(688a) Invited Speaker: Engineering Cupriavidus Necator for Valorization of Waste-Derived Substrates

Bioconversion of high-volume waste streams to value-added products offers a promising approach to bolster manufacturing resilience and support circularity in the bioeconomy. While microbial hosts have been utilized extensively for production of chemicals and fuels from “first-generation” feedstocks, such as glucose, production of the same compounds from alternative feedstocks has received less attention, likely due to the heterogeneity and potential toxicity of these “second-generation” streams. Cupriavidus necator H16 is a soil microbe with broad metabolic flexibility and tolerance, as well as genetic tractability, making it ideal for conversion of waste feedstocks. Promising waste-derived substrates for upcycling by C. necator include organic acids such as lactate and succinate, as well as volatile fatty acids (VFAs) like acetate, propionate, butyrate, valerate, and hexanoate. These products can be derived from arrested anaerobic digestion of organic waste streams (e.g., dairy waste, food waste) and electrochemical reduction of waste gases such as CO₂ and CO. Previous work has shown that C. necator can produce >60% of its dry cell weight as a bio-polyester, polyhydroxybutyrate (PHB), when cultivated on VFAs. In this work, we sought to eliminate PHB biosynthesis as a carbon sink and thereby improve the yield of other target products. We discovered that deletion of PHB biosynthesis led to a significant growth defect on VFA substrates, but we implemented evolution and engineering strategies to overcome this defect and generate efficient strains for production of food-grade protein from food waste. We incorporated process modeling, technoeconomic analysis (TEA), and life cycle assessment (LCA) to assess the efficacy of waste conversion to protein by C. necator. Overall, this work provides a roadmap for the integration of waste feedstocks in biomanufacturing systems, building toward sustainable alternatives to first-generation feedstocks.