A <i>Clostridioides difficile</i> cell-free gene expression system for prototyping and gene expression analysis

<i>Clostridioides difficile</i> is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of <i>C. difficile</i> under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of <i>C. difficile</i>. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and <i>in vitro</i> screening. In this study, we developed a <i>C. difficile</i> CFE system capable of <i>in vitro</i> transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the protein yield significantly. Furthermore, our observations indicated that this system exhibited higher protein yield using linear DNA templates than circular plasmids for <i>in vitro</i> expression. The prototyping capability of the <i>C. difficile</i> CFE system was assessed using a series of synthetic <i>Clostridium</i> promoters, demonstrating a good correlation between <i>in vivo</i> and <i>in vitro</i> expression. Additionally, we tested the expression of <i>tcdB</i> and <i>tcdR</i> from clinically relevant <i>C. difficile</i> strains using the CFE system, confirming higher toxin expression of the hypervirulent strain R20291. We believe that the CFE system can not only serve as a platform for <i>in vitro</i> protein synthesis and genetic part prototyping but also has the potential to be a simplified model for studying metabolic regulations in <i>Clostridioides difficile</i>.IMPORTANCE<i>Clostridioides difficile</i> has been listed as an urgent threat due to its antibiotic resistance, and it is crucial to conduct gene expression analysis to understand gene functionality. However, this task can be challenging, given the need to maintain the bacterium in an anaerobic environment and the inefficiency of introducing genetic material into <i>C. difficile</i> cells. Conversely, the <i>C. difficile</i> cell-free gene expression (CFE) system enables <i>in vitro</i> transcription and translation in the presence of oxygen within just half an hour. Furthermore, the composition of the CFE system is adaptable, permitting the addition or removal of elements, regulatory proteins for example, during the reaction. As a result, this system could potentially offer an efficient and accessible approach to accelerate the study of gene expression and function in <i>Clostridioides difficile</i>.