Development of carbon monoxide-tolerant fluorescent reporter system functional under anoxic conditions in Eubacterium...

This study aimed to develop a fluorescent reporter system optimized for acetogen Eubacterium callanderi KIST612, addressing key challenges such as autofluorescence and CO exposure. To support reporter selection, autofluorescence in E. callanderi KIST612 was characterized. Fluorescence-activating and absorption-shifting tags (FAST), particularly FAST1-HMBR, was identified as the most suitable candidate due to its spectral properties, kinetics, and compatibility with the anaerobic strain. Under strict anoxic environments, FAST1-expressing strains exhibited significantly greater fluorescence intensity than controls-4.55-fold in glucose and 4.54-fold in CO conditions. Functional validation using a CO-sensing system showed a proportional increase in fluorescence intensity with the increasing CO partial pressure (R², 0.97), confirming the system's sensitivity and capability in anaerobic CO conditions.