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

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.

Authors:
 [1];  [2];  [3];  [4];  [2];  [5];  [6];  [1];  [7];  [2]; ORCiD logo [8]; ORCiD logo [6]; ORCiD logo [9]
  1. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
  2. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; USDOE Agile BioFoundry, Emeryville, CA (United States)
  4. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology; Instituto Tecnologico y de Estudios superiores de Monterrey, Monterrey (Mexico). Centro de Biotecnologia FEMSA
  5. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology; USDOE Agile BioFoundry, Emeryville, CA (United States)
  6. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Comparative Biochemistry Graduate Group
  7. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Joint Program in Bioengineering
  8. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology; USDOE Agile BioFoundry, Emeryville, CA (United States); Basque Center for Applied Mathematics (BCAM), Bilbao (Spain)
  9. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Div.; Univ. of California, Berkeley, CA (United States). Joint Program in Bioengineering; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering; Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability; Shenzhen Univ. (China)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
OSTI Identifier:
1782137
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

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, and Keasling, Jay D.. Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida. United States: N. p., 2019. Web. https://doi.org/10.1021/acssynbio.9b00292.
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.. Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida. United States. https://doi.org/10.1021/acssynbio.9b00292
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, and Keasling, Jay D.. Mon . "Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida". United States. https://doi.org/10.1021/acssynbio.9b00292. https://www.osti.gov/servlets/purl/1782137.
@article{osti_1782137,
title = {Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida},
author = {Thompson, Mitchell G. and Pearson, Allison N. and Barajas, Jesus F. and Cruz-Morales, Pablo and Sedaghatian, Nima and Costello, Zak and Garber, Megan E. and Incha, Matthew R. and Valencia, Luis E. and Baidoo, Edward K. and Martin, Hector Garcia and Mukhopadhyay, Aindrila and Keasling, Jay D.},
abstractNote = {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.},
doi = {10.1021/acssynbio.9b00292},
journal = {ACS Synthetic Biology},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {12}
}

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