Performance upgrade of a microbial explosives' sensor strain by screening a high throughput saturation library of a transcriptional regulator

Lidor David; Etai Shpigel; Itay Levin; Shaked Moshe; Lior Zimmerman; Shilat Dadon-Simanowitz; Benjamin Shemer; Shon A Levkovich; Liraz Larush; Shlomo Magdassi; Shimshon Belkin

doi:10.1016/j.csbj.2023.08.017

Performance upgrade of a microbial explosives' sensor strain by screening a high throughput saturation library of a transcriptional regulator

Comput Struct Biotechnol J. 2023 Aug 22:21:4252-4260. doi: 10.1016/j.csbj.2023.08.017. eCollection 2023.

Authors

Affiliations

¹ Enzymit Ltd. 3 Pinhas Sapir St., Ness Ziona 7403626, Israel.
² Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
³ George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.
⁴ Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.

Abstract

We present a methodology for a high-throughput screening (HTS) of transcription factor libraries, based on bacterial cells and GFP fluorescence. The method is demonstrated on the Escherichia coli LysR-type transcriptional regulator YhaJ, a key element in 2,4-dinitrotuluene (DNT) detection by bacterial explosives' sensor strains. Enhancing the performance characteristics of the YhaJ transcription factor is essential for future standoff detection of buried landmines. However, conventional directed evolution methods for modifying YhaJ are limited in scope, due to the vast sequence space and the absence of efficient screening methods to select optimal transcription factor mutants. To overcome this limitation, we have constructed a focused saturation library of ca. 6.4 × 107 yhaJ variants, and have screened over 70 % of its sequence space using fluorescence-activated cell sorting (FACS). Through this screening process, we have identified YhaJ mutants exhibiting superior fluorescence responses to DNT, which were then effectively transformed into a bioluminescence-based DNT detection system. The best modified DNT reporter strain demonstrated a 7-fold lower DNT detection threshold, a 45-fold increased signal intensity, and a 40 % shorter response time compared to the parental bioreporter. The FACS-based HTS approach presented here may hold a potential for future molecular enhancement of other sensing and catalytic bioreactions.

Keywords: Bioluminescence; Bioreporters; Escherichia coli; High-throughput screen (HTS); Landmines; NGS; Precision Mutant Library; Protein engineering; Whole-cell biosensors.