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. 1991 Jul;173(14):4379–4385. doi: 10.1128/jb.173.14.4379-4385.1991

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

W P Revill 1, P F Leadlay 1
PMCID: PMC208099  PMID: 2066335

Abstract

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.

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

  1. Aplin R. T., Baldwin J. E., Fujishima Y., Schofield C. J., Green B. N., Jarvis S. A. Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry. FEBS Lett. 1990 May 21;264(2):215–217. doi: 10.1016/0014-5793(90)80251-d. [DOI] [PubMed] [Google Scholar]
  2. Aplin R. T., Baldwin J. E., Fujishima Y., Schofield C. J., Green B. N., Jarvis S. A. Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry. FEBS Lett. 1990 May 21;264(2):215–217. doi: 10.1016/0014-5793(90)80251-d. [DOI] [PubMed] [Google Scholar]
  3. Barton G. J., Sternberg M. J. A strategy for the rapid multiple alignment of protein sequences. Confidence levels from tertiary structure comparisons. J Mol Biol. 1987 Nov 20;198(2):327–337. doi: 10.1016/0022-2836(87)90316-0. [DOI] [PubMed] [Google Scholar]
  4. Beremand P. D., Hannapel D. J., Guerra D. J., Kuhn D. N., Ohlrogge J. B. Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene. Arch Biochem Biophys. 1987 Jul;256(1):90–100. doi: 10.1016/0003-9861(87)90428-0. [DOI] [PubMed] [Google Scholar]
  5. Bibb M. J., Biró S., Motamedi H., Collins J. F., Hutchinson C. R. Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis. EMBO J. 1989 Sep;8(9):2727–2736. doi: 10.1002/j.1460-2075.1989.tb08414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Cortes J., Haydock S. F., Roberts G. A., Bevitt D. J., Leadlay P. F. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature. 1990 Nov 8;348(6297):176–178. doi: 10.1038/348176a0. [DOI] [PubMed] [Google Scholar]
  9. Delo J., Ernst-Fonberg M. L., Bloch K. Fatty acid synthetases from Euglena gracilis. Arch Biochem Biophys. 1971 Apr;143(2):384–391. doi: 10.1016/0003-9861(71)90225-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  12. Fenn J. B., Mann M., Meng C. K., Wong S. F., Whitehouse C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. doi: 10.1126/science.2675315. [DOI] [PubMed] [Google Scholar]
  13. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  14. Gold L. Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988;57:199–233. doi: 10.1146/annurev.bi.57.070188.001215. [DOI] [PubMed] [Google Scholar]
  15. Guerra D. J., Browse J. The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester. Arch Biochem Biophys. 1989 May 15;271(1):246–253. doi: 10.1016/0003-9861(89)90275-0. [DOI] [PubMed] [Google Scholar]
  16. Hale R. S., Jordan K. N., Leadlay P. F. A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus. FEBS Lett. 1987 Nov 16;224(1):133–136. doi: 10.1016/0014-5793(87)80436-2. [DOI] [PubMed] [Google Scholar]
  17. Henry K. D., Williams E. R., Wang B. H., McLafferty F. W., Shabanowitz J., Hunt D. F. Fourier-transform mass spectrometry of large molecules by electrospray ionization. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9075–9078. doi: 10.1073/pnas.86.23.9075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hopwood D. A., Sherman D. H. Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet. 1990;24:37–66. doi: 10.1146/annurev.ge.24.120190.000345. [DOI] [PubMed] [Google Scholar]
  20. KANEDA T., BUTTE J. C., TAUBMAN S. B., CORCORAN J. W. Actinomycete antibiotics. III. The biogenesis of erythronolide, the C-21 branched chain lactone in erythromycin. J Biol Chem. 1962 Feb;237:322–328. [PubMed] [Google Scholar]
  21. Kaneda T. Biosynthesis of branched long-chain fatty acids from the related short-chain -keto acid substrates by a cell-free system of Bacillus subtilis. Can J Microbiol. 1973 Jan;19(1):87–96. doi: 10.1139/m73-013. [DOI] [PubMed] [Google Scholar]
  22. Kauppinen S., Siggaard-Andersen M., von Wettstein-Knowles P. beta-Ketoacyl-ACP synthase I of Escherichia coli: nucleotide sequence of the fabB gene and identification of the cerulenin binding residue. Carlsberg Res Commun. 1988;53(6):357–370. doi: 10.1007/BF02983311. [DOI] [PubMed] [Google Scholar]
  23. Kawaguchi A., Okuda S. Fatty acid synthetase from Brevibacterium ammoniagenes: formation of monounsaturated fatty acids by a multienzyme complex. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3180–3183. doi: 10.1073/pnas.74.8.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kieser T. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 1984 Jul;12(1):19–36. doi: 10.1016/0147-619x(84)90063-5. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  27. Mohamed A. H., Chirala S. S., Mody N. H., Huang W. Y., Wakil S. J. Primary structure of the multifunctional alpha subunit protein of yeast fatty acid synthase derived from FAS2 gene sequence. J Biol Chem. 1988 Sep 5;263(25):12315–12325. [PubMed] [Google Scholar]
  28. Pazirandeh M., Chirala S. S., Huang W. Y., Wakil S. J. Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli. J Biol Chem. 1989 Oct 25;264(30):18195–18201. [PubMed] [Google Scholar]
  29. Rackwitz H. R., Zehetner G., Frischauf A. M., Lehrach H. Rapid restriction mapping of DNA cloned in lambda phage vectors. Gene. 1984 Oct;30(1-3):195–200. doi: 10.1016/0378-1119(84)90120-3. [DOI] [PubMed] [Google Scholar]
  30. Rossi A., Corcoran J. W. Identification of a multienzyme complex synthesizing fatty acids in the actinomycete Streptomyces erythreus. Biochem Biophys Res Commun. 1973 Feb 5;50(3):597–602. doi: 10.1016/0006-291x(73)91286-2. [DOI] [PubMed] [Google Scholar]
  31. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schweizer E., Werkmeister K., Jain M. K. Fatty acid biosynthesis in yeast. Mol Cell Biochem. 1978 Nov 1;21(2):95–107. doi: 10.1007/BF00240280. [DOI] [PubMed] [Google Scholar]
  35. Schweizer M., Takabayashi K., Laux T., Beck K. F., Schreglmann R. Rat mammary gland fatty acid synthase: localization of the constituent domains and two functional polyadenylation/termination signals in the cDNA. Nucleic Acids Res. 1989 Jan 25;17(2):567–586. doi: 10.1093/nar/17.2.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sherman D. H., Malpartida F., Bibb M. J., Kieser H. M., Bibb M. J., Hopwood D. A. Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22. EMBO J. 1989 Sep;8(9):2717–2725. doi: 10.1002/j.1460-2075.1989.tb08413.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Spaink H. P., Weinman J., Djordjevic M. A., Wijffelman C. A., Okker R. J., Lugtenberg B. J. Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein. EMBO J. 1989 Oct;8(10):2811–2818. doi: 10.1002/j.1460-2075.1989.tb08427.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Stoops J. K., Awad E. S., Arslanian M. J., Gunsberg S., Wakil S. J., Oliver R. M. Studies on the yeast fatty acid synthetase. Subunit composition and structural organization of a large multifunctional enzyme complex. J Biol Chem. 1978 Jun 25;253(12):4464–4475. [PubMed] [Google Scholar]
  40. Tabor S., Huber H. E., Richardson C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed] [Google Scholar]
  41. 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]
  42. Vanaman T. C., Wakil S. J., Hill R. L. The complete amino acid sequence of the acyl carrier protein of Escherichia coli. J Biol Chem. 1968 Dec 25;243(24):6420–6431. [PubMed] [Google Scholar]
  43. Vanden Boom T., Cronan J. E., Jr Genetics and regulation of bacterial lipid metabolism. Annu Rev Microbiol. 1989;43:317–343. doi: 10.1146/annurev.mi.43.100189.001533. [DOI] [PubMed] [Google Scholar]
  44. Witkowska H. E., Green B. N., Smith S. The carboxyl-terminal region of thioesterase II participates in the interaction with fatty acid synthase. Use of electrospray ionization mass spectrometry to identify a carboxyl-terminally truncated form of the enzyme. J Biol Chem. 1990 Apr 5;265(10):5662–5665. [PubMed] [Google Scholar]
  45. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  46. Yuan Z. Y., Liu W., Hammes G. G. Molecular cloning and sequencing of DNA complementary to chicken liver fatty acid synthase mRNA. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6328–6331. doi: 10.1073/pnas.85.17.6328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhang H., Scholl R., Browse J., Somerville C. Double stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 1988 Feb 11;16(3):1220–1220. doi: 10.1093/nar/16.3.1220. [DOI] [PMC free article] [PubMed] [Google Scholar]

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