Biotechnology and Bioprocess Engineering

, Volume 23, Issue 6, pp 693–703 | Cite as

Biosynthesis of Methoxymalonyl-acyl Carrier Protein (ACP) as an Extender Unit for Bafilomycin Polyketide in Streptomyces griseus DSM 2608

  • Nguyen Phan Kieu Hanh
  • Jae Yoon Hwang
  • Doo Hyun NamEmail author
Research Paper


Bafilomycins belong to the 16-membered macrolactone family plecomacrolide antibiotics. In the bafilomycin biosynthetic gene cluster of Streptomyces griseus DSM 2608, five polyketide synthase (PKS) genes coding 12 PKS modules responsible for bafilomycin polyketide backbone were found. Based on the chemical structure of bafilomycin polyketide having two methoxy side chains, it was assumed that PKS module 6 and module 12 can utilize the methoxymalonyl-acyl carrier protein (ACP) as an extender unit to incorporate methoxy group. At the downstream of PKS genes, five putative genes responsible for the generation of methoxymalonyl-ACP from 1,3-bis-phosphoglycerate were located. In order to confirm the biochemical role of those genes, five putative genes (bafAI, bafAII, bafAIII, bafAIV, and bafAV) were amplified, cloned into the pET-32a(+) plasmid, and expressed in Escherichia coli Rosetta(DE3). The produced soluble recombinant proteins were purified through nickel-affinity column chromatography, and the biochemical reactions of recombinant proteins were investigated. Firstly the purified BafAII protein, a putative apo-ACP, was converted to active form by attaching 4’-phosphopantetheinyl group of coenzyme A (CoA) by the action of Bacillus subtilis Sfp protein. The linking of glyceryl group on functional BafAII, a holo-ACP, from 1,3-bis-phosphoglycerate (1,3-BPG) was confirmed by the reaction of the recombinant BafAIV protein, a putative glyceryl-ACP synthase. The next oxidation step of glyceryl-BafAII to 2-hydroxy-3-oxopropanoyl- BafAII was identified, which was catalyzed by the recombinant BafAI, a putative glyceryl-ACP dehydrogenase, in the presence of NAD+. Further oxidation of 2-hydroxy- 3-oxopropanoyl-BafAII to 2-hydroxymalonyl-BafAII was also verified by the recombinant BafAIII, a putative 3- oxoacyl-ACP dehydrogenase, in the presence of FAD. The final methylation of 2-hydroxymalonyl-BafAII by the recombinant BafAV, a putative 2-hydroxymalonyl-ACP O-methyltransferase, was proven to be mediated using S-adenosylmethionine as a methyl donor. Throughout checking the biochemical characteristics of five putative genes, the biosynthetic pathway of methoxymalonyl-ACP as an extender unit for bafilomycin polyketide synthesis was clearly evidenced.


methoxymalonyl-ACP glyceryl-ACP bafilomycin Streptomyces griseus 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

12257_2018_427_MOESM1_ESM.pdf (458 kb)
Supplementary material, approximately 459 KB.


  1. 1.
    Werner, G., H. Hagenmaier, K. Albert, H. Kohlshorn, and H. Drautz (1983) The structure of the bafilomycins, a new group of macrolide antibiotics. Tetrahedron Lett. 24: 5193–5196.CrossRefGoogle Scholar
  2. 2.
    Werner, G., H. Hagenmaier, H. Drautz, A. Baumgartner, and H. Zähner (1984) Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J. Antibiot. 37: 110–117.Google Scholar
  3. 3.
    Kretschmer, A., M. Dorgerloh, M. Deeg, and H. Hagenmaier (1985) The structures of novel insecticidal macrolides: bafilomycins D and E, and oxohygrolidin. Agric. Biol. Chem. 49: 2509–2511.Google Scholar
  4. 4.
    Hatfield, G. M., R. W. Woodard, and J.–K. Son (1992) Isolation and structure determination of new macrolide antibiotics. J. Nat. Prod. 55: 753–759.CrossRefGoogle Scholar
  5. 5.
    Ohta, E., S. Ohta, N. K. Kubota, M. Suzuli, T. Ogawa, A. Yamasaki, and S. Ikegami (2001) Micromonospolide A, a new macrolide from Micromonospora sp. Tetrahedron Lett. 42: 4179–4181.CrossRefGoogle Scholar
  6. 6.
    Moon, S., W. Hwang, Y. R. Chung, and J. Shin (2003) New cytotoxic bafilomycin C1–amide produced by Kitasatospora cheerisanensis. J. Antibiot. 56: 856–861.CrossRefGoogle Scholar
  7. 7.
    Carr, G., D. E. Williams, A. R. Díaz–Marrero, B. O. Patrick, H. Bottriell, A. D. Balgi, E. Donohue, M. Roberge, and R. J. Andersen (2010) Bafilomycins produced in culture by Streptomyces spp. isolated from marine habitats are potent inhibitors of autophagy. J. Nat. Prod. 73: 422–427.CrossRefGoogle Scholar
  8. 8.
    Ndejouong, B. L. S. T., I. Sattler, A. Maier, G. Kelter, K. D, Menzel, H. H. Fiebig, and C. Hertweck (2010) Hygrobafilomycin, a cytotoxic and antifungal macrolide bearing a unique monoalkylmaleic anhydride moiety, from Streptomyces varsoviensis. J. Antibiot. 63: 359–363.CrossRefGoogle Scholar
  9. 9.
    Bowman, E. J., A. Siebers, and K. Altendorf (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl Acad. Sci. USA 85, 7972–7976.CrossRefGoogle Scholar
  10. 10.
    Frändberg, E, C. Petersson, L. N. Lundgren, and J. Schnüre (2000) Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1. Can. J. Microbiol. 46: 753–757.CrossRefGoogle Scholar
  11. 11.
    Goetz, M. A., P. A. Mccormick, R. L. Monaghan, and D. A. Ostlind (1985) L–155,175: a new antiparasitic macrolide. Fermentation, isolation and structure. J. Antibiot. 38: 161–168.Google Scholar
  12. 12.
    Vanek, Z., J. Mateju, and E. Curdova (1991) Immunomodulators isolated from microorganisms. Folia Microbiol. 36: 99–111.CrossRefGoogle Scholar
  13. 13.
    Wilton, J. H., G. C. Hokanson, and J. C. French (1985) PD 118,576: a new antitumor macrolide antibiotic. J. Antibiot. 38: 1449–1452.CrossRefGoogle Scholar
  14. 14.
    Ichikawa, N., A. Oguchi, H. Ikeda, J. Ishikawa, S. Kitani, Y. Watanabe, S. Nakamura, Y. Katano., E. Kishi, M. Sasagawa, A. Ankai, S. Fukui, Y. Hashimoto, S. Kamada, M. Otoguro, S. Tanikawa, T. Nihira, S. Horinouchi, Y. Ohnishi, M. Hayakawa, T. Kuzuyama, A. Arisawa, F. Nomoto, H. Miura, Y. Takahashi, and N. Fujita (2010) Genome sequence of Kitasatospora setae NBRC 14216: An evolutionary snapshot of the family Streptomycetaceae. DNA Res. 17: 393–2010.CrossRefGoogle Scholar
  15. 15.
    Hwang, J. Y., H. S. Kim, S. H. Kim, H. R. Oh, and D. H. Nam (2013) Organization and characterization of a biosynthetic gene cluster for bafilomycin from Streptomyces griseus DSM 2608. AMB Express 3: 24.CrossRefGoogle Scholar
  16. 16.
    Hwang, J. Y., S. H. Kim, H. R. Oh, E. J. Kwon, and D. H. Nam (2015) Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics. J. Microbiol. 53: 84–89.CrossRefGoogle Scholar
  17. 17.
    Zhang, W., J. L. Fortman, J. C. Carlson, J. Yan, Y. Liu, F. Bai, W. Guan, J. Jia, T. Matainaho, D. H. Sherman, and S. Li (2013) Characterization of the bafilomycin gene cluster from Streptomyces lohii, ChemBioChem. 14: 301–306.Google Scholar
  18. 18.
    Hanh, N. P. K., J. Y. Hwang, H. R. Oh, G. J. Kim, H. Choi, and D. H. Nam (2018) Biosynthesis of 2–amino–3–hydroxycyclopent–2–enone moiety of bafilomycin in Kitasatospora cheerisanensis KCTC2395. J. Microbiol. 56: 571–578.CrossRefGoogle Scholar
  19. 19.
    Chan, Y. A., A. M. Prodevels, B. M. Kevany, and M. G. Thomas (2009) Biosynthesis of polyketide synthase extender units. Nat. Prod. Rep. 26: 90–114.CrossRefGoogle Scholar
  20. 20.
    Chan, Y. A., M. T. Boyne II, A. M. Podevels, A. K. Klimowicz, J. Handelsman, N. L. Kelleher, and M. G. Thomas (2006) Hydroxymalonyl–acyl carrier protein (ACP) and aminomaonyl–ACP are two additional type I polyketide synthase extender units. PNAS 103: 14349–14354.CrossRefGoogle Scholar
  21. 21.
    Chan, Y. A. and M. G. Thomas (2010) Recognition of (2S)–aminomalonyl–acyl carrier protein (ACP) and (2R)–hydroxymalonyl–ACP by acyltransferases in zwittermicin A biosynthesis. Biochemistry 49: 3667–3677.CrossRefGoogle Scholar
  22. 22.
    Sun, Y., H. Hog, F. Gillies, J. B. Spencer, and P. F. Leadlay (2008) Glyceryl–S–acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics. ChemBioChem. 9: 150–156.CrossRefGoogle Scholar
  23. 23.
    Wu, K., L. Chung, W. P. Revill, L. Katz, and C. D. Reeves (2000) The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251: 81–90CrossRefGoogle Scholar
  24. 24.
    Schuhmann, T. and S. Grond (2004) Biosynthetic investigation of the V–type ATPase inhibitors bafilomycin A1, B1 and cocanamycin A. J. Antibiot. 57: 655–661.CrossRefGoogle Scholar
  25. 25.
    Schuhmann, T., D. Vollmar, and S. Grond (2007) Biosynthetic origin of the methoxyl extender unit in bafilomycin and concanamycin using stereospecifically labeled precursors. J. Antibiot. 60: 52–60.CrossRefGoogle Scholar
  26. 26.
    Lee, J. S., M. G. Vladimirova, A. V. Demirev, B. G. Kim, S. K. Lim, and D. H. Nam (2008) Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus. J. Microbiol. Biotechnol. 18: 427–433.Google Scholar
  27. 27.
    Sambrook, J. and D. W. Russell (2001) Molecular Cloning: a Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  28. 28.
    Charbonnier, F., T. Köhler, J. C., Pechère, and A. Ducruix (2001) Overexpression, refolding, and purification of the histidinetagged outer membrane efflux protein OprM of Pseudomonas aeruginosa. Protein Exp. Purif. 23: 121–127.CrossRefGoogle Scholar
  29. 29.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72: 248–254.CrossRefGoogle Scholar
  30. 30.
    Quadri, L. E., P. H. Weinreb, M. Lei, M. M. Nakano, P. Zuber, and C. T. Walsh (1998) Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37: 1585–1595.CrossRefGoogle Scholar
  31. 31.
    Rieger, D. (1997). Batch analysis of the ATP content of bovine sperm, oocytes, and early embryos using a scintillation counter to measure the chemiluminescence produced by the luciferinluciferase reaction. Anal. Biochem. 246: 67–70.Google Scholar
  32. 32.
    Berridge, M. V., P. M. Herst, and A. S. Tan (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Ann. Rev. 11: 127–152.CrossRefGoogle Scholar
  33. 33.
    Zhu, A., R. Romero, and H. R. Petty (2010) A sensitive fluorimetric assay for pyruvate. Anal. Biochem. 396: 146–151.CrossRefGoogle Scholar
  34. 34.
    Hudec, R., K. Hamada, and K. Mikoshiba (2013) A fluorescencebased assay for the measurement of S–adenosylhomocysteine hydrolase activity in biological samples. Anal. Biochem. 433: 95–101.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Nguyen Phan Kieu Hanh
    • 1
  • Jae Yoon Hwang
    • 1
  • Doo Hyun Nam
    • 1
    Email author
  1. 1.College of PharmacyYeungnam UniversityGyeongsanKorea

Personalised recommendations