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Title: Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase

Abstract

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60%more » yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.« less

Authors:
ORCiD logo [1];  [1]; ORCiD logo [1];  [1];  [1];  [2];  [1]; ORCiD logo [2];  [1];  [1]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States); Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); USDOE Office of Science (SC), Workforce Development for Teachers and Scientists (WDTS) (SC-27); Defense Threat Reduction Agency (DTRA) (United States)
OSTI Identifier:
1506612
Alternate Identifier(s):
OSTI ID: 1511227
Report Number(s):
NREL/JA-5100-73635; LA-UR-18-20530
Journal ID: ISSN 2161-5063
Grant/Contract Number:  
AC36-08GO28308; 89233218CNA000001; CBCALL12-LS-6-0622
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 8; Journal Issue: 4; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; 4-hydroxybenzoate; biosensor; cis,cis-muconic acid; PobR; product inhibition; Pseudomonas putida KT2440; UbiC

Citation Formats

Jha, Ramesh K., Narayanan, Niju, Pandey, Naresh, Bingen, Jeremy M., Kern, Theresa L., Johnson, Christopher W., Strauss, Charlie E. M., Beckham, Gregg T., Hennelly, Scott P., and Dale, Taraka. Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase. United States: N. p., 2019. Web. doi:10.1021/acssynbio.8b00465.
Jha, Ramesh K., Narayanan, Niju, Pandey, Naresh, Bingen, Jeremy M., Kern, Theresa L., Johnson, Christopher W., Strauss, Charlie E. M., Beckham, Gregg T., Hennelly, Scott P., & Dale, Taraka. Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase. United States. doi:10.1021/acssynbio.8b00465.
Jha, Ramesh K., Narayanan, Niju, Pandey, Naresh, Bingen, Jeremy M., Kern, Theresa L., Johnson, Christopher W., Strauss, Charlie E. M., Beckham, Gregg T., Hennelly, Scott P., and Dale, Taraka. Tue . "Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase". United States. doi:10.1021/acssynbio.8b00465.
@article{osti_1506612,
title = {Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase},
author = {Jha, Ramesh K. and Narayanan, Niju and Pandey, Naresh and Bingen, Jeremy M. and Kern, Theresa L. and Johnson, Christopher W. and Strauss, Charlie E. M. and Beckham, Gregg T. and Hennelly, Scott P. and Dale, Taraka},
abstractNote = {Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.},
doi = {10.1021/acssynbio.8b00465},
journal = {ACS Synthetic Biology},
number = 4,
volume = 8,
place = {United States},
year = {2019},
month = {3}
}

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