Genome-wide analysis of the l-methionine biosynthetic pathway in Corynebacteriumglutamicum by targeted gene deletion and homologous complementation

Abstract

The genome sequence of Corynebacterium glutamicum, a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis of l-methionine. The functions of candidate ORFs were derived by gene deletion and, if necessary, by homologous complementation of suitable mutants. Of nine candidate ORFs (four of which were known previously), seven ORFs ( cg0754 ( metX), cg0755 ( metY), cg1290 ( metE), cg1702 ( metH), cg2383 ( metF), cg2536 ( aecD), and cg2687 ( metB)) were demonstrated to be part of the pathway while two others ( cg0961 and cg3086) could be excluded. C. glutamicum synthesizes methionine in three, respectively four steps, starting from homoserine. C. glutamicum possesses two genes with similarity to homoserine acetyltransferases but only MetX can act as such while Cg0961 catalyzes a different, unknown reaction. For the incorporation of the sulfur moiety, the known functions of MetY and MetB could be confirmed and AecD was proven to be the only functional cystathionine β-lyase in C. glutamicum, while Cg3086 can act neither as cystathionine γ-synthase nor as cystathionine β-lyase. Finally, MetE and MetH, which catalyze the conversion of l-homocysteine to l-methionine, could be newly identified, together with MetF which provides the necessary N 5-methyltetrahydrofolate.