L-Lysine-producing corynebacteria and process for the preparation of L-lysine

#The invention relates to L-lysine-producing strains of corynebacteria with enhanced lysE gene (lysine export carrier gene), in which strains additional genes, chosen from the group comprising the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the dapB gene (dihydrodipicolinate reductase gene) and the pyc gene, but especially the dapA gene and the lysC gene (aspartate kinase gene), are amplified and, in particular, over-expressed, and to a process for the preparation of L-lysine.

#L-Lysine is a commercially important L-amino acid which is used especially as a feed additive in animal nutrition. The need has been steadily increasing in recent years.

#L-Lysine is prepared by a fermentation process with L-lysine-producing strains of corynebacteria, especially Corynebacterium glutamicum. Because of the great importance of this product, attempts are constantly being made to improve the preparative process. Improvements to the process may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.

#The following Figures are attached:

#FIG. 1: Plasmid pEC7lysE

#FIG. 2: Plasmid pEC7dapB

#The abbreviations used in the Figures are defined as follows: Cm: chloramphenicol resistance gene dapB: dapB gene from C. glutamicum lysE: lysE gene from C. glutamicum pyc: pyc gene from C. glutamicum OriE: plasmid-coded origin of replication of E. coli pBL: DNA fragment of plasmid pBL1 EcoRI: cleavage site of the restriction enzyme EcoRI EcoRV: cleavage site of the restriction enzyme EcoRV HincII: cleavage site of the restriction enzyme HincII HindIII: cleavage site of the restriction enzyme HindIII KpnI: cleavage site of the restriction enzyme KpnISalI: cleavage site of the restriction enzyme SalISmaI: cleavage site of the restriction enzyme SmaISphI: cleavage site of the restriction enzyme SphI PvuII: cleavage site of the restriction enzyme PvuII BamHI: cleavage site of the restriction enzyme BamHI.

#Preparation of the DNA Coding for lysE

#Chromosomal DNA was isolated from the strain ATCC13032 by the conventional methods (Eikmanns et al., Microbiology 140: 1817–1828 (1994)). The polymerase chain reaction (PCR) was used to amplify a DNA fragment carrying the lysE gene. The following primer oligonucleotides were chosen for the PCR on the basis of the lysE gene sequence known for C. glutamicum (Vrljic et al., Molecular Microbiology 22(5), 815–826 (1996)) (accession number X96471):