De Graeve, S. | bioRxiv Science

bioRxiv2025

Development and characterisation of pNarsenic: a naringenin-inducible biosensor for arsenic in Escherichia coli

Whole-cell biosensors detecting the heavy metal arsenic have been widely studied for their potential in environmental monitoring. And while inducible biosensors have been shown to be an effective tool to tune the operational range, a thoroughly characterised inducible biosensor is currently lacking. Here, we present an Escherichia coli biosensor for arsenic in which the transcription factor gene arsR is inducible by naringenin, a plant-derived secondary metabolite. Increasing naringenin concentrations reduced the basal output while increasing both the dynamic range and sensing threshold of the biosensor dose-response curves, but the operational ranges appeared constrained by a fixed upper limit. Comparison with a previously published phenomenological model revealed good overall agreement between experimental data and model predictions, except for the behaviour of the maximum output and threshold. This work expands the biosensor toolbox with a profoundly characterised arsenic biosensor and raises a potential practical limit to dose-response curve engineering by tuning transcription factor expression alone. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/678503v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c15051org.highwire.dtl.DTLVardef@180214forg.highwire.dtl.DTLVardef@10afa84org.highwire.dtl.DTLVardef@1c51bba_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Reprogramming the EnvZ-OmpR two-component system confers ethanol tolerance in Escherichia coli by stabilizing the outer membrane and altering iron homeostasis.

Ethanol is a fermentation product widely used as a fuel and chemical precursor in various applications. However, its accumulation imposes severe stress on the microbial producer, leading to significant production losses. To address this, improving a strains ethanol tolerance is considered an effective strategy to enhance production. In our previous research, we conducted an adaptive evolution experiment with Escherichia coli growing under gradually increasing concentrations of ethanol, which gave rise to multiple hypertolerant populations. Based on the genomic mutational data, we demonstrated in this work that adaptive alleles in the EnvZ-OmpR two-component system drive the development of ethanol tolerance in E. coli. Specifically, when a single leucine was substituted for a proline residue within the periplasmic domain using CRISPR, the mutated EnvZ osmosensor caused a significant increase in ethanol tolerance. Through promoter fusion assays, we showed that this particular mutation stabilizes EnvZ in a kinase-dominating state, which reprograms signal transduction involving its cognate OmpR response regulator. Whole-genome proteomics analysis revealed that this altered signaling pathway predominantly maintains outer membrane stability by upregulating global porin levels and attenuating iron metabolism in the tolerant envZ*L116P mutant. Moreover, we demonstrated that the hypertolerant envZ*L116P allele also promotes ethanol productivity in fermentation, providing valuable insights for enhancing industrial ethanol production. AUTHOR SUMMARYEthanol is a versatile chemical with many applications, but producing it in high quantities remains a challenge. This is because Escherichia coli, a candidate ethanol production strain, is naturally sensitive to this short-chain alcohol, especially when levels are gradually accumulating during fermentation. To resolve this bottleneck, we have investigated how E. coli can acquire tolerance to its own toxic fermentation product. Our research indicated that a single amino acid substitution in EnvZ-a key sensor protein that normally protects E. coli against extreme osmotic stress-is sufficient to confer ethanol tolerance. Further analysis revealed that the mutation perturbs the EnvZ-mediated signaling cascade, which, in turn, changes the transporter composition in the outer membrane and attenuates the cells iron metabolism. These adaptations enable E. coli to survive under high-ethanol conditions, thereby promoting its ethanol production efficiency. This discovery provides a suitable strategy to increase ethanol titers in industrial settings using fermentation.

microbiology↗