A regulatory toolkit of arabinose-inducible artificial transcription factors for Gram-negative bacteria

Abstract

AbstractThe Gram-negative bacteriaSalmonellaTyphimurium andEscherichia coliare important model organisms, powerful prokaryotic expression platforms for biotechnological applications, and pathogenic strains constitute major public health threats. To facilitate new approaches for research, biomedicine, and biotechnological applications, we developed a set of arabinose-inducible artificial transcription factors (ATFs) using CRISPR/dCas9 andArabidopsis-derivedDNA-binding proteins, allowing to control gene expression inE. coliandSalmonellaover a wide inducer concentration range. As a proof-of-concept, we employed the developed ATFs to engineer aSalmonellabiosensorstrain, SALSOR 0.2 (SALmonella biosenSOR 0.2), which responds to the presence of alkaloid drugs with quantifiable fluorescent output. We demonstrated that SALSOR 0.2 was able to detect the presence of the antitussive noscapine alkaloid with ~2.3-fold increased fluorescent signal over background noise compared to a previously described biosensor. Moreover, we used plant-derived ATFs to control β-carotene biosynthesis inE. coli, which resulted in ~1.6-fold higher β-carotene production compared to expression of the biosynthesis pathway using a strong constitutive promoter. The arabinose-inducible ATFs reported here thus enhance the synthetic biology repertoire of transcriptional regulatory modules that allow tuning protein expression in the Gram-negative model organismsSalmonellaandE. coli.