Engineering the Bacillus Transcription Factor CcpC to Construct Citrate-Responsive Biosensors in Escherichia coli

The tricarboxylic acid (TCA) cycle is an essential part of the central metabolic hub that provides energy and biosynthetic precursors. Efficient regulation of central carbon flux is critical for maintaining optimal productivity of microbial cell factories (MCFs). However, biosensors capable of sensing TCA intermediates remain limited. Here, we engineered the catabolite control protein C (CcpC) from Bacillus species to reconstruct citrate-responsive biosensors inEscherichia coli. Through hybrid promoter engineering, we systematically characterized and identified the functional roles of two CcpC binding sites. By applying the hybrid promoter, the engineered biosensor BcCcpC-PLBs exhibited the broadest dynamic range and highest expression level among its counterparts. Ligand profiling revealed the diverse responsiveness of BcCcpC to multiple metabolites of the TCA cycle. By structure-guided mutagenesis of BcCcpC, the obtained variant BcCcpC(S138L) exhibited an improved dynamic range of up to 3.02-fold under 80 mM citrate induction. This work establishes the first transcription factor (TF)-based citrate-responsive biosensor, which broadens the regulatory toolkit for central metabolism engineering.