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Construction method and application of metabolic engineering escherichia coli strain for producing hydracrylic acid from acetic acid

A technology of hydroxypropionic acid and Escherichia coli, applied in the field of bioengineering, can solve the problems of slow growth, low production, and rapid inactivation of Klebsiella

Active Publication Date: 2018-04-06
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, using K.pneumoniae to anaerobically produce 3-HP with glycerol as a substrate, there are still many problems: DhaB is very sensitive to oxygen and is quickly inactivated under aerobic conditions; the catalytic effect of DhaB is strictly dependent on coenzyme B 12 , although Klebsiella can produce coenzyme B12, it produces very little under aerobic conditions and must be added additionally; Klebsiella grows slowly, and the fermentation conditions are not easy to control, etc. Due to the limitations of these factors, 3 - HP industrialization is limited
However, there are still no reports on the strain construction and fermentation conditions for producing lactic acid using acetic acid

Method used

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  • Construction method and application of metabolic engineering escherichia coli strain for producing hydracrylic acid from acetic acid
  • Construction method and application of metabolic engineering escherichia coli strain for producing hydracrylic acid from acetic acid
  • Construction method and application of metabolic engineering escherichia coli strain for producing hydracrylic acid from acetic acid

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Embodiment 1

[0043] Example 1. Construction of 3-hydroxypropionic acid, lactic acid and simultaneous production of 3-hydroxypropionic acid and lactic acid metabolic pathway

[0044] Using acetic acid as a carbon source to produce 3-hydroxypropionic acid, lactic acid, and 3-hydroxypropionic acid and lactic acid at the same time, acetic acid is first converted into acetyl-CoA, part of acetyl-CoA is converted into target metabolites, and part of acetyl-CoA enters the central metabolism way. To convert acetyl-CoA to different metabolites of interest, different plasmids were constructed. The specific operation is as follows:

[0045] Construction of 3-hydroxypropionic acid production pathway: Acetyl-CoA first generates malonyl-CoA under the action of acetyl-CoA carboxylase, and then generates 3-HP under the action of malonyl-CoA reductase. Therefore, genes encoding acetyl-CoA carboxylase and malonyl-CoA reductase from different sources can be selected for overexpression. This patent selects ...

Embodiment 2

[0048] Embodiment 2. to host bacterium related gene (ack, pta, ldh) promoter replacement

[0049] For the use of MG1655 as the starting bacterium to produce 3-HP and lactic acid with acetic acid as the sole carbon source and to produce 3-HP and lactic acid simultaneously, the expression of ack and pta was enhanced by replacing the promoters of ack and pta, thereby increasing the uptake rate of acetic acid. The promoter replacement method adopts the Red recombination method, and the replaced promoter is derived from the mutation of the trc promoter and is constitutively expressed. The resulting strain was named HY01, and the strain was preserved by the laboratory.

[0050] For the production of lactic acid and simultaneous production of lactic acid and 3-hydroxypropionic acid, the promoter of the lactate dehydrogenase coding gene (ldh) was replaced by Red recombination on the basis of HY01 strain, and the strain was named HY011. The specific operation is as follows: first, the...

Embodiment 3

[0055] Example 3. Knockout of related genes to increase the yield and yield of the target product

[0056] For the production of lactic acid, the respiratory chain strength was reduced by knocking out ubiX to accumulate lactic acid, and ubiX was knocked out in the HY011 strain to obtain the HY012 strain.

[0057] Since the gene expression of the gluconeogenesis pathway is up-regulated in the medium with acetic acid as the carbon source, excess carbon source flows to the gluconeogenesis pathway. Therefore, the red recombination method was used to knock out the genes pckA, adhE, and sfcA. The pckA knockout strains on the basis of HY 01 and HY012 were named HY 02 and HY 022, and the icdA knockout strains on the basis of HY 02 and HY 022 were named HY 03 and HY 032. For the production of 3-hydroxypropionic acid , further knocked out maeB on the basis of HY 02, and named the strain HY 031.

[0058] The specific operation of gene knockout is as follows: For the knockout of gene ub...

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Abstract

The invention discloses a construction method of a metabolic engineering escherichia coli strain for producing hydracrylic acid from acetic acid. Escherichia coli reformed by metabolic engineering isutilized for producing hydracrylic acid by fermenting acetic acid; and construction ways are as follows: constructing a metabolic way for producing hydracrylic acid from acetyl CoA, and / or over-expressing relevant genes of an acetic acid intake way so as to increase the transmission rate of acetic acid, and / or blocking or decreasing TCA circle so as to increase metabolic flow of acetyl CoA flowingtowards a target metabolite, and / or reducing decarboxylic reaction between malic acid or oxaloacetic acid so as to delete a byproduct generation way, and / or deleting key genes in an alcohol production way so as to regulate metabolic flow of an acetyl CoA node and / or regulating intracellular oxidation reduction balance through coenzyme engineering. According to the construction method, a production way for exogenously expressing hydracrylic acid is constructed, the metabolic ways, the adjustment and control are analyzed, and escherichia coli is reformed by virtue of a metabolic engineering measure, so that the prepared strain can be utilized for producing hydracrylic acid in a culture medium taking acetic acid as a carbon source.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to the construction of a recombinant Escherichia coli strain using acetic acid to produce hydroxypropionic acid. Background technique [0002] 3-Hydroxypropionic acid (3-HP) is an important platform compound. In 2004, the US Department of Energy (DOE) proposed 12 important platform compounds, and 3-HP ranked fourth. 3-HP is a three-carbon compound with active chemical properties and a carboxyl group and a hydroxyl group at both ends, making it widely used. 3-HP can be used to produce many compounds, such as acrylic acid, 1,3-propanediol, acrylamide, malonic acid, etc. It can also be used as a cross-linking agent for polymeric coatings and metal lubricants and an antistatic agent for textiles. At present, the methods for producing 3-HP are mainly chemical synthesis methods, such as bromo-3-hydroxypropionaldehyde oxidation method, propionic acid hydrolysis meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/42C12N15/53C12R1/19
CPCC12N9/0006C12N9/0008C12N15/70C12P7/42C12Y101/01C12Y101/05006C12Y102/07001
Inventor 吴辉黄兵李志敏杨昊叶勤
Owner EAST CHINA UNIV OF SCI & TECH
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