Method for the fermentative production of L-amino acids
The present invention 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-omithine and new measures for the fermentative production of proteinogenic L-amino acids and L-ornithine by such bacteria.
1. A bacterium of the genus Corynebacterium , wherein:
a) the bacterium has the ability to excrete an L-amino acid selected from proteinogenic L-amino acids and L-ornithine;
b) the bacterium comprises a chromosome with a polynucleotide that, prior to modification, encodes a polypeptide which is at least 90% identical to the amino acid sequence of SEQ ID NO:8 and which confers upon Escherichia coli a resistance to at least one antibiotic, selected from erythromycin, tetracycline, puromycin and bleomycin; and
c) said chromosome has been modified by a modification comprising either:
i) a deletion of part or all of the polynucleotide sequence coding for a portion of the polypeptide corresponding to amino acids 149 to 251, 41 to 344, or 14 to 435 of SEQ ID NO:8; or
ii) a deletion of the complete polynucleotide sequence coding for said polypeptide.
2. The bacterium of claim 1 , wherein the modification comprises a deletion of at least the complete nucleotide sequence coding for said polypeptide and the adjoining stop codon.
3. The bacterium of claim 1 , wherein the modification further comprises an insertion of a recognition site for the restriction enzyme EcoRV.
4. The bacterium of claim 1 , wherein said bacterium belongs to the species Corynebacterium glutamicum.
5. The bacterium of claim 1 , wherein said L-amino acid is selected from the proteinogenic L-amino acids L-lysine, L-valine, L-threonine, L-isoleucine, L-histidine and L-proline.
6. The bacterium of claim 5 , wherein said L-amino acid is L-lysine.
7. The bacterium of claim 1 , wherein, prior to modification, the amino acid sequence of the polypeptide comprises 459 amino acids.
8. The bacterium of claim 7 , wherein, prior to modification, the polypeptide comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 prior.
9. The bacterium of claim 8 , wherein prior to modification, the polypeptide comprises the amino acid sequence of SEQ ID NO:8.
10. The bacterium of claim 9 , wherein, prior to modification, the nucleotide sequence coding for said polypeptide comprises positions 1001 to 2377 of the nucleotide sequence of SEQ ID NO:7 or positions 1001 to 2377 of SEQ ID NO:7, wherein at position 2341 cytosine (c) is replaced by thymine (t).
11. A method for the fermentative production of an L-amino acid, selected from proteinogenic L-amino acids and L-ornithine, comprising the steps of:
a) cultivating the bacterium of claim 1 in a medium under conditions suitable for the production of said L-amino acid;
b) accumulating said L-amino acid in the medium to form an L-amino acid containing fermentation broth.
12. The method of claim 11 , wherein said L-amino acid is selected from the proteinogenic L-amino acids L-lysine, L-valine, L-threonine, L-isoleucine, L-histidine and L-proline.
13. The method of claim 12 , wherein said L-amino acid is L-lysine.
14. The method of claim 11 , further comprising concentration of the L-amino acid containing fermentation broth.
15. The method of claim 11 , further comprising drying the L-amino acid containing fermentation broth.
16. The method of claim 11 , further comprising purifying the L-amino acid from said L-amino acid containing fermentation broth.