Germination metrics in all strains.

<div><p>We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the <i>hag</i> locus, a σ<sup>D</sup>-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous <i>hag</i> promoter, following a deletion of the gene encoding a post-translational regulator of σ<sup>D</sup>, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the <i>hag</i> locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a <i>Cupriavidus necator</i> aldehyde dehydrogenase gene (<i>acoD</i>) into the <i>hag</i> locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics.</p></div>