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.