While dietary sugars can alter the bacteriophage community in the gut ecosystem, the underlying mechanisms driving these changes remain elusive. Until we have filled these voids in our knowledge base, we will not be in a position to comprehend the interplay between dietary sugars, and probiotics and their viruses; this hampers the development of rational approaches to use diet to promote the efficacy of (engineered) probiotics. The long- term goals are (i) to unravel the mechanisms that drive the interplay between diet, and probiotic bacteria and their viruses, and (ii) to develop microbial therapeutics. The objectives of this research program are (1) to eluci- date the mechanisms by which sucrose promotes phage production in the probiotic gut symbiont Lactobacillus reuteri, and (2) to exploit diet-induced phage production to promote colonization and the release of therapeutics. The overarching hypothesis is that sucrose increases phage production, which consequently promotes coloni- zation and the release of recombinant proteins from engineered probiotics. The rationale of the work proposed is that its successful completion is expected to result in a paradigm shift of our understanding how diet impacts phage production, which will open up new and exciting avenues to modulate the gut microbiota composition, promote probiotic growth, and to tailor therapeutic delivery. The overarching hypothesis will be tested by pursuing three specific aims: (1) To characterize the sucrose metabolism pathway in relation to phage production; (2) To determine the role of phage on L. reuteri gut fitness in response to a diet enriched in sucrose; and (3) To use dietary sugar to control phage-mediated lysis and therapeutic delivery. In the first aim, targeted mutagenesis is used to dissect the sucrose metabolism pathway and their products to determine the triggers of sucrose-driven phage production in L. reuteri. Under the second aim, bacterial competition assays in gnotobiotic mice will de- termine the ecological ramifications of sucrose-driven phage production; isogenic mutants with reduced ability to metabolize sucrose, and to produce phage, are expected to reveal causation. Under the third aim, L. reuteri will be engineered to lyse—and release recombinant interleukin-22—in response to metabolism of a specific sugar. In an animal model of fatty liver disease therapeutic efficacy in response to diet will be investigated. This research is innovative because an important mutualistic gut symbiont is used to unravel the mechanisms by which sucrose boosts phage production in the gut ecosystem, which can be applied towards the development of next-generation probiotics. The research is significant because understanding how a dietary sugar boosts phage production in the gut ecosystem, and what the ecological ramifications are, opens up previously unexplored opportunities to alter the composition of the gut microbiota, which may include enrichment and/or engraftment of probi...