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Method for improving capacity of producing pyruvic acid with engineering bacteria by knocking out pyruvic acid transport protein genes

A transporter, pyruvate technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc.

Inactive Publication Date: 2020-09-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is no report on the method of improving microbial fermentation to produce pyruvate by knocking out the pyruvate transporter gene

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  • Method for improving capacity of producing pyruvic acid with engineering bacteria by knocking out pyruvic acid transport protein genes
  • Method for improving capacity of producing pyruvic acid with engineering bacteria by knocking out pyruvic acid transport protein genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Knockout of protein-coding genes cstA and yjiY predicted to have pyruvate transport function

[0082] (1) Knockout of the predicted pyruvate transporter gene cstA

[0083] The predicted gene cstA encoding pyruvate transporter has a sequence length of 2108 bases, and its nucleotide sequence is shown in SEQ ID NO.4.

[0084] Knockout vector construction: Genomic DNA of PDL-7 ΔpflBΔldhD::nox was prepared using conventional methods. The process referred to the method for small-scale preparation of bacterial genomes in the "Guide to Molecular Biology" published by Science Press; using synthetic The primers "cstA1-f and cstA1-r" and "cstA2-f and cstA2-r" PCR amplified the upstream and downstream homology arms of cstA coding gene. Use the obtained upper and lower homology arms as templates for recombinant PCR, and then use "cstA1-f and cstA2-r" primers to amplify the recombinant fragment by PCR to obtain a truncated fragment of cstA, which contains EcoRI restrictio...

Embodiment 2

[0102] Example 2: Verification that the predicted proteins CstA and YjiY are pyruvate transporters

[0103] (1) Plate culture: Streak the recombinant strains Klebsiella oxytocaPDL-7ΔpflBΔldhD::nox and Klebsiella oxytocaPDL-CY respectively on the LB medium containing 1.6-1.8% agar by mass volume ratio, culture at 37±1°C for 10±1 hours ;

[0104] (2) Seed culture: under sterile conditions, use a sterile gun tip to pick a single colony from the two strain plates in step (1), and then inoculate it into 5 mL of LB liquid medium at 37±1°C Incubate on a shaking table for 10 ± 1 hour; then re-inoculate into 5 mL of LB liquid medium at an inoculum size of 1% (v / v), and culture on a shaking table at 37 ± 1°C for 10 ± 1 hour;

[0105] (3) Using pyruvate as the only carbon source, shake flask culture: under sterile conditions, take the bacterial solution obtained in step (2), and inoculate it into the KO inorganic salt medium according to the inoculum amount of 1% (v / v). Wherein, the cu...

Embodiment 3

[0109] Example 3: Recombinant oxytoca Klebsiella oxytocaPDL-CY uses glucose as a substrate to ferment and produce pyruvate

[0110] (1) Plate culture: Streak the recombinant strain Klebsiella oxytocaPDL-CY on the LB medium containing 1.6-1.8% agar in mass volume ratio, culture at 37±1°C for 10±1 hours;

[0111] (2) Seed culture: under sterile conditions, use a sterile gun tip to pick a single colony on the plate in step (1), then inoculate it into 5 mL of LB liquid medium, and culture it on a shaking table at 37±1°C 10±1 hours; then inoculate 50 mL of LB liquid medium at 37±1°C with shaking for 10±1 hours at an inoculation amount of 1% (v / v);

[0112] (3) 1L fermenter culture: under aseptic conditions, take the bacterial liquid obtained in step (2), and inoculate it into the fermentation medium containing it according to the inoculum amount of 5% (v / v). Wherein, the fermentation conditions are as follows: the culture temperature is 37°C, the culture method is stirring culture...

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Abstract

The invention discloses a method for improving the capacity of producing pyruvic acid with engineering bacteria by knocking out pyruvic acid transport protein genes. Recombinant klebsiella oxytoca PDL-7 Delta pflB Delta ldhD::nox is taken as an original strain; the pyruvic acid transport protein genes cstA and yjiY of the original strain are knocked out to construct a klebsiella oxytoca engineering strain which can increase the fermentation production capacity of the pyruvic acid, and the klebsiella oxytoca engineering strain is named klebsiella oxytoca PDL-CY; experiments confirm that the yield of the pyruvic acid fermented-produced by the constructed engineering strain is 103.31g / L by taking glucose as a substrate, and the substrate conversion rate reaches 0.90g / g; and compared with theoriginal strain, the yield of the pyruvic acid and the substrate conversion rate of the constructed engineering strain are increased by 67.8% and 76.8% respectively. The method of the invention has asimple process; product components are single and are easy to separate; and the method has important economic benefits and social meaning.

Description

technical field [0001] The present invention relates to a method for increasing microbial fermentation to produce pyruvate, in particular to a method for increasing the fermentation and production of pyruvate by Klebsiella oxytoca engineering strains by knocking out the pyruvate transporter, which belongs to the biological fermentation preparation technology of carboxylic acid field. Background technique [0002] Pyruvate is an important intermediate in the biochemical metabolic network in organisms [1] . At the same time, as a basic chemical raw material, pyruvic acid is widely used in various fields such as chemistry, medicine, food, agriculture and environmental protection. [2,3] . In addition, pyruvic acid is also the precursor of some high value-added products such as alanine, isobutanol and carotenoids, and has broad application prospects [3] . [0003] At present, the main source of commercial pyruvate is the chemical synthesis method based on non-renewable resou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P7/40C12R1/22
CPCC07K14/26C12N15/74C12P7/40
Inventor 高超曹梦豪马翠卿许平
Owner SHANDONG UNIV
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