(500a) Cell-Free Biosynthesis to Inform Metabolic Engineering of Non-Model Bacteria

The accelerating climate crisis requires sustainable technologies, such as biological systems, to reduce greenhouse gas emissions across industrial sectors. A promising approach has emerged in pursuit of this goal using Clostridium autoethanogenum as a chassis for biological carbon capture. Although this anaerobic, non-model bacterium natively produces ethanol from carbon monoxide, efforts to expand the product scope have been limited due to slow growth and a lack of well-developed genetic tools. To facilitate metabolic engineering in C. autoethanogenum to produce value-added products from waste gases, we implemented cell-free biosynthesis for metabolic pathway prototyping. Cell-free systems comprising crude cell extracts enable the exploration of biochemical conversions in the absence of growth and viability constraints with greater throughput and flexibility than living cells, which facilitates rapid design-build-test cycles. We have demonstrated the production of diverse metabolites in cell-free systems derived from E. coli with enzymes expressed either during cell growth or directly in the cell extract, which enables prototyping of heterologous biosynthetic enzymes and competing endogenous pathways to maximize flux to the desired product. This cell-free prototyping approach accelerated the engineering of C. autoethanogenum by identifying multiple gene knockouts that resulted in highly productive strains converting greenhouse gases to acetone and isopropanol. Such iterative prototyping and strain optimization would have taken at least 12 months longer using in vivo methods alone. Continuous culture of engineered acetone production strains at industrial pilot scale produced up to 3 g/L-h while demonstrating significant greenhouse gas savings compared to conventional processes. Together, cellular and cell-free metabolism offer a promising path toward the rapid development of efficient and sustainable biosynthesis platforms.