Novel FNR-Dependent Oxygen-Responsive Promoters in <i>Escherichia coli</i>: Design, Characterization, and Metabolic Engineering Applications

Promoters responsive to changes in cultivation conditions are essential tools for dynamic metabolic engineering. Oxygen-responsive promoters, in particular, exhibit significant application potential in oxygen-limited fermentation processes. However, currently reported oxygen-dependent promoters exhibit limited dynamic ranges, and notably, there remains a lack of research on oxygen-responsive negatively regulated promoters. In this study, we designed and characterized a series of dissolved oxygen-responsive promoters in <i>Escherichia coli</i> under the regulation of the transcription factor fumarate-nitrate reduction (FNR). Anaerobically activated promoters were constructed by inserting FNR binding site (FBS) upstream of inducible core promoters, while anaerobically repressed promoters were developed by inserting FBS downstream of or flanking constitutive promoters. The most effective anaerobically activated promoters showed 24-138-fold higher activity under anaerobic conditions compared to aerobic conditions. Under anaerobic conditions, promoters with DNA looping-mediated anaerobic repression maintained only 8-17% of the activity observed under aerobic conditions. These promoters were specifically regulated by FNR, as confirmed by tests in a DH5α Δ<i>fnr</i> strain, and responded rapidly to oxygen depletion (within 30 min). The utility of these genetic tools was demonstrated by applying them to enhance pyruvate production in <i>E. coli</i>. An engineered strain with anaerobic-repressed <i>aceE</i> and anaerobic-activated <i>atpAGD</i> genes produced 5.76 g/L pyruvate at 55.7% yield in shake flask fermentations. This study offers an expanded toolbox of oxygen-responsive promoters that enable precise gene regulation based on dissolved oxygen levels, providing novel genetic strategies for developing efficient two-stage fermentation processes with separated growth and production phases.