Escherichia coli K12 strains producing L-phenylalanine were converted to L-tyrosine-producing strains using a novel genetic method for gene replacement. We deleted a region of the E. coli K12 chromosome including the pheA gene encoding chorismate mutase/prephenate dehydratase, its leader peptide (pheL), and its promoter using a new polymerase chain reaction-based method that does not leave a chromosomal scar. For high level expression of tyrA, encoding chorismate mutase/prephenate dehydrogenase, its native promoter was replaced with the strong trc promoter. The linked Delta pheLA and Ptrc-tyrA::Kan(R) genetic modifications were moved into L-phenylalanine producing strains by generalized transduction to convert L-phenylalanine-producing strains to L-tyrosine-producing strains. Moreover, introduction of a plasmid carrying genes responsible for sucrose degradation into these strains enabled L-tyrosine-production from sucrose.