Cell-free Metabolic Engineering Strategies for Accelerated Biomanufacturing

Biomanufacturing propels the bioeconomy. Accelerating bioeconomic growth thus requires the expedited development of biomanufacturing processes that can expand the current bioproduct portfolio. Lysate-based cell-free systems provide unique advantages for simplified metabolic pathway construction. Their central metabolic pathways and transcriptional-translational (TX-TL) machineries are free from genome regulation and are amenable to direct manipulation, enabling the streamlined construction of biomanufacturing processes. While their utility as prototyping platforms for accelerating cellular metabolic engineering has been demonstrated, the potential to rapidly build commercial “cell-free factories” capable of sophisticated bioconversion has not been fully realized. Lysates with high-yield pathways are projected to enable commercialized cell-free biomanufacturing of high-value chemicals. However, strategies that can be incorporated into frameworks for lysate pathway yield optimization rely on traditional cell-based metabolic engineering techniques that are cumbersome to extracts’ source strains (e.g., gene knockouts and cell-based overexpression) and thus lengthen design-build-test-learn (DBTL) cycles. The work in this dissertation introduces new strategies for cell-free pathway engineering that can benefit conversion yields in lysates, with a focus on 1) restructuring the endogenous metabolic proteome post-lysis and 2) optimizing the lysate TX-TL machinery for the cell-free overexpression of complex heterologous enzymes. By minimizing the involvement of cell-based engineering, these strategies enable faster endogenous and heterologous pathway build cycles compared to previous approaches. Part one describes the development of the first lysate flux-rewiring approach that enables conversion through a native pathway at 100% of the theoretical yield. Part two reports on the design and development of a plate reader assay for troubleshooting the expression of biosynthetic enzymes in lysates. The assay allows the generalizable screening of cell-free expression conditions with higher throughput than previous approaches and is leveraged to improve the lysate-based expression of natural product forming megasynthases. Refining and integrating these approaches into cost-effective cell-free metabolic engineering (CFME) workflows will enable rapid high-yield metabolic pathway construction, advancing lysates as sustainable biomanufacturing platforms.