The biosynthesis of 5-hydroxy-4-oxo-L-norvaline (HON) inStreptomycesakiyoshiensishas been investigated using13C-labelled substrates. Incorporations of13C label from sodium [1-13C]-, [2-13C]-, and [1,2-13C2]acetate indicated that HON was formed from a four-carbon compound derived from the citric acid cycle and the methyl carbon of acetate. Feeding experiments usingDL-[4-13C]- andDL-[2-13C,15N]aspartate demonstrated that aspartate served as the four-carbon precursor to HON. Both enantiomers of aspartate were metabolized byS.akiyoshiensis, but theDisomer was consumed at a slower rate. The distribution of13C label in the intracellularL-glutamic acid isolated in these feeding experiments is consistent with the operation of the citric acid cycle inS.akiyoshiensis. A biosynthetic hypothesis that involves a condensation reaction between acetyl or malonyl CoA and the β-carboxyl group of aspartate, and subsequent oxidative decarboxylation, is proposed to account for the incorporation results. An analogous condensation step has been proposed for the biosynthesis of other natural products, including the carbapenem antibiotics.DL-[2-13C,15N]Aspartate was synthesized from [2-13C]diethylmalonate and potassium [15N]phthalimide via diethyl [2-13C,15N]phthalimidomalonate.