1. Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida
- Author
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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 EK, Martin, Hector Garcia, Mukhopadhyay, Aindrila, and Keasling, Jay D
- Subjects
Biological Sciences ,Industrial Biotechnology ,Biosensing Techniques ,Caprolactam ,Escherichia coli ,Lactams ,Ligands ,Metabolic Engineering ,Plasmids ,Pseudomonas putida ,Medicinal and Biomolecular Chemistry ,Biochemistry and Cell Biology ,Biomedical Engineering ,Biochemistry and cell biology ,Bioinformatics and computational biology - Abstract
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.
- Published
- 2020