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A kind of Escherichia coli using triglyceride as carbon source and its application in the synthesis of bio-based chemicals

A technology of Escherichia coli and triglyceride, applied in the field of genetic engineering bacteria, can solve the problems of environmental pollution, impact on food safety, high cost, and achieve the effect of easy gene manipulation and clear genetic background

Active Publication Date: 2016-04-13
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, my country's waste edible oils and fats have not been rationally utilized. On the contrary, waste edible oils and fats have become an environmental pollutant and impact food safety.
[0003] The main component of waste oil is triglyceride. At present, its main utilization technology is to prepare biodiesel (Chinese invention patent 201210329282.0), but its cost is too high, which limits the development of biodiesel industry

Method used

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  • A kind of Escherichia coli using triglyceride as carbon source and its application in the synthesis of bio-based chemicals
  • A kind of Escherichia coli using triglyceride as carbon source and its application in the synthesis of bio-based chemicals
  • A kind of Escherichia coli using triglyceride as carbon source and its application in the synthesis of bio-based chemicals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning and expression vector construction of Alcaligeneseutrophus H16 secreted lipase gene (slipase, Genbank accession number: AM260479REGION: 965025..966251), the specific process is as follows:

[0033] Oligonucleotide 5'-CCC AAGCTT GATGTCTCGCGCTCCTGGCATG-3' and 5'-CCG GAA TTC CTATGGGCACTCGGATACCGC-3' was used as a primer, and the total DNA of Alcaligenes eutropha H16 (purchased from the German Culture Collection of Microorganisms, DSMNo.:428) was used as a template to amplify the eutrophic The secreted lipase gene (sequence 1) of Alcaligenes, and HindIII and EcoRI restriction sites were introduced at the 5' end and 3' end respectively. The amplification system and conditions are as follows:

[0034] PCR amplification system:

[0035]

[0036] PCR program:

[0037] The PCR amplification product was recovered by agarose gel electrophoresis, and then the PCR product and the pUC19 empty vector (purchased from Invitrogen) were digested with restriction endonu...

Embodiment 2

[0046] The knockout of Escherichia coli BW25113fadR gene, the specific process is as follows:

[0047] The fadR gene (GeneID: 948652) was knocked out using the Red recombination system (Datsenko et al., PNAS, 2000, 97(12): 6640-6645), and the primers (5 '-ATGGTCATTAAGGCGCAAAGCCCGGCGGGTTTCGCGGAAGAGTACATTATgtgtaggctggagctgcttc-3' and 5'-TTATCGCCCCTGAATGGCTAAATCACCCGGCAGATTTTTCTGCATCCGGTatgggaattagccatggtcc-3'), with pKD4 plasmid (preserved by our laboratory, Cao et al., PLoSONE amplified as a template, 2013, 7( Mycin-resistant recombinant fragments. The amplified product was digested with DpnI endonuclease, recovered and concentrated by agarose gel electrophoresis. The concentrated amplified fragment was transformed into Escherichia coli BW25113 competent cells by electroshock transformation method, and the electroshock conditions were as follows:

[0048] Voltage: 2.5kV

[0049] Resistance: 200Ω

[0050] Capacitance: 25μF

[0051] The electroporated cells were coated with a ...

Embodiment 3

[0053] Construction of engineering strain BW25113ΔfadR / pUC-slipase:

[0054] The pUC-slipase plasmid was extracted from the strain BW25113 / pUC-slipase by alkaline lysis, and the recombinant plasmid pUC-slipase was transformed into the competent cells of the strain BW25113ΔfadR in Example 2 to obtain the final engineering strain BW25113ΔfadR / pUC-slipase.

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Abstract

The invention discloses escherichia coli with triglyceride as a carbon source and an application of the escherichia coli in bio-based chemicals synthesis. A secretory type lipase (slipase) gene from alcaligenes entruphus is constructed on a prokaryotic expression vector pUC19, and then is guided into the escherichia coli of which the fadR gene is knocked out, so as to obtain recombinant bacteria, the bacteria strain is subjected to fermentation with triglyceride as a substrate, and the results show that in a culture medium in which triglyceride is used as the only one carbon source, the strain can grow normally. A new way is provided for the biological application of lipid compounds.

Description

technical field [0001] The invention relates to a method for constructing Escherichia coli using triglyceride as a carbon source, a genetically engineered bacterium obtained by the construction, and an application of the genetically engineered bacterium in fermenting and producing a target product using triglyceride as a carbon source. Background technique [0002] Waste oil refers to the non-edible animal and vegetable oil produced in the production process of catering industry and food processing industry, including frying waste oil, swill oil and gutter oil. Waste oil mainly refers to the common vegetable oil and animal oil residues and the waste oil residue and swill discharged. According to expert calculations, the amount of these waste oils accounts for about 20-30% of the total consumption of edible oil. Based on the fact that the average annual consumption of edible oil in my country is 21 million tons, 4-6 million tons of waste oil will be produced every year. At p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
Inventor 咸漠曹玉锦张汝兵曹志峰
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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