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Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering
Vikas D Trivedi1, Karishma Mohan1, Todd C Chappell1
1Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts 02155, United States.
ACS Synthetic Biology
|December 16, 2021
Summary
To combat false positives in cell engineering, researchers desensitized biosensors and used a prescreen. This improved screening accuracy, enabling the isolation of high-performance enzyme variants for phenylketonuria treatment and flavonoid production.
Area of Science:
- Synthetic Biology
- Biotechnology
- Enzyme Engineering
Background:
- Transcription factor (TF)-based biosensors are crucial for high-throughput strain engineering.
- Cellular crosstalk, where detected molecules leak between cells, leads to false positives and complicates screening.
- Passive diffusion of molecules like trans-cinnamic acid exacerbates crosstalk, hindering effective biosensor application.
Purpose of the Study:
- To develop strategies to mitigate crosstalk and reduce false-positive cell enrichment in TF-based biosensor screens.
- To improve the efficiency of directed evolution for enzymes like phenylalanine ammonia-lyase (PAL).
- To enable the isolation of superior enzyme variants for applications in phenylketonuria treatment and flavonoid biosynthesis.
Main Methods:
- Desensitizing the trans-cinnamic acid (tCA) biosensor by increasing its limit of detection.
- Implementing an orthogonal prescreen to eliminate true negative cells before the main sort.
- Utilizing fluorescence-activated cell sorting (FACS) to enrich for desired cell populations.
Main Results:
- Desensitization of the biosensor effectively suppressed the enrichment of false-positive/cheater cells.
- The combination of biosensor desensitization and orthogonal prescreening significantly reduced cheater populations during sorting.
- Isolation of PAL variants with approximately 70% higher catalytic efficiency (kcat) after a single sorting round.
Conclusions:
- Biosensor desensitization and orthogonal prescreening are effective strategies to overcome crosstalk limitations in TF-based biosensor applications.
- This approach enhances the accuracy and efficiency of directed evolution for enzyme engineering.
- The isolated high-activity PAL variants hold significant promise for therapeutic and industrial applications, including phenylketonuria treatment and enhanced flavonoid production.
