Pathway engineering is the use of recombinant DNA technology to modify existing, or to introduce entirely new, metabolic pathways and regulatory systems within cells in order to improve their capacity to overproduce a desired molecule [10,15,26,41,43]. By channeling metabolic pathways in organisms toward a desired metabolite through rational introduction, modication, and removal of genes, a wide range of valuable products can be produced. Unfortunately, many desired compounds are produced only in small amounts in their native or engineered hosts. In the past, the improvement of metabolic pathways has been done primarily by evolutionary breeding methods or repeated rounds of mutagenesis and selection of a desired phenotype [61,66]. Recent advances in recombinant DNA technology allow more rational approaches to be applied to metabolic pathway engineering. Productivity of the desired metabolite can be increased by carefully balancing the expression of the genes and metabolic ux, both within the metabolic pathway and between the pathway and the host’s native metabolism. Pathway engineering also involves improvement of the overall cellular physiology, extension of the host’s substrate range, and deletion or reduction of by-product formation [24]. In this chapter, we review the most important considerations for successful metabolic pathway engineering and illustrate the concepts with selected examples.