STUDYING AND ENGINEERING MEMBRANE-BOUND AND MEMBRANELESS ORGANELLES FOR BIOTECHNOLOGY

Abstract

This thesis consists of three chapters that dive into the biology and engineering of membrane-bound and membraneless organelles. While these are two very different groups of cellular organelles, they both contribute to cellular architecture, are complex in their own right, and are employed in biotechnology for therapeutic, commercial, and industrial purposes. The first chapter gives a technical overview of membraneless and membrane-bound organelles. In part one of the first chapter, we give background about phase separation, the proteins that undergo phase separation to form membraneless organelles, and how biotechnology employs these organelles. In the second part of chapter one, we give an overview of mitochondrial engineering that serves as the basis for chapter three. The second chapter of the thesis dives into developing a high-throughput method to study phase separation interactions. We developed a novel method to screen for weak phase-like protein interactions. Lastly, we perform validation studies and show instances of droplet formation and colocalization of our hits in yeast. In the second chapter, we take a step back and, in this case, engineer mitochondrial morphology to increase chemical production. We show that engineering mitochondrial morphology can significantly improve the activity of some biosynthetic pathways targeted to this organelle (isobutanol, by over 2-fold) but not others (geraniol). Our results suggest that the difference depends on the involvement of acetyl-CoA. Pathways that do not depend on acetyl-CoA are more likely to benefit from altered mitochondrial morphology. This study lays the groundwork for how organelle morphology can be engineered to improve the production of valuable chemicals. Taken together, we show two different approaches of how both membraneless and membrane-bound organelles may be used and studied for biotechnological applications.