An Anaerobic Fluorescent Reporter System and CRISPR-Cas12a Enable High-Throughput Metabolic Engineering of Clostridium butyricum

Clostridium butyricum is an important probiotic and industrial organism with significant potential for anaerobic bioproduction. However, the lack of efficient genetic tools, particularly for high-throughput screening under strict anaerobic conditions, has hindered its metabolic engineering. To address this, we first established a highly efficient conjugation method, significantly improving exogenous DNA transformation efficiency. Leveraging time-resolved transcriptomic data, we then mapped the dynamic activity of native promoters and developed a robust anaerobic fluorescent protein reporter system. This system overcomes the oxygen-dependent limitation of traditional reporters like GFP, enabling high-throughput and quantitative screening of promoter strength in live anaerobes. Furthermore, we constructed a CRISPR-Cas12a-based genome editing platform for scarless gene manipulation and a two-plasmid curing strategy to generate markerless and genetically stable engineered strains. The power of this integrated toolkit was demonstrated by engineering the acetaldehyde metabolic pathway. Overexpression of aldehyde dehydrogenase (ALDH) resulted in a 79.29% increase in enzyme activity, indicating an enhanced catalytic capacity for acetaldehyde oxidation. This proof-of-concept module, together with the anaerobic fluorescent reporter and CRISPR–Cas12a platform, supports a streamlined workflow for genetic part characterization and metabolic engineering in C. butyricum under strict anaerobic conditions.