Holomycin is a dithiolopyrrolone antibiotic natural product produced by Streptomyces clavuligerus. ATCC 27064. This paper reports on the identification of a gene cluster from S. clavuligerus that directs holomycin biosynthesis. Heterologous expression of the cluster in S. albus then induced the production of holomycin. Bioinformatics analysis of the gene cluster revealed that holomycin was assembled by a single multidomain non-ribosomal peptide synthetase (NRPS) consisting of heterocyclization, adenylation, and thiolation domains (Cy-A-T), a free-standing condensation domain, two thioesterases, five tailoring enzymes involved in oxidative reactions and two regulatory and transcriptional genes. Knock out of the gene, Homl, completely abolished the production of holomycin, which is consistent with its role as a key biosynthetic gene and is likely involved in the formation of the disulfide bond generating the final precursor, holothin. (C) 2010 Elsevier Ltd. All rights reserved.