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Method for improving glycerol microbial fermentation production of 1,3-propanediol by constructing gene engineering bacterium

A technology of genetically engineered bacteria and propylene glycol is applied in the field of constructing genetically engineered bacteria to enhance microbial production, so as to achieve the effects of reducing production costs, improving production efficiency, and increasing concentration and yield.

Active Publication Date: 2011-09-28
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] At present, there is no report on the construction of genetically engineered bacteria to enhance the expression of malic enzyme gene to enhance microbial fermentation of glycerol to produce 1,3-propanediol

Method used

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  • Method for improving glycerol microbial fermentation production of 1,3-propanediol by constructing gene engineering bacterium
  • Method for improving glycerol microbial fermentation production of 1,3-propanediol by constructing gene engineering bacterium
  • Method for improving glycerol microbial fermentation production of 1,3-propanediol by constructing gene engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The construction of embodiment 1 genetically engineered bacteria

[0051] (1) Strain: the starting strain Klebsiella pneumoniae (CGMCC1.9131);

[0052] (2) Using Klebsiella pneumoniae (CGMCC1.9131) genomic DNA as a template, clone the malic enzyme gene by PCR (polymerase chain reaction); cloning primers are: upstream primer ME-F: 5' -gcgaattcatggatgagcagttaaaacag-3', downstream primer ME-R: 5'-cagtcgacttacagcggttcggtttgcg-3'; PCR conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 3 minutes, 35 cycles, 72°C Extend for 10 minutes. The obtained malic enzyme gene PCR product sequence is shown in SEQ ID No.1.

[0053] (3) Purify the gene fragment of the malic enzyme gene PCR product and connect it to the cloning vector pMD18-T to transform the competent cell DH5α;

[0054] (4) Screen positive clones, cultivate, extract plasmids, digest with EcoR I and Sal I, reclaim, connect the expr...

Embodiment 2

[0057] The fermentation culture of embodiment 2 genetically engineered bacteria

[0058] (1) culture medium

[0059] LB medium (g·L -1 ): yeast powder 5, peptone 10, NaCl 10, agar 10, adjusted to pH 7.0, for short-term preservation and activation of Klebsiella species. The composition of seeds and fermentation medium is shown in Table 1:

[0060] Table 1 Medium Composition

[0061]

[0062] (2) Training method:

[0063] (i) Seed activation

[0064] The recombinant Klebsiella KP-pET-ME strain in Example 1 preserved in a glycerol tube was inoculated into LB medium slant for activation, and the seeds were incubated at 37° C. for 12 hours to activate the seeds.

[0065] (ii) Seed culture

[0066] Seed culture: 250mL triangular flask sealed with 9 layers of gauze, filled with 100mL of liquid, connected to a ring of slant lawn, aerobic seed culture in a shaker, temperature 30°C, speed 150r min -1 , add 50mg / l of kanamycin antibiotic in the culture medium.

[0067] (iii) F...

Embodiment 3

[0074] The fermentation culture of embodiment 3 genetically engineered bacteria

[0075] The recombinant Klebsiella KP-pET-ME strain constructed in Example 1 was used for fermentation and culture.

[0076] (1) Culture medium: the composition of the seed medium is the same as in Example 2, and the fermentation medium is the same as in Example 2 except that the initial glycerol concentration is 20g / l.

[0077] (2) Training method:

[0078] (i) seed activation, with embodiment 2;

[0079] (ii) seed culture, with embodiment 2;

[0080] (iii) Fermentation culture

[0081] When carried out in a 5L stirred fermenter, the liquid volume is 4L, the inoculum size is 1%, and 0.5vvm air is introduced to carry out the microaerobic fermentation culture, and the stirring speed is 250rpm. The fermentation temperature was kept constant at 37°C; the pH was adjusted to 6.8 by NaOH, and the pH of the system was regulated by feeding 40% NaOH solution during the fermentation process. After the ...

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Abstract

The invention provides a method for improving the microbial production of 1,3-propanediol by constructing a gene engineering bacterium. The method comprises the following steps: constructing an expression vector with the inserted malic enzyme gene; delivering the expression vector in host bacteria generating 1,3-propanediol; adding an inducer to induce the overexpression of the malic enzyme gene in the fermentation and culture process; and adopting the aerobic fermentation means and performing the fed batch of substrate glycerol to produce 1,3-propanediol. The method is characterized in that the constructed gene engineering bacterium can express more malic enzyme than the original strain in the fermentation process, thus the convertion from pyruvic acid to malic acid can be promoted, the circulation of tricarboxylic acid can be promoted, the bacterium can generate more nicotinamide adenine dinueleotide (NADH) and energy (ATP), the activity of 1,3-propanediol oxidation-reduction enzymein the bacterium and the glycerol conversion rate can be increased. The invention has the following advantages: the substrate glycerol utilization rate of the producing bacterium can be increased, the concentration and production strength of the fermented 1,3-propanediol can be obviously increased, the yield of 1,3-propanediol can be increased and the production cost can be reduced.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for constructing genetically engineered bacteria to enhance the production of 1,3-propanediol by microorganisms. Background technique [0002] 1,3-Propanediol (PDO) is an important chemical raw material, which can be used as an organic solvent in high-pressure lubricants, dyes, inks, antifreeze and other industries. The main reason why PDO attracts the attention of the industry and develops rapidly is that it can be used as a monomer for the synthesis of polyester PTT. Butylene glycol formate (PBT) is a new fiber-forming polyester polymer material that can be realized on an industrial scale, and it is a new type of polyester material with great development prospects. Due to the many excellent properties of PTT, it is widely used in the carpet industry, clothing materials, engineering thermoplastics and many other fields. [0003] The key to pro...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/18C12N15/74C12R1/22
Inventor 刘德华周胜罗吉安
Owner TSINGHUA UNIV
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