Tabor, Jeffrey JThyer, Ross2024-01-242024-01-242023-082023-09-06August 202Lazar, John Tyler. "Engineering Optogenetic Control of Bacterial Metabolism in Stationary Phase of Growth." (2023). PhD diss., Rice University. https://hdl.handle.net/1911/115382https://hdl.handle.net/1911/115382Genetically-encoded sensors are used to induce metabolite production in bacterial fermentations. However, these sensors are typically optimized for exponential growth phase rather than stationary phase where the majority of metabolite production occurs. In the first portion of this work, we find that our exponential phase-optimized green light-activated E. coli two-component gene regulatory system CcaSR is effectively non-functional in stationary phase. We show that the major causes of failure are stationary-phase specific mutation of the plasmid-borne biosynthetic pathway used to produce the required chromophore phycocyanobilin (PCB) and accumulation of the response regulator CcaR leading to very high leaky target gene expression. To address these problems, we move the PCB biosynthetic pathway into the chromosome and re-optimize expression of the component enzymes, and re-balance CcaR expression for stationary phase . The resulting CcaSRstat system exhibits low levels of leakiness and an 80-fold activation of target gene expression in stationary phase. Notably, our stationary phase-optimized CcaSRstat system is not functional in exponential phase, a feature that may have benefits for metabolic engineering and other applications. In the second portion of this work, we combine CcaSRstat with static and periodic illumination patterns to achieve high levels of production of several industrially-relevant phenylpropanoid metabolites such as p-coumaric acid and bisdemethoxycurcumin. We then proceed to demonstrate that our optimal light signals at the 0.5 mL light plate apparatus (LPA) volume scale to 25 mL optogenetic bioreactors. CcaSRstat is a useful tool for optimizing bacterial metabolite production and could be used to control bacterial behaviors in other non-growth environments such as the gastrointestinal tract or soil. Our work lays the foundation for increasing the exploration space of dynamic control of metabolic pathways while also providing valuable insight into design considerations of biosensors in the stationary phase of growth.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.Synthetic BiologyTwo-component SystemsMetabolic EngineeringOptogeneticsThesis2024-01-24