Establishing a Cell-Free Transcription–Translation Platform for Cutibacterium acnes to Prototype Engineered Metabolic and Synthetic Biology

[Image: see text] In the past few years, new bacterial-cell-free transcription–translation systems have emerged as potent and quick platforms for protein production as well as for prototyping of DNA regulatory elements, genetic circuits, and metabolic pathways. The Gram-positive commensal Cutibacterium acnes is one of the most abundant bacteria present in the human skin microbiome. However, it has recently been reported that some C. acnes phylotypes can be associated with common inflammatory skin conditions, such as acne vulgaris, whereas others seem to play a protective role, acting as possible “skin probiotics”. This fact has made C. acnes become a bacterial model of interest for the cosmetic industry. In the present study we report for the first time the development and optimization of a C. acnes-based cell-free system (CFS) that is able to produce 85 μg/mL firefly luciferase. We highlight the importance of harvesting the bacterial pellet in mid log phase and maintaining CFS reactions at 30 °C and physiological pH to obtain the optimal yield. Additionally, a C. acnes promoter library was engineered to compare coupled in vitro TX-TL activities, and a temperature biosensor was tested, demonstrating the wide range of applications of this toolkit in the synthetic biology field.