Yeast Mitochondrial Engineering: Targeting the Powerhouse of the Cell for Advanced Biofuel Production

Yeast metabolic engineering has mostly focused on the construction of metabolic pathways in the cell cytoplasm. However, there is huge potential in harnessing the diversity of environments, metabolites and enzymes that exist in the different organelles of eukaryotic cells, to enhance engineered metabolic pathways. We developed new molecular tools to expedite the simultaneous construction of multiple metabolic pathways targeted to either the cytoplasm or the mitochondria of yeast. With these tools, we show that targeting biosynthetic pathways to the mitochondria of the yeast Saccharomyces cerevisiae improved the production of advanced biofuels by as much as 500%, compared to identical pathways targeted to the cytoplasm. The mechanisms of this enhancement include (1) the elimination of metabolic bottlenecks, (2) increased availability of intermediates to the engineered pathways, and (3) increased local concentrations of enzymes due to their confinement inside the smaller volume of mitochondria. Our study opens multiple new avenues to target metabolic pathways to yeast mitochondria and affect mitochondrial physiology for the benefit of engineered pathways.