Engineering a probiotic Bacillus subtilis for acetaldehyde removal: A hag locus integration to robustly express acetaldehyde dehydrogenase

We have identified solutions to key challenges in probiotic design to create a commercially viable strain for the removal of the intestinal toxin acetaldehyde. Here we report engineering of a {sigma}D-dependent flagellin expression locus, hag, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter following a deletion of the gene encoding a post-translational regulator of {sigma}D, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate hag locus activity after germination and show a steady increase in heterologous expression throughout outgrowth and resumption of vegetative growth. To test the chassis as a spore-based probiotic solution we identified the physiologically relevant pathway of ethanol metabolism and the buildup of gut-derived acetaldehyde after alcohol consumption. Herein, we describe the integration of a robustly expressed Cupriavidus necator aldehyde dehydrogenase, AcoD, under a flagellin protein promoter at the hag locus and report the rapid reduction of acetaldehyde levels after germination in gut simulated conditions.