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[Display omitted] • Cell-free systems enable biochemical reactions in the absence of cell viability constraints. • Cell-free metabolic engineering presents opportunities for biomanufacturing diverse chemical products. • Native metabolism in cell extracts can be coupled with exogenous or artificial pathways to use renewable carbon sources. • The open, cell-free reaction environment is amenable to electrochemical and photosynthetic energy regeneration modules. • Increasing cell-free reactions beyond the prototyping scale will enable sustainable biomanufacturing. Industrial biotechnology is an attractive approach to address the need for low-cost fuels and products from sustainable resources. Unfortunately, cells impose inherent limitations on the effective synthesis and release of target products. One key constraint is that cellular survival objectives often work against the production objectives of biochemical engineers. Additionally, industrial strains release CO 2 and struggle to utilize sustainable, potentially profitable feedstocks. Cell-free biotechnology, which uses biological machinery harvested from cells, can address these challenges with advantages including: (i) shorter development times, (ii) higher volumetric production rates, and (iii) tolerance to otherwise toxic molecules. In this review, we highlight recent advances in cell-free technologies toward the production of non-protein products beyond lab-scale demonstrations and describe guiding principles for designing cell-free systems. Specifically, we discuss carbon and energy sources, reaction homeostasis, and scale-up. Expanding the scope of cell-free biomanufacturing practice could enable innovative approaches for the industrial production of green chemicals. [ABSTRACT FROM AUTHOR]

Engineering metabolism for the synthesis of bio-based products in non-model organisms can be challenging. One specific challenge is that biosynthetic pathways are often built from enzyme candidates sourced from diverse organisms, which can prove difficult to implement in recombinant hosts due to differences in their cellular environments (e.g. pH, cofactor balance). To address this problem, we report a cell-free synthetic biology approach for understanding metabolism in a range of environmental conditions, specifically under varied pH. The key idea is to control the pH of Escherichia coli -based cell-free systems for assessing pathway performance using enzymes sourced from organisms other than E. coli. As a model, we apply this approach to study the impact of pH on the n -butanol biosynthesis pathway derived from clostridia in E. coli lysates. Specifically, we exploit the open, cell-free reaction environment to explore pH outside the habitable range of E. coli , revealing insights into how chemical context impacts the interaction between native metabolism and heterologous enzymes. We find that the pH optimum for butanol production from acetyl-CoA is substantially lower than the optimal pH of glycolysis in E. coli -based crude lysates. In addition, pH is an essential factor to consider when activating metabolic pathways in the cell-free environment due to its effect on reaction yield or enzyme activity, the latter of which is demonstrated in this work for alcohol dehydrogenases from a range of extremophiles. Ultimately, altering metabolism through pH control will allow cell-free systems to be used in studying the metabolic state of organisms and identify suitable enzymes for pathway engineering. [ABSTRACT FROM AUTHOR]