The gut microbiome plays an important role in determining host metabolic health, largely through the production of metabolites. Bile acids are one of the most abundant and variable gut microbial metabolites; however, the details of gut microbial bile acid metabolism remain poorly understood. A key pathway in gut microbial bile acid metabolism is the conversion of conjugated primary bile acids to secondary bile acids. This is a multi-step process that can be distilled down to two key steps: deconjugation and 7-α-dehydroxylation. We have found that treatment of patients with obesity, but without metabolic compromise, with fecal microbiota transplantation (FMT) derived from a lean donor delays the development of glucose intolerance which is associated with increased bile acid deconjugation. In a complementary line of research, we have found that dietary fiber supplementation in mice improves glucose metabolism and increases gut microbial 7-α-dehydroxylation. However, the genes and bacterial species involved in gut microbial bile acid metabolism are incompletely defined, which limits our ability to refine our FMT design for future clinical testing. We hypothesize that fiber supplementation in mice and FMT in humans alters gut microbial bile acid metabolism through novel bacterial species and/or genes. In aim 1, we will define the bacterial species and genes responsible for gut microbial bile acid metabolism in response to fiber supplementation in mice. To this end, we will perform metagenomics and metatranscriptomics of the gut microbiome from high fat diet-fed mice receiving fiber or an isocaloric diet. Further, metagenomic DNA from fiber- and isocaloric-treated groups will be used to generate a fosmid library which will be screened to identify the genes involved in gut microbial bile acid metabolism. In aim 2, we will define the bacterial species and genes responsible for gut microbial bile acid metabolism in response to FMT in humans. To this end, we will perform metagenomics, metatranscriptomics and a functional metagenomics screen on fecal samples from patients receiving FMT or placebo. These data will enable future work to optimize multimodal FMT, prebiotic and probiotic combination therapies aimed at enhancing gut microbial bile acid metabolism for type 2 diabetes treatment and prevention.