Dynamic expression range expansion of ECF sigma factor-dependent synthetic regulons by promoter context engineering
- 1Philipps-University Marburg
Abstract
Alternative {sigma} factors enable bacteria to reprogram transcription in response to environmental cues and provide powerful tools for synthetic gene regulation. Extracytoplasmic function (ECF) {sigma} factors have recently been implemented as orthogonal transcriptional switches in several bacteria, including the -proteobacterium Sinorhizobium meliloti. Here, we investigated how promoter sequence context influences the activity and specificity of heterologous ECF-dependent core promoters. Combining ECF core promoters from Pseudomonas syringae and Escherichia coli with flanking sequences derived from strong promoters of S. meliloti and other proteobacteria increased promoter activity by up to 45-fold while preserving {sigma} factor specificity. We identified a short upstream AT motif, resembling a minimal UP element, as a key determinant of promoter strength. Targeted mutagenesis confirmed its role in supporting transcription initiation. Engineering promoter-flanking sequences together with the AT motif generated promoter libraries spanning up to a 75-fold range of activities. Importantly, promoter activity hierarchies were maintained within synthetic multi-gene regulons controlled by a single ECF master regulator, demonstrating modular and predictable gene expression. Our results establish promoter environment engineering as a robust strategy for expanding the dynamic range of orthogonal ECF regulatory systems and facilitate the scalable design of synthetic transcriptional programs in bacteria.
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BibTeX
@misc{rauch2026dynamic,
title = {{Dynamic expression range expansion of ECF sigma factor-dependent synthetic regulons by promoter context engineering}},
author = {C. Rauch and D. Meier and V. Seiler and E. M. Elsayed and A. Becker},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.15.751809},
url = {https://www.biorxiv.org/content/10.64898/2026.09.15.751809v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Dynamic expression range expansion of ECF sigma factor-dependent synthetic regulons by promoter context engineering
AU - C. Rauch
AU - D. Meier
AU - V. Seiler
AU - E. M. Elsayed
AU - A. Becker
PY - 2026
DA - 2026/09/15
PB - bioRxiv
DO - 10.64898/2026.09.15.751809
UR - https://www.biorxiv.org/content/10.64898/2026.09.15.751809v1
AB - Alternative {sigma} factors enable bacteria to reprogram transcription in response to environmental cues and provide powerful tools for synthetic gene regulation. Extracytoplasmic function (ECF) {sigma} factors have recently been implemented as orthogonal transcriptional switches in several bacteria, including the -proteobacterium Sinorhizobium meliloti. Here, we investigated how promoter sequence context influences the activity and specificity of heterologous ECF-dependent core promoters. Combining ECF core promoters from Pseudomonas syringae and Escherichia coli with flanking sequences derived from strong promoters of S. meliloti and other proteobacteria increased promoter activity by up to 45-fold while preserving {sigma} factor specificity. We identified a short upstream AT motif, resembling a minimal UP element, as a key determinant of promoter strength. Targeted mutagenesis confirmed its role in supporting transcription initiation. Engineering promoter-flanking sequences together with the AT motif generated promoter libraries spanning up to a 75-fold range of activities. Importantly, promoter activity hierarchies were maintained within synthetic multi-gene regulons controlled by a single ECF master regulator, demonstrating modular and predictable gene expression. Our results establish promoter environment engineering as a robust strategy for expanding the dynamic range of orthogonal ECF regulatory systems and facilitate the scalable design of synthetic transcriptional programs in bacteria.
ER -