Genetic engineering strain for efficiently producing 1,3-propanediol, construction method for genetic engineering strain and application of genetic engineering strain

A technology of genetically engineered bacteria and propylene glycol, applied in the biological field, can solve problems such as abnormal termination of the fermentation process

Active Publication Date: 2020-11-27
QILU UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the process of producing 1,3-propanediol by Klebsiella pneumoniae fermentation method, the high concentration accumulation of 3-HPA w

Method used

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  • Genetic engineering strain for efficiently producing 1,3-propanediol, construction method for genetic engineering strain and application of genetic engineering strain
  • Genetic engineering strain for efficiently producing 1,3-propanediol, construction method for genetic engineering strain and application of genetic engineering strain
  • Genetic engineering strain for efficiently producing 1,3-propanediol, construction method for genetic engineering strain and application of genetic engineering strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Construction of plasmid pET28a-dhat:

[0064] Replace the T7 promoter on the vector plasmid pET28a with the promoter dhat that starts 1,3-propanediol oxidoreductase in Klebsiella pneumoniae, and replace the lactose operon with the operon corresponding to the promoter dhat, construct pET28a-dhat plasmid, the map of pET28a-dhat plasmid is as follows figure 1 As shown in A. The specific operation steps are as follows:

[0065] The Klebsiella pneumoniae genome was used as a template for PCR amplification, and bioinformatics software was used to design amplification primers for the promoter dhat sequence, and the promoter dhat sequence was amplified. The primer sequences are as follows:

[0066] Primer1: 5′-CGAGCTCATGAGCTATCGTATGTTTGA-3′ (containing BglII restriction site),

[0067] Primer2: 5'-CCCAAGCTTTCAGAATGCCTGGCGGAA-3' (containing SacI restriction site);

[0068] The PCR amplification system was prepared according to the kit instructions;

[0069] PCR ...

Embodiment 2

[0071] Embodiment 2: Construction of plasmid pET28a-dhat-Dhat:

[0072] The 1,3-propanediol oxidoreductase gene Dhat of Klebsiella pneumoniae was cloned into the pET28a-dhat plasmid, and the 1,3-propanediol oxidoreductase gene Dhat was located downstream of the promoter dhat to construct the pET28a-dhat-Dhat plasmid, The map of pET28a-dhat-Dhat plasmid is as follows figure 1 Shown in B. The specific operation steps are as follows:

[0073] Using Klebsiella pneumoniae genome as a template for PCR amplification, according to the Klebsiella pneumoniae 1,3-propanediol oxidoreductase gene Dhat sequence and the characteristics of the multiple cloning sites on the expression vector pET28a-dhat, using biological information Amplification primers were designed by scientific software, and the 1,3-propanediol oxidoreductase gene Dhat sequence (accession number MT674525) was amplified, and its nucleotide sequence was shown in SEQ ID NO.1; the primer sequences were as follows:

[0074] ...

Embodiment 3

[0079] Embodiment 3: Construction of plasmid pET28a-dhat-YqhD

[0080] The 1,3-propanediol oxidoreductase isozyme gene YqhD in Escherichia coli was cloned into the pET28a-dhat plasmid, and the 1,3-propanediol oxidoreductase isozyme gene YqhD was located downstream of the promoter dhat to construct pET28a-dhat- YqhD plasmid, the map of pET28a-dhat-YqhD plasmid is as follows figure 1 C shown. The specific operation steps are as follows:

[0081] PCR amplification was performed using the Escherichia coli genome as a template, and the amplification was designed using bioinformatics software according to the sequence of the Escherichia coli 1,3-propanediol oxidoreductase isozyme gene YqhD and the characteristics of the multiple cloning sites on the expression vector pET28a-dhat Primers, amplified to obtain 1,3-propanediol oxidoreductase isoenzyme gene YqhD sequence (accession number MT674524), its nucleotide sequence is shown in SEQ ID NO.2; primer sequence is as follows:

[008...

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Abstract

The invention relates to a genetic engineering strain for efficiently producing 1,3-propanediol, a construction method for the genetic engineering strain and application of the genetic engineering strain. According to the genetic engineering strain, the construction method therefor and the application of the genetic engineering strain, the enzyme activity of 1,3-propanediol oxidoreductase is improved through strengthening expression of the 1,3-propanediol oxidoreductase in Klebsiella pneumoniae, meanwhile, a NADH regeneration way is constructed, thus, the accumulation of 3-hydroxypropanal is lowered, the conversion of the 1,3-propanediol from the 3-hydroxypropanal is strengthened, and thus, the yield of the 1,3-propanediol is increased. The enzyme activity of the genetic engineering strain, i.e., the 1,3-propanediol oxidoreductase, constructed by the method is 3.25 to 3.5 times that of a starting strain; and the enzyme activity of formate dehydrogenase is 0.414U/mg to 0.45U/mg. Throughcarrying out fed-batch fermentation for 29 hours by employing the genetic engineering strain constructed by the method, the yield of the 1,3-propanediol is increased by 43.89%-97.93% compared with that of the starting strain; and due to a dual-gene synergistic effect, the output and yield of the 1,3-propanediol are increased remarkably.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium for efficiently producing 1,3-propanediol and its construction method and application. Background technique [0002] 1,3-Propanediol (1,3-propanediol, 1,3-PD) is an important chemical raw material. In addition to being used as a solvent, its main function is as a monomer for the synthesis of polyester and polyurethane. - Polytrimethylene terephthalate (PTT), a polymer synthesized from propylene glycol and terephthalic acid, is a new type of polyester material, which has the characteristics of easy dyeing, good elasticity, antistatic, degradable, good softness, etc. features have also attracted people's attention in recent years. The production methods of 1,3-propanediol include chemical method and microbial fermentation method. With the depletion of fossil fuel resources, chemical synthesis based on petroleum resources is severely limi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/18C12R1/22
CPCC12P7/18C12N9/0008C12N9/0006C12Y102/01002C12Y101/01202C12N15/74
Inventor 马春玲王瑞明李丕武王艳霞
Owner QILU UNIV OF TECH
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