Attenuated live Salmonella enterica serovar Typhimurium is a versatile organism for the generation of live recombinant vaccines for mucosal immunization and various approaches were devised for the stable and efficient expressions of heterologous antigens by attenuated S. enterica strains. Phage lamda Red recombinase has recently been devised for gene replacements in S. enterica after introduction of PCR products as a one-step deletion approach and FLP-mediated recombination allows the subsequent removal of antibiotic resistance markers. As an extension of this method, we have developed an approach that allows the sequential integration of multiple recombinant expression cassettes for heterologous antigens into the chromosome of S. enterica. We observed the stable expression of model antigens without selective pressure. In addition, the method allows the simultaneous generation of double-attenuating mutations by gene deletions. This approach allows the rapid and efficient construction of recombinant Salmonella strains as vaccine carriers.