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.

自养细菌（如梭状芽孢杆菌）的气体发酵为从一系列当地、丰富的废物和低成本原料（如工业烟道气或由生物质或城市废物产生的合成气）生产众多生物产品提供了可持续途径。不幸的是，设计和工程化梭菌仍然费力且缓慢。在体外对个体遗传部分功能、基因表达模式和生物合成途径性能进行原型设计的能力在单元中实施设计之前，可以通过加速设计来帮助解决这些瓶颈。不幸的是，尚未开发出来自梭菌的高产无细胞基因表达 (CFE) 系统。在这里，我们报告了来自工业相关厌氧菌Clostridium autoethanogenum的高产 (236 ± 24 μg/mL) 批次 CFE 平台的开发和优化。该平台的一个关键特点是环状和线性 DNA 模板都可以直接应用于 CFE 反应以编程蛋白质合成。我们展示了原型基因表达的能力，并定量绘制了来自该系统的裂解物中的有氧无细胞代谢。我们预计C. autoethanogenum CFE 平台不仅将扩展用于合成生物学的蛋白质合成工具包，还将作为一个平台，加速非模型、工业相关微生物中基因调控元件的筛选和原型设计。