Optimization of Malonyl Coenzyme A Biosensors in a Reconstituted Cell-Free System for Detecting Acetyl-CoA Carboxylase Activity.

Malonyl coenzyme A (malonyl-CoA) is a key precursor in the biosynthesis of fatty acids and polyketides, critical for industrial applications such as biofuel and pharmaceutical productions. Optimizing acetyl-CoA carboxylase (ACC), the enzyme that converts acetyl-CoA to malonyl-CoA, is essential for advancing metabolic engineering. Effective biosensors that detect malonyl-CoA levels are vital for high-throughput screening and directed evolution of ACC. Earlier efforts utilized the Bacillus subtilis FapR/FapO biosensor system in vivo to convert malonyl-CoA concentrations into fluorescent signals. However, B. subtilis biosensors suffered from narrow detection ranges, impeding accurate quantification across the concentrations needed to evaluate ACC activity, and were further limited by inconsistent cell viability, variable protein expression, and inability to directly supply acetyl-CoA. To address these challenges, we optimized a FapR/FapO biosensor tailored for the reconstituted cell-free protein synthesis system. By engineering the spacer sequence between the T7 promoter and the FapO operator, we developed an in vitro malonyl-CoA biosensor system with a broad detection range (50-1500 μM) with a boost in the maximum dynamic range reaching 95.3-fold at 1500 μM. Furthermore, we screened homologous FapR/FapO pairs from various Bacillota species, identifying the Bacillus cytotoxicus pair sensitive to low malonyl-CoA concentrations, exhibiting a maximum dynamic range of 96.6-fold at 500 μM. This renovated in vitro cell-free biosensor system enabled highly sensitive detection and precise quantification of single-chain, multidomain ACC-fusion protein activity in a reconstituted cell-free protein synthesis system, with the capacity to detect malonyl-CoA produced from as little as 100 pM of ACC-encoding DNA template. Overall, this platform offers a robust tool for the directed evolution and high-throughput screening of ACC, with a broad potential to enhance metabolic engineering and synthetic biology.