High-yield propionic acid propionibacterium jensenii engineering bacterium and application thereof

A technology of Propionibacterium jannaschii and engineering bacteria, applied in the field of genetic engineering, can solve the problems of low utilization rate of glycerol, low product efficiency and high cost of propionic acid, and achieve the effect of improving the production capacity of propionic acid fermentation

Inactive Publication Date: 2014-08-27
JIANGNAN UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the microbial production of propionic acid still has the disadvantages of high cost and low yield of propionic acid.
The main carbon source of Propionibacterium is glycerol. However, in its metabolic pathway, the utilization rate of glycerol by Propionibacterium janesii is not high, which leads to low efficiency of converting carbon source into products, and finally leads to low production of propionate

Method used

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  • High-yield propionic acid propionibacterium jensenii engineering bacterium and application thereof
  • High-yield propionic acid propionibacterium jensenii engineering bacterium and application thereof
  • High-yield propionic acid propionibacterium jensenii engineering bacterium and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 The construction of the acquisition of Klebsiella pneumoniae gldA gene and mdh gene and expression plasmid

[0031] The gldA gene and the mdh gene can be obtained by chemical total synthesis, and can also be obtained by the following PCR method.

[0032] (1) Using the whole genome sequence of Klebsiella pneumonia subsp.pneumoniae ATCC 12657 as a template, the target fragments of gldA gene and mdh gene with the expected size were obtained by PCR amplification.

[0033] (2) When two genes are co-expressed: the target gene is connected to the expression vector pZGX04 through blunt ends to obtain the recombinant expression plasmid pZGX04-gldA-mdh. After sequencing verification, the connection was proved to be correct.

[0034] (3) When only the malate dehydrogenase gene is overexpressed: the target gene is connected to the expression vector pZGX04 through blunt ends to obtain the recombinant expression plasmid pZGX04-mdh. After sequencing verification, the co...

Embodiment 2

[0037] Example 2 Construction and Identification of Recombinant Bacteria

[0038] Due to the restrictive modification in Propionibacterium jensenii, after the recombinant plasmid was amplified by E. coli JM109, it was first transformed into E. coli JM110, and the recombinant plasmid extracted from E. coli JM110 was electrotransformed into P. jensenii CCTCC No. :M 2013071, which was spread to seed medium containing chloramphenicol resistance. Immediately, the transformant was selected for colony PCR verification, and a positive transformant was obtained, and the plasmid was further isolated for sequencing verification to confirm that the recombinant Propionibacterium janesii was obtained.

Embodiment 3

[0039] Embodiment 3 Recombinant bacteria and control bacteria fermentation experiment

[0040] Separate the original bacteria P.jensenii CCTCC NO:M 2013071 and the recombinant bacteria P.jensenii CCTCC NO:M2013071-gldA-mdh, P.jensenii CCTCC NO:M 2013071-gldA, P.jensenii CCTCC NO:M2013071-mdh Fed-batch fermentation, the fermentation parameters are shown in Table 1, and the four bacteria were compared: (1) recombinant bacteria overexpressing glycerol dehydrogenase, recombinant bacteria overexpressing malate dehydrogenase and co-expressing glycerol dehydrogenase, apple The propionic acid output of the recombinant bacterium of acid dehydrogenase is respectively 34.62, 36.09 and 39.43g / L, which is 28.46, 33.91 and 46.3% higher than the starting strain (26.95g / L); (2) co-expression of glycerol dehydrogenase The specific enzyme activity of glycerol dehydrogenase and malate dehydrogenase of recombinant bacteria with malate dehydrogenase increased significantly, indicating that the ove...

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Abstract

The invention discloses a high-yield propionic acid propionibacterium jensenii engineering bacterium and application thereof, and belongs to the field of genetic engineering. A molecular method is adopted. The propionibacterium jensenii engineering bacterium serves as a host, and overexpression is carried out on malate dehydrogenase (mdh) from Klebsiella pneumoniae, or overexpression is simultaneously carried out on glycerol dehydrogenase (gldA) from the Klebsiella pneumoniae. Compared with an original strain, the propionic acid yield of recombination and mixing coexpression glycerol dehydrogenase of overexpression malate dehydrogenase and the propionic acid yield of a recombinant strain of the malate dehydrogenase are 36.09 g/L and 39.43 g/L respectively, and are improved by 33.91 % and 46.3 % respectively than that of the original strain. The method provides new ideas for modifying the propionibacterium jensenii and improving productivity of propionic acid fermentation in the industrial biotechnology.

Description

technical field [0001] The invention relates to a high-propionate-producing P. janesii engineering bacterium and its application, belonging to the field of genetic engineering. Background technique [0002] Propionic acid and its derivatives are widely used in industry, mainly used in food preservation, feed storage, synthesis of pharmaceutical intermediates, synthesis of agricultural herbicides, organic synthesis intermediates, etc. In 2006, the world's total production capacity of propionic acid was about 350,000 tons / year, and in 2008 it reached 500,000 tons / year. The United States is the world's largest producer and consumer of propionic acid. At present, the actual annual output of propionic acid in my country is only about 200 tons, which is far from meeting the actual market demand, and a large amount of imports are still needed to make up for the domestic vacancy. [0003] Propionibacterium jensenii belongs to the genus Propionibacterium and is an anaerobic bacteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/52C12R1/01
Inventor 陈坚堵国成刘龙诸葛鑫
Owner JIANGNAN UNIV
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