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Enhanced promoter and method for producing L-lysine usinge the same

A promoter and transformant technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic material, etc., to achieve high promoter activity, improve aminotransferase activity, and increase production efficiency.

Active Publication Date: 2013-04-24
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there have been no reports of high expression levels in coryneform bacteria by improving the promoter of the aspartate aminotransferase (EC; 2.6.1.1; aspB) gene. Aspartate aminotransferase (EC; 2.6. 1.1; aspB) is thought to play an important role in supplying the lysine precursor, aspartic acid, to the lysine biosynthetic pathway

Method used

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  • Enhanced promoter and method for producing L-lysine usinge the same
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  • Enhanced promoter and method for producing L-lysine usinge the same

Examples

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Effect test

Embodiment 1

[0052] Example 1: Identification of the transcription initiation site of the aspB gene

[0053] In this example, an unknown transcription start site of the aspB gene was identified.

[0054]In order to identify the transcription initiation site of the aspB gene, the 5'-RACE (rapid amplification of 5' cDNA ends) technique (Sambrook, J., Russell, D.W. Molecular Cloning: a laboratory manual) was used 3 rd ed. 8.54). 5'-RACE is a method of cloning an unknown 5' upstream region using known mRNA, and this method is performed using 5'-Full Race core set (Cat. No. 6122) obtained from Takara Corporation.

[0055] In order to carry out the 5'-RACE experiment, 5 kinds of primers (Table 1, SEQ ID NO: 5) were synthesized based on the base sequence of the aspB gene (NCBI Accession No. to 9).

[0056] Table 1

[0057] Primer

base sequence

SEQ ID NO:

aspB(race) / RT

P-ccaagacctgctcc (5' phosphorylated)

5

aspB(race) / F1

Tactcgcggtaagccttcg

...

Embodiment 2

[0061] Embodiment 2: the construction of the recombinant vector that is used for promoter improvement

[0062] Construction of vector (pDZ) for chromosomal insertion

[0063] In this example, the vector pDZ for insertion into the chromosome of Corynebacterium was constructed based on the Escherichia coli cloning vector pACYC177 (New England Biolab, GenBank accession number #X06402).

[0064] The pACYC177 vector was treated with restriction enzymes Acul and BanI, followed by klenow treatment to obtain blunt ends. The lacZ gene of Escherichia coli origin used as a screening marker was amplified from the genomic DNA of Escherichia coli K12W3110 by PCR, the PCR was designed so that the amplified fragment included the promoter of the gene, and T4 DNA polymerase and polynucleoside Acid kinase treatment to generate blunt ends and phosphorylation of the 5' end. The above two DNA fragments are connected to each other to generate a circular DNA molecule, and then an artificially sy...

Embodiment 3

[0073] Embodiment 3: the recombinant vector is inserted in the Corynebacterium glutamicum bacterial strain

[0074] In this example, in order to improve the aspB promoter on the corynebacterium chromosome, the constructed recombinant vector was transformed into lysine-producing Corynebacterium glutamicum KFCC10881, and the promoter sequence of the vector was replaced by homologous recombination A promoter sequence on a chromosome such that an enhanced promoter sequence is inserted into the chromosome.

[0075] Each of the recombinant vectors pDZ-aspBP1, pDZ-aspBP2, and pDZ-aspBP3 for base substitution in Corynebacterium was designed to include a DNA fragment having the enhanced promoter sequence in Example 2, by electric pulse The above vector was transformed into Corynebacterium glutamicum KFCC10881 (transformed according to Appl. Microbiol. Biotechnol. (1999) 52: 541-545), and the transformant was screened on a medium containing 25 mg / L kanamycin to obtain Transformants i...

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Abstract

The present invention provides a nucleic acidmolecule having enhanced promoter activity, which is operably linked to a gene encoding aspartate aminotransferase and derived from Corynebacterium glutamicum, a vector comprising the nucleic acid molecule, a transformant transformed with the vector, and a method for producing L-lysine using the transformant.

Description

technical field [0001] The present invention relates to an enhanced promoter and a method for producing L-lysine using the promoter. Background technique [0002] Coryneform bacteria are industrial microorganisms widely used for the production of amino acids and various nucleic acids. Coryneform bacteria are Gram-positive bacteria used to produce chemicals with diverse industrial applications in animal feed, pharmaceuticals, food processing, etc., including such substances as L-lysine, L-threonine, L-arginine and glutamic acid amino acids and various nucleic acids, the growth of coryneform bacteria requires biotin. Coryneform bacteria are characterized by snapping division and rarely degrade the metabolites produced. Examples of such coryneform bacteria are: Corynebacterium, including Corynebacterium glutamicum; Brevibacterium, including Brevibacterium flavum; Arthrobacter sp. and Microbacterium sp.). [0003] As a type of L-amino acid, L-lysine is used commercially as a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12P13/08C12R1/13C12R1/15
CPCC12P13/08C12N9/1096C12N15/77
Inventor 权刀显金亨俊张在佑文准玉林相曹
Owner CJ CHEILJEDANG CORP
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