1887

Abstract

6-Deoxyerythronolide B synthase (DEBS) is a large multifunctional enzyme that catalyses the biosynthesis of the erythromycin polyketide aglycone. DEBS is organized into six modules, each containing the enzymic domains required for a single condensation of carboxylic acid residues which make up the growing polyketide chain. Module 1 is preceded by loading acyltransferase (AT-L) and acyl carrier protein (ACP-L) domains, hypothesized to initiate polyketide chain growth with a propionate-derived moiety. Using recombinant DNA technology several mutant strains of were constructed that lack the initial AT-L domain or that lack both the AT-L and ACP-L domains. These strains were still able to produce erythromycin, although at much lower levels than that produced by the wild-type strain. In addition, the AT-L domain expressed as a monofunctional enzyme was able to complement the deletion of this domain from the PKS, resulting in increased levels of erythromycin production. These findings indicate that neither the initial AT-L nor the ACP-L domains are required to initiate erythromycin biosynthesis; however, without these domains the efficiency of erythromycin biosynthesis is decreased significantly. It is proposed that in these mutants the first step in erythromycin biosynthesis is the charging of KS1 with propionate directly from propionyl-CoA.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-144-2-543
1998-02-01
2021-05-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/144/2/mic-144-2-543.html?itemId=/content/journal/micro/10.1099/00221287-144-2-543&mimeType=html&fmt=ahah

References

  1. Aigle B., Schneider D., Morilhat C., Vandewiele D., Daty A., Holl A. -C., Simonet J. -M., Decaris B. 1996; An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes.. Microbiology 142:2815–2822
    [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool.. J Mol Biol 215:403–410
    [Google Scholar]
  3. Aparicio J. F., Caffrey P., Marsden A. F. A., Stauton J., Leadlay P. F. 1994; Limited proteolysis and active-site studies of the first multienzyme component of the erythromycin-producing polyketide synthase.. J Biol Chem 269:8524–8528
    [Google Scholar]
  4. Bedford D., Jacobsen J. R., Luo G., Cane D. E., Khosla C. 1996; A functional chimeric modular polyketide synthase generated via domain replacement.. Chem Biol 3:827–831
    [Google Scholar]
  5. Bevitt D., J, Cortes J., Haydock S. F., Leadlay P. F. 1992; 6-Deoxyerythronolide-B synthase 2 from Saccharopolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme.. Fur J Biochem 204:39–49
    [Google Scholar]
  6. Bibb M. J., White J., Ward J. M., Janssen G. R. 1994; The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site.. Mol Microbiol 14:533–545
    [Google Scholar]
  7. Caffrey P., Bevitt D. J., Staunton J., Leadlay P. F. 1992; Identification of DEBS1, DEBS2 and DEBS3, the multienzyme polypeptides of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea . FFBS Lett 304:225–228
    [Google Scholar]
  8. Cortes J., Haydock S. F., Roberts G. A., Bevitt D. J., Leadlay P. F. 1990; An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea . Nature 348:176–178
    [Google Scholar]
  9. 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]
  10. Dewitt J. P. 1985; Evidence for a sex factor in Streptomyces erythreus . J Bacteriol 164:969–971
    [Google Scholar]
  11. Donadio S., Katz L. 1992; Organization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea . Gene 111:51–60
    [Google Scholar]
  12. Donadio S., Staver M., McAlpine J. B., Swanson S. J., Katz L. 1991; Modular organization of genes required for complex polyketide biosynthesis.. Science 252:675–679
    [Google Scholar]
  13. Donadio S., McAlpine J. B., Shelden P. J., Jackson M., Katz L. 1993; An erythromycin analog produced by reprogramming polyketide synthesis.. Proc Natl Acad Sci USA 90:7119–7123
    [Google Scholar]
  14. Hopwood D. A., Sherman D. H. 1990; Molecular genetics of polyketides and its comparison to fatty acid biosynthesis.. Annu Rev Genet 24:37–66
    [Google Scholar]
  15. Hopwood 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., Schempf H. 1985 Genetic Manipulation of Streptomyces: a Laboratory Manual Norwich: John Innes Foundation;
    [Google Scholar]
  16. Hsieh Y. -J., Kolattukudy P. E. 1994; Inhibition of erythromycin synthesis by disruption of malonyl-coenzyme A decarboxylase gene eryM in Saccharopolyspora erythraea . J Bacteriol 176:714–724
    [Google Scholar]
  17. Hutchinson C. R., Fujii I. 1995; Polyketide synthase gene manipulation: a structure–function approach in engineering novel antibiotics.. Annu Rev Microbiol 49:201–238
    [Google Scholar]
  18. Jacoben J. R., Hutchinson C. R., Cane D. E., Khosla C. 1997; Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase.. Science 277:367–369
    [Google Scholar]
  19. Kao C. M., Katz, L & Khosla C. 1994; Engineered biosynthesis of a complete macrolactone in a heterologous host.. Science 265:509–512
    [Google Scholar]
  20. Katz L., Donadio S. 1993; Polyketide synthesis: prospect for hybrid antibiotics.. Annu Rev Microbiol 47:875–912
    [Google Scholar]
  21. Kuhstoss S., Huber M., Turner J. R., Paschal J. W., Rao N. 1996; Production of a novel polyketide through the construction of a hybrid polyketide synthase.. Gene 183:231–236
    [Google Scholar]
  22. McAlpine J. B., Tuan J. S., Brown D. P., Grebner K. D., Whittern D. N., Buko A., Katz L. 1987; New antibiotics from geneticaIly engineered actinomycetes.. J Antibiot 40:1115–1122
    [Google Scholar]
  23. Marsden A. F. A., Caffrey P., Aparicio J. F., Loughran M. S., Staunton J., Leadlay P. F. 1994; Stereospecific acyl transfers on the erythromycin-producing polyketide synthase.. Science 263:378–380
    [Google Scholar]
  24. Mathur M., Kolattukudy P. E. 1992; Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guerin.. J Biol Chem 267:19388–19395
    [Google Scholar]
  25. Molnar I., Aparicio J. F., Haydock S. F., Khaw L E., Schwecke T., O’Hagan D. 1993; Biosynthesis of fatty acid and polyketide metabolites.. Nat Prod Rep 10:593–624
    [Google Scholar]
  26. Olinynyk I., Brown M. J. B., Cortes J., Staunton J., Leadlay P. F. 1996; A hybrid modular polyketide synthase obtained by domain swapping.. Chem Biol 3:833–839
    [Google Scholar]
  27. Osakabe Y., Osakabe K., Kawai S., Katayama Y., Morohoshi N. 1995; Characterization of the structure and determination of mRNA levels of the phenylalanine ammonia-lyase gene family from Populus kitakamiensis . Plant Mol Biol 28:1133–1141
    [Google Scholar]
  28. Pieper R., Ebert-Khosla S., Cane D., Khosla C. 1996a; Erythromycin biosynthesis: kinetic studies on a fully active modular polyketide synthase using natural and unnatural substrates.. Biochemistry 35:2054–2060
    [Google Scholar]
  29. Pieper R., Kao C., Khosla C., Luo G., Cane D. E. 1996b; Specificity and versatility in erythromycin biosynthesis.. Chem Soc Rev 25:297–302
    [Google Scholar]
  30. Ruan X., Stassi D., Lax S., Katz L. 1997a; A second type-I PKS gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-producing strain.. Gene 184:1–9
    [Google Scholar]
  31. Ruan X., Pereda A., Stassi D., Zeidner D., Summers R. G., Jackson M., Shivakumar A., Kakavas S., Staver M. J., Donadio S., Katz L. 1997b; Acyltransferase domain substitutions in the erythromycin polyketide synthase yield novel erythromycin derivatives.. J Bacteriol 179:6416–6425
    [Google Scholar]
  32. Ruby C. L., Danis S. J. 1992; Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.. Gene 115:119–125
    [Google Scholar]
  33. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual 2nd edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. Schwecke T., Aparicio J. F., Molnar I., Konig A., Khaw L. E., Haydock S. F., Oliynyk M., Caffrey P., Cortes J. B., Lester J. B., Bohm G. A., Staunton J., Leadlay P. F. 1995; The biosynthethic gene cluster for the polyketide immunosuppressant rapamycin.. Proc Natl Acad Sci USA 92:7839–7843
    [Google Scholar]
  35. Swan D. G., Rodriguez A. M., Vilches C., Mendez C., Salas J. A. 1994; Characterization of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence.. Mol Gen Genet 242:358–362
    [Google Scholar]
  36. Tsukamoto N., Chuck J. -A., Luo G., Kayo C. M., Khosla C., Cane D. E. 1996; 6-Deoxyerythronolide B synthase 1 is specifically acylated by a diketide intermediate at the beta-ketoacyl-acyl carrier protein synthase domain of module 2.. Biochemistry 35:15244–15248
    [Google Scholar]
  37. Vara J. A., Lewandowska-Skarbek M., Wang Y. -G., Donadio S., Hutchinson C. R. 1989; Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus).. J Bacteriol 171:5872–5881
    [Google Scholar]
  38. Weber J. M., 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]
  39. Weber J. M., Leung J. O., Swanson S. J., Idler K. B., McAlpine J. B. 1991; An erythromycin derivative produced by targeted gene disruption in Saccharopolyspora erythraea . Science 252:114–117
    [Google Scholar]
  40. Yamamoto H., Maurer K. H., Hutchinson C. R. 1986; Transformation of Streptomyces erythreus . J Antibiot 39:1304–1313
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-144-2-543
Loading
/content/journal/micro/10.1099/00221287-144-2-543
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error