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Process for the fermentative production of L-amino acids using coryneform bacteria
| Details |
Inventors: Marx, Achim; Mockel, Bettina; Pfefferle, Walter; Sahm, Hermann; De Graaf, Albert; Eggeling, Lothar;
Assignee: Degussa Huls AG (Hanau, DE)
Primary Examiner: Achutamurthy; Ponnathapu
Assistant Examiner: Kerr; Kathleen
Attorney, Agent or Firm: Pillsbury Winthrop LLP
This invention relates to a process for the production of L-amino acids using coryneform bacteria, in which the glutamate dehydrogenase gene is amplified. |
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DETAILED DESCRIPTION OF THE INVENTION The present invention is illustrated in greater detail by the following practical examples.
To this end, testing was performed with amino acid producing strains, in which the superiority of the claimed process is demonstrated: a) the L-lysine producing strain Corynebacterium glutamicum DSM5715, (EP-B- 0435 132) and b) the L-threonine and L-isoleucine producing strain Brevibacterium flavum DSM5399 (EP-B- 0385 940) and c) the L-valine producing, isoleucine-requiring strain ATCC13032.
DELTA.
ilvA, which has been deposited as DSM12455 with Deutschen Sammlung fur Mikroorganismen und Zellkulturen in Braunschweig (Germany) in accordance with the Budapest Treaty.
EXAMPLE 1 Production of L-amino acid producers with amplified glutamate dehydrogenase Plasmid pEK1.
9gdh-1 corresponds to the plasmid pEK1.
9gdh described by Bormann et al.
(Molecular Microbiology 6, 317-326 (1992)).
It was isolated from ATCC13032/pEK1.
9gdh-1.
The known plasmid pEKExpgdh (Marx et al.
, Metabolic Engineering 1, 35-48 (1999)), which bears the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus (Snedecor et al.
, Journal of Bacteriology 173, 6162-6167 (1991)) was isolated in the same manner from E.
coli strain DH5.
alpha.
/pEKExpgdh.
Strains DSM5715, DSM5399 and ATCC13032.
DELTA.
ilvA were transformed with plasmid pEK1.
9gdh-1 as described by Liebl et al.
(FEMS Microbiology Letters 65, 299-304 (1989)).
The transformants were selected on brain/heart agar from Merck (Darmstadt, Germany) which had been supplemented with 50 mg/l of kanamycin.
In this manner, strains DSM5715/pEK1.
9gdh-1, DSM5399/pEK1.
9gdh-1 and ATCC13032.
DELTA.
ilvA/pEK1.
9gdh-1 were obtained.
Strain DSM5715 was transformed in the same manner with plasmid pEKExpgdh and strain DSM5715/pEKExpgdh obtained.
EXAMPLE 2 Production of L-lysine Strain DSM5715/pEK1.
9gdh-1 was precultivated in complex medium 2TY consisting of 16 g/l of tryptone, 10 g/l of yeast extract and 5 g/l of NaCl.
To this end, 60 ml of medium 2TY, contained in a 500 ml Erlenmeyer flask with 2 flow spoilers, were inoculated with an inoculating loop of the strain and the culture incubated for 12 hours at 150 rpm and 30
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Molecular Biology 
