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Method for preparing recombinant L-glutamic acid producing strain, strain prepared by method and application method of strain

A technology for the production of strains, Corynebacterium glutamicum, applied in the field of genetic engineering, can solve the problem of low acid production performance

Active Publication Date: 2017-04-05
COFCO NUTRITION & HEALTH RES INST +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the existing L-glutamic acid producing strains, especially the wild-type L-glutamic acid producing strains, have low acid production performance. Therefore, it is urgent to develop genetically engineered bacteria with high acid production performance.

Method used

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  • Method for preparing recombinant L-glutamic acid producing strain, strain prepared by method and application method of strain
  • Method for preparing recombinant L-glutamic acid producing strain, strain prepared by method and application method of strain
  • Method for preparing recombinant L-glutamic acid producing strain, strain prepared by method and application method of strain

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preparation example Construction

[0033] In the method for preparing recombinant L-glutamic acid producing strains of the present invention, the available L-glutamic acid producing strains can be, for example, wild-type Corynebacterium glutamicum, including but not limited to: Corynebacterium glutamicum wild-type Strain ATCC13032 (China Industrial Microorganism Culture Collection Management Center, preservation number CICC20213) or Corynebacterium glutamicum wild-type strain S9114 (China Industrial Microbiology Culture Collection Management Center, preservation number CICC20935).

[0034] In some embodiments of the present invention for preparing recombinant L-glutamic acid producing strains, the expression level of the target gene (such as lpdA gene and optional odhA gene and / or sucB gene) of the present invention can be obtained by the following method: Recombinant L-glutamic acid production strain: use the transformed gene sequence fragment to construct a plasmid with gene knockout or gene expression level w...

Embodiment 1

[0065] This example is used to illustrate the method for preparing the recombinant L-glutamic acid producing strain of the present invention.

[0066] (1) Design and synthesis of ΔlpdA-Pwt::PS100a6-LR homology arm sequence:

[0067] According to the sequence of the wild-type promoter of the lpdA gene in Corynebacterium glutamicum CICC20935 and its upstream and downstream sequences, a homology arm sequence (ΔlpdA -Pwt::PS100a6-LR, as shown in SEQ ID NO: 1), and was synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0068] Wherein, the sequence of the wild-type promoter of the lpdA gene in Corynebacterium glutamicum CICC20935 and its upstream and downstream sequences are respectively represented by SEQ ID NO: 12-SEQ ID NO: 14, and the sequence of the S100a6 gene promoter of Chinese hamster ovary (CHO) cells is represented by represented by SEQ ID NO: 15.

[0069] (2) Construction of pK18mobsacB-ΔlpdA-Pwt::PS100a6-LR plasmid:

[0070] The synthesized ΔlpdA-Pwt::PS10...

Embodiment 2

[0082] This example is used to illustrate the method for preparing the recombinant L-glutamic acid producing strain of the present invention.

[0083] (1) Design and synthesis of ΔlpdA-LR homology arm sequence:

[0084] According to the sequence of the lpdA gene and its upstream and downstream sequences in Corynebacterium glutamicum CICC20935, a homology arm sequence (ΔlpdA-LR) for knocking out the lpdA gene was designed: the upstream and downstream homology arms of the lpdA gene were amplified using CICC20935 genomic DNA as a template. The source arm fragments, and then the upstream and downstream homology arm fragments were connected by overlapping PCR to obtain ΔlpdA-LR (shown by SEQ ID NO: 2).

[0085] The specific steps are as follows: using the genomic DNA of Corynebacterium glutamicum CICC20935 as a template, amplify lpdA with forward primer P3 (SEQ ID NO: 5) and reverse primer P4 (SEQ ID NO: 6) as shown in Table 1 The upstream homology arm of the gene (ΔlpdA-L, 1009bp...

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Abstract

The invention relates to a method for preparing a recombinant L-glutamic acid producing strain with the high acid-producing property, the recombinant L-glutamic acid-producing strain prepared by the method and a method of producing L-glutamic acid through the recombinant L-glutamic acid producing strain in a fermentation manner. In the recombinant L-glutamic acid-producing strain prepared by the method, the expression level of an lpdA gene and an optional odhA gene and / or sucB gene is reduced by at least 10% relative to the wild type strain. Compared with the wild type strain, the acid producing concentration and the glucose-glutamic acid conversion rate of the recombinant L-glutamic acid producing strain in the L-glutamic acid production both are increased remarkably, that is, the recombinant L-glutamic acid-producing strain has the remarkably-improved acid production property for producing the L-glutamic acid; and the L-glutamic acid is produced through the recombinant L-glutamic acid in the fermentation manner, and the yield of the L-glutamic acid can be increased greatly.

Description

technical field [0001] The invention relates to the field of genetic engineering. Specifically, the present invention relates to a method for preparing a recombinant L-glutamic acid producing strain with high acid production performance, a recombinant L-glutamic acid producing strain prepared by the method, and the production of L - Glutamate method. Background technique [0002] In industrial production and academic research, bacteria of the genus Corynebacterium (Corynebacterium spp.) or Brevibacterium (Brevibacterium spp.) with L-glutamic acid production capacity are generally used (now it is generally believed that bacteria of the genus Brevibacterium also belong to the genus Corynebacterium ) to produce L-glutamic acid by fermentation. In order to improve the productivity of these L-glutamic acid-producing bacteria, bacterial strains isolated from nature or their artificially mutated strains or recombinant strains are generally used to produce L-glutamic acid. [000...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P13/14C12R1/15
Inventor 臧传刚王春才王宏龄王晓建熊强陈博林海龙韩隽朱威宇
Owner COFCO NUTRITION & HEALTH RES INST
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