Microbial systems have gained increased importance in biotechnology as platforms for a wide range of applications, including but not limited to biomedical applications, cell factories, and bioremediation. To date, bacterial systems have led the way due to their simplicity of their biological network compared to eukaryotic systems. Furthermore, the wide range of genetic manipulation tools and fast generation times allows for a rapid turnover between hypothesis and experimental validation. However, because of their simplicity, bacterial hosts are often restricted to a specified range of environmental stress. Eukaryotic systems, on the other hand, have a more complex biological network that allows them to adapt more readily to higher levels of stress compared to bacterial systems. Here the physiology of three yeast species, Pichia pastoris, Schizosaccharomyces pombe, and Kluyveromyces marxianus, are investigated through their metabolic network and analyzed using the reconstructed metabolic model of the respective species. In P. pastoris, the physiology of the yeast while producing heterologous proteins was investigated. S. pombe was validated extensively using the single gene knockout library and refined iteratively, resulting in an increased accuracy in predicting mutant phenotype. Finally, K. marxianus physiology was explored to develop systems metabolic engineering strategies for achieving high levels of an industrially important compound, 3-hydroxypropionate. Furthermore, three different versions of the K. marxianus genome-scale metabolic model with changes to charge and mass balance for the reactions to represent the metabolism at three different pH levels.