The g-lactam-b-lactone natural product salinosporamide A (1) is a potent proteasome inhibitor produced by the marine bacterium Salinispora tropica. Herein we report the genome-inspired discovery and characterization of salinosporamide K (8) from a new source, "Salinispora pacifica" strain CNT-133, that provides insight into the evolution of the salinosporamide biosynthetic pathway. A draft genome of "S. pacifica" strain CNT-133 revealed a truncated biosynthetic gene cluster (Sp_sal) related to the S. tropica sal locus but lacking all genes coding for the chloroethylmalonyl-CoA pathway enzymes. Transcript analyses by semiquantitative RT-PCR confirmed the expression of representative Sp_sal genes under laboratory growth conditions. A novel compound with a salinosporamide-like UV profile was isolated from a 20 L culture, yielding 23 mg of salinosporamide K (8) with the molecular composition C13H17NO4 as established by high-resolution ESI-MS. Structural characterization via NMR and single-crystal X-ray analysis revealed salinosporamide K as a novel salinosporamide with an unsubstituted C-2. Salinosporamide K exhibited 20S proteasome (chymotrypsin-like) inhibitory activity (4.6±0.4 nM) comparable to salinosporamide B (3) and cytotoxicity against the human colon carcinoma cell line HCT-116 (988±155 nM), which was less potent than salinosporamide A (1) but more potent than salinosporamide B (3). Isotope feeding experiments with [1,2-13C]acetate showed intact incorporation into salinosporamide K, supporting malonyl-CoA as the polyketide synthase extender unit. Phylogenetic analysis of the SalA acyltransferase domain (SalA-AT1) suggested convergent evolution for malonyl-CoA specificity in "S. pacifica". The Sp_sal cluster lacks chloroethylmalonyl-CoA biosynthetic genes, which are replaced by transposases in "S. pacifica", indicating that the pathway evolved to utilize readily available cellular pools of malonyl-CoA and methylmalonyl-CoA. This study provides the first glimpse of pathway evolution in the salinosporamides by dictating precursor supply and gives insight into g-lactam-b-lactone pathway engineering for designer proteasome inhibitors.