The Biosynthetic Gene Cluster for the 26-Membered Ring Polyene Macrolide Pimaricin

Abstract

The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis. The pimS0 gene encodes a relatively small acetate-activating PKS (∼193 kDa) that appears to work as a loading protein which “presents” the starter unit to the second PKS subunit. The pimS1 gene encodes a giant multienzyme (∼710 kDa) harboring 15 activities responsible for the first four cycles of chain elongation in pimaricin biosynthesis, resulting in formation of the polyene chromophore. The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis. The pimS0 gene encodes a relatively small acetate-activating PKS (∼193 kDa) that appears to work as a loading protein which “presents” the starter unit to the second PKS subunit. The pimS1 gene encodes a giant multienzyme (∼710 kDa) harboring 15 activities responsible for the first four cycles of chain elongation in pimaricin biosynthesis, resulting in formation of the polyene chromophore. polyketide synthase acyl carrier protein acyltransferase β-ketoacyl ACP synthase kilobase(s) Polyketides are a large and highly diverse group of natural products (1O′Hagan D. The Polyketide Metabolites. Ellis Horwood Ltd., Chichester, UK1991Google Scholar) that includes antibacterial, antifungal, anticancer, antiparasitic, and immunosuppressant activities, among others. Despite their structural diversity, these metabolites are synthesized by a common pathway in which units derived from acetate, propionate, or butyrate are condensed onto the growing chain by a polyketide synthase (PKS)1 in a process resembling fatty acid biosynthesis (2Hopwood D.A. Sherman D.H. Annu. Rev. Genet. 1990; 24: 37-66Crossref PubMed Google Scholar, 3Staunton J. Angew. Chem. Int. Ed. Engl. 1991; 30: 1302-1306Crossref Scopus (44) Google Scholar, 4Katz L. Donadio S. Annu. Rev. Microbiol. 1993; 47: 875-912Crossref PubMed Scopus (321) Google Scholar), except that the β-keto function introduced at each elongation step may undergo all, part, or none of a reductive cycle comprising β-ketoreduction, dehydration, and enoyl reduction (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (399) Google Scholar). The structural variety in this class of products arises from choice of monomers, the extent of β-ketoreduction, and dehydration and the stereochemistry of each chiral center. Yet further diversity is produced by functionalization of the polyketide chain by the action of glycosylases, methyltransferases, and oxidative enzymes. Macrocyclic polyketides are produced mainly by Streptomycesand related filamentous bacteria through the action of so-called type I modular PKSs (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (399) Google Scholar, 6Donadio S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Science. 1991; 252: 675-679Crossref PubMed Scopus (752) Google Scholar, 7Cortés J. Haydock S.F. Roberts G.A. Bevitt D.J. Leadlay P.F. Nature. 1990; 348: 176-178Crossref PubMed Scopus (515) Google Scholar, 8Aparicio J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar). These enzymes usually consist of several extremely large polypeptides in which different modules (sets) of enzymic activities catalyze each successive round of elongation (6Donadio S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Science. 1991; 252: 675-679Crossref PubMed Scopus (752) Google Scholar, 7Cortés J. Haydock S.F. Roberts G.A. Bevitt D.J. Leadlay P.F. Nature. 1990; 348: 176-178Crossref PubMed Scopus (515) Google Scholar). Characterization of such PKS systems is not only an important scientific challenge but a necessary start to the study of factors that control the specificity and extent of chain extension, which are largely unknown (4Katz L. Donadio S. Annu. Rev. Microbiol. 1993; 47: 875-912Crossref PubMed Scopus (321) Google Scholar, 9Marsden A.F.A. Caffrey P. Aparicio J.F. Loughran M.S. Staunton J. Leadlay P.F. Science. 1994; 263: 378-380Crossref PubMed Scopus (145) Google Scholar, 10Aparicio J.F. Caffrey P. Marsden A.F.A. Staunton J. Leadlay P.F. J. Biol. Chem. 1994; 269: 8524-8528Abstract Full Text PDF PubMed Google Scholar). Polyene macrolides are a group of macrocyclic polyketides that interact with membrane sterols and are, therefore, active against fungi but not bacteria (11Martı́n J.F. Annu. Rev. Microbiol. 1977; 31: 12-38Crossref Scopus (60) Google Scholar, 12Gil J.A. Martı́n J.F. Strohl W.R. Biotechnology of Antibiotics. 2nd Ed. Marcel Dekker, Inc., New York1997: 551-576Google Scholar). Pimaricin (Fig. 1) represents a prototype molecule of glycosylated polyenes (13Omura S. Tanaka H. Omura S. Macrolide Antibiotics, Chemistry, Biology, and Practice. Academic Press, Inc., NY1984: 351-404Google Scholar) important for antifungal therapy and promising for antiviral activity, stimulation of the immune response, and action in synergy with other antifungal drugs or antitumor compounds (14Bolard J. Biochim. Biophys. Acta. 1986; 719: 11-17Google Scholar). It is produced byStreptomyces natalensis and widely utilized in the food industry to prevent mold contamination of cheese and other nonsterile foods (i.e. cured meats). The macrolide rings of polyene antibiotics are larger (up to twice in size) than those of standard 14- or 16-membered nonpolyene macrolides (11Martı́n J.F. Annu. Rev. Microbiol. 1977; 31: 12-38Crossref Scopus (60) Google Scholar). Their rings include a chromophore of conjugated double bonds (the characteristic polyene structure). The genetic determination of the length of the polyene and the chromophore and the ring size of these giant macrolide rings is intriguing. Despite the general interest of polyene macrolides, very little is known about their biosynthetic routes and the gene clusters encoding them. We have, therefore, undertaken the cloning of a largeStreptomyces gene cluster involved in the biosynthesis of the polyketide backbone that forms the 26-membered tetraene macrolide ring of pimaricin. pimS1, the gene responsible for the first four rounds of chain elongation has been sequenced, and its modular nature has been established. pimS0, a gene required for chain initiation has been also studied. Gene disruption studies provide evidence for the involvement of pimS0 and pimS1in pimaricin biosynthesis. S. natalensis ATCC 27448 was used as the source of DNA in the construction of the genomic library. Escherichia coli strain XL1-Blue MR was used for obtaining SuperCos 1 cosmid (Stratagene) recombinant derivatives and also served as a host for plasmid subcloning in plasmids pBluescript (Stratagene) pUC18 and pUC19.Candida utilis (synonym Pichia jadinii) CECT 1061 was used for bioassay experiments. Phage KC515 (c + attP::tsr::vph), a ØC31-derived phage (15Rodicio M.R. Bruton C.J. Chater K.F. Gene. 1985; 34: 283-292Crossref PubMed Scopus (54) Google Scholar), was used for gene disruption experiments (16Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985Google Scholar). Streptomyces lividans JII 1326 (17Chater K.F. Bruton C.J. Suárez J.E. Gene. 1981; 14: 183-194Crossref PubMed Scopus (25) Google Scholar) served as a host for phage propagation and transfection. Standard conditions for culture ofStreptomyces species and isolation of phages were as described by Hopwood et al. (16Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985Google Scholar). S. natalensis was routinely grown in YEME medium (16Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985Google Scholar). Sporulation was achieved in TBO medium (2% (w/v) tomato paste, 2% (w/v) oats flakes, 2.5% (w/v) agar) at 30 °C. For pimaricin production, the strain was grown in phosphate-limited SPG medium (2.5% (w/v) soya peptone, 0.5 mmZnSO4·7H2O, 2% (w/v) glucose, pH 7.5) as described by Martı́n and Demain (18Martı́n J.F. Demain A.L. Biochem. Biophys. Res. Commun. 1975; 71: 1103-1109Crossref Scopus (34) Google Scholar). Standard genetic techniques withE. coli and in vitro DNA manipulations were as described by Sambrook et al. (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). E. coli was transformed by the procedure of Hanahan (20Hanahan D. Glover D.M. DNA Cloning: A Practical Approach. IRL Press at Oxford University Press, Oxford1985: 109-132Google Scholar). Recombinant DNA techniques in Streptomyces species and isolation ofStreptomyces total and phage DNA were performed as described previously (16Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985Google Scholar). The library of size-fractionated genomic DNA was screened using probes derived from the rapamycin PKS genes from Streptomyces hygroscopicus (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (399) Google Scholar, 8Aparicio J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar) and from the candicidin genes ofStreptomyces griseus (21Criado L.M. Martı́n J.F. Gil J.A. Gene. 1993; 126: 135-139Crossref PubMed Scopus (32) Google Scholar). A positive clone (Cos 9) containing PKS genes was used as the starting point for chromosome walking. Cosmid clones were obtained that extended over a contiguous 110-kb region of the S. natalensis chromosome, and individual inserts were subcloned into plasmid vectors. Sequencing templates were obtained by random subcloning of fragments generated by controlled partial HaeIII digestions. DNA sequencing was accomplished by the dideoxynucleotide chain termination method (22Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (56781) Google Scholar) on double-stranded DNA templates with an Applied Biosystems model 310 sequencer (Foster City, CA.). Subclone junctions were verified by direct sequencing of cosmid clones, using as sets of synthesized was sequenced a of on of DNA and protein DNA were and by using from DNA and protein were with the University of J. P. Res. PubMed Scopus Google Scholar). pimaricin in culture 0.5 of culture was with of and the was in to the at in the of to of pimaricin The were used as the of a at and was routinely in a For the of pimaricin was by bioassay using utilis CECT 1061 as The pimaricin biosynthetic genes were by using as probes DNA from the PKS genes of S. hygroscopicus that direct the biosynthesis of the macrocyclic polyketide immunosuppressant rapamycin (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (399) Google Scholar, 8Aparicio J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar) and the PKS genes griseus that are involved in the biosynthesis of the macrolide candicidin (21Criado L.M. Martı́n J.F. Gil J.A. Gene. 1993; 126: 135-139Crossref PubMed Scopus (32) Google Scholar). A cosmid library was and a total of 110 of contiguous DNA was the cosmid of this the were with enzymes and fragments were the size as their fragments of S. natalensis total that the DNA was not and fragments of the were with a from a in was for a of a type I PKS (the in modular 8Aparicio J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar), as by with other modular PKSs of the modular nature of PKS genes, the of a highly at a of the extent of the about of the of (Fig. not with the that the of the PKS of the gene cluster is cosmid also that of the of (Fig. were the PKS of the was in the of the cluster (Fig. that a region is sets of PKS genes in this these the fragments the 110 of DNA were subcloned into and their were the function to which from the and the of The first of PKS genes pimS1 (Fig. were fully The second of PKS genes are in to the first and appears to modular were to the large sets of PKS further the of these activities with the to of other Streptomyces activities with in the protein a a and a these genes in pimaricin biosynthesis. the of the a gene was also and fully F. A. J. and J. F. other with known were on the of the 110-kb region studied. The involvement of the DNA region in pimaricin biosynthesis was by gene S. natalensis ATCC 27448 has to highly to with used Streptomyces a different for of DNA into this strain was We of the of phage (15Rodicio M.R. Bruton C.J. Chater K.F. Gene. 1985; 34: 283-292Crossref PubMed Scopus (54) Google Scholar), to S. natalensis to DNA into this A in pBluescript containing an region to PKS on the of the cluster in was used as a source of DNA for subcloning into KC515 (Fig. A of the and of the of was into and the recombinant phage DNA was into S. lividans as described (16Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate D.J. Smith C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces: A Laboratory Manual. John Innes Foundation, Norwich, UK1985Google Scholar). of S. natalensis with the recombinant phage the for the KC515 phage only by into the chromosome (Fig. of S. natalensis were obtained by for and their pimaricin was by bioassay against was in the (Fig. as with the of antifungal produced by the type of these disrupted was and its culture was with It of pimaricin by the (Fig. that of the phage DNA a the used to the KC515 was used as a as in for genomic DNA with the of the in the disrupted a also with the (Fig. that a The to those to the in the DNA used for disruption in was sequenced and of the of the sequenced region is the of an large pimS1 encoding a protein of the The of the protein was to as a of protein with of the protein a to known fatty acid and polyketide the of gene was with evidence was obtained for that of modules for chain 1 and ACP and that for a of contiguous enoyl in the pimaricin molecule of is a with the from synthase and the from the of the rapamycin synthase J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google the D.J. Cortés J. Haydock S.F. Leadlay P.F. J. Biochem. PubMed Scopus Google Scholar, S. Katz L. Gene. Scopus Google Scholar) is are highly over the and the and of the as previously for other PKS 8Aparicio J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google and The of a for at the of the A. Nature. 1990; PubMed Scopus Google Scholar), as with the previously except for those of the rapamycin synthase J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar). ACP in are also highly ACP from 1 has the previously have to the of type I modular PKSs into the for the is A.F.A. Caffrey P. Aparicio J.F. Loughran M.S. Staunton J. Leadlay P.F. Science. 1994; 263: 378-380Crossref PubMed Scopus (145) Google Scholar) or as in S. Aparicio J.F. Molnár I. Schwecke T. König A. Marsden A.F.A. Staunton J. Leadlay P.F. 1995; PubMed Scopus Google Scholar). were with the in of type I a was with the and in that these units into the growing polyketide of other of the cluster derived from the DNA of the the of that to the the of and to to the known chain length of and that units and on the biosynthetic units of pimaricin (11Martı́n J.F. Annu. Rev. Microbiol. 1977; 31: 12-38Crossref Scopus (60) Google Scholar) (Fig. the polyketide synthase only such the in and responsible for the of and the group that undergo to the function of the as in biosynthesis J. PubMed Scopus Google Scholar). The has been to as a for in Streptomyces J.M. Chater K.F. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). is such in the pimS1 which may this to control of of pimS1 and is encoding a protein of The of the protein by pimS0 was to as a in its a PKS with for reduction or dehydration (i.e. only and the (Fig. the of its first in the containing about acid to acid to from from from E. and and the chain fatty from among others. ACP the the of this protein (Fig. the ACP has the of the the that pimS0 a of a gene with involvement in pimaricin biosynthesis, its disruption was A the region for of the and the of the was subcloned into The method to disruption was as described for of pimaricin that pimS0 is required for pimaricin biosynthesis. Polyene macrolides very large macrolide rings to It is unknown such large macrolides are synthesized by PKSs as with 14- or 16-membered this the first genetic of a polyene macrolide biosynthetic gene experiments were used as to and cosmid clones containing the S. natalensis gene cluster for the 26-membered ring of the macrocyclic polyketide pimaricin. The of the DNA region is that the polyketide synthase genes to into separated by genes involved in the functionalization of the polyketide is the first of PKSs required for the biosynthesis of different fragments of a the rapamycin biosynthetic gene the PKS genes are separated by a gene that is involved in the formation of the of rapamycin (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. 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Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar). modular each includes the activities required for a round of polyketide chain elongation and The of the modules the the in which each is used for polyketide chain (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (399) Google Scholar, 6Donadio S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Science. 1991; 252: 675-679Crossref PubMed Scopus (752) Google Scholar, 7Cortés J. Haydock S.F. Roberts G.A. Bevitt D.J. Leadlay P.F. Nature. 1990; 348: 176-178Crossref PubMed Scopus (515) Google Scholar, 8Aparicio J.F. Molnár I. Schwecke T. König A. Haydock S.F. Khaw L.E. Staunton J. Leadlay P.F. Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar, D.J. T. S.J. Gene. 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Gene. 1996; 169: 9-16Crossref PubMed Scopus (202) Google Scholar). the of a PKS with for reduction or It is that the the a this the with its It that this of the formation of bonds and the studies in the strain Streptomyces in which the was by the J. 1995; PubMed Google Scholar), the that this is and of that catalyze a dehydration of the β-keto group by the activities in the modules (6Donadio S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Science. 1991; 252: 675-679Crossref PubMed Scopus (752) Google Scholar), double bonds in the polyketide of pimaricin to the polyene chromophore. this only process the to a group in the growing polyketide chain is only of the pimaricin polyketide backbone in which the first four chain elongation 1 catalyze the of a starter unit to the unit modular the starter unit is by a loading at the of the first as with synthase 1 J.F. Caffrey P. Marsden A.F.A. Staunton J. Leadlay P.F. J. Biol. Chem. 1994; 269: 8524-8528Abstract Full Text PDF PubMed Google Scholar, J. Caffrey P. Aparicio J.F. G.A. Leadlay P.F. Biol. 1996; PubMed Scopus Google Scholar) or the rapamycin synthase 1 (5Schwecke T. Aparicio J.F. Molnár Y. König A. Khaw L.E. Haydock S.F. Oliynyk M. Caffrey P. Cortés J. Lester J.B. Böhm G.A. Staunton J. Leadlay P.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7839-7843Crossref PubMed Scopus (399) Google Scholar). such loading necessary the of 1 with with a loading protein that the starter unit to the The gene such a loading first acetate, the that by the ACP to the of not catalyze but the of the growing polyketide chain to the which the to 1 of for the first elongation step (Fig. The cloning and of the biosynthesis genes for this polyene the of with antifungal We M. for KC515 phage and P. F. Leadlay and J. A. Gil for the of probes from the rapamycin and the candicidin biosynthesis