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A kind of genetic engineering bacteria for efficient production of 1,3-propanediol and its construction method and application

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, and achieve the effects of improving induction effect, high glycerol tolerance, and reducing accumulation

Active Publication Date: 2022-07-08
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of producing 1,3-propanediol by Klebsiella pneumoniae fermentation method, the high concentration accumulation of 3-HPA will seriously inhibit the growth of cells and the synthesis of metabolites, resulting in abnormal termination of the fermentation process

Method used

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  • A kind of genetic engineering bacteria for efficient production of 1,3-propanediol and its construction method and application
  • A kind of genetic engineering bacteria for efficient production of 1,3-propanediol and its construction method and application
  • A kind of genetic engineering bacteria for efficient production of 1,3-propanediol and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0064] The T7 promoter on the vector plasmid pET28a was replaced with the 1,3-propanediol oxidoreductase promoter dhat in Klebsiella pneumoniae, and the lactose operon was replaced with the operon corresponding to the promoter dhat to construct pET28a-dhat plasmid, the map of pET28a-dhat plasmid is as follows figure 1 A shown. 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 the amplification primers of the promoter dhat sequence, and the promoter dhat sequence was obtained by amplification. The primer sequences are as follows:

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

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

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

[0069] PCR am...

Embodiment 2

[0071] Example 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 shown in figure 1 shown in B. The specific operation steps are as follows:

[0073] PCR amplification was performed using the Klebsiella pneumoniae genome as a template, and the biological information was used according to the Dhat sequence of the Klebsiella pneumoniae 1,3-propanediol oxidoreductase gene and the characteristics of the multi-cloning site on the expression vector pET28a-dhat. The amplification primers were designed by the software, and the 1,3-propanediol oxidoreductase gene Dhat sequence (accession number MT674525) was obtained by amplification, and its nucleotide sequence was shown in SEQ ID NO.1; the primer s...

Embodiment 3

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

[0080] The 1,3-propanediol oxidoreductase isoenzyme gene YqhD in E. coli was cloned into the pET28a-dhat plasmid, and the 1,3-propanediol oxidoreductase isoenzyme 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 shown in C. The specific operation steps are as follows:

[0081] PCR amplification was carried out using the E. coli genome as a template. According to the E. coli 1,3-propanediol oxidoreductase isoenzyme gene YqhD sequence and the characteristics of the multi-cloning site on the expression vector pET28a-dhat, the amplification was designed using bioinformatics software. The primer was amplified to obtain the 1,3-propanediol oxidoreductase isoenzyme gene YqhD sequence (accession number MT674524), and its nucleotide sequence was shown in SEQ ID NO.2; the primer sequence was as follows:

[0082] Primer5: ...

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Abstract

The invention relates to a genetically engineered bacterium capable of efficiently producing 1,3-propanediol and a construction method and application thereof. The present invention strengthens the expression of 1,3-propanediol oxidoreductase in Klebsiella pneumoniae to enhance the enzymatic activity of 1,3-propanediol oxidoreductase, and simultaneously constructs a NADH regeneration pathway to reduce the accumulation of 3-hydroxypropionaldehyde, Enhances the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, thereby increasing the yield of 1,3-propanediol. The enzymatic activity of the 1,3-propanediol oxidoreductase of the genetically engineered bacteria constructed by the present invention is 3.25-3.5 times that of the starting strain; the enzymatic activity of the formate dehydrogenase is 0.414-0.45 U / mg. Using the genetically engineered bacteria constructed by the present invention to carry out fed-batch fermentation for 29 hours, the yield of 1,3-propanediol is increased by 43.89%-97.93% compared with the starting strain, and the synergistic effect of the double genes significantly increases the yield of 1,3-propanediol and yield.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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