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Microorganism gene modification method, genetically engineered bacteria for producing higher aliphatic alcohol and higher aliphatic hydrocarbon and application of genetically engineered bacteria

A technology of genetically engineered bacteria and genetic modification, applied in the field of molecular biology, can solve the problems of limiting maximum yield and high cytotoxicity

Inactive Publication Date: 2016-04-06
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The defect of these two transformation methods is that the product hydrocarbons are synthesized through fatty acid and fatty acyl-CoA, because fatty acid synthesis and β-oxidation are all completed in the cytoplasm, and the accumulation of fatty acid is relatively toxic to cells and will not Accumulates at high concentrations, which limits the maximum yield possible for this type of pathway

Method used

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  • Microorganism gene modification method, genetically engineered bacteria for producing higher aliphatic alcohol and higher aliphatic hydrocarbon and application of genetically engineered bacteria
  • Microorganism gene modification method, genetically engineered bacteria for producing higher aliphatic alcohol and higher aliphatic hydrocarbon and application of genetically engineered bacteria
  • Microorganism gene modification method, genetically engineered bacteria for producing higher aliphatic alcohol and higher aliphatic hydrocarbon and application of genetically engineered bacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The preparation of embodiment 1 culture medium

[0056] The composition of LB medium is 5 g of yeast powder, 10 g of tryptone and 10 g of NaCl per liter of water. The solid medium is 1.5% agar powder added to the liquid base. In order to increase the selectivity of the medium, the concentration of ampicillin (Amp) was 50 μg / mL.

Embodiment 2

[0057] Example 2 Extraction of the Total DNA of Marine Bacteria VT8 and Synechococcus elongatus PCC7942

[0058] The cells were lysed with SDS-proteinase K, cetyltrimethylammonium bromide (CTAB) was used to precipitate cell debris and polysaccharides, and then precipitated with isopropanol to extract total DNA.

[0059] 1) A single colony of Marine Bacteria was inoculated in 100 mL of DSMZ514 medium and cultured at a suitable temperature for 2 days; Synechococcus elongatus was cultured in 100 mL of medium BG11 and cultured at a suitable temperature for 8 days.

[0060] 2) Take 20mL of bacterial liquid in a centrifuge tube, centrifuge at 13,000rpm for 2min, and discard the supernatant.

[0061] 3) The pellet was resuspended in 1.0 mL of 0.85% NaCl.

[0062] 4) Centrifuge at 13,000 rpm for 2 minutes at room temperature, and discard the supernatant.

[0063] 5) Resuspend the pellet in 550 μL 1×TE.

[0064] 6) Add 17 μL of lysozyme (35 mg / mL), and incubate at 37° C. for 30 minu...

Embodiment 3

[0082] The construction of embodiment 3 recombinant plasmid pTr-FAR

[0083] Cloning of the FAR gene

[0084] Using the total DNA extracted from MarinobacteraquaeoleiVT as a template for PCR reaction

[0085] The PCR reaction system is:

[0086]

[0087] The PCR reaction program is: 94°C 4min

[0088] 94°C 30s, 64°C 30s, 72°C 1min35cycles,

[0089] 72℃5min

[0090] The enzyme digestion system of the DNA fragment containing the FAR enzyme gene is composed according to the following ratio:

[0091]

[0092]

[0093] The digestion reaction was carried out overnight, and the degree of digestion was checked by electrophoresis. Recover the cleaved nucleic acid with a DNA recovery kit.

[0094] The enzyme digestion system of pTrcHisA plasmid is composed as follows:

[0095]

[0096] After the digestion reaction was carried out overnight, the degree of digestion was checked by electrophoresis. Recover the digested plasmid.

[0097] The digested plasmid and the DNA...

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Abstract

The invention relates to the field of molecular biology, in particular to a microorganism gene modification method, genetically engineered bacteria for producing higher aliphatic alcohol and higher aliphatic hydrocarbon and an application of the genetically engineered bacteria. The microorganism gene modification method includes the step of deleting paths for acyl-ACP to generate fatty acid in microbial cells to reduce generation of free fatty acid in cytoplasm. The method for deleting the paths for acyl-ACP to generate fatty acid in the microbial cells is a method for knocking out thioesterase TesC encoding genes in microorganisms. The yield of higher aliphatic alcohol and higher hydrocarbon of original escherichia coli can be increased through the microorganism gene modification method and the genetically engineered bacteria, and the genetically engineered bacteria are recombinant genetically engineered bacteria with the higher yield of higher aliphatic alcohol and higher hydrocarbon.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular, the present invention relates to a method for genetically modifying microorganisms, genetically engineered bacteria for producing higher fatty alcohols and higher fatty hydrocarbons, and applications thereof. Background technique [0002] The energy problem in today's world has become the bottleneck of the world's economic development, and the use of renewable resources such as biomass for material production has become a hot spot in current scientific research. [0003] Higher fatty alcohols are not only efficient biodiesel, but also have important uses in medicine and daily chemical products. With the depletion of petroleum resources, the biological production of fatty alcohols has received more and more attention. Long-chain alkanes and alkenes are the most promising biofuels. Its physical properties have a low melting point and are most suitable for use as energy subs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/02C12P5/00C12R1/19
Inventor 邢建民刘谊兰杨茂华王钦宏马延和
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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