Escherichia coli for producing isobutanol and ethanol and preparation method of escherichia coli

A technology of Escherichia coli and isobutanol, applied in the biological field, can solve problems such as imbalance of redox power supply

Active Publication Date: 2017-07-25
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

When the strain is fermented under anaerobic conditions, it can produce 35mM isobutanol with a conversion rate of 0.66mol / mol, but there is still an imbalance of redox power supply in anaerobic fermentation

Method used

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  • Escherichia coli for producing isobutanol and ethanol and preparation method of escherichia coli
  • Escherichia coli for producing isobutanol and ethanol and preparation method of escherichia coli
  • Escherichia coli for producing isobutanol and ethanol and preparation method of escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0056] Embodiment 1, construction and application of recombinant Escherichia coli ZL004

[0057] 1. Construction of recombinant Escherichia coli ZL002

[0058] 1. Construction of plasmid pXZ021C.

[0059] In the first step, using the pXZ020 plasmid DNA (Shi, et al., Metab Eng, 2013, 16:1-10) as a template, PCR amplification was performed using primers XZ-adhE-1 and XZ-adhE-2 to obtain a long 4662bp DNA fragment, called fragment I;

[0060] The above amplification system is: 10 μl of NewEngland Biolabs Phusion 5X 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 High-Fidelity DNA polymerase (2.5 U / μl) , 33.5 μl of distilled water, the total volume is 50 μl.

[0061] The above amplification conditions are 98°C pre-denaturation for 2 minutes (1 cycle); 98°C denaturation for 10 seconds, 56°C annealing for 10 seconds, 72°C extension for 30 seconds (30 cycles); 72°C extension for 5 minutes (1 cycle) .

[0062] In t...

Embodiment 2

[0134] Regulation of zwf gene in embodiment 2, recombinant escherichia coli ZL004

[0135] Starting from ZL004, the glucose-6-phosphate dehydrogenase coding gene zwf is regulated through the RBS library, including the following steps:

[0136] 1. Construction of recombinant Escherichia coli ZL004-zwf by homologous recombination for the first time

[0137] (1) Preparation of DNA fragment I for homologous recombination

[0138] Using the pXZ-CS plasmid DNA as a template, a 2722bp DNA fragment I was amplified using primers zwf-cat-sacB-up / down.

[0139] The nucleotide sequence of the DNA fragment I of 2722bp is sequence 5 in the sequence listing, and this fragment comprises about 50bp zwf gene regulatory region upstream homology arm (the 1st-53rd nucleotide of sequence 5), cat-sacB gene fragment (sequence 5 Nucleotides 54-2672), and the initial 50 bp of the zwf gene as a downstream homology arm (nucleotides 2673-2722 in Sequence 5).

[0140] (2) The first homologous recombinat...

Embodiment 3

[0169] Regulation of the adhE gene in embodiment 3, recombinant escherichia coli ZL004

[0170] Starting from ZL004, the alcohol dehydrogenase encoding gene adhE is regulated through the RBS library, including the following steps:

[0171] 1. Construction of recombinant Escherichia coli ZL004-adhE by homologous recombination for the first time

[0172] (1) Preparation of DNA fragment I for homologous recombination

[0173]Using the pXZ-CS plasmid DNA as a template, a 2719bp DNA fragment I was amplified using primers adhE-cat-sacB-up / down.

[0174] The nucleotide sequence of the DNA fragment I of 2719bp is sequence 11 in the sequence listing, and this fragment comprises about 50bp adhE gene regulatory region upstream homology arm (the 1st-50th nucleotide of sequence 11), cat-sacB fragment (sequence 11 Nucleotides 51-2669), the initial 50 bp sequence of the adhE gene is the downstream homology arm (nucleotides 2670-2719 of sequence 11).

[0175] (2) The first homologous recom...

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Abstract

The invention discloses escherichia coli for producing isobutanol and ethanol and a preparation method of the escherichia coli. The invention provides a method for constructing recombinant bacteria for producing isobutanol and / or ethanol, wherein the method comprises the following steps: 1) expressing an ethanol dehydrogenase adhE and a pyruvate formate-lyase coding gene pflB in the escherichia coli AS108, so as to obtain a recombinant bacterium which is named as A; and 2) reducing enzymatic activity of ethanol dehydrogenase in the recombinant bacterium which is named as A and / or improving enzymatic activity of glucose-6-phosphate dehydrogenase in the recombinant bacterium which is named as A, so as to obtain a recombinant bacterium which is named as B. Experiments prove that excessive NADH can be consumed through a mode of introducing ethanol production in the AS108 strain based upon researches, so that toxicity to cells due to accumulation of the NADH is relieved; and meanwhile, NADPH supply can be promoted by activating a pentose phosphate pathway (PPP), so that an isobutanol production capacity is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an Escherichia coli producing isobutanol and ethanol and a preparation method thereof. Background technique [0002] At present, liquid transportation fuels in the world are basically refined from petroleum. The burning of these fuels will generate a large amount of greenhouse gases, which will increase the global temperature. Global warming will cause a series of problems, such as rising sea levels, expanding deserts in subtropical regions, melting glaciers, thawing permafrost, shrinking Arctic rainforests and boreal forests, increasing tsunamis and hurricane density, and species extinction. Biomass is a widespread and renewable clean resource. Through biomanufacturing, biomass can be converted into a renewable alternative energy source. In recent decades, the research and development of bioenergy has mainly focused on ethanol, butanol, biogas, hydrogen and biodiesel. Alcohols a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/16C12P7/06C12R1/19
CPCC12N9/0006C12N9/1029C12P7/065C12P7/16C12Y101/01001C12Y101/01049C12Y203/01054Y02E50/10
Inventor 张学礼刘萍萍刘子纯
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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