BME PhD Defense: Cristian E. Coriano-Ortiz

Title: " Engineering optogenetic tools for protein degradation in Escherichia coli."

Advisory Committee: Dr. Ahmad (Mo) Khalil (Chair) Dr. Mary Dunlop (Advisor) Dr. John Ngo Dr. Joe Larkin Dr. Zeba Wunderlich

Abstract: Optogenetics has had a significant impact in synthetic biology by providing precise spatiotemporal control over cellular processes. However, most existing bacterial optogenetic tools control transcriptional regulation, which is inherently limited by delays associated with gene expression and a dependence on cell growth for protein dilution. To address these limitations, this dissertation focuses on the development of post-translational optogenetic tools that offer rapid, modular, and growth-independent control of protein abundance and activity. We first describe the engineering of LOVdeg, a modular, blue-light-responsive protein degradation tag. By engineering the Avena sativa LOV2 domain (AsLOV2) to expose a degron upon light induction, we target proteins of interest for active degradation via endogenous Escherichia coli proteases. We demonstrate the versatility of LOVdeg by controlling the degradation of proteins from different classes, including transcription factors, enzymes, and transmembrane proteins. We also showcase the utility of LOVdeg in metabolic engineering by dynamically regulating fatty acid synthesis. Next, we explore the development of allosteric control in the exogenous Mesoplasma florum Lon (mf-Lon) protease, which operates in an orthogonal fashion to native E. coli degradation machinery. Using a high-throughput domain-insertion approach combined with Bayesian inference, we identify novel variants in which enzymatic activity is modulated by light, demonstrating the possibility of engineering allosteric control within the complex structure of a hexameric protease. Together, the tools and methods presented in this work expand the optogenetic toolbox for post-translational regulation and provide new strategies for dynamic control of biological networks.