Construction of lysine-producing strains by gene disruption and replacement in Brevibacterium divaricatum

Gene disruption and replacement techniques were applied to block the biosynthesis of threonine and methionine and thus to construct genetically stable lysine producers in a glutamate-producing bacteria, Brevibacterium divaricatum. The homoserine dehydrogenase gene (hom), homoserine kinase gene (thrB) and hom-thrB operon were amplified as 1.8, 1.25 and 2.8 kb fragments from B. divaricatum by polymerase chain reaction (PCR) and cloned in an E. coli-coryneform bacteria shuttle vector, pSUMN18. These genes were disrupted by inserting a kanamycin resistant gene (kan) from pUC4-KISS into the structural genes. Integrative plasmids were constructed and transformed into B. divaricatum. Integrative transformants could be obtained only when the integrative plasmids were constructed from the plasmids which had escaped from the restriction barrier of the hosts. The resulting integrative transformants showed kanamycin resistance and contained no plasmids. About 1-10% of the transformants were auxotrophs. By checking the nutritional requirement, it was found that all of these transformants required threonine and/or homoserine as expected. Southern blot analysis confirmed the integrations, and both single and double crossover homologous recombination mechanisms were proposed to explain the integration and replacement. These auxotrophic integrative transformants which were derived from double crossover events accumulated 1-3% lysine in culture broth only when the added threonine was limited. Integrative transformants which were site-specifically inactivated in hom or hom-thrB genes produced more lysine than did those only inactivated at the thrB gene. These transformants were extremely stable, and the reversion frequency was below 10(-9) per generation. It is suggested that this technique will be useful in the construction of stable auxotrophic mutants.