Ever since the industrial revolution, humankind has made tremendous scientific progress in an exponentially growing way. However, the dependency of processes resulting from this progress on the burning of fossil fuels causes pollution, environmental problems and, most importantly, climate change. Because of these downsides, a huge interest arose in the development of industrially relevant, green production processes based on renewable resources. Industrial, or white, biotechnology plays an important role herein by using microorganisms and their enzymes for the sustainable production of industrially relevant compounds. Moreover, the emerging of metabolic engineering, synthetic biology and systems engineering allows to further expand the use of these microorganisms to product targets beyond their own metabolites and to express new-to-nature pathways leading to a wide variety of interesting molecules. An attractive production host in these processes is the eukaryotic host Saccharomyces cerevisiae. However, many challenges still remain in rewiring this host into a so-called microbial cell factory which is relevant on an industrial scale. The conversion of renewable resources to the desired product through the overexpression of heterologous pathways often leads to metabolic burden and suboptimal production. Furthermore, imbalances in the pathway may lead to the accumulation of (toxic) intermediates and the loss of intermediates to competing pathways, thereby reducing the efficiency. Additional crosstalk between the heterologous pathway and other cellular processes may also lead to unwanted reactions. In nature, two distinct strategies can be found to limit this metabolic crosstalk: the formation of an enzyme complex or metabolon and the use of subcellular compartments or organelles in eukaryotes. The latter also offers the advantage of sequestration of possible toxic intermediates, thereby shielding other cellular processes from these compounds. In both cases, the enzymes are co-localized to a smaller subspace in the cell, leading to higher local enzyme and metabolite concentrations and hence higher pathway fluxes. This doctoral thesis aimed at investigating several co-localization strategies to minimize this crosstalk and avoid the accumulation of (toxic) intermediates by focusing on the flavonoid pathway as case-study.