PROJECT SUMMARY/ABSTRACT The human gut is a complex ecosystem, where trillions of microbes interact with dietary and host-derived molecules, mediating biotransformations that significantly affect both the microbial community and their human hosts. Central to this metabolic interplay are ene-reductases, a versatile class of oxidoreductases that facilitate the reduction of carbon-carbon double bonds to single bonds. Stool metabolomics data implicates the existence of numerous unidentified ene-reductases, constituting a gap in our understanding of gut microbial metabolism. While these enzymes are pivotal in modulating metabolite bioavailability, impacting both microbial physiology and human health, most remain unidentified. This research proposal aims to systematically characterize novel gut microbial ene-reductases based on the hypothesis that these enzymes play a critical role in shaping the chemical milieu of the gut. To achieve this goal, we will develop a platform that fuses microbiology, biochemistry, and bioinformatics in a four-stage strategy to systematically identify and characterize gut microbial ene- reductases responsible for the reduction of a target metabolite. First, we will identify microbial strains capable of metabolite reduction. Second, we will identify candidate reductase genes through comparative genomics and RNA-seq. Third, we will experimentally validate the candidate enzymes for their reductase activity. Fourth, studies will be performed to understand the mechanistic basis of the novel enzymes. Through this research, we aim to deepen our understanding of the chemical dialogues occurring between gut microbes and their host. Specifically, the study will illuminate the roles that ene-reductases play in performing consequential biotransformations and shaping microbiome community structure. While this proposal focuses on fundamental characterization of key gut microbial enzymes, it lays the groundwork for translational advancement by uncovering metabolic mechanisms that will ultimately facilitate precise, personalized microbiome modulation.