New macrolactam natural product OL44.465 was discovered from Streptomyces sp. OL44 by genomic and spectroscopic signature-based methods. To discover novel macrolactams, actinomycete strains collected from Olleung, a traditional Korean royal tomb in Yangju, Gyeonggido, were screened using primers targeting adenylation enzyme and proline aminopeptidase, which are two core enzymes in biosynthesis of macrolactam moiety, respectively. The fermentation of OL44, one of the hit strains, resulted in production of a novel compound with molecular weight of 465. After purification of the compound by HPLC, the planar structure of OL44.465 was identified as a macrolactam compound bearing two 5-membered rings by 1D and 2D NMR spectroscopy. The stereochemistry of OL44.465 was elucidated using ROESY spectra, calculation, and Mosher\'s method using (R)- and (S)-MTPA chlorides.