Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida

• Published in: ACS synthetic biology 2019-12, Vol.9 (1)
• Main Authors: Thompson, Mitchell G., Pearson, Allison N., Barajas, Jesus F., Cruz-Morales, Pablo, Sedaghatian, Nima, Costello, Zak, Garber, Megan E., Incha, Matthew R., Valencia, Luis E., Baidoo, Edward K., Martin, Hector Garcia, Mukhopadhyay, Aindrila, Keasling, Jay D.
• Format: Article
• Language: English
• Subjects: INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
• ISSN: 2161-5063; 2161-5063

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 Pseudomonas putida KT2440 which is > 1000 × more sensitive to an exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd 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 in vivo 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.