In the past few decades, concerns on climate change have contributed to the development of biotechnological alternatives for petrochemical processes. In order to make these processes economically competitive, there has been an intensive search for improved, more efficient production organisms. In the past decade, genetic engineering of microorganisms has undergone a tremendous development. The radical modification or replacement of a key route in metabolism requires a strategy in which the essential cellular function of this route is not interrupted while it is being replaced. In this thesis, such a strategy for radical reprogramming of metabolism is developed and applied to the sugar metabolism (glycolysis) of baker's yeast (Saccharomyces cerevisiae). The application of such a large number of changes in the yeast genome implied the exploration of a new scale of genetic modification and demanded the further development of several modification techniques. The results presented in this thesis demonstrate that it is possible to integrally replace an essential metabolic pathway, which is essential for the viability of the cell. This research therefore provides a basis for a new, modular approach for metabolic engineering, in which entire metabolic routes can be quickly replaced and optimized. The ability to quickly replace the yeast glycolysis by alternative routes supplies also a wonderful tool for fundamental research into the regulation of the glycolytic "flux". This may eventually contribute to the further development of S. cerevisiae to a more efficient industrial platform for the production of chemicals from sugars. In addition, the present study provides an illustration of the rapid developments and new possibilities in the field of genetic modification.