Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from <i>Pseudomonas putida</i>

Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from <i>Pseudomonas putida</i> KT2440 which is >1000× more sensitive to an exogenous ligand than previously reported sensors. Manipulating the expression of the sensor <i>oplR</i> (PP_3516) substantially altered the sensing parameters, with various vectors showing <i>K</i>_d values ranging from 700 nM (79.1 μg/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect <i>in vivo</i> production of caprolactam above background at ∼6 μg/L. The high sensitivity and range of OplR is a powerful tool toward the development of novel routes to the biological synthesis of caprolactam.