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Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof

A cyanobacteria, hydrocarbon gene technology, applied in the application, bacteria, genetic engineering and other directions, can solve the problem of not reaching industrial application, and achieve the effect of increasing carbon emissions

Active Publication Date: 2016-05-18
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the level of aliphatic hydrocarbon synthesis by genetically engineered cyanobacteria is far from the level of industrial application. The mining of germplasm resources and genetic resources of hydrocarbon-producing cyanobacteria is an important factor to further improve the efficiency of aliphatic hydrocarbon synthesis in cyanobacteria and enrich the diversity of hydrocarbon production. one

Method used

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  • Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof
  • Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof
  • Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1: the cultivation and biomass determination of cyanobacteria

[0105] Cultivation and physiological index determination of cyanobacteria:

[0106] The cyanobacterial strains in Table 1 were cultured by aeration culture, and the algae strains were inoculated into a 500mL Erlenmeyer flask containing 300mL of BG11 medium, at 30°C, 30μE.m -2 .s -1 Grow in continuous light for 8 to 10 days. In order to measure its growth, 10ml of the well-mixed cyanobacteria suspension was passed through a 0.45μm pre-weighed nitrocellulose membrane (ddH 2 O washed three times, 110 ℃ high temperature drying) after suction filtration, put 110 ℃, dry 24 hours, and weigh its dry weight. Finally, the dry weight of the bacteria was used as a reference index to measure the biomass. The strains and their sources are listed in Table 1.

[0107] Algae (bacteria) strains used in Table 1 and their sources

[0108]

[0109] a FACHB: FreshwaterAlgalCultureCollectionofTheInstituteOfHy...

Embodiment 2

[0111] Embodiment 2: the aliphatic hydrocarbon composition and content determination of cyanobacteria

[0112] Each of the above-mentioned 200mL of cyanobacteria (in Table 1) that was in the plateau phase and well mixed was collected, and mixed with 10mLddH 2 O resuspended the above-mentioned cyanobacteria respectively, and then placed them on an ice-water bath, and ultrasonically disrupted them for 10 minutes (10son, 10soff). After crushing, add 30 μg of n-eicosane as an internal standard, and after mixing, add 10 mL of chloroform:methanol (V:V is 2:1), shake and mix vigorously for 1-2 hours. Then centrifuge at 6,000 g for 10 min, transfer the lower organic phase to a new test tube, and dry it with nitrogen at 55°C. Add 1ml of n-hexane to dissolve the extracted aliphatic hydrocarbons, then filter through a 0.22μm membrane filter, and transfer to a 2ml gas phase vial. Use Agilent7890A gas chromatography-mass spectrometry (GC-MS) to measure aliphatic hydrocarbons. The GC-MS t...

Embodiment 3

[0119] Example 3: Sequencing of the cyanobacteria genome and sequence retrieval of the key gene ado-aar for hydrocarbon production

[0120] (1) The extraction steps of the cyanobacteria genome are as follows:

[0121] Collect about 50ml of each bacterial solution that is fully mixed, centrifuge at 5,000g, and use 1.8ml of solution A (50mM Tris-Cl+50mMNa 2 EDTA+1MNaCl), and then moderately homogenized with a 2ml tissue homogenizer to disperse the filamentous cell homogenate into a single cell state.

[0122] Then proceed as follows: the cell suspension of each of the above-mentioned cyanobacteria and algal strains is divided into 6 Eppendorf tubes (300 μl / tube), and 10% sodium lauryl sarcosine aqueous solution (Sarkosyl solution) is added to each tube. The final concentration is 0.1%, and then stored at 4°C for 1 hr, centrifuged at 10,000g for 15 min to pellet the cells; wash the cells twice with 1ml solution A;

[0123] After washing, resuspend the cyanobacteria and algae ce...

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Abstract

The invention discloses a cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof. The aliphatic hydrocarbon synthesis gene includes the coding genes of fatty acyl-acyl carrier protein reductase (AAP) and fatty aldehyde deformylating oxygenase (ADO). The invention also relates a contractor, wherein the contractor comprises a promoter having activity and the aliphatic hydrocarbon key synthesis gene controlled by the promoter. By converting the contractor into escherichia coli, the genetic engineering escherichia coli can have an aliphatic hydrocarbon synthesis capacity. By converting the at least one contractor into the cyanobacteria, the aliphatic hydrocarbon yield of the genetic engineering cyanobacteria can be significantly improved. The cyanobacteria hydrocarbon production key gene disclosed by the invention can provide a new candidate gene resource for directly synthesizing aliphatic hydrocarbon novel bio-fuels from microorganisms.

Description

technical field [0001] The invention relates to the field of energy microbial germplasm resources and gene resources, in particular to a key gene for cyanobacteria aliphatic hydrocarbon synthesis. Background technique [0002] Energy is the pillar of modern industry. The increasing shortage of traditional energy represented by fossil fuels is the bottleneck restricting the sustainable development of my country's economy. The application of renewable biofuels has become one of the best choices to alleviate the energy crisis and improve the ecological environment. Fossil fuels such as gasoline, diesel and aviation kerosene widely used in the transportation industry are mainly composed of aliphatic hydrocarbons of a specific chain length range, which have high energy density, low hygroscopicity, low volatility, and are compatible with existing engines It is the best substitute for traditional petrochemical liquid fuels (Keasling and Chou2008). [0003] Petroleum, ancient sedim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/63C12N1/21C12R1/19
Inventor 吕雪峰朱涛莫慧琳
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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