(265e) Principles of Metabolic Pathway Control By Biomolecular Condensates in Cells

Intracellular phase separation (PS) is now recognized as an important biophysical phenomenon that regulates a wide variety of cellular processes. One of its consequences is the creation of membraneless microcompartments, where selective biomolecules are enriched. Through this enrichment, the concentrations of these molecules are much higher than those outside the compartment. Inspired by this observation, PS has emerged as an effective method for regulating enzymatic reactions in a cell by clustering selective enzymes in microcompartments, which is expected to increase reaction fluxes towards products and decreases those towards byproducts. However, past studies have shown mixed success, which requires us to critically evaluate conditions when PS will be beneficial. In this study, we have developed mathematical models for common reaction networks and identified a simple metric to predict outcomes of PS. Through the metric, we identified two parameters that govern the overall outcome: enzymatic activity in the PS microcompartment and the fractions of enzymes partitioned to the microcompartment. Specifically, an enzyme activity needs to increase inside microcompartment to see the product gain through PS. If this criterion is met, the gain from implementing PS increases as the fraction of enzymes in microcompartment increases. As proof of concept, we engineered S. cerevisiae to produce acetoin, where corresponding enzymes were engineered to undergo PS. By changing the fraction of enzymes inside the compartment, we were able to change the acetoin biosynthesis accordingly. Thus, the derived metric will serve as an effective guideline to design and optimize PS compartmentalization strategy to optimize metabolic fluxes.