Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase
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. 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.
- Research Organization:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States); Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
- Sponsoring Organization:
- USDOE National Nuclear Security Administration (NNSA); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office (BETO); USDOE Office of Science (SC), Workforce Development for Teachers and Scientists (WDTS); Defense Threat Reduction Agency (DTRA) (United States)
- Grant/Contract Number:
- AC36-08GO28308; 89233218CNA000001; CBCALL12-LS-6-0622
- OSTI ID:
- 1506612
- Alternate ID(s):
- OSTI ID: 1511227
- Report Number(s):
- NREL/JA-5100-73635; LA-UR-18-20530
- Journal Information:
- ACS Synthetic Biology, Vol. 8, Issue 4; ISSN 2161-5063
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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