SHORT CONTRIBUTION C. Ferna ´ndez-Gonza ´lez · J. A. Gil · L. M. Mateos A. Schwarzer · A. Scha ¨fer · J. Kalinowski A. Pu ¨hler · J. F. Martı ´n Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996 Abstract The mobilization of plasmids from gram-ne- gative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked speci- fically in the homoserine branch of the aspartate path- way. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B. lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mu- tants do not overproduce lysine because R31 is an ala- nine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of ala- nine. Introduction Brevibacterium lactofermentum and related species such as Brevibacterium flavum and Corynebacterium glutami- cum are gram-positive bacteria used for the industrial production of amino acids. Cloning systems and trans- formation procedures for this group of microorganisms have already been described (Santamarı ´a et al. 1984; Katsumata et al. 1984; Martı ´n et al. 1987; Martı ´n 1989). Recently, the classical strain-improvement methods of random mutagenesis and screening were complemented by the application of recombinant DNA technology to this group of microorganisms. Techniques like gene disruption and gene replacement have been developed in C. glutamicum (Schwarzer and Pu ¨ hler 1991; Scha ¨ fer et al. 1994a) taking advantage of the conjugal transfer of mobilizable plasmids from E. coli to C. glutamicum (Scha ¨ fer et al. 1990). These techniques have been applied to other coryneform bacteria and, in all cases, mutants obtained were stable; and, therefore this strategy might be applicable to the construction of industrial strains to be used in fermentation processes. In this paper we describe the transfer of conjugative plasmids from E. coli to B. lactofermentum using plas- mids carrying internal fragments of the B. lacto- fermentum hom and thrB genes (Mateos et al. 1987a, b) coding for homoserine dehydrogenase and homoserine kinase respectively. The goal was to study the con- sequences of hom and thrB disruption in B. lacto- fermentum and, as was expected, we isolated stable B. lactofermentum mutants in the hom and thrB genes showing increased lysine production. Materials and methods Bacterial strains, plasmids, and culture conditions Bacterial strains and plasmids are described in Table 1. E. coli cells were grown in Luria-Bertani broth (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) at 37 °C with aeration. When necessary, ka- namycin and chloramphenicol were added to a final concentration of 50 g/ml. Coryneform cells were grown in trypticase/soy broth (TSB, Difco) or TSB containing 2% agar at 30°. For testing auxotrophs, minimal medium for corynebacteria (Kaneko and Sakaguchi 1979) was used and, when needed, supplemented with 0.3 mM threonine, homoserine and methionine. DNA isolation and manipulation Plasmid DNA was isolated from E. coli according to the method of Birnboim and Doly (1979) and from corynebacteria as described by Kieser (1984). E. coli cells were transformed by the method of Appl Microbiol Biotechnol (1996) 46: 554–558  Springer-Verlag 1996 C. Ferna ´ndez-Gonza ´lez · J. A. Gil L. M. Mateos · J. F. Martin (&) Area de Microbiologı ´a, Departamento de Ecologı ´a, Gene ´tica y Microbiologı ´a, Facultad de Biologı ´a, Universidad de Leo ´n, Spain. Fax: +34 87 291506 A. Schwarzer · A. Scha ¨fer · J. Kalinowski · A. Pu ¨ hler Department of Genetics, University of Bielefeld, P.O. Box 100131, D-33501 Bielefeld, Germany