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

Christopher Johnson, Gregg Beckham, Ramesh Jha, Niju Narayanan, Naresh Pandey, Jeremy Bingen, Theresa Kern, Charlie Strauss, Scott Hennelly, Taraka Dale

Research output: Contribution to journalArticlepeer-review

22 Scopus Citations

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% 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.

Original languageAmerican English
Pages (from-to)775-786
Number of pages12
JournalACS Synthetic Biology
Volume8
Issue number4
DOIs
StatePublished - 19 Apr 2019

Bibliographical note

Publisher Copyright:
© 2019 American Chemical Society.

NREL Publication Number

  • NREL/JA-5100-73635

Keywords

  • 4-hydroxybenzoate
  • biosensor
  • cis, cis-muconic acid
  • PobR
  • product inhibition
  • Pseudomonas putida KT2440
  • UbiC

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