Alleles of the zwf gene from coryneform bacteria

a technology of alleles and genes, applied in the field of alleles of the zwf gene of coryneform bacteria, can solve problems such as inability to predi

Inactive Publication Date: 2007-04-26
DEGUSSA AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is therefore not possible to predict which mutation alters the catalytic activity of glucose 6-phosphate dehydrogenase in such a way that production of L-amino acids is improved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Mutagenesis of the L-Lysine-Producing Strain DM1797

[0237] The Corynebacterium glutamicum strain DM1797 was used as a starting strain for mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The DM1797 strain is an aminoethylcysteine-resistant mutant of Corynebacterium glutamicum ATCC13032 and has been deposited under the name DSM16833 with the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Brunswick, Germany).

[0238] The DM1797 strain was cultured in 10 ml of LB broth (Merck, Darmstadt, Germany) contained in a 100 ml Erlenmeyer flask on a Certomat BS-1 rotary shaker (B. Braun Biotech International, Melsungen, Germany) at 33° C. and 200 rpm for 24 hours. The culture was subsequently removed by centrifugation, the sediment was resuspended in 10 ml of 0.9% NaCl solution, the suspension obtained was again removed by centrifugation and the sediment obtained was taken up in 10 ml of 0.9% NaCl solution. 5 ml of this cell suspension were treated with 400 μg / ml MNNG...

example 2

Performance Test of the DM1797 Strain Mutants

[0239] The mutants obtained in example 1 were cultured in a nutrient medium suitable for lysine production, and the lysine content was determined in the culture supernatant.

[0240] For this purpose, the clones were first propagated on brain-heart agar plates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. Starting from these agar plate cultures, in each case a preculture was inoculated (10 ml of medium in a 100 ml Erlenmeyer flask). The medium used for said preculture was MM medium. The preculture was incubated on a shaker at 33° C. and 240 rpm for 24 hours. From this preculture, a main culture was inoculated in such a way that the starting OD (660 nm) of said main culture was 0.1 OD. The MM medium was likewise used for the main culture.

Medium MMCSL5g / lMOPS20g / lGlucose (autoclaved separately)50g / lSalts:(NH4)2SO4)25g / lKH2PO40.1g / lMgSO4 * 7H2O1.0g / lCaCl2 * 2H2O10mg / lFeSO4 * 7H2O10mg / 1MnSO4 * H2O5.0mg / lBiotin (sterile-filtered)0.3mg / ...

example 3

Sequencing of the Zwf Gene of the DM1816 Mutant

[0244] Chromosomal DNA was isolated from the DM1816 clone by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). A DNA section carrying the zwf gene was amplified with the aid of the polymerase chain reaction. To this end, the following oligonucleotides were used as primers:

zwf-L1:5′ agaagctgac gctgtgttct 3′(SEQ ID NO:19)zwf-L2:5′ cattggtgga ctcggtaact 3′(SEQ ID NO:20)

[0245] The primers depicted were synthesized by MWG Biotech (Ebersberg, Germany). They enable an approx. 1.95 kb DNA section carrying the zwf gene to be amplified. The zwf-L1 primer binds to the region corresponding to positions 59 to 78 of the strand complementary to SEQ ID NO:3. The zwf-L2 primer binds to the region corresponding to positions 2026 to 2007 of the strand according to SEQ ID NO:3.

[0246] The PCR reaction was carried out using the Phusion High Fidelity DNA polymerase (New England Biolabs, Frankfurt, Germany). The reaction mixture was prep...

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Abstract

The invention relates to mutants and alleles of the zwf gene of coryneform bacteria, which encode variants of the Zwf subunit of glucose 6-phosphate dehydrogenase (EC: 1.1.1.49), and to processes for preparing amino acids, in particular L-lysine and L-tryptophan, by using bacteria which harbor said alleles.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to German application 10 2005 013 676.1, filed on Mar. 24, 2005, and to U.S. provisional application 60 / 709,094, filed on Aug. 18, 2005. These previous applications are incorporated herein in their entirety by reference.FIELD OF THE INVENTION [0002] The invention relates to mutants and alleles of the zwf gene of coryneform bacteria, which encode variants of the Zwf subunit of glucose 6-phosphate dehydrogenase (EC: 1.1.1.49), and to processes for preparing amino acids, in particular L-lysine and L-tryptophan, by using bacteria which harbor said alleles. BACKGROUND OF THE INVENTION [0003] Amino acids are applied in human medicine, in the pharmaceutical industry, in the food industry and especially in animal nutrition. [0004] Amino acids are known to be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, continuous efforts...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P13/22C12P13/08C07H21/04C12N15/74C12N1/21
CPCC07K14/34C12P13/08C12P13/227
Inventor BATHE, BRIGITTESCHISCHKA, NATALIETHIERBACH, GEORG
Owner DEGUSSA AG
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