Project Summary The metabolism of corticosteroids by host-associated microbiomes rivals that of host peripheral tissues. Side- chain metabolism of corticosteroids is capable of changing the functional class of steroid. The focus of the current project is elucidating the microbial enzymes involved in (Aim 1) the side-chain cleavage of cortisol and reduction of 17-keto resulting in 11-oxy-androgens (Aim 2) 21-dehydroxylation yielding 11-oxy-progesterone derivatives. These pathways are significant because they are hypothesized to potentiate diseases relevant to the mission of NIH. The formation of 11-oxy-androgens in the intestine and urinary tract is hypothesized to affect host immune function, and may be a risk factor for prostate cancer. The formation of 11-oxy-progesterone derivatives has been shown to be pro-hypertensive through a well-established mechanism involving the ‘dehydrogenase hypothesis’. Our preliminary data demonstrates that we have established the side-chain cleavage enzyme, steroid-17,20-desmolase, from both gut and urinary tract bacteria. The end-product 17-keto steroid can then be reduced to either testosterone or epitestosterone derivatives. We have cultured anaerobes that encode the enzymes for (epi)testosterone formation, and our preliminary data demonstrates our expertise and the feasibility of locating and characterizing these enzymes. We also show a cultured anaerobe capable of corticosteroid 21- dehydroxylation and formulated a hypothesis and preliminary data that a putative corticosteroid dehydroxylation (csd) gene cluster encodes 21-dehydroxylation. Once located, we will apply previously established bioinformatics approaches to uncover the diversity of host-associated bacteria encoding homologous sterolbiome enzymes (phylogenetic analysis, sequence similarity networks). However, to address evolutionarily analogous sterolbiome enzymes with the same functions described above, we will utilize an unbiased functional metagenomic approach. We present additional robust preliminary data demonstrating the feasibility of our studies that include (1) collection of 180 stool samples for functional metagenomic screening (2) utilization of RNA-Seq for steroid- inducible gene discovery (3) heterologous expression and characterization of numerous microbial sterolbiome enzymes (3) establishment of enzyme assays and steroid metabolomics. The research proposed in this application is innovative, in our opinion, because it represents a new and substantive departure from the status quo established four decades ago by combining transcriptomics, heterologous protein expression, enzyme assays, phylogenetic and sequence similarity networks, synthetic biology, and functional metagenomic screening to discover the precise nucleic acid sequences encoding corticosteroid side-chain metabolizing enzymes. Successful completion will potentiate research into the cause and effect relationships between the gut sterolbiome and host diseases, and is expected ...