Carothers, James MZalatan, Jesse GCardiff, Ryan Asher Levine2025-08-012025-08-012025-08-012025Cardiff_washington_0250E_28066.pdfhttps://hdl.handle.net/1773/53348Thesis (Ph.D.)--University of Washington, 2025The chemical industry’s reliance on fossil fuels drives significant carbon emissions, emphasizing the urgent need for sustainable alternatives. Microbial bioproduction presents a promising solution by enabling the synthesis of valuable chemicals from renewable feedstocks, such as CO2 and biomass waste. However, the ability to address wide chemical markets with bioproduction is limited by insufficient tools for metabolic pathway engineering and precise gene regulation. This work describes several strategies to improve bioproduction by prototyping engineered metabolic pathways and complex gene regulatory programs. We utilize an E. coli-based cell-free gene expression system to engineer high-performing synthetic promoters and investigate pathways for carbon-conserving bioproduction of industrially relevant chemicals. We then discuss recent progress and opportunities at the intersection of CRISPR-based metabolic engineering and systems-level modeling approaches for improved bioproduction. Finally, we adapt the RNA-targeting CRISPR-dCas13 system as a next-generation CRISPR tool to improve metabolic regulation in bacteria. Collectively, these works describe the development of several tools that advance metabolic engineers’ ability to construct complex gene expression programs, prototype engineered carbon-conserving pathways, and precisely regulate metabolism across bacterial genomes. These tools offer new approaches to engineer microorganisms that can incorporate renewable carbon feedstocks and efficiently upcycle them into value-added chemicals, advancing the goals of sustainable bioproduction and a reduced global reliance on fossil fuels.application/pdfen-USCC BY-NCbioproductionCRISPRgenetic engineeringmetabolic engineeringsynthetic biologyMolecular biologyMicrobiologyChemical engineeringMolecular engineeringThesis