Fluorescent protein-based anaerobic reporter for construction of promoter libraries in Clostridium autoethanogenum

Clostridium autoethanogenum, a key organism for syngas fermentation, has great industrial potential as an anaerobic microbe. However, tools for monitoring and characterizing gene expression, such as fluorescent protein-based anaerobic reporters (FPARs), and promoter libraries for regulating expression intensity, are lacking. In this study, we developed a fluorescent protein-based anaerobic reporter (FPAR) tailored for C. autoethanogenum. The FPAR enabled intuitive and precise assessment of promoter activity, facilitating the creation of libraries of constitutive promoters with varying expression strengths, as well as lactose-inducible promoter libraries. The strongest constitutive promoter exhibited approximately 7.5-fold greater activity than the weakest, while the strongest inducible promoter demonstrated a 10-fold increase compared to the weakest. This work not only establishes an efficient FPAR system for C. autoethanogenum, but also provides key genetic elements for advancing metabolic engineering and optimizing industrial processes involving this microbe.