Project Summary The Sterolbiome Laboratory focuses on working out host-associated bacterial steroidogenic pathways. The focus of the parent R01 is in identifying bacterial 17α-HSDH and 17β-HSDH involved in conversion of androstenedione to the stereoisomers epitestosterone and testosterone, respectively. So far, we have discovered a 17α-HSDH from the gut bacterium, Clostridium scindens, and 17β-HSDH from urinary isolates of Propionimicrobium lymphophilum. These initial discoveries led to a manuscript that is currently under review in the journal Nature. However, we are now focusing on identifying additional isoforms of bacterial 17α-HSDH and 17β-HSDH in human stool samples (>200 samples). To accomplish this, we will use sequence agnostic and culture independent functional metagenomic library screening. In this approach, community DNA is partially digested and ligated into an expression vector before transformation into E. coli. Colonies are transferred to wells of microplates containing bacterial medium and steroid substrates. After extraction of steroid products, hundreds to thousands of wells need to be analyzed by liquid chromatography mass spectrometry. The Sterolbiome lab at Illinois has a much larger collection of steroids than the Metabolomics Core on campus, and even core labs at other institutions that specialize in steroid quantification. Using core labs is both time consuming, and results in inconsistencies as different facilities utilize different instruments, columns, and solvent systems. The throughput we are approaching with functional metagenomic subaims makes a dedicated instrument a necessity. Since functional metagenomics is expected to result in identification of novel enzymatic reactions, we require MS/MS application to produce unique daughter ions which act as “fingerprints” to assist in identifying unknowns. Knowing the mass(es) of each peak is critical to differentiating steroid ions from other compounds and provides immediate information on likely products. Without MS/MS, we would still need to submit most samples to core labs which is the problem to begin with. Instead, we can develop a custom standardized workflow with the ThermoFisher Vanquish Flex UHPLC coupled with VWD & IR, and TSQ Fortis Plus electrospray ionization (ESI) Triple Quadrupole MS. PI Ridlon has support from both his Department and College and with matching funds, the overall cost to NIGMS for the equipment has been significantly reduced.