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Method for the fermentative production of l-amino acids

一种氨基酸、发酵液的技术，应用在细菌领域，能够解决没有公开核苷酸序列具体应用等问题

Inactive Publication Date: 2019-03-26
EVONIK OPERATIONS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific application of the nucleotide sequence in the production of specific fine chemicals is not disclosed

Method used

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  • Method for the fermentative production of l-amino acids
  • Method for the fermentative production of l-amino acids
  • Method for the fermentative production of l-amino acids

Examples

Experimental program
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Effect test

Embodiment 1

[0213] cmr gene sequences of C. glutamicum strains DM1933 ​​and DM1797

[0214] Strain DM1933 ​​is an L-lysine producer as described by Blombach et al. (Applied and Environmental Microbiology 75(2), 419-427, 2009). It was deposited with the DSMZ under the Budapest Treaty under the accession number DSM25442.

[0215] The nucleotide sequence of the chromosome of strain DM1933 ​​was determined by Illumina whole genome sequencing technology (Illumina Inc., San Diego, CA, US). See, for example, Whole-Genome Sequencing for Comparative Genomics and Derivatives by Benjak et al. (2015) in (Parish T., Roberts D. (eds) Mycobacteria Protocols, Methods in Molecular Biology, Vol. 1285, Humana Press, NY, US). Article by Novo Genome Assembly and Bennet, S (Pharmacogenomics 5(4), 433-438, 2004).

[0216] The nucleotide sequence of the cmr coding sequence, including its upstream and downstream nucleotide sequences, was found to be identical to the nucleotide sequence of ATCC13032 shown in SEQ...

Embodiment 2

[0222] Construction of plasmid pK18mobsacB_Dcmr

[0223] Plasmid pK18mobsacB_Dcmr was constructed to allow the introduction into the chromosome of the desired C. glutamicum strain of a deletion comprising the cmr coding sequence and its adjacent stop codon and insertion of a restriction enzyme EcoRV recognition site. The plasmid is based on The mobile vector pK18mobsacB described by et al. (Gene 145, 69-73, 1994)). pK18mobsacB_Dcmr was constructed using the Gibson Assembly method.

[0224] For this purpose, three polynucleotides or DNA molecules were generated separately: one called cmr_up containing the upstream sequence (5'-flanking sequence) and a second called cmr_down containing the downstream sequence of the cmr coding sequence (3'-flanking sequence) polynucleotide. The third polynucleotide was plasmid pK18mobsacB linearized with the restriction endonuclease Xbal. Polynucleotides cmr_up and cmr_down were fused during the Gibson Assembly process to generate polynucle...

Embodiment 3

[0242] Construction of strain DM1933_Δcmr::EcoRV

[0243] The plasmid pK18mobsacB_Dcmr was used to introduce the deletion of the entire cmr coding sequence and its adjacent stop codon into the chromosome of L-lysine producing strain DM1933, accompanied by the insertion of the recognition site for the restriction endonuclease EcoRV.

[0244] The deletion of the entire cmr coding sequence with its adjacent stop codon and the concomitant insertion of a recognition site for the restriction enzyme EcoRV is abbreviated as Δcmr::EcoRV or deltacmr::EcoRV.

[0245] Chemically competent cells of E. coli strain S17-1 were transformed with the plasmid DNA of pK18mobsacB_Dcmr obtained in Example 2. Materials and methods described in The modified conjugation method of et al. (Journal of Bacteriology 172, 1663-1666, 1990) was used for conjugative transfer into strain DM1933 ​​and for selection of transzygote clones by their sucrose resistance and kanamycin sensitivity phenotypes.

[0246]...

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Abstract

The present invention provides provides a bacterium of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, having the ability to excrete an L-amino acid selected from proteinogenic L-amino acids and L-ornithine and new measures for the fermentative production of proteinogenic L-amino acids and L-ornithine by such bacteria.

Description

technical field [0001] The present invention provides a bacterium of the genus Corynebacterium, in particular a bacterium of the species Corynebacterium glutamicum, which has the ability to secrete a protein selected from proteinogenic (proteinogenic) L-amino acids and L-ornithine. Capability of L-amino acids, and a new method for the production of protein-derived L-amino acids and L-ornithine by fermentation of said bacteria. Background of the invention [0002] L-amino acids are used in human medicine, the pharmaceutical industry, the food industry and especially in animal nutrition. [0003] L-amino acids such as L-lysine are produced by fermentation of strains of the genus Corynebacterium, particularly Corynebacterium glutamicum. Due to its economic importance, continuous efforts are being made to improve production methods. Improvements may relate to fermentation techniques such as agitation and oxygenation, or to the composition of the nutrient medium such as sugar c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/10C12P13/04C12R1/15
CPCC07K14/34C12P13/04C12P13/08C12P13/10C12P13/12C12P13/24C12N15/70
Inventor G·蒂尔巴赫T·贝克尔K·福斯
Owner EVONIK OPERATIONS GMBH
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