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Construction method and application of metabolic engineering escherichia coli strain for producing (R)-3-hydroxybutyric acid by using acetic acid or salt thereof

A technology of hydroxybutyric acid and metabolic engineering, applied in the field of bioengineering to achieve the effects of improving yield and yield, less energy consumption and fewer steps

Pending Publication Date: 2021-03-26
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is still no report on the use of metabolic engineering strains to produce 3-HB from acetic acid

Method used

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  • Construction method and application of metabolic engineering escherichia coli strain for producing (R)-3-hydroxybutyric acid by using acetic acid or salt thereof
  • Construction method and application of metabolic engineering escherichia coli strain for producing (R)-3-hydroxybutyric acid by using acetic acid or salt thereof
  • Construction method and application of metabolic engineering escherichia coli strain for producing (R)-3-hydroxybutyric acid by using acetic acid or salt thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Construction of (R)-3-hydroxybutyric acid production pathway

[0022] The enzymes that generate (R)-3-hydroxybutyrate from acetyl-CoA are β-ketothiolase (encoded by phaA from Ralstonia eutropha), acetoacetyl-CoA reductase (encoded by phaB from Ralstonia eutropha) and Propionyl CoA transferase (by pct derived from Clostridium beijeriinckii8052 2040 or pct 4543 coding). Using the pTrc99a plasmid as a vector, the recombinant plasmid pTrc99a-phaA-RBS-phaB (pTrc99a-phaAB for short) was obtained by overexpressing phaA and phaB derived from Ralstonia eutropha at the same time. Using pBAD33 plasmid as vector, overexpress pct derived from Clostridium beijeriinckii 8052 2040 or pct 4543 At the same time, the Trc promoter was added before the gene to obtain the recombinant plasmid pBAD33-Trc-pct 2040 or pBAD33-Trc-pct 4543 . In addition, the pTrc99a vector has a gene lacI encoding the lac repressor protein, which can block the transcription of the target gene cau...

Embodiment 2

[0031] Embodiment 2. optimal host and pct gene screening shake flask fermentation

[0032] Using Escherichia coli MG1655, BW25113, and W3110 as starting bacteria (MG1655, BW25113, and W3110 are provided by The ColiGenetic Stock Center), the plasmids for constructing (R)-3-hydroxybutyric acid production pathways were transferred by calcium transfer, and MG1655 (pTrc -99a-phaAB,pBAD33-Trc-pct 2040 ), MG1655 (pTrc-99a-phaAB, pBAD33-Trc-pct 4543 ), BW25113 (pTrc-99a-phaAB, pBAD33-Trc-pct 2040 ), BW25113 (pTrc-99a-phaAB, pBAD33-Trc-pct 4543 ), W3110 (pTrc-99a-phaAB, pBAD33-Trc-pct 2040 ), W3110 (pTrc-99a-phaAB, pBAD33-Trc-pct 4543 ). In Table 3, pTrc-99a-phaAB is abbreviated as phaAB, pBAD33-Trc-pct 2040 abbreviated as pct 2040 , pBAD33-Trc-pct 4543 abbreviated as pct 4543 .

[0033] Shake flask fermentation operation: Pick a single colony on the plate and inoculate it in a test tube containing 3mL LB for overnight culture, then transfer to a conical flask containing 50mL...

Embodiment 3

[0040] Embodiment 3. Fermentation temperature optimizes shake flask fermentation

[0041] Using Escherichia coli BW25113 or W3110 as the starting bacteria, the plasmids for the construction of (R)-3-hydroxybutyric acid production pathway were transferred by calcium transfer, and BW25113 (pTrc-99a-phaAB, pBAD33-Trc-pct 4543 ), W3110 (pTrc-99a-phaAB, pBAD33-Trc-pct 2040 ). In Table 4, pTrc-99a-phaAB is abbreviated as phaAB, pBAD33-Trc-pct 2040 abbreviated as pct 2040 , pBAD33-Trc-pct 4543 abbreviated as pct 4543 .

[0042] The composition of M9 medium, the determination method of (R)-3-hydroxybutyric acid and acetic acid, and the determination method of cell concentration are the same as those described in Example 2. The shaking flask fermentation operation is the same as that described in Example 2, except that it is transferred to different temperature culture (25° C., 30° C. or 37° C.) after induction.

[0043] The shake flask fermentation results are shown in Table 4....

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Abstract

The invention provides a construction method and application of a metabolic engineering escherichia coli strain for producing (R)-3-hydroxybutyric acid by using acetic acid or salt thereof. The metabolic pathway is as follows: the acetic acid or salt thereof is used for producing the (R)-3-hydroxybutyric acid by using beta-ketothiolase and acetoacetyl CoA reductase, and finally the (R) 3hydroxybutyric acid is produced by using the non-specificity of propionyl CoA transferase. According to the method, a modified path is an exogenous metabolic path for producing (R)-3-hydroxybutyric acid by acetyl CoA, and the path utilizes the non-specificity of propionyl CoA transferase, so that the steps are fewer than those of the traditional path, and the energy consumption is less. According to the method, the (R)-3-hydroxybutyric acid is produced by taking acetic acid as a carbon source in escherichia coli, the yield and yield of the (R)-3-hydroxybutyric acid are improved by using different propionyl CoA transferase and host escherichia coli, and the metabolic pathway is proved to have universality in escherichia coli. According to the method, the (R)-3-hydroxybutyric acid is produced by taking the acetic acid-containing synthesis gas fermentation liquor as a culture medium through fermentation.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to a construction method and application of a metabolic engineering Escherichia coli strain for producing (R)-3-hydroxybutyric acid by utilizing acetic acid or a salt thereof. Background technique [0002] Acetic acid is a cheap and easy-to-obtain carbon source. At present, it is mainly synthesized by chemical methods. The synthesis processes mainly include acetaldehyde method, butane (or light oil) liquid-phase oxidation method and methanol carbonylation method. Among them, the catalytic efficiency of methanol carbonylation method is High, low raw material price, simple operation process, and milder reaction conditions than other processes, it is the main method for the chemical synthesis of acetic acid. Nearly 40% of acetic acid in the world is prepared by methanol carbonylation, while butane (or light oil ) liquid phase oxidation method has advantages. In ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/42C12R1/19
CPCC12N15/70C12N15/52C12N9/0006C12N9/13C12N9/1029C12P7/42C12Y101/01036C12Y208/03001C12Y203/01009
Inventor 吴辉费鹏赖宁玉
Owner EAST CHINA UNIV OF SCI & TECH
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