Quantifying the efficiency of Saccharomyces cerevisiae translocation tags

Compartmentalization of metabolic pathways into organelles of the yeast Saccharomyces cerevisiae has been used to improve chemical production. Pathway compartmentalization aids chemical production by bringing enzymes into close proximity to one another, placing enzymes near key starting metabolites or essential co-factors, increasing the effective concentration of metabolic intermediates, and providing a more suitable chemical environment for enzymatic activity. Although several translocation tags have been used to localize enzymes to different yeast organelles, their translocation efficiencies have not been quantified. Here, we systematically quantify the translocation efficiencies of 10 commonly used S. cerevisiae tags by localizing green fluorescent protein (GFP) into three yeast organelles: the mitochondrion (4 tags), the vacuole (3 tags), and the peroxisome (3 tags). Further, we investigate whether plasmid copy number or mRNA levels vary with tag translocation efficiency. Quantification of the efficiencies of S. cerevisiae translocation tags provides an important resource for bioengineering practitioners when choosing a tag to compartmentalize their desired protein. Finally, these efficiencies can be used to determine the percentage of enzyme compartmentalization and, thus, help better quantify effects of compartmentalization on metabolic pathway efficiency.