PS10-4. Effect of Mutating Porcine CYP17A1 for Altering Pregnenolone Metabolism to Control Boar Taint.

Abstract Cytochrome P450 17A1 (CYP17A1) interacts with cytochrome b5A (CYB5A) to regulate the metabolism of pregnenolone into 17α-hydroxypregnenolone (17OHP) and dehydroepiandrosterone (DHEA), androgens associated with superior growth performance in boars, or 5α-androst-16-ene-3-one (androstenone), a 16-androstene (16A) steroid and primary contributor to boar taint. Because CYB5A also regulates cytochrome P450 enzymes involved in other metabolic processes, CYP17A1 serves as a feasible target for producing gene edited boars with superior growth performance and minimal boar taint. This study aimed to identify mutations in CYP17A1 that could reduce 16A steroid production while maintaining androgen synthesis, and to establish an optimized cell culture system for validating these mutations. Mutations were selected by first identifying key regions of the CYP17A1 amino acid sequence which may influence its interaction with CYB5A and metabolism of pregnenolone. These regions involved in CYB5A binding, electron transfer, and steroid binding pocket formation, were then compared across species which produce 16A steroids, pigs, humans and bovines, and a species which does not, rats. The selected mutations were Q122H, A128V, N200T, K211D, V304I, S309T and L319V, and were inserted into CYP17A1 expression vectors using site-directed mutagenesis for future analysis. To optimize a cell culture system, human embryonic kidney 293FT cells were transfected with varying amounts of expression vectors for CYP17A1, CYB5A, and other relevant components of the cytochrome P450 system, cytochrome b5 reductase (CYB5R3) and cytochrome P450 oxidoreductase (POR). Transfected amounts ranged from 1.00-1.85 µg for CYP17A1, 3.00-3.40 µg for CYB5A, 0.00-0.15 µg for CYB5R3, and 0.00-0.30 µg for POR. Formation of 17OHP, DHEA and 16A steroids from radiolabeled pregnenolone was quantified using high-performance liquid chromatography and the mean production of each metabolite was compared across doses using an ANOVA in SAS. Transfections with 1.60 µg of CYP17A1 increased 16A steroid production relative to 1.35 µg (p < 0.05), 3.10 µg of CYB5A increased pregnenolone metabolism relative to 3.40 µg (p < 0.05), 0.00 µg of CYB5R3 increased pregnenolone metabolism relative to 0.10 µg (p < 0.05) and 0.00 µg of POR increased 16A steroid production relative to 0.30 µg (p < 0.01), and were therefore selected as the optimal dosages. Future work will use this optimized system to evaluate the effects of the selected mutations on pregnenolone metabolism. Western blotting will confirm equal expression of the wild type and mutant CYP17A1 proteins, while an artificial intelligence software, AlphaFold3, and deep learning biomolecular emulator, BioEmu, will be used to predict the effect of mutations on protein stability and help define other potentially useful mutation sites. Using a well-defined cell culture system to identify targets for producing gene edited pigs without boar taint could not only improve pork production but also give insight into a highly complex cytochrome P450 system.