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The novel genetically modified probiotic Bacillus subtilis ZB423 was assessed in a 90‐day repeated‐dose oral toxicity study adhering to Good Laboratory Practice (GLP) and Organization for Economic Cooperation and Development (OECD) guidelines. Spray‐dried spores at a concentration of 1.1E12 CFU/g were administered at doses of 130, 260, and 519 mg/kg body weight/day correlating to 1.43 × 1011, 2.86 × 1011, and 5.71 × 1011 CFU/kg/day, respectively, by oral gavage to Wistar rats for a period of 90 consecutive days. Results showed no toxicologically relevant findings for B. subtilis ZB423 from measured parameters. The no observed adverse effect level (NOAEL) of B. subtilis ZB423 is 519 mg/kg body weight/day corresponding to 5.71 × 1011 CFU/kg/day for lyophilized B. subtilis ZB423 spores under the test conditions employed. The novel genetically modified probiotic B. subtilis ZB423 was assessed in a 90‐day repeated‐dose oral toxicity study. Results showed no toxicologically relevant findings for B. subtilis ZB423 from measured parameters. The no observed adverse effect level (NOAEL) of the B. subtilis ZB423 is 519 mg/kg body weight/day corresponding to 5.71 × 1011 CFU/kg/day for lyophilized B. subtilis ZB423 spores under the test conditions employed. [ABSTRACT FROM AUTHOR]

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 hag locus, a σD-dependent flagellin expression site, 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 σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag 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 Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag 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. [ABSTRACT FROM AUTHOR]