Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jul;177(14):3946–3952. doi: 10.1128/jb.177.14.3946-3952.1995

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant.

W P Revill 1, M J Bibb 1, D A Hopwood 1
PMCID: PMC177122  PMID: 7608065

Abstract

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.

Full Text

The Full Text of this article is available as a PDF (439.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bibb M. J., Cohen S. N. Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen Genet. 1982;187(2):265–277. doi: 10.1007/BF00331128. [DOI] [PubMed] [Google Scholar]
  2. Bibb M. J., Findlay P. R., Johnson M. W. 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. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  3. Buttner M. J., Chater K. F., Bibb M. J. Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J Bacteriol. 1990 Jun;172(6):3367–3378. doi: 10.1128/jb.172.6.3367-3378.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caballero J. L., Martinez E., Malpartida F., Hopwood D. A. Organisation and functions of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. Mol Gen Genet. 1991 Dec;230(3):401–412. doi: 10.1007/BF00280297. [DOI] [PubMed] [Google Scholar]
  5. Chater K. F., Bruton C. J. Mutational cloning in Streptomyces and the isolation of antibiotic production genes. Gene. 1983 Dec;26(1):67–78. doi: 10.1016/0378-1119(83)90037-9. [DOI] [PubMed] [Google Scholar]
  6. Chater K. F., Bruton C. J., Suarez J. E. Restriction mapping of the DNA of the Streptomyces temperate phage phi C31 and its derivatives. Gene. 1981 Aug;14(3):183–194. doi: 10.1016/0378-1119(81)90114-1. [DOI] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fernández-Moreno M. A., Caballero J. L., Hopwood D. A., Malpartida F. The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces. Cell. 1991 Aug 23;66(4):769–780. doi: 10.1016/0092-8674(91)90120-n. [DOI] [PubMed] [Google Scholar]
  9. Fernández-Moreno M. A., Martínez E., Boto L., Hopwood D. A., Malpartida F. Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. J Biol Chem. 1992 Sep 25;267(27):19278–19290. [PubMed] [Google Scholar]
  10. Fernández-Moreno M. A., Martínez E., Caballero J. L., Ichinose K., Hopwood D. A., Malpartida F. DNA sequence and functions of the actVI region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2). J Biol Chem. 1994 Oct 7;269(40):24854–24863. [PubMed] [Google Scholar]
  11. Gramajo H. C., Takano E., Bibb M. J. Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol Microbiol. 1993 Mar;7(6):837–845. doi: 10.1111/j.1365-2958.1993.tb01174.x. [DOI] [PubMed] [Google Scholar]
  12. Hallam S. E., Malpartida F., Hopwood D. A. Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor. Gene. 1988 Dec 30;74(2):305–320. doi: 10.1016/0378-1119(88)90165-5. [DOI] [PubMed] [Google Scholar]
  13. Harder M. E., Beacham I. R., Cronan J. E., Jr, Beacham K., Honegger J. L., Silbert D. F. Temperature-sensitive mutants of Escherichia coli requiring saturated and unsaturated fatty acids for growth: isolation and properties. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3105–3109. doi: 10.1073/pnas.69.11.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Janssen G. R., Bibb M. J. Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies. Gene. 1993 Feb 14;124(1):133–134. doi: 10.1016/0378-1119(93)90774-w. [DOI] [PubMed] [Google Scholar]
  15. Katz L., Donadio S. Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol. 1993;47:875–912. doi: 10.1146/annurev.mi.47.100193.004303. [DOI] [PubMed] [Google Scholar]
  16. Khosla C., Ebert-Khosla S., Hopwood D. A. Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein. Mol Microbiol. 1992 Nov;6(21):3237–3249. doi: 10.1111/j.1365-2958.1992.tb01778.x. [DOI] [PubMed] [Google Scholar]
  17. Kieser H. M., Kieser T., Hopwood D. A. A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome. J Bacteriol. 1992 Sep;174(17):5496–5507. doi: 10.1128/jb.174.17.5496-5507.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim E. S., Cramer K. D., Shreve A. L., Sherman D. H. Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species. J Bacteriol. 1995 Mar;177(5):1202–1207. doi: 10.1128/jb.177.5.1202-1207.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Magnuson K., Jackowski S., Rock C. O., Cronan J. E., Jr Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev. 1993 Sep;57(3):522–542. doi: 10.1128/mr.57.3.522-542.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Magnuson K., Oh W., Larson T. J., Cronan J. E., Jr Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. FEBS Lett. 1992 Mar 16;299(3):262–266. doi: 10.1016/0014-5793(92)80128-4. [DOI] [PubMed] [Google Scholar]
  22. Malpartida F., Hopwood D. A. Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host. 1984 May 31-Jun 6Nature. 309(5967):462–464. doi: 10.1038/309462a0. [DOI] [PubMed] [Google Scholar]
  23. McDaniel R., Ebert-Khosla S., Hopwood D. A., Khosla C. Engineered biosynthesis of novel polyketides. Science. 1993 Dec 3;262(5139):1546–1550. doi: 10.1126/science.8248802. [DOI] [PubMed] [Google Scholar]
  24. Meurer G., Biermann G., Schütz A., Harth S., Schweizer E. Molecular structure of the multifunctional fatty acid synthetase gene of Brevibacterium ammoniagenes: its sequence of catalytic domains is formally consistent with a head-to-tail fusion of the two yeast genes FAS1 and FAS2. Mol Gen Genet. 1992 Mar;232(1):106–116. doi: 10.1007/BF00299143. [DOI] [PubMed] [Google Scholar]
  25. Meurer G., Hutchinson C. R. Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens. J Bacteriol. 1995 Jan;177(2):477–481. doi: 10.1128/jb.177.2.477-481.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rodicio M. R., Bruton C. J., Chater K. F. New derivatives of the Streptomyces temperate phage phi C31 useful for the cloning and functional analysis of Streptomyces DNA. Gene. 1985;34(2-3):283–292. doi: 10.1016/0378-1119(85)90137-4. [DOI] [PubMed] [Google Scholar]
  27. Ruch F. E., Vagelos P. R. Characterization of a malonyl-enzyme intermediate and identification of the malonyl binding site in malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli. J Biol Chem. 1973 Dec 10;248(23):8095–8106. [PubMed] [Google Scholar]
  28. Ruch F. E., Vagelos P. R. The isolation and general properties of Escherichia coli malonyl coenzyme A-acyl carrier protein transacylase. J Biol Chem. 1973 Dec 10;248(23):8086–8094. [PubMed] [Google Scholar]
  29. Seno E. T., Bruton C. J., Chater K. F. The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gylDNA. Mol Gen Genet. 1984;193(1):119–128. doi: 10.1007/BF00327424. [DOI] [PubMed] [Google Scholar]
  30. Smith S. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. FASEB J. 1994 Dec;8(15):1248–1259. [PubMed] [Google Scholar]
  31. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stephen D., Jones C., Schofield J. P. A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing. Nucleic Acids Res. 1990 Dec 25;18(24):7463–7464. doi: 10.1093/nar/18.24.7463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tsay J. T., Oh W., Larson T. J., Jackowski S., Rock C. O. Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12. J Biol Chem. 1992 Apr 5;267(10):6807–6814. [PubMed] [Google Scholar]
  35. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  36. Wright F., Bibb M. J. Codon usage in the G+C-rich Streptomyces genome. Gene. 1992 Apr 1;113(1):55–65. doi: 10.1016/0378-1119(92)90669-g. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES