The antibiotic alaremycin (5-acetamido-4-oxo-5-hexenoic acid, ), isolated from sp. A012304, structurally resembles 5-aminolevulinic acid (ALA), a precursor in porphyrin biosynthesis, and inhibits porphobilinogen synthase, the enzyme responsible for catalyzing the first common step of this pathway. In our previous study, the biosynthetic gene cluster responsible for alaremycin production-composed of (ALA synthase homologue), (-acetyltransferase), (oxidoreductase), and (MFS-type transporter)-was identified, and a potential biosynthetic pathway was proposed. In this study, the biosynthetic pathway of was confirmed by detecting intermediates using the liquid chromatography-mass spectrometry/MS (LC-MS/MS) analysis of extracts from cells transformed with the biosynthetic genes, followed by reconstitution of the biosynthetic reactions using purified enzymes. AlmA catalyzed the condensation of l-serine and succinyl-CoA to produce 5-amino-6-hydroxy-4-oxohexanoic acid (), AlmB catalyzed the -acetylation of to produce 5-acetamido-6-hydroxy-4-oxohexanoic acid (), and AlmC catalyzed the dehydration of to form . The AlmC-catalyzed reaction may involve a two-step mechanism including reduction by NADH and oxidation by Fe. Additionally, a novel derivative of was identified in the culture broth of the producer strain, and its structure was determined as 5,6-dihydroalaremycin (5-acetamido-4-oxohexanoic acid, ). It was revealed that is synthesized via the same biosynthetic pathway but with AlmA and AlmB utilizing l-alanine as the amino acid precursor instead of l-serine.