Glycolysis, a biochemical pathway that oxidizes glucose to pyruvate, is at the core of sugar metabolism in Saccharomyces cerevisiae (bakers’ yeast). Glycolysis is not only a catabolic route involved in energy conservation, but also provides building blocks for anabolism. From an applied perspective, several glycolytic intermediates are key precursors for the production of a wide range of highly valuable compounds. The most obvious case is the production of ethanol from pyruvate. Its ability to rapidly ferment sugars to ethanol has made S. cerevisiae the major microbial player in large scale biofuel production. Because of its importance in cellular processes and in the biotechnology industry, glycolysis in S. cerevisiae has been studied in detail. However, despite the large amount of information generated about all the components in glycolysis, the limited understanding on how these components interact and are co-ordinately regulated to ensure a robust and balanced pathway, has to date defied all metabolic engineering attempts to significantly accelerate glycolytic flux. So far, the mechanisms that govern the glycolytic flux are not fully known. A particularly poorly understood factor in glycolysis is its high genetic redundancy. This phenomenon is observed in many organisms, but is highly pronounced in S. cerevisiae which, for most glycolytic reactions, harbours multiple isoenzymes and corresponding paralogous genes. The contribution of the glycolytic paralogs to the glycolytic flux is unknown and the simultaneous presence of different isoenzymes – with potentially different kinetic and regulatory properties - complicates the mathematical modelling that is required for a deeper understanding of the regulation of this industrially relevant pathway. Saccharomyces cerevisiae’s glycolytic pathway consist of ten biochemical reactions. In this Crabtree positive yeast, glycolysis is under most growth conditions linked to ethanol formation. Without considering the very complex transport of glucose from the extracellular environment to the intracellular compartment, glycolysis and ethanol fermentation together encompass 12 biochemical reactions. These biochemical reactions are catalysed by enzymes encoded in 27 glycolytic genes, separated in eight paralog families and four unique structural genes. S. cerevisiae evolved from an ancestor that, approximately 100 million years ago, underwent a whole genome duplication (WGD). Many of its hallmark phenotypic characteristics have been proposed to be the result of this duplication event and the subsequent genome rearrangement, gene loss and gains through evolution. In S. cerevisiae, no fewer than eight of the ten enzyme reactions in glycolysis are represented by multiple paralogs genes. This incidence of paralogous combinations represents a significant overrepresentation relative to the ca. 26 % of the yeast genome that consists of paralogous combinations. The WGD event and resulting duplication of glycolytic genes has been proposed to have contributed to the strong tendency of this yeast to produce ethanol under aerobic conditions (Crabtree effect) and to its high glycolytic capacity. However, the impact of reducing the number of glycolytic paralogs on these and other physiological characteristics of S. cerevisiae has not been systematically explored. All glycolytic reactions are equally essential for yeast growth on glucose. However, for all paralogs gene sets in yeast glycolysis, with the notable exception of phosphofructokinase, gene expression and gene deletion studies support the definition of a single, major paralog and one to four minor paralogs. Additionally, except for the pseudogenes GPM2 and GPM3, all paralogs have retained their original catalytic function, although their context-dependent expression profiles differ. Furthermore, deletion of minor paralogs for individual glycolytic enzymes has minor effects on enzyme activities in cell extracts and on specific growth rate under standard laboratory conditions. Reduction of the genetic complexity in S. cerevisiae’s glycolysis could deliver a more predictable and malleable glycolytic pathway. These characteristics could greatly benefit the biotechnology industry and our understanding about glycolysis. Thus, the main goal of this thesis was the construction and analysis of a strain with a minimal set of glycolytic enzymes. Despite the astonishing genetic accessibility of S. cerevisiae and its broad genetic toolbox, large-scale deletion strategies, like the deletion of 13 genes undertaken in the present thesis, were a challenging endeavour at the outset of this project, that only few previous studies had tackled. Despite the large collection of selectable marker genes for genetic modification available for S. cerevisiae, marker availability still presented a hurdle when dozens of genetic deletions were required. Additionally, the presence of the selectable markers in the host genome can influence the fitness or performance of the strain, thus, different methods for marker removal were required. Deletion of the hexose transporter genes (HXT’s) in S. cerevisiae has, for over a decade, represented the paradigm for elimination of complexity in a large, redundant paralog family. This yeast harbours a large group of tightly controlled transporters with different characteristics for glucose uptake. This family is composed of genes with similar function but that are expressed under different conditions, thereby allowing yeast to grow and cope with large and dynamic changes in glucose concentration. Interestingly, deletion of single or several HXT’s does not abolish growth on glucose. In 1999, Eckhard Boles and his team took on the enormous endeavour of removing all transporters capable to import glucose in S. cerevisiae, resulting in the strain EBY.VW4000. Ever since its construction, EBY.VW4000 has become a widely used platform for the discovery and characterization of transporters from a wide range of organisms and as a platform strain for metabolic engineering approaches. Additionally, EBY.VW4000, with its reduced genetic redundancy in glucose transport, could serve as an splendid platform for the construction of a minimal glycolytic pathway. Despite the extensive usage of this strain, the genome of EBY.VW4000 had hitherto not been characterized in detail. Chapter 2 addresses this information gap and presents the whole-genome sequence of EBY.VW4000. To abolish glucose uptake in this strain, 21 genes (including all HXT’s) had been knocked-out across 16 successive deletion rounds with the LoxP/Cre system. Based on a combination of whole-genome sequencing, karyotyping and molecular confirmation, we demonstrated that the construction of EBY.VW4000 resulted in gene losses and chromosomal rearrangements guided by LoxP/Cre. In contrast, only 13 single nucleotide variations (SNV’s) were identified. Recombinations between LoxP scars led to the assembly of four neo-chromosomes, truncation of two chromosomes and the loss of two telomeric regions. By karyotyping the EBY.VW4000 lineage, it became clear that its current chromosomal architecture has resulted from four translocations events that occurred between the 6th and the 12th rounds of deletion/marker recycling. Additionally, sporulation and spore germination were found to be severely impaired in EBY.VW4000. This work also demonstrated that, due to the massive LoxP/Cre-induced genome modifications observed, neither EBY.VW4000 nor LoxP/Cre were suitable for the construction of a minimal glycolysis strain. Therefore, a combination of classical genetic deletion and novel tools and methodologies were developed and implemented. In Chapter 3 the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS) is presented. This module confers laboratory, wild and industrial Saccharomyces strains the ability to use acetamide as sole nitrogen source. Direct repeats flanking the marker cassette allow for its efficient recombinative excision. This cassette loss can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in Saccharomyces strains. Furthermore, using astute cassette design, amdSYM excision could be performed without leaving a scar or heterologous sequences in the targeted genome. The amdSYM cassette is available for the scientific community via the Euroscarf collection. Including the amdSYM marker cassette, only four counter-selectable markers for S. cerevisiae are available. Extensive strain engineering is severely hampered by this limited marker availability and by the reduced genome stability that occurs upon repeated use of heterologous recombinase-based marker removal methods such as LoxP/Cre system. Chapter 4 introduces an efficient method to recycle multiple markers in S. cerevisiae simultaneously, thereby circumventing shortcomings of existing techniques and substantially accelerating the process of selection-excision. This method relies on artificial generation of double strand breaks around the selection marker cassette by the meganuclease I-SceI and the subsequent repair of these breaks by the yeast homologous recombination machinery, guided by direct repeats. Simultaneous removal of up to three marker cassettes was achieved with high efficiencies (up to 56%). This locus- and marker-independent method can be used for both dominant and auxotrophy-complementing marker genes. Chapter 5 describes the experimental exploration of genetic redundancy in yeast glycolysis by cumulative deletion of minor paralogs and presents a new experimental platform for fundamental yeast research by constructing a yeast strain with a functional minimal glycolysis. The construction of this strain was performed with a combination of classical genetics tools and the newly developed methodologies presented in this thesis. It encompassed the deletion of 13 glycolytic paralogs considered to have a minor contribution to flux. After thorough experimental analysis, using quantitative and systems approaches, under growth conditions leading to a high glycolytic flux and after semi-quantitative analysis under a wide range of growth conditions, the most remarkable feature of the minimal glycolysis strain was the lack of visible phenotypic response to the deletion of 13 genes. The high glycolytic rates in anaerobic cultures of the minimal glycolysis strain and the small effect on the transcriptome argue against gene dosage or back-up effects as means for fixing minor glycolytic paralogs in the yeast genome. The near-wild type growth kinetics of the minimal glycolysis strain in aerobic and anaerobic cultures are difficult to reconcile with the hypothesis that duplication of glycolytic genes during the WGD event played a major role in increasing its glycolytic capacity or in causing the Crabtree effect. This reduction of genetic complexity contributed to the understanding of yeast glycolysis by, for the first time, studying the synergetic effects of multiple deletions of glycolytic genes. Moreover, it eliminated intrinsic uncertainties caused by the simultaneous, context-dependent expression of different isoenzymes, facilitating the formulation and validation of mathematical models that describe the kinetics of this key metabolic pathway. Analysis of the physiology and fitness of the minimal glycolysis strain under dynamic man-made and natural conditions may contribute to the daunting but exciting task of resolving the origin, fate, evolution and role of glycolytic paralogs. Additionally, it will serve as a basis for a more extreme genetic and pathway engineering strategy, in which different glycolytic pathways can be redesigned and interchanged in two simple steps.