Malleicyprol, a virulence factor of notorious animal and human pathogens of the Burkholderia pseudomallei (BP) group, features a molecular cyclopropanol warhead linked to a reactive butenolide core. Biosynthetic considerations suggested that this heterocycle was formed by the merger of two individual polyketide chains, but the precise mechanism has remained elusive. By combining chemical synthesis, complete in vitro reconstitution of the biotransformation, and mutational analysis, we show that two individually generated polyketide chains are joined by a noncanonical condensation domain of the PKS-NRPS hybrid synthetase BurF, which forms an ester bond. By mutagenesis, biochemical assays, and trapping of the aldehyde generated from a substrate surrogate, we found that the terminal reductase domain mediates a reductive chain release with concomitant ring formation. The feasibility of the proposed Knoevenagel-type intramolecular cyclization into the butenolide moiety was confirmed by a biomimetic synthesis of malleicyprol. The elucidation of the unprecedented thiotemplated butenolide biosynthesis by head-to-head fusion of two polyketide chains not only expands the synthetic biology toolbox but may also inspire the development of antivirulence strategies against BP pathogen infections.