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

  • Author(s): 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;
  • Keasling, Jay D
  • et al.
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.

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