacteria for increasing yield of succinic acid and construction method thereof
  • Recombinant bacteria for increasing yield of succinic acid and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the construction of Escherichia coli ATCC8739 mutant strain recombinant Escherichia coli Suc-T108

[0054] 1. Construction of plasmid pXZ-CS

[0055] There are four steps in the plasmid construction procedure:

[0056] In the first step, the plasmid pACYC184 (Mok, Y.K., Clark, D.R., Kam, K.M. and Shaw, P.C. BsiY I, a novel thermophilic restriction endonuclease that recognizes 5'CCNNNNNNNGG3' and the discovery of a wrongly sequenced site in pACYC177.Nucleic Acids .1991,19:2321-2323; the public can obtain from Tianjin Institute of Industrial Biotechnology) DNA as a template, using primers 184-cat-up / 184-cat-down, amplified to obtain chloramphenicol resistance gene, gene fragment The size is 994bp, including the chloramphenicol gene promoter sequence, called fragment I.

[0057] The amplification system is: 10 μl of NewEngland Biolabs Phusion5X buffer, 1 μl of dNTP (10 mM for each dNTP), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion ...

Embodiment 2

[0157] Example 2, Improve PPC and PCK enzyme activity in Escherichia coli ATCC8739 and construct recombinant bacterium ZT-020

[0158] The recombinant strain ZT-020 replaces the regulatory element (sequence 3) of the PCK gene in Escherichia coli ATCC8739 with an artificial regulatory element RBSL-2 (sequence 12), and replaces the regulatory element (sequence 9) of the PPC gene with an artificial regulatory element M1-46 (SEQ ID NO: 6), the obtained recombinant bacteria;

[0159] The recombinant bacterium ZT-018 that only improves the PCK enzyme activity in Escherichia coli ATCC8739 is a recombinant bacterium obtained by replacing the regulatory element (sequence 3) of the PCK gene in Escherichia coli ATCC8739 with the artificial regulatory element RBSL-2 (sequence 12);

[0160] The recombinant bacterium ZT-019 that only improves the PPC enzyme activity in Escherichia coli ATCC8739 is a recombinant bacterium obtained by replacing the regulatory element (sequence 9) of the PPC g...

Embodiment 3

[0187] Embodiment 3, improve the activity of PCK enzyme in recombinant Escherichia coli Suc-T108 and construct the recombinant bacterium

[0188] (1) Construction of recombinant Escherichia coli ZT-001 to ZT-005

[0189] 1. Construction of recombinant Escherichia coli ZT-001 to ZT-005

[0190] Recombinant Escherichia coli ZT-001 is a recombinant bacterium obtained by replacing the regulatory element (sequence 3) of the PCK gene in the recombinant bacterium Suc-T108 with M1-12 (sequence 4);

[0191] Recombinant Escherichia coli ZT-002 is a recombinant bacterium obtained by replacing the regulatory element (sequence 3) of the PCK gene in the recombinant bacterium Suc-T108 with M1-30 (sequence 5);

[0192] Recombinant Escherichia coli ZT-003 is a recombinant bacterium obtained by replacing the regulatory element of the PCK gene in the recombinant bacterium Suc-T108 with M1-46 (sequence 6);

[0193] Recombinant Escherichia coli ZT-004 is a recombinant bacterium obtained by repla...

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Abstract

The invention discloses recombinant bacteria for increasing the yield of succinic acid and a construction method thereof. The recombinant bacteria disclosed by the invention are obtained so as to increase the enzyme activities of phosphoenolpyruvate carboxylase (PPC) and phosphoenolpyruvate carboxykinase (PCK) in escherichia coli or mutant strains of the escherichia coli. Experiments prove that the expressions of ppc and pck genes of the E.coli are respectively regulated and controlled by using constitutive controlling elements with different expression intensity, and the law between the enzyme activities of the PPC and the PCK and the succinic acid production is explored; and on the basis, the PPC and PCK catalyzing enzymes are simultaneously used for giving play to respective catalyzing advantages through coordinated regulation of the ppc and pck genes of the E.coli, so that the yield and the conversion rate of the E.coli succinic acid are obviously increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacterium and a construction method for improving the production of succinic acid. Background technique [0002] Succinic acid, also known as succinic acid, is an important intermediate metabolite in the TCA cycle process in organisms, and also has important application value. Succinic acid can be used as a precursor raw material for many important compounds, such as 1,4-butanediol, tetrahydrofuran, etc., and is widely used in food, medicine, cosmetics, and synthesis of degradable plastics. One of the 12 most valuable bulk chemicals. At present, succinic acid is mainly synthesized by chemical methods from non-renewable petroleum raw materials. Due to the depletion of petroleum resources, the use of microorganisms to produce succinic acid has attracted great attention. Compared with the chemical synthesis method, the microbial fermentation method has many advantages, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/46C12N15/70C12R1/19
Inventor 张学礼谭在高朱欣娜徐洪涛
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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