A triple mutant (strain KA57) of Streptomyces rochei 7434AN4 produces an azoxy-alkene compound, KA57A, which was not detected in a parent strain or other single and double mutants. This strain accumulated several additional minor components, whose structures were elucidated. HPLC analysis of strain KA57 indicated the presence of two UV active components (KA57D1 and KA57D2) as minor components. They exhibited a maximum UV absorbance at 218 nm, whereas a UV absorbance of azoxy-alkene KA57A was detected at 236 nm, suggesting that both KA57D1 and KA57D2 contain a different chromophore from KA57A. KA57D1 has a molecular formula of C12H22N2O2, and NMR analysis revealed KA57D1 is a novel hydrazide-alkene compound, (Z)-N-acetyl-N'-(hex-1-en-1-yl)isobutylhydrazide. Labeling studies indicated that nitrogen Nβ of KA57D1 is derived from l-glutamic acid, and the isobutylamide unit (C-1 to C-3, 2-Me, and Nα) originates from valine. KA57D2 has a molecular formula of C13H24N2O2, and its structure was determined to be (Z)-N-acetyl-N'-(hex-1-en-1-yl)-2-methylbutanehydrazide, in which a 2-methylbutanamide unit was shown to originate from isoleucine. Different biogenesis of the Nα atom (l-serine for KA57A, l-valine for KA57D1, and l-isoleucine for KA57D2) indicates the relaxed substrate recognition for nitrogen-nitrogen bond formation in the biosyntheses of KA57A, KA57D1, and KA57D2.