Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering

Abstract

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false-positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection) suppresses cheater population enrichment. Furthermore, we show that, if we couple the biosensor-based screen with an orthogonal prescreen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting. Using the approach developed here, we were successfully able to isolate PAL variants with ∼70% higher k cat after a single sort. These mutants have tremendous potential in phenylketonuria (PKU) treatment and flavonoid production.

Construction and Application of a High-Throughput In Vivo Screening Platform for the Evolution of Nitrile Metabolism-Related Enzymes Based on a Desensitized Repressive Biosensor

“…As expected, the function of the NAsensor has been slightly improved in the pncA -deficient strain (Figure S2), which supports our hypothesis. In fact, ultra-sensitivity is a characteristic of many natural regulators, which prevents artificial genetic circuits built on natural regulators from working as expected. , Therefore, desensitization of BsNadR was essential to modify the response of this regulatory system to make it functional at the desired niacin concentration.…”

A Silk-Based Platform to Stabilize Phenylalanine Ammonia-lyase for Orally Administered Enzyme Replacement Therapy

“…The purified enzyme was analyzed using SDS-PAGE and assayed as described previously. 20,21 Silk Fibroin Extraction. Regenerated silk fibroin solution was extracted from B. mori cocoons as previously reported.…”

Enzyme directed evolution using genetically encodable biosensors

“…To address cheating by intercellular crosstalk, Trivedi et al used carbon catabolite repression of the hca operon to lower basal transcription levels of the fluorescent reporter (Table 1 entry 12). 71 As glucose is the preferred carbon source for E. coli, the expression of other catabolic pathways is repressed Fig. 6 Cheating by inter-cellular small molecule diffusion (crosstalk).…”

“…6b). 71 Additionally, the authors incorporated a prescreen to eliminate inactive mutants from the library by growing cells encoding PAL variants on minimal medium containing phenylalanine.…”

“…These modifications produced an A. variabilis PAL mutant with ∼70% improvement in k cat , increasing from 0.9 to 1.5 s −1 , and a relatively unaltered K m (168 μM). 71…”