Alpha-aminoadipate delta-semialdehyde synthase (AASS) is the bifunctional enzyme containing the lysine alpha-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first 2 steps in the irreversible catabolism of lysine. A rare disease in humans, familial hyperlysinemia, can be caused by very low LKR activity and, as expected, reduces the lysine "requirement" of the individual. This concept was applied to a murine hepatic cell line (ATCC, FL83B) utilizing RNA interference (RNAi) to achieve AASS mRNA knockdown. Cells were antibiotic selected for stable transfection of 2 plasmids that express different short hairpin RNA sequences for AASS knockdown. Compared with the wild-type cell line, AASS mRNA abundance was reduced 79.0 +/- 6.4% (P < 0.05), resulting in a 29.8 +/- 5.2% (P < 0.05) reduction in AASS protein abundance, 41.3 +/- 10.0% (P < 0.05) less LKR activity, and a reduction in lysine oxidation by 50.7 +/- 11.8%. To determine the effect of AASS knockdown on the lysine requirement, cells were grown in media containing 12.5, 25.0, 50.0, 100, or 200 micromol/L lysine. Using a segmented model approach for growth rate analysis, the lysine requirement of the cell line with AASS silencing was 43.4 +/- 1.7 micromol/L, approximately 26% lower (P < 0.05), than the lysine requirement of the wild-type cell line. These results indicate AASS knockdown decreases the lysine requirement of the cell via a reduction of lysine catabolism through the saccharopine pathway, providing the initial proof in principle that RNAi can be used to reduce the nutrient requirement of a system.