Pollution-intensive industrial manufacturing processes threaten the health of ecosystems and societies through toxic waste streams and energy intensive processes that lead to greenhouse gas emissions. Biological systems present more sustainable routes to many useful industrial chemicals by using enzymes at low temperatures, but the time and effort required to optimized cellular biomanufacturing platforms typically makes these strategies slow and expensive to develop. As an effort to accelerate biological production approaches, my doctoral research implemented cell-free systems from Escherichia coli and Saccharomyces cerevisiae to reconstitute biosynthetic pathways in vitro for rapid biochemical production. Cell-free synthesis of proteins and metabolites relies on the crude extracts from lysed cells that contain active biological machinery in the absence of viability constraints, genomic regulation, or membrane barriers. Cell extracts are extremely flexible systems with an open reaction environment that enables greater control over variables than cell cultures to produce proteins and metabolites, generating a wide variety of enzymes by changing DNA templates or altering an extract’s metabolic potential by changing the source strain or growth conditions. This strategy of cell-free optimization and prototyping enabled me to produce more than 10 unique biochemical products in 5 years, which is much greater than traditional metabolic engineering approaches, and it contributed to the development of higher efficiency and carbonnegative biomanufacturing efforts. Overall, my research exploited the flexibility of cell-free metabolism for characterization, prototyping, and biomanufacturing to gain insights into extract-based systems and apply them toward sustainable biosynthesis strategies.