Method for improving yield of butanol produced by escherichia coli

A technology for producing butanol and Escherichia coli, applied in the biological field, can solve the problems of Clostridium not showing sufficient advantages and the like

Active Publication Date: 2016-04-20
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the following years, a lot of related work was gradually carried out and published. The highest butanol production of Escherichia coli is currently reported to be 14-15g / L, which is close to the production of Clostridium, but compared with Clostridium, it does not show sufficient Advantages, it is far from reaching the level of industrialization with market competitiveness, and more improvements are still needed, such as output, yield, substrate utilization, etc.

Method used

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  • Method for improving yield of butanol produced by escherichia coli
  • Method for improving yield of butanol produced by escherichia coli
  • Method for improving yield of butanol produced by escherichia coli

Examples

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Effect test

Embodiment 1

[0034] Embodiment 1, preparation of recombinant bacteria by knocking out the mdh gene in Escherichia coli EB205

[0035] The following examples use the λ-red homologous recombination system to knock out the mdh gene, involving the plasmids:

[0036] pKD4 is described in the following documents: Datsenko, Kirill A., and Barry L. Wanner. One-step in activation of chromosomal genes in Escherichiacoli K-12 using PCR products. Proceeding of the National Academy of Sciences 97.12 (2000): 6640-6645;

[0037] pKD46 is described in the following documents: Datsenko, Kirill A., and Barry L. Wanner. One-step in activation of chromosomal genes in Escherichiacoli K-12 using PCR products. Proceeding of the National Academy of Sciences 97.12 (2000): 6640-6645;

[0038] The nucleotide sequence of the mdh gene is sequence 1 in the sequence listing, and the amino acid sequence of the protein malate dehydrogenase encoded by it is sequence 3 in the sequence listing.

[0039] 1. Preparation of re...

Embodiment 2

[0063] Embodiment 2, fermentation of recombinant bacteria EB205△mdh::kan

[0064] The recombinant bacteria EB205△mdh::kan obtained in Example 1 and the control strain EB205 were inoculated into 10ml of M9Y medium in a centrifuge tube with a capacity of 15ml with a sterilized toothpick, and placed in a constant temperature incubator at 37°C after inoculation with a toothpick. Place and ferment for 2 days to obtain a fermented liquid. Experiments were repeated three times.

[0065] The above-mentioned M9Y medium is composed of 17.1g / lNa 2 HPO 4 12H 2 O, 3.0g / lKH 2 PO 4 , 0.5g / lNaCl, 2.5g / lNH 4 Cl, 2g / l YeastExtract, 22g / lC 6 h 12 o 6 ·H 2 O, 2mM MgSO 4 ·7H 2 O, 0.1mM CaCl 2 and water composition.

[0066] The supernatant after the centrifugation of the above-mentioned fermentation broth was filtered through a filter with a pore size of 0.22 μm and then analyzed by high-performance liquid chromatography. The analysis used an Agilent 1260 liquid chromatograph, a diff...

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PUM

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Abstract

The invention discloses a method for improving yield of butanol produced by escherichia coli. According to the method for constructing recombinant bacteria, expression of a malate dehydrogenase gene (mdh) on the genome of an original strain for producing butanol is inhibited or silenced to obtain the recombinant bacteria. Experiments prove that the modified target capable of promoting escherichia coli to produce butanol is the malate dehydrogenase gene (Genbank: mdh gene (b3236) in the NC_000913.3 sequence). By a lembda-red homogenous recombinant system knockout technology, after the mdh gene of the engineering strain of escherichia coli for producing butanol is knocked out, the butanol output can be increased by 283 percent, and the yield can be increased by 89 percent.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving butanol production by Escherichia coli. Background technique [0002] Butanol is an important chemical that can be directly used as an organic solvent and a precursor for the synthesis of various ester compounds, and it is also a more advantageous biofuel than ethanol. Bio-butanol can be produced by some Clostridium strains through ABE (Acetone-Butanol-Ethanol) fermentation under anaerobic conditions, and it has a history of more than one hundred years. However, because the Clostridium strain itself is a strictly anaerobic Gram-positive bacterium, genetic manipulation is difficult, and due to its own complex metabolic regulation mechanism, it is difficult to improve the ability of Clostridium to ferment and produce butanol, thus limiting the bio-butanol market Competitiveness. With the help of modern biotechnology, scientists have begun to use the easy-to-ope...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P7/16C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12N2800/101C12N2800/80C12N2810/10C12P7/16C12Y101/01037Y02E50/10
Inventor 董红军赵春华张延平李寅
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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