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Construction body for efficiently synthesizing ethylene, and construction method and application thereof

A technology of constructs and ethylene, applied in the field of genetic engineering and biosynthesis, can solve the problems of instability and inability to synthesize ethylene stably

Active Publication Date: 2015-05-27
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research in this experiment found that the efe gene was unstable in Synechococcus sp. PCC7942, and the multiple mutation hot spots (hot spots) carried by the gene itself made the obtained genetically engineered strain unable to stably synthesize ethylene; Ungerer et al. used Synechocystis sp. ( Synechocystis sp.) PCC6803 was used as the host bacterium, and the ethylene production reached 5650 μL / L / h (Ungerer et al., 2012) through the optimization of the gene codon, the increase of the copy number of the efe gene and the optimization of the culture conditions of the engineered strain (Ungerer et al., 2012), which is significantly higher Related reports on other heterotrophic microorganisms in the early stage, which shows that the use of genetically engineered cyanobacteria to synthesize ethylene has great potential for research and development applications

Method used

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  • Construction body for efficiently synthesizing ethylene, and construction method and application thereof
  • Construction body for efficiently synthesizing ethylene, and construction method and application thereof
  • Construction body for efficiently synthesizing ethylene, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1: Selection against ethylene synthase gene

[0131] In order to ensure the continuous and stable expression of the ethylene synthase gene (efe gene) in cyanobacteria, by comparing the ethylene synthases from different sources, the ethylene synthase derived from Pseudomonas syringae pv. sesame, which can efficiently synthesize ethylene, was finally selected. Enzyme gene, and then obtain the efe gene codon-optimized and masked mutation hotspots (CTATG) (see eg SEQ ID NO: 1).

Embodiment 2

[0132] Example 2: Construction of vectors for driving the expression of ethylene synthase with different promoters.

[0133] In order to realize the expression of ethylene synthase (efe gene) driven by different promoters in Synechocystis sp. PCC6803, the expression plasmid pXX29 (P 6803psbA1 ), pXX32 (P 7942psbA1 ), pXX33 (P 7942psbA2 ) pXX47 (P cpcB ), pXX48 (P rbcL ), pXX49 (P ci ), pXX50 (P psbD ), pXX53 (P glnA ), pXX54 (P groESL ).

[0134] 1. Plasmid pXX29 (P 6803psbA1 ), pXX32 (P 7942psbA1 ), pXX33 (P 7942psbA2 ) build

[0135] Respectively, pTZ32 (derived from the pXT37b plasmid (see Chinese invention patent application 201010213758.5), which contains the 7942psbA1 promoter through conventional genetic engineering operations), pTZ33 (derived from the pXT37b plasmid, which contains the 7942psbA2 promoter through conventional genetic engineering operations) and pTZ34 ( Derived from the pXT37b plasmid, the 6803psbA2 promoter was used as a template through con...

Embodiment 3

[0152] Example 3: For P cpcB Construction of related vectors driving the expression of efe at the sll1981 and slr0370 sites of Synechocystis sp. PCC6803

[0153] In order to confirm the influence of integrating ethylene synthase at the sll1981 and slr0370 sites on the synthesis of ethylene, and confirm that the expression of ethylene synthase can be increased by increasing the copy number of the efe gene, thereby increasing the production of ethylene, the following construction is used for the synthesis of ethylene by P cpcB The driven ethylene synthase gene (efe gene) was integrated into the vector pXX64 at the sll1981 site in the PCC6803 genome, and used to convert the cpcB The driven ethylene synthase gene (efe gene) was integrated into the vector pXX65 at the slr0370 site in the PCC6803 genome.

[0154] 1. Construction of vector pXX64

[0155] Synechocystis PCC6803 genomic DNA was used as a template, PCR amplification was carried out with 1981-1 (SEQ ID NO:42) and 1981-2...

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Abstract

The invention relates to the fields of gene engineering and biosynthesis, and concretely relates to a construction body for efficiently synthesizing ethylene, and a construction method and an application thereof. The construction body comprises an active promoter and an ethylene synthetase gene (efe) controlled by the promoter. Carbon dioxide can be immobilized by above engineering bacterium through using solar energy in order to synthesize ethylene. An ethylene synthesis approach is constructed in cyanobacterial cells in order to realize immobilization of carbon dioxide in photosynthetic microbal cyanobacterial cells through using solar energy and synthesis of ethylene, wherein energy for ethylene synthesis is from the solar energy, and a carbon source is carbon dioxide. The method can be used to prepare bioethylene without restriction of petroleum shortage, and the bioethylene does not increase carbon emission.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biosynthesis, in particular to a construct for efficiently synthesizing ethylene, its construction method and application. Background technique [0002] Energy crisis and environmental degradation are the core issues facing the sustainable development of mankind. The massive consumption of fossil energy represented by oil, coal and natural gas will inevitably lead to the depletion of non-renewable energy and climate change caused by the release of carbon dioxide, and the ethylene industry, which uses oil as the main raw material, will also face great challenges. Ethylene is one of the chemical products with the largest output in the world. It is widely used in the production of polyethylene, ethylbenzene, polystyrene and ethanol. It is a very important basic raw material for organic chemicals. Therefore, finding a sustainable development path to solve the negative impact of the lack of pet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P5/02C12R1/01
CPCY02P20/133Y02P20/59
Inventor 吕雪峰解晓慢朱涛
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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