Already for millennia, microbial fermentation is used for the production of dairy products, alcoholic beverages and bread. In the last decades, the field of biotechnology has tremendously expanded and nowadays, a wide range of compounds ranging from biofuels to chemicals and pharmaceuticals is produced using microbial cell factories. The development of genetic engineering tools has greatly contributed to this rapid development. Catalysing the conversion of renewable carbohydrate feedstocks into fuels and chemicals, microbial cell factories offer a sustainable alternative to fossil resources-based production, and thereby contribute to reduce greenhouse gas emissions. The yeast Saccharomyces cerevisiae plays an important role in industrial biotechnology. Its popularity for applied research and industrial production can be attributed to several factors as its fast fermentative metabolism, its tolerance to low pH, high sugar and alcohol concentrations and its genetic tractability. S. cerevisiae possesses one of the best furbished molecular toolboxes, which makes it possible to assemble complex heterologous pathways, as was recently illustrated by the successful biosynthesis of opioids in yeast. Despite this great progress, extensive genetic remodelling of native pathways remains challenging. This can largely be explained by the high genetic redundancy present in the yeast genome, in which multiple genes encode proteins with redundant functions, and by the fact that the genes belonging to a pathway are scattered over the entire genome. The goal of this thesis was to design, set up and validate a strategy aiming at facilitating the remodelling of (essential) pathways, based on simplifying and reorganizing the yeast genome. The starting point of this research is the central carbon metabolism and in particular, as proof of concept, the glycolytic pathway.@en

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.@en