The introduction of new metabolic activities into a host organism is an important assignment of metabolic engineering. Multi-copy plasmids may be best choice for the cloning and expression of recombinant genes, in particular for the maximum overproduction of one desired protein. However, the strong gene expression could be unfavorable for a increased productivity in metabolic engineering applications. To overcome this problem, heterologous genes can be cloned and expressed e.g. on low-copy plasmids. But the general disadvantage of recombinant plasmids is their possible structural instability and the need of selection markers to maintain the vectors in the host cell. In this presentation we describe a fast and efficient method for the integration of heterologous expression cassettes into a specific locus on the E. coli chromosome using the l-Red recombinase (1). The biosynthesis genes needed for the formation of lycopene and of tocochromanol-derivatives in E. coli, respectively, were integrated into redundant genes in the E. coli chromosome. As preferred locus we used genes responsible for the degradation of rare sugars, like fucIK, rhaBAD, rbsDK and others. By this way the integration into these loci can be confirmed easily using a fast screening on differential indicator medium (MacConkey-agar plates containing the corresponding sugar). The heterologous biosynthesis of lycopene as well as of tocochromanol will be presented. Plasmid-coded and chromosomal-integrated gene expression will be compared and their disadvantages and advantages will be discussed. (1) K.A. Datsenko and B.L. Wanner (2000) One-step inactivation of chromosomal genes in E.coli K12 using PCR products. PNAS, 97, 6640-5.