Carothers, James M.Sugianto, Widianti2025-10-022025-10-022025Sugianto_washington_0250E_28839.pdfhttps://hdl.handle.net/1773/53929Thesis (Ph.D.)--University of Washington, 2025Engineered biological systems, such as microbes and cell-free systems, have been harnessed for chemical bioproduction. However, their long-term use and field-deployment, especially in resource-limited environments, remain challenging: traditional microbial fermentations are often constrained by cellular limitations and genetic instability, while cell-free bioproduction is hindered by operational instability and complex downstream processing. This work outlines efforts to create robust bioproduction platforms by integrating engineered microbes and cell-free systems with biomaterial supports. We first demonstrated that engineered living materials from input-responsive microbes encapsulated in pluronic hydrogels maintained inducible responses over multiple weeks of continuous cultures and exhibited 10-times longer genetic activity than planktonic cells – enabling long-term, switchable bioproduction. Next, we developed reusable biocatalysts by co-immobilizing multiple cell-free enzymes in poly(ethylene glycol) diacrylate-glycerol hydrogels for bioproduction of malic acid, an industrially versatile chemical. Immobilized enzymes can be retained for at least a week and reused for multiple biocatalytic cycles, achieving up to 1.6-fold higher activity and 2-fold longer lifetimes than free enzymes in liquid reactions. Finally, to address poor expression of plant transmembrane cytochrome P450s, we devised an approach that supplements tailored liposomes to accommodate transmembrane protein expression and translocation in cell-free systems. Notably, supplying tailored liposomes from natural and synthetic lipids led to more than 2-fold increase in the expression of three plant P450s that could be used for bioproduction of bioactive natural products. Together, these advances provide a framework for developing durable, reusable, and versatile bioproduction platforms, paving the way for next-generation, on-demand chemical biomanufacturing.application/pdfen-USCC BY-NC-NDBiomaterialsBioproductionCell-free systemsEngineered Living MaterialsOn-demandChemical engineeringBioengineeringChemical engineeringThesis