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

Mitchell G. Thompson, Allison N. Pearson, Jesus F. Barajas, Pablo Cruz-Morales, Nima Sedaghatian, Zak Costello, Megan E. Garber, Matthew R. Incha, Luis E. Valencia, Edward E.K. Baidoo, Hector Garcia Martin, Aindrila Mukhopadhyay, Jay D. Keasling*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
JournalACS Synthetic Biology
Volume9
Issue number1
Pages (from-to)53-62
Number of pages10
ISSN2161-5063
DOIs
Publication statusPublished - 2020

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