Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways |
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| Institution: | 1. Department of Chemical Engineering, Kyungpook National University, Daegu 702-701, South Korea;2. Department of Chemical Engineering, College of Engineering, Dhofar University, Salalah 211, Oman;1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA;2. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA;3. Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA;4. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA;5. Simpson Querrey Institute, Northwestern University, Chicago, IL 60611, USA;1. Department of Chemical Engineering, Stanford University, 443 Via Ortega, Stanford, CA 95305-4300, USA;2. Department of Bioengineering, Stanford University, 443 Via Ortega, Stanford, CA 95305-4300, USA;1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA;2. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA;3. Robert H. Lurie Comprehensive Cancer Center Northwestern University, Chicago, IL 60611, USA;4. Simpson Querrey Institute Northwestern University, Chicago, IL 60611, USA |
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| Abstract: | Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes. |
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| Keywords: | Clostridia Cell-free gene expression Cell-free protein synthesis Cell-free metabolic engineering Synthetic Biology Prototyping genetic parts |
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