Skip to main content
Log in

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

The gene cluster (ery) responsible for production of the macrolide antibiotic erythromycin by Saccharopolyspora erythraea is also known to contain ermE, the gene conferring resistance to the antibiotic. The nucleotide sequence has been determined of a 4.5 kb portion of the biosynthetic gene cluster, from a region lying between 3.7 kb and 8.2 kb 3′ of ermE. This has revealed the presence of four complete open reading frames, including the previously known ery gene eryG, which catalyses the last step in the biosynthetic pathway. Comparison of the amino acid sequence of EryG with the sequence of other S-adenosylmethionine (SAM)-dependent methyltransferases has revealed that one of the sequence motifs previously suggested to be part of the SAM-binding site is present not only in EryG but also in many other recently sequenced SAM-dependent methyltransferases. Previous genetic studies have shown that this region also contains gene(s) involved in hydroxylation of the intermediate 6-deoxyerythronolide B. One of the three other open reading frames (eryf) in fact shows very high sequence similarity to known cytochrome P450 hydroxylases. An adjacent gene (ORF5) shows a strikingly high degree of similarity to prokaryotic and eukaryotic acyltransferases and thioesterases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bibb MJ, Findlay PR, Johnson MW (1984) The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein coding sequences. Gene 30:157–166

    Google Scholar 

  • Chou PY, Fasman GD (1978) Empirical predictions of protein conformations. Annu Rev Biochem 47:251–276

    Google Scholar 

  • Cortes J, Haydock SF, Roberts GA, Bevitt DJ, Leadlay PF (1990) An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature 348:176–178

    Google Scholar 

  • Crouzet J, Cameron B, Cauchois L, Rigault S, Rouyez M-C, Blanche F, Thibaut D, Debussche L (1990) Genetic and sequence analysis of an 8.7-kilobase Pseudomonas denitrificans fragment carrying eight genes involved in transformation of precorrin-2 to cobyrinic acid. J Bacteriol 172:5980–5990

    Google Scholar 

  • Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395

    Google Scholar 

  • Dhillon N, Hale RS, Cortes J, Leadlay PF (1989) Molecular characterisation of a gene from Saccharopolyspora erythraea (Streptomyces erythreus) which is involved in erythromycin biosynthesis. Mol Microbiol 3:1405–1414

    Google Scholar 

  • Donadio S, Hutchinson CR (1991) Cloning and characterisation of the Saccharopolyspora erythraea fdxA ferredoxin gene. Gene (in press)

  • Eggink G, Engel H, Vriend G, Terpstra P, Witholt B (1990) Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. J Mol Biol 212:135–142

    Google Scholar 

  • Frischauf AM, Lehrach H, Poutska A, Murray N (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842

    Google Scholar 

  • Gold L, Stormo G (1978) Translational initiation. In: Niedhardt F (ed) Escherichia coli and Salmonella typhimurium. Cellular and molecular biology. American Society for Microbiology, Washington, DC pp 1302–1307

    Google Scholar 

  • Guschelbauer W (1988) The DNA and S-adenosylmethionine-binding regions of EcoDam and related methyltransferases. Gene 74:211–214

    Google Scholar 

  • Henikoff S (1984) Unidirectional digestion with exonuclease III creates targetted breakpoints for DNA sequencing. Gene 28:351–359

    Google Scholar 

  • Hopwood, DA, Sherman D (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Gen 24:37–66

    Google Scholar 

  • Horii M, Ishizaki T, Paik S-Y, Manome T, Murooka Y (1990) An operon containing the genes for cholesterol oxidase and a cytochrome P-450 like protein from a Streptomyces sp. J Bacteriol 172:3644–3653

    Google Scholar 

  • Ingrosso D, Fowler AV, Bleibaum J, Clarke S (1989) Sequence of the d-aspartyl/l-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for the protein, DNA, RNA and small molecule S-adenosylmethionine dependent methyltransferases. J Biol Chem 264:20131–20139

    Google Scholar 

  • Kaneda T, Butte JC, Taubman SB, Corcoran JW (1962) Actinomycete antibiotics III. The biogenesis of erythronolide, the C21 branched chain lactone in erythromycin. J Biol Chem 237:322–327

    Google Scholar 

  • Kieser T (1984) Factors affecting the isolation of cccDNA from S. lividans and E. coli. Plasmid 12:19–36

    Google Scholar 

  • Kolodner R, Fishel RA, Howard M (1985) Genetic recombination of bacterial plasmid DNA: Effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol 163:1060–1066

    Google Scholar 

  • Kratschmar J, Krause M, Marahiel MA (1989) Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J Bacteriol 171:5422–5429

    Google Scholar 

  • Lamperti ED, Villa-Komaroff L (1990) Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases. Anal Biochem 185:187–193

    Google Scholar 

  • Lauster R (1989) Evolution of type II DNA methyltransferases. A gene duplication model. J Mol Biol 206:313–321

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Naggert J, Witkowski J, Mikkelson J, Smith S (1988) Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase. J Biol Chem 263:1146–1150

    Google Scholar 

  • Paulus TJ, Tuan J, Luebke VE, Maine GT, DeWitt JP, Katz L (1990) Mutation and cloning of eryG, the structural gene for erythromycin O-methyltransferase from Saccharopolyspora erythraea, and expression of eryG in Escherichia coli. J Bacteriol 172:2541–2546

    Google Scholar 

  • Poulos TL, Finzel BC, Howard AJ (1987) High resolution crystal structure of cytochrome P450CAM. J Mol Biol 195:687–700

    Google Scholar 

  • Poulose AJ, Rogers L, Cheesbrough TM, Kolattukudy PE (1985) Cloning and sequencing of cDNA for S-acyl fatty acid synthetase from uropygial gland of mallard duck J Biol Chem 260:15953–15958

    Google Scholar 

  • Rackwitz HR, Zehetner G, Frischauf AM, Lehrach H (1984) Rapid restriction mapping of DNA cloned in lambda phage vectors. Gene 30:195–200

    Google Scholar 

  • Salminen M, Lundstrom K, Tilgman C, Savolainen R, Kalkkinen N, Ulmanen I (1990) Molecular cloning and characterisation of rat liver catechol-O-methyltransferase. Gene 93:241–247

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Scrutton NS, Berry A, Perham R (1990) Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343:38–43

    Google Scholar 

  • Shafiee A, Hutchinson CR (1988) Purification and reconstruction of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis. J Bacteriol 170:1548–1553

    Google Scholar 

  • Staden R (1984) Graphic methods to determine the function of nucleic acid sequences. Nucleic Acids Res 12:521–528

    Google Scholar 

  • Thompson CJ, Kieser T, Ward JM, Hopwood DA (1982) Physical analysis of antibiotic resistance genes from Streptomyces and their use in vector construction. Gene 20:51–62

    Google Scholar 

  • Tuan JS, Weber JM, Staver MJ, Leung JO, Donadio S, Katz L (1990) Cloning of genes involved in erythromycin biosynthesis from Saccharopolyspora erythraea using a novel actinomycete-Escherichia coli cosmid. Gene 90:21–29

    Google Scholar 

  • Vara J, Hutchinson CR (1988) Purification of thymidine-diphospho-d-glucose 4,6-dehydratase from an erythromycin-producing strain of Saccharopolyspora erythraea by high resolution liquid chromatography. J Biol Chem 263:14992–14995

    Google Scholar 

  • Vara J, Lewandowska-Skarbek M, Wang Y, Donadio S, Hutchinson CR (1989) Cloning of genes governing the deoxy sugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea. J Bacteriol 171:5872–5881

    Google Scholar 

  • Vogelstein B, Gillespie D (1979) Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA 76:615–619

    Google Scholar 

  • Weber JM, Losick R (1988) The use of a chromosome integration vector to map erythromycin resistance and production genes in Saccharopolyspora erythraea (Streptomyces erythreus). Gene 68:173–180

    Google Scholar 

  • Weber JM, Wierman CK, Hutchinson CR (1985) Genetic analysis of erythromycin production of Streptomyces erythreus. J Bacteriol 164:425–433

    Google Scholar 

  • Weber JM, Schoner B, Losick R (1989) Identification of a gene required for the terminal step in erythromycin A biosynthesis in Saccharpolyspora erythraea (Streptomyces erythreus). Gene 75:235–241

    Google Scholar 

  • Weber JM, Leung JO, Maine GT, Potenz RHB, Paulus TJ, De Witt JP (1990) Organisation of a cluster of erythromycin genes in Saccharopolyspora erythraea. J Bacteriol 172:2372–2383

    Google Scholar 

  • Weber JM, Leung JO, Swanson SJ, Idler KB, McAlpine JB (1991) An erythromycin derivative produced by targeted gene disruption in Saccharopolyspora erythraea. Science 252:114–117

    Google Scholar 

  • White PC, New MI, Dupon B (1986) Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci USA 83:5111–5115

    Google Scholar 

  • Wierenga RK, Terpstra P, Hol WGJ (1986) Prediction of the occurrence of the ADP-binding βαβ fold in protein, using an amino sequence fingerprint. J Mol Biol 187:101–107

    Google Scholar 

  • Yabusaki Y, Murakami H, Ohkawa H (1988) Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleic acid sequence of its cloned gene. J Biochem 103:1004–1010

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

The nucleotide sequence presented here has been submitted to the EMBL Data Library under accession number X60379

Rights and permissions

Reprints and permissions

About this article

Cite this article

Haydock, S.F., Dowson, J.A., Dhillon, N. et al. Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases. Molec. Gen. Genet. 230, 120–128 (1991). https://doi.org/10.1007/BF00290659

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00290659

Key words

Navigation