Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis not yet clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage, and a 3’-deoxyuridine nucleoside attached to DABA3 of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood due to the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. In this work, a minimum set of enzymes were identified in generating the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly, as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. Based on the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogs were produced using in vitro total biosynthesis.