Construction method and application of genetic engineering escherichia coli for increasing content of lactic acid components in polyhydroxybutyrate lactate

A technology of polyhydroxybutyrate lactate and Escherichia coli, applied in the field of bioengineering, can solve the problem that the enzyme that degrades LA-CoA has not been reported, and achieves the effect of increasing the content of lactic acid components and reducing waste

Inactive Publication Date: 2020-07-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It shows that there is a degradative enzyme active on LA-CoA, which leads to the degradation of LA-CoA, and the absence of this degradative enzyme will be of great significance for increasing the content of lactic acid components in P(3HB-co-LA)
But in Escherichia coli, there is no report about the enzyme that degrades LA-CoA

Method used

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  • Construction method and application of genetic engineering escherichia coli for increasing content of lactic acid components in polyhydroxybutyrate lactate
  • Construction method and application of genetic engineering escherichia coli for increasing content of lactic acid components in polyhydroxybutyrate lactate
  • Construction method and application of genetic engineering escherichia coli for increasing content of lactic acid components in polyhydroxybutyrate lactate

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Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Knockout gene dld prevents lactate from being converted into pyruvate

[0030] In the later stage of fermentation, lactic acid may synthesize pyruvate under the action of D-lactate dehydrogenase (dld), which reduces the lactic acid component in P(3HB-co-LA). Therefore, the gene dld was knocked out by Red recombination method, and the obtained strain was named WXJ01. The specific operation of gene knockout is as follows:

[0031] For the knockout of the gene dld, primers were firstly designed (primer sequences are shown in the table below), and a DNA fragment of about 1700 bp with kanamycin resistance was cloned by PCR. The plasmid pKD46 was introduced into the host bacteria through calcium transformation, and the recombinants were selected with ampicillin. Recombinant bacteria introduced with pKD46 were cultured at 30°C to OD 600 At about 0.3, L-arabinose was added for induction for 1 hour, and then 10% glycerol was used to prepare electroporation competen...

Embodiment 2

[0033] Example 2. Knockout gene ydiI weakens the degradation pathway of LA-CoA

[0034] The thioesterase in Escherichia coli has a degradative effect on LA-CoA, making LA-CoA move towards the fatty acid synthesis pathway, resulting in a decrease in the flux of LA-CoA synthetic polymers, which is ultimately unfavorable for the P(3HB-co- Synthesis of LA). Therefore, the present invention knocks out the endogenous thioesterase ydiI of Escherichia coli, weakens the degradation pathway of LA-CoA, and makes LA-CoA, as the direct precursor of P(3HB-co-LA), more towards the polymer synthesis pathway . On the basis of strain WXJ01, the gene ydiI was knocked out by Red recombination method, and the obtained strain was named WXJ02. The specific operation of gene knockout is the same as that of gene dld.

Embodiment 3

[0035] Example 3. Knockout gene yciA weakens the degradation pathway of LA-CoA

[0036] The thioesterase yciA and ydiI in Escherichia coli have similar properties, and both can degrade LA-CoA to form the corresponding fatty acid. Therefore, the present invention knocks out the gene yciA on the basis of the strain WXJ01, and the obtained strain is named WXJ021, which weakens LA-CoA. -The degradation of CoA makes it more towards the synthetic pathway of P(3HB-co-LA). The specific operation of gene knockout is the same as that of gene dld.

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Abstract

The invention discloses a construction method and an application of genetic engineering escherichia coli for increasing the content of lactic acid components in polyhydroxybutyrate lactate. The transformation pathway is that construction of a pyruvic acid synthesized polyhydroxybutyrate lactate metabolic pathway is carried out, and over-expression of related gene in 2-hydroxy propionyl-CoA synthesizing path is carried out to increase synthesis of LA-CoA; and the transformation pathway also comprises weakening of one or two of escherichia coli endogenous thioesterase ydiI and yciA, so as to reduce flowing of LA-CoA to a lactic acid synthesis pathway. According to the method disclosed by the invention, the La-CoA in escherichia coli can effectively flow to a synthesis module of a target product, the waste of the target product synthesis precursor La-CoA is reduced, the escherichia coli is transformed by analysis and regulation of the metabolic pathway via a gene engineering means, and the content of the lactic acid components in the polyhydroxy alkanoate produced by an obtained recombinant strain is obviously increased.

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 genetically engineered Escherichia coli strain for producing polyhydroxybutyrate lactate by utilizing glucose or xylose. Background technique [0002] Currently, commonly used plastics are mostly synthesized from fossil fuels such as oil and natural gas, leading to environmental problems such as global warming and accumulation of solid waste, which urge us to develop and utilize sustainable raw materials to produce bio-based polymers. Polyhydroxyalkanoates (Polyhydroxy alkanoates, PHA) is a kind of polyester produced by microorganisms, which has attracted extensive attention of researchers due to its excellent properties such as biodegradability, optical properties and biocompatibility. [0003] Polylactic acid is a promising biomass-derived polymer because it can replace petroleum-based plastics and has several de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N9/1029C12N9/13C12P7/625C12Y101/01036C12Y203/01009C12Y203/01016C12Y208/03001
Inventor 吴辉魏香菊吴榉郭鹏业
Owner EAST CHINA UNIV OF SCI & TECH
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