High-flux screening method of aliphatic hydrocarbon generation gene, obtained mutant and application

A screening method, technology of hydrocarbon genes, applied in the field of bioengineering

Active Publication Date: 2016-07-27
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

Limited by the analysis method, there is no precedent for the implementati...

Method used

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  • High-flux screening method of aliphatic hydrocarbon generation gene, obtained mutant and application
  • High-flux screening method of aliphatic hydrocarbon generation gene, obtained mutant and application
  • High-flux screening method of aliphatic hydrocarbon generation gene, obtained mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of Escherichia coli aliphatic hydrocarbon synthesis pathway and determination of aliphatic hydrocarbon content

[0054] Strains:

[0055] Escherichia coli BL21(DE3)ΔfadE

[0056] Carrier:

[0057] Plasmid 1 carrying fatty aldehyde deformyloxygenase ADO and fatty acyl-ACP reductase AAR: pACYCduet-ADO-AAR ( figure 1 )

[0058] Medium:

[0059] LB medium (containing tryptone 10g / L, yeast extract powder 5g / L, NaCl10g / L, if it is a solid medium, add agar 15g / L per liter of medium)

[0060] Improved M9 medium (containing 6g / LNa 2 HPO 4 ,3g / LKH 2 PO 4 ,0.5g / LNaCl,2g / LNH 4 Cl,0.25g / LMgSO 4 ·7H 2 O,11mg / LCaCl 2 ,27mg / LFeCl 3 ·6H 2 O,2mg / LZnCl 2 4H 2 O,2mg / LNa 2 MoO 4 2H 2 O,1.9mg / LCuSO 4 ·5H 2 O,0.5mg / LH 3 BO 3 , 1mg / L vitamin B1, 200mM Bis-Tris (pH7.25) and 0.1% (v / v) Triton-X100)

[0061] Implementation steps: transform plasmid 1 into Escherichia coli BL21(DE3)ΔfadE, culture the obtained transformant in resistant LB medium (contai...

Embodiment 2

[0063] Example 2 Using aliphatic hydrocarbon components to detect the synthesis of aliphatic hydrocarbons in cells

[0064] Strains:

[0065] Escherichia coli BL21(DE3)ΔfadE; Escherichia coli DH5α

[0066] Carrier:

[0067] Plasmid 1 carrying fatty aldehyde deformyloxygenase ADO and fatty acyl-ACP reductase AAR: pACYCduet-ADO-AAR; plasmid 2 carrying aliphatic hydrocarbon detection element AlkR-PalkM::GFP ( image 3 ):: pCom8-AlkR-PalkM::GFP

[0068] Medium:

[0069] LB medium (containing tryptone 10g / L, yeast extract powder 5g / L, NaCl10g / L, if it is a solid medium, add agar 15g / L per liter of medium)

[0070] Improved M9 medium (containing 6g / LNa 2 HPO 4 ,3g / LKH 2 PO 4 ,0.5g / LNaCl,2g / LNH 4 Cl,0.25g / LMgSO 4 ·7H 2 O,11mg / LCaCl 2 ,27mg / LFeCl 3 ·6H 2 O,2mg / LZnCl 2 4H 2 O,2mg / LNa 2 MoO 4 2H 2 O,1.9mg / LCuSO 4 ·5H 2 O,0.5mg / LH 3 BO 3 , 1mg / L vitamin B1, 200mM Bis-Tris (pH7.25) and 0.1% (v / v) Triton-X100)

[0071] Implementation steps: Plasmid 1 and plasmid 2 a...

Embodiment 3

[0073] Example 3 High-throughput screening of ADO mutant genes with high aliphatic hydrocarbon production ability

[0074] The ADO gene was amplified with primers ADO-For and ADO-Rev, and an error-prone PCR reaction system was used to introduce random base mutations. The primer sequences used are as follows (the underlined part is the restriction site):

[0075] ADO-For: TATA CCATGG CGCAGCTTGAAGCCAGCCT

[0076] ADO-Rev: CCTG GAATTC AAACGGCCGCAA

[0077] Specifically, in the PCR reaction, the plasmid carrying the ADO gene was used as a template, ADO-For and ADO-Rev were used as upstream and downstream primers respectively, and rTaqDNA polymerase from Takara Company was used. The reaction system is shown in the table below:

[0078] Table 1 PCR reaction system

[0079]

[0080]

[0081] The PCR reaction conditions are as follows: first 95°C for 2min; then 94°C for 1min, 55°C for 1min, 72°C for 1min, a total of 30 cycles; finally 72°C for 10min.

[0082] After the ...

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Abstract

The invention discloses a high-flux screening method of an aliphatic hydrocarbon generation gene, an obtained mutant and application, and belongs to the technical field of biological engineering.By means of the method, a carrier carrying a potential aliphatic hydrocarbon generation gene is introduced into a host cell carrying a detection element, the host cell is cultured, the expression situation of the target gene is authenticated by responding to a signal of the detection element, and the mutant with high aliphatic hydrocarbon yield is separated out.Meanwhile, the invention further provides the aldehyde deformylating oxygenase mutant obtained through the screening method.The aldehyde deformylating oxygenase mutant has higher aliphatic hydrocarbon synthesis efficiency compared with an enzyme wild type.By means of the method, the specific gene or mutant with higher aliphatic hydrocarbon yield can be easily and rapidly screened from candidate genes with potential aliphatic hydrocarbon generation capacity and a mutant library of the candidate genes.

Description

technical field [0001] The invention relates to a high-throughput screening method for aliphatic hydrocarbon-producing genes, the obtained mutant and its application, and belongs to the technical field of bioengineering. Background technique [0002] The development of biofuels is one of the effective ways to solve the problems of resources, energy and environment faced by human beings. Since aliphatic hydrocarbons are the main components of gasoline, diesel, aviation kerosene and other engine fuels, they have high energy density, low hygroscopicity and low volatility, and are compatible with existing transportation facilities and engine systems, bio-aliphatic hydrocarbons have been considered as a The most promising high-quality biofuel. [0003] In recent years, it has been found in some organisms that some specific enzymes can convert intracellular aliphatic compounds (such as fatty acids and fatty aldehydes) into aliphatic hydrocarbons. For example, in cyanobacteria, t...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/81C12N15/74C12N1/21C12N1/19C12N9/02C12N9/88C12P5/02
CPCC12N9/0008C12N9/0069C12N9/88C12P5/02C12Y102/0108
Inventor 吕雪峰吴伟梁雅静张磊
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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