Real-Time Optogenetic Control of Intracellular Protein Concentration and its Application in Increasing Ethanol Production Efficiency in S. cerevisiae

Abstract

The ability to precisely control the intracellular concentration of a protein without otherwise impacting the cellular environment has important implications for biological research and industrial applications. In this thesis, we describe a novel method for regulating the concentration of a single protein in real time in S. cerevisiae. We enhance the CIB1-CRY2 optogenetic induction system with an exogenous zinc finger DNA binding motif to generate simple and highly specific regulation of genetic induction. This control combined with real-time sampling and fluorescent imaging under the automatic regulation of an in silico feedback control suite allows us to generate and maintain precise adherence to both static and oscillating target protein concentration profiles. We applied this optogenetic technology to the optimization of ethanol production efficiency through the reduction of fermentation byproduct production. The production of glycerol was placed under the control of optogenetic induction through the regulation of GPD1 and GPD2, which enable glycerol production in response to cellular stress. We showed that light-based control of glycerol concentration is a potential means of increasing ethanol production efficiency during fermentation while maintaining cellular viability.