DOI:10.1002/cbic.200500325 DecipheringtheBiosynthesisPathwayofthe AntitumorThiocoralinefromaMarine ActinomyceteandItsExpressioninTwo Streptomyces Species Felipe Lombó, [a] Ana Velasco, [b] Angelina Castro, [a] Fernando de la Calle, [b] Alfredo F. BraÇa, [a] JosØ M. Sµnchez-Puelles, [b, c] Carmen MØndez, [a] and JosØ A. Salas* [a] Introduction Bacteria and fungi synthesize a wide variety of biologically active peptides of nonribosomal origin, including antitumor compounds (bleomycin), antibiotics (vancomycin, b-lactams), immunosuppressive agents (cyclosporin), siderophores (entero- bactin), biosurfactants (surfactin), and toxins (microcystin). The biosynthesis of this family of compounds is carried out by non- ribosomal peptide synthetases (NRPSs), which are large multi- functional enzymes with a modular organization of catalytic domains. [1–5] Each of these modules carries out an elongation cycle, that is, it activates and incorporates a selected amino acid into the structure of the final compound. A minimal module is composed of three domains: 1) an adenylation domain (A, approximately 550 residues) which selects a specific amino acid and generates its aminoacyl adenylated version by using adenosine triphosphate (ATP); 2) a peptidyl-carrier pro- tein domain (P, approximately 80 residues) which contains a 4’- phosphopantetheine (PP) prosthetic group for covalent thio- ester binding of the activated adenylated amino acid; and 3) a condensation domain (C, approximately 450 residues) which generates a new peptide bond between the two aminoacyl adenylated residues located on consecutive P domains. The P domains need a post-translational conversion into the active holoforms by a specific PP transferase. [6,7] Some NRPSs possess extra domains for carrying out specific activities, such as epi- merizations to form D-amino acids, N- or C-methylations, or cyclizations acting on L-Cys or L-Ser residues. Generally, a final thioesterase domain (TE, approximately 250 residues) located after the last module is in charge of enzyme-intermediate cleavage, thereby generating a linear peptide (for example, chloroeremomycin) or a cyclic one (such as bacitracin). As an almost general rule, the structure of the different modules re- Thiocoraline is a thiodepsipeptide antitumor compound produced by two actinomycetes Micromonospora sp. ACM2-092 and Mi- cromonospora sp. ML1, isolated from two marine invertebrates (a soft coral and a mollusc) found of the Indian Ocean coast of Mozambique. By using oligoprimers derived from nonribosomal peptide synthetase (NRPS) consensus sequences, six PCR frag- ments containing putative NRPS adenylation domains were am- plified from the chromosome of Micromonospora sp. ML1. Inser- tional inactivation of each adenylation domain showed that two of them generated nonproducing mutants, thereby indicating that these domains were involved in thiocoraline biosynthesis. Se- quencing of a 64.6 kbp DNA region revealed the presence of 36 complete open reading frames (ORFs) and two incomplete ones. Heterologous expression of a region of about 53 kbp, containing 26 of the ORFs, in Streptomyces albus and S. lividans led to the production of thiocoraline in these streptomycetes. Surprisingly, the identified gene cluster contains more NRPS modules than ex- pected on the basis of the number of amino acids of thiocoraline. TioR and TioS would most probably constitute the NRPS involved in the biosynthesis of the thiocoraline backbone, according to the colinearity of the respective modules. It is proposed that two other NRPSs, TioY and TioZ, could be responsible for the biosyn- thesis of a small peptide molecule which could be involved in regulation of the biosynthesis of thicoraline in Micromonospora sp. ML1. In addition, a pathway is proposed for the biosynthesis of the unusual starter unit, 3-hydroxy-quinaldic acid. [a] Dr. F. Lombó, Dr. A. Castro, Dr. A. F. BraÇa, Dr. C. MØndez, Prof. J.A. Salas Departamento de Biología Funcional e Instituto Universitario de Oncología del Principado de Asturias (I.U.O.P.A), Universidad de Oviedo 33006 Oviedo (Spain) Fax: (+ 34) 985103652 E-mail: jasalas@uniovi.es [b] Dr. A. Velasco, Dr. F. de la Calle, Dr. J. M. Sµnchez-Puelles Drug Discovery Department, PharmaMar, S.A. 28770 Colmenar Viejo, Madrid (Spain) [c] Dr. J. M. Sµnchez-Puelles Current address: Dep. Farmacología Molecular Fundación Valenciana de Investigaciones BiomØdicas 46010 Valencia (Spain) 366 # 2006 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2006, 7, 366 – 376