Recombinant microorganism with few byproducts and high yield of 1, 3-propylene glycol and application thereof

A technology of recombinant microorganisms and propylene glycol, which is applied in the fields of genetic engineering and biological fermentation, can solve the problems of poor biological safety of Escherichia coli, large price fluctuations, and complicated post-extraction processes, and achieve biological safety, less by-products, and cost reduction Effect

Active Publication Date: 2022-03-01
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages of this process route are: 1. Klebsiella pneumoniae is an opportunistic pathogen, and its biological safety needs to be strictly controlled in the production process; 2. A large number of by-products such as acetic acid, lactic acid, succinic acid, 2,3 -The synthesis of butanediol makes the whole post-extraction process very complicated; 3. The source of glycerin is limited, and the price fluctuates greatly with the market
The disadvantage of this process route is that Escherichia coli has poor biological safety, and the fermentation and cultivation of Escherichia coli requires the use of expensive raw material yeast powder, so this technology is not conducive to industrial scale production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Constructing a synthetic pathway that can utilize sugars to synthesize 1,3-propanediol in Corynebacterium glutamicum

[0046] Corynebacterium glutamicum cannot use sugar to synthesize 1,3-propanediol. In order to make it able to synthesize 1,3-propanediol, it is necessary to introduce glycerol and 1,3-propanediol synthesis modules, mainly including: a glycerol-3-phosphate desorbent The fusion protein gene (gpd-gpp) of hydrogenase and glycerol-3-phosphatase can catalyze dihydroxyacetone phosphate to produce glycerol, diol dehydratase and its activator gene pduCDEGH and alcohol dehydrogenase gene yqhD.

[0047] The specific construction method is as follows:

[0048] A fusion protein of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase is artificially designed, its amino acid sequence is shown in SEQ ID No.4, and its nucleotide sequence is shown in SEQ ID No.5. A fragment comprising the trc promoter and the above-mentioned fusion protein coding sequ...

Embodiment 2

[0052] Example 2 Knockout of key genes affecting 3-hydroxypropionic acid synthesis in Corynebacterium glutamicum

[0053] The present invention finds that a large amount of 3-hydroxypropionic acid will be accumulated in the process of producing 1,3-propanediol by recombinant Corynebacterium glutamicum C.glutamicum-fusion fermentation, which will lead to the reverse consumption of 1,3-propanediol (see Example 3) . Through a large number of bioinformatics and transcriptomics analysis, the present invention found that the acetaldehyde dehydrogenase gene ald, the alcohol dehydrogenase gene adhA, or its regulatory gene ramA affect the synthesis of 3-hydroxypropionic acid and the synthesis of 1,3-propanediol Anti-consumption. Then use the gene knockout method to construct different gene knockout strains, the method is as follows:

[0054] 1) Knockout of the ald gene (SEQ ID No.1) in C. glutamicum-fusion: Using the genome of C. glutamicum-fusion as a template, primers ald-up-F (aca...

Embodiment 3

[0060] The fermentation culture of embodiment 3 recombinant Corynebacterium glutamicum

[0061] In LB Plate overnight. Single bacterium colonies were inoculated from the fresh plate into a 250 ml shaker flask with baffles containing 30 ml of seed medium, and cultured at 32° C. at 200 rpm for 12 hours.

[0062] The formulation of the seed medium includes (g / L): Glucose 25, (NH 4 ) 2 SO 4 5.0,K 2 HPO 4 1.5,MgSO 4 1.0,MnSO 4 0.005,FeSO 4 0.005, corn steep liquor 30, kanamycin 0.025.

[0063] Inoculate the seed liquid into 2L fermentation medium with 10% inoculation amount, use a 5L fermenter for fermentation, control the temperature at 30°C, and the ventilation rate at 1vvm, adjust the speed to keep the dissolved oxygen level above 5%. Ammonia water controls the pH value to be stable at about 7.0. Add the glucose of 600g / L to keep the glucose concentration in the fermentation broth higher than 10g / L.

[0064] The fermentation medium formula includes (g / L): glucos...

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PUM

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Abstract

The invention relates to the technical field of genetic engineering and biological fermentation, and particularly discloses a recombinant microorganism with few byproducts and high yield of 1, 3-propylene glycol and application of the recombinant microorganism. The invention provides a recombinant microorganism, and the recombinant microorganism has reduced expression and/or enzymatic activity of ethanol dehydrogenase adhA and/or acetaldehyde dehydrogenase ald compared with an original strain; the original strain is corynebacterium glutamicum capable of synthesizing 1, 3-propylene glycol. The reduction of expression and/or enzyme activity can be realized by knocking out or down-regulating one or more of the following genes: 1) an ethanol dehydrogenase adhA gene; 2) an acetaldehyde dehydrogenase ald gene; and 3) regulatory protein genes ramA of the adhA and the ald. When the recombinant microorganism is used for producing 1, 3-propylene glycol, the biological safety is high, byproducts are few, the yield is high, the operation is simple and convenient, and the cost is low.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and biological fermentation, in particular to a recombinant microorganism with few by-products and high yield of 1,3-propanediol and its application. Background technique [0002] 1,3-Propanediol is an important chemical raw material, which can be used as an organic solvent in ink, printing and dyeing, paint, lubricant, antifreeze and other industries. Its main use is as a monomer for the synthesis of polyester and polyurethane. Especially polymerized with terephthalic acid to form polytrimethylene terephthalate (PTT). Compared with PET (polyethylene terephthalate), PBT (polybutylene terephthalate), PTT has better properties, such as better stain resistance, toughness and resilience, and UV resistance In addition, it has the advantages of wear resistance, low water absorption and low static electricity. Therefore, PTT is considered as an upgraded product of PET and has broad market p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/77C12P7/18C12R1/15
CPCC12N9/0006C12N9/0008C12N15/77C12P7/18
Inventor 陈振刘德华
Owner TSINGHUA UNIV
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