Escherichia coli for synthesis of propane through pathway of valine and establishing method of escherichia coli

A technology of Escherichia coli and valine, applied in the field of genetic engineering, can solve problems such as restricting propane production, achieve efficient gene knockout programs, reduce the activity of aldehyde reductase, and weaken the activity of aldehyde reductase

Inactive Publication Date: 2016-02-03
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the n-butyraldehyde provided by the fatty acid synthesis system in thi...

Method used

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  • Escherichia coli for synthesis of propane through pathway of valine and establishing method of escherichia coli
  • Escherichia coli for synthesis of propane through pathway of valine and establishing method of escherichia coli
  • Escherichia coli for synthesis of propane through pathway of valine and establishing method of escherichia coli

Examples

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Embodiment 1

[0080] Example 1 Construction of gene knockout plasmid

[0081] In this example, a two-step seamless method is used to knock out multiple genes of E. coliBW25113. For each target gene knocked out in this method, two corresponding plasmids containing homology arms of the target gene to be knocked out need to be constructed. These two plasmids served as PCR templates, providing two fragments for homologous recombination during the knockout process. Therefore, the following figure 1 pEBS-yqhD in and figure 2 Taking the pEBS-yqhD-Cat-sacB plasmid as an example, the construction steps of the knockout plasmid are described in detail. The other 12 gene knockout plasmids were constructed in the same way.

[0082]Find the nucleic acid sequence of the yqhD gene in the genome of E. coliBW25113 in NCBI, and design two pairs of primers, yqhD-F / yqhD-fusion-R, yqhD-R / yqhD-fusion-F. Using the E.coliBW25113 genome as a template, the primers yqhD-F / yqhD-fusion-R were used to carry out PCR ...

Embodiment 2

[0085] Example 2 Construction of plasmid pBAD33-alsS-Kivd

[0086] The acetolactate synthase gene alsS from Bacillus subtilis and the 2-keto acid decarboxylase gene Kivd from Lactococcus lactis were synthesized by Shanghai Yingweijieji Company and connected to the pMD18-T vector to obtain pMD18-T-alsS and pMD18- T-Kivd plasmid. The alsS gene fragment was amplified from pMD18-T-alsS using primers alss-SacI / alss-fusion-R, and the Kivd gene fragment was amplified from pMD18-T-Kivd using Kivd-SphI / Kivd-fusion-F. The two fragments were purified and recovered by gel tapping respectively, and a fusion PCR reaction was performed. The fused fragment was used as a template, and then the primers alsS-SacI / Kivd-SphI were used to amplify. Finally, the amplified fusion fragment alsS-Kivd was purified and recovered by gel cutting, and digested with SacI and SphI. The digested alsS-Kivd fusion fragment was connected with the pBAD33 vector that was also double digested by SacI and SphI, and ...

Embodiment 3

[0087] Example 3 Construction of plasmid pAL96-ilvCD

[0088] Using the BW25113 genome as a template, primers ilvC-F / ilvC-fusion-R were used to perform PCR reaction to obtain ilvC gene fragments; primers ilvD-fusion-F / ilvD-R were used to perform PCR reactions to obtain ilvD gene fragments. The above-mentioned two fragments were recovered by gel-tapping purification, and a fusion PCR reaction was carried out. The fused fragments were used as templates to amplify the fusion fragments using primers ilvC-F / ilvD-R. Finally, the amplified fusion fragment ilvCD was purified and recovered by gel cutting, and digested with SacI and Spll. The digested ilvCD fusion fragment was connected with the pAL96 vector that was also double digested by SacI and SplI, and the ligation product was transformed into E.coliDH5α and screened on the LB plate containing ampicillin to obtain the pAL96-ilvCD plasmid ( Figure 28 ). The primer sequences used are shown in SEQ ID NO.57-SEQ ID NO.60.

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Abstract

The invention discloses escherichia coli for synthesis of propane through the pathway of valine and an establishing method of the escherichia coli, and belongs to the technical field of genetic engineering. According to the escherichia coli, nine sorts of aldose reductase genes, a lactic dehydrogenase gene, a fumarate reductase gene, a formate-lyase gene and an overall transcription factor gene are eliminated. Meanwhile, overexpression of an acetolactate synthetase gene, a ketol-acid reductoisomerase gene, a DHAD gene, a 2-ketoacid decarboxylase gene and an aldehyde deformylating oxygenase gene is achieved. The invention provides the establishing method of the escherichia coli at the same time. Through overexpression of the propane synthesis pathway in an improved strain, the strain successfully achieves biosynthesis of propane.

Description

technical field [0001] The invention relates to an Escherichia coli that uses a valine metabolic pathway to synthesize propane and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] The continuous growth of the global population and the economic development of developing countries have promoted the exploitation and use of fossil energy, leading to serious environmental pollution and climate change problems. Therefore, it is of great significance to develop environmentally friendly clean energy. [0003] Propane is one of the main components of liquefied petroleum gas, which has a high boiling point and combustion calorific value, so it is easy to liquefy and transport. Its combustion process is sufficient to be compatible with existing internal combustion engines and energy transport systems. In addition, propane can be used as a refrigerant and spray propellant, replacing chlorofluorocarbons that are destr...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P5/02C12R1/19
Inventor 吕雪峰张磊谈晓明吴伟
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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