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Construction method of escherichia coli synthetic strain capable of efficiently fermenting isobutanol in low-residual-sugar manner

A technology of Escherichia coli and construction methods, applied in the direction of microorganism-based methods, fermentation, biochemical equipment and methods, etc., can solve the problems of undiscovered patent reports, high concentration of residual sugar, ignoring the mechanism of influence, etc.

Inactive Publication Date: 2017-05-31
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent: CN201480041525.8, 2014-07-22), thus ignoring the influence mechanism of the overall metabolic process in the cell on the absorption and utilization of carbon sources
Although the above methods can improve the utilization efficiency of raw materials to a certain extent, they cannot completely solve the problem of excessive residual sugar concentration in the fermentation process.
[0004] At present, there are not enough patent reports on the mechanism of residual sugar phenomenon

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of isobutanol-synthesizing strain LA09 and analysis of rate-limiting steps affecting residual sugar conversion

[0026] (1) The construction of the isobutanol synthetic strain LA09 was carried out strictly according to the method described in the patent CN201410814025.5.

[0027] (2) Taking the isobutanol-synthesizing strain LA09 as the research object, a series of batch fermentation experiments with initial sugar concentrations of 35, 40, 45, 50, 55, and 60 g / L were carried out to determine the isobutanol synthesis. The optimal initial glucose concentration of strain LA09 was 45g / L. Culture method: Bacteria were cultured at a culture temperature of 37°C and a rotational speed of 250rpm. When the cell OD600 increased to 0.6-0.8, 0.1-1mM IPTG was added for isobutanol induction, and the subsequent culture conditions were 30°C and 200rpm.

[0028] (3) Extract the metabolites inside and outside the cells for GC-MS and LC-MS analysis, and use the buil...

Embodiment 2

[0029] Example 2 Construction of artificial ED upstream and downstream pathway fragments

[0030] (1) Using the ED downstream pathway gene fragment ZMO0368 (SEQ ID NO: 1) of Mobilis Z. mobilis ZM4 as a template to design primers z368-F / z368-R (the primer sequence number is SEQ ID NO: 10) , obtain the ZMO0368 gene fragment controlled by the promoter BBa_J23119 through PCR amplification, and amplify another gene from the ED downstream pathway of Molecularis ZM4 with primer z997-F / z997-R (the sequence number of the primer is SEQ ID NO: 11) Fragment ZMO0997 (SEQ ID NO: 2).

[0031] Promoter construction: The promoter BBa_J23119 sequence is included in the primer z368-F, which is directly synthesized by Huada Gene, and the promoter BBa_J23119 is used to regulate the downstream pathway of ED (ZMO0368-ZMO0997).

[0032] PCR reaction system: 1 μL of upstream and downstream primers (10 μM), 1 μL of template, 2 μL of dNTP (2.5 mM) solution, 2 μL of 10× buffer, 0.5 μL of DNA polymerase,...

Embodiment 3

[0041] Example 3 Construction of Escherichia coli Isobutanol Synthesizing Strain E.coli ED Containing Heterologous ED Sugar Metabolism Pathway

[0042] (1) Construction of E.coli LA09 recombinant system and competent preparation.

[0043] Construction of Red recombination system: Introduce plasmid pKD46 into E.coli LA09, inoculate in LB liquid medium, add ampicillin, and culture overnight at 30°C and 200rpm. Insert 1% inoculum into 50 mL LB liquid medium, add ampicillin resistance (100 μg / mL) and add L-arabinose with a final concentration of 2 mM, cultivate at 30 ° C and 200 rpm until the OD600 is 0.7, and stop the culture.

[0044]Competent preparation: the bacterial solution was pre-cooled on ice for 20 minutes, centrifuged at 4°C and 4000 rpm for 10 minutes, and the bacterial cells were collected. Add 20 mL of ice-cold 10% (v / v) glycerol solution, keep the low temperature, gently resuspend the cells, centrifuge at 4000 rpm for 10 min at 4 °C, and repeat washing twice. The...

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Abstract

The invention provides a construction method of an escherichia coli synthetic strain capable of efficiently fermenting isobutanol in a low-residual-sugar manner. Escherichia coli MG 1655 is taken as a host, an isobutanol synthesis strain E.coli LA09 is constructed, and the fact that a conversion reaction from intracellular 6-phosphofructose into 1,6-fructose bisphosphate is a key limitation factor of residual sugar conversion is determined with a metabonomics method. Heterogenous artificial ED metabolic pathway upstream and downstream fragments derived from a strain Zymomonas mobilis ZM4 are constructed, ED upstream and downstream pathways are matched and adjusted with an artificial promoter in combination of synthetic biology, and one escherichia coli synthetic strain E.coli ED capable of efficiently fermenting isobutanol in the low-residual-sugar manner is constructed; with the initial glucose concentration being 45 g / L, fermentation is completed within 30 h, the yield of isobutanol reaches 13.67 g / L, and the fermenting residual sugar concentration is 0.87 g / L, thereby proving that the method is practical.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and bioenergy, and specifically relates to taking Escherichia coli MG1655 as the research object, after determining the limiting factors of residual sugar conversion of Escherichia coli isobutanol synthesis strains according to the detection and analysis of metabolomics data, using synthetic biology Methods Heterologously expressing the ED sugar metabolism pathway from Mobilis and using artificial promoters to regulate the upstream and downstream of the ED pathway, and finally obtain a synthetic strain of E. coli that can ferment isobutanol with high efficiency and low residual sugar. Background technique [0002] As an important platform compound, isobutanol is widely used in the fields of food, medicine and chemical industry, and has the advantages of high energy density, high octane number, low volatility and corrosion. Compared with the first-generation fuel ethanol, bio-based isobut...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P7/16C12R1/19
CPCC12N9/0006C12N9/18C12N9/88C12N15/70C12N2800/101C12P7/16C12Y101/01049C12Y301/01031C12Y401/02028C12Y402/01012Y02E50/10
Inventor 闻建平陈红刘蛟
Owner TIANJIN UNIV
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