Recombinant corynebacterium glutamicum, construction method thereof and method for producing tetrahydropyrimidine by same

A technology of Corynebacterium glutamicum and recombinant bacteria, which is applied in the field of genetic engineering and biological fermentation, can solve the problems of increasing the difficulty of downstream purification processes, ectoine restricting the industrial production and large-scale application of ectoine, and increasing production costs. Achieve the effect of solving biological safety problems, good industrial application prospects, and reducing production costs

Active Publication Date: 2020-07-10
BEIJING BIOINNO BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

This method has high requirements on the stability of the reactor material, and the discontinuous production process and high concentration of salt increase the difficulty of the downstream purification process. In addition, the high concentration of salt is easy to cause corrosion to the equipment, and also affects The growth of bacteria affects the output of ectoine, which leads to the increase of production cost and affects the large-scale application of ectoine
[0004] At present, the existing strains and methods for producing ectoine have seriously restricted the industrial production and large-scale application of ectoine. Therefore, a novel high-yield ectoine-producing strain is developed to simplify the production process, improve synthesis efficiency, and reduce production costs. It has important practical significance for the application of ectoine

Method used

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  • Recombinant corynebacterium glutamicum, construction method thereof and method for producing tetrahydropyrimidine by same
  • Recombinant corynebacterium glutamicum, construction method thereof and method for producing tetrahydropyrimidine by same
  • Recombinant corynebacterium glutamicum, construction method thereof and method for producing tetrahydropyrimidine by same

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preparation example Construction

[0046] In the method for preparing recombinant Corynebacterium glutamicum of the present invention, wild-type Corynebacterium glutamicum includes but not limited to: Corynebacterium glutamicum wild-type strain ATCC13032 or Corynebacterium glutamicum wild-type strain S9114. In some embodiments, when the wild-type Corynebacterium glutamicum used contains the ppc gene itself, for example, the strain ATCC13032 is used as the starting strain, then the introduction into the starting strain for expressing phosphoenolpyruvate carboxyl This step is the expression cassette of the enzyme gene. In some embodiments, when the expression level of phosphoenolpyruvate carboxykinase, homoserine dehydrogenase and / or dihydropyrimidinedicarboxylate synthetase of wild-type Corynebacterium glutamicum itself is low , the step of reducing the expression level of the corresponding enzyme can be omitted.

[0047] In some embodiments of the construction method of the present invention, the knockout and / ...

Embodiment 1

[0132] The construction of embodiment 1 recombinant Corynebacterium glutamicum

[0133] (1) Knock out the pck gene and introduce the ppc gene at the same time

[0134] Using Corynebacterium glutamicum wild-type strain S9114 as the starting strain, the pck gene on the genome of the wild-type Corynebacterium glutamicum is replaced with the ppc gene, so as to realize the knockout of the pck gene and the introduction of the ppc gene. Artificially synthesize the polynucleotide sequence shown in SEQID NO.:6 for replacing the pck gene with the ppc gene. SEQ ID NO.: 6 consists of 1001 bp upstream of the pck gene, SEQ ID NO.: 2 (ppc gene) and 999 bp downstream of the pck gene.

[0135] The Corynebacterium glutamicum suicide plasmid pK18mobsacB and the above polynucleotide sequence were double digested with Sma I / Sal I restriction endonuclease according to the enzyme digestion reaction system and reaction conditions in Table 2. Recover the digested fragments respectively, and then use...

Embodiment 2

[0149] Example 2 uses CG.ECT-2 to ferment and produce ectoine

[0150] The recombinant Corynebacterium glutamicum CG.ECT-2 obtained in Example 1 was cultured overnight at 30° C. on an LB plate. The next day, a single colony was picked from the plate and inoculated into a 250 mL shaker flask with baffles containing 30 mL of seed medium (containing 15 mg / L kanamycin), and cultivated at 32° C. and 200 rpm for 12 hours to obtain a seed liquid.

[0151] Inoculate the seed solution into a 2L fermentation medium (containing 15mg / L kanamycin) with an inoculation amount of 10v / v%, use a 5L fermenter for fermentation, control the temperature at 32°C, the ventilation rate is 1vvm, and the initial rotation speed is 350 -500rpm, adjust the rotation speed during the fermentation process to keep the dissolved oxygen level above 10%, and control the pH value to be stable at about 7.0 by feeding 13% ammonia water. 1 mM IPTG was added at 3 h of fermentation. Samples were taken at different fe...

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Abstract

The invention relates to recombinant corynebacterium glutamicum, a method for constructing the recombinant corynebacterium glutamicum and a method for producing tetrahydropyrimidine by using the recombinant corynebacterium glutamicum. Compared with wild corynebacterium glutamicum, the recombinant corynebacterium glutamicum has phosphoenolpyruvate carboxylase, homoserine dehydrogenase and dihydropyrimidinedicarboxylate synthetase with reduced expression level, and also has a gene expression cassette for expressing phosphoenolpyruvate carboxylase and a gene expression cassette for expressing ectA enzyme, ectB enzyme and ectC enzyme, so that tetrahydropyrimidine can be efficiently produced. The tetrahydropyrimidine produced by using the recombinant corynebacterium glutamicum has high yield, the safety of tetrahydropyrimidine products is improved, the production cost is greatly reduced, and the recombinant corynebacterium glutamicum has good market application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biological fermentation, and in particular relates to a recombinant Corynebacterium glutamicum, a method for constructing the recombinant Corynebacterium glutamicum, and a method for producing ectoine by using the recombinant strain. Background technique [0002] Ectoine is a compatible solute produced in cells by many salt-tolerant and halophilic microorganisms to maintain osmotic pressure balance. , proteins, cell membranes and nucleic acids provide protection. In addition, ectoine has a certain effect on neurological diseases such as Alzheimer's disease and Parkinson's disease, and the latest research has found that ectoine can improve skin regeneration and delay skin aging. Therefore, ectoine will have broad application prospects in industries such as fine chemical industry and biomedicine. [0003] At present, the production method of ectoine is mainly obtained by high-densi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12N15/60C12N15/53C12N15/54C12P17/12C12R1/15
CPCC12N9/88C12N9/0006C12N9/1096C12N9/1029C12N15/77C12P17/12C12Y401/01031C12Y101/01003C12Y206/01076C12Y203/01178C12Y402/01108C12Y401/01
Inventor 于淼郭元亨臧传刚张媛周卫强刘安妮叔谋王小艳丁子元彭超陈博江俊杰李榕榕
Owner BEIJING BIOINNO BIOTECHNOLOGY CO LTD
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