Engineering Optogenetic Control of Bacterial Metabolism in Stationary Phase of Growth

dc.contributor.advisorTabor, Jeffrey Jen_US
dc.contributor.advisorThyer, Rossen_US
dc.creatorLazar, John Tyleren_US
dc.date.accessioned2024-01-24T17:20:41Zen_US
dc.date.available2024-01-24T17:20:41Zen_US
dc.date.created2023-08en_US
dc.date.issued2023-09-06en_US
dc.date.submittedAugust 2023en_US
dc.date.updated2024-01-24T17:20:41Zen_US
dc.description.abstractGenetically-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.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationLazar, John Tyler. "Engineering Optogenetic Control of Bacterial Metabolism in Stationary Phase of Growth." (2023). PhD diss., Rice University. https://hdl.handle.net/1911/115382en_US
dc.identifier.urihttps://hdl.handle.net/1911/115382en_US
dc.language.isoengen_US
dc.rightsCopyright 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.en_US
dc.subjectSynthetic Biologyen_US
dc.subjectTwo-component Systemsen_US
dc.subjectMetabolic Engineeringen_US
dc.subjectOptogeneticsen_US
dc.titleEngineering Optogenetic Control of Bacterial Metabolism in Stationary Phase of Growthen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentChemical and Biomolecular Engineeringen_US
thesis.degree.disciplineEngineeringen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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