Applied Microbiology and Biotechnology

, Volume 72, Issue 4, pp 763–769 | Cite as

Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae

  • Won Seok Jung
  • Sang Kil Lee
  • Jay Sung Joong Hong
  • Sung Ryeol Park
  • Soon Jeong Jeong
  • Ah Reum Han
  • Jae Kyung Sohng
  • Byung Gee Kim
  • Cha Yong Choi
  • David H. Sherman
  • Yeo Joon Yoon
Applied Genetics and Molecular Biotechnology

Abstract

Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.

References

  1. Bierman M, Logan R, O’Brien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43–49CrossRefPubMedGoogle Scholar
  2. Borisova SA, Zhao L, Sherman DH, Liu HW (1999) Biosynthesis of desosamine: construction of a new macrolide carrying a genetically designed sugar moiety. Org Lett 1:133–136CrossRefPubMedGoogle Scholar
  3. Butler AR, Bate N, Kiehl DE, Kirst HA, Cundliffe E (2002) Genetic engineering of aminodeoxyhexose biosynthesis in Streptomyces fradiae. Nat Biotechnol 20:713–716CrossRefPubMedGoogle Scholar
  4. Elhai J, Wolk CP (1988) A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68:119–138CrossRefPubMedGoogle Scholar
  5. Furumai T, Maezawa I, Matsuzawa N, Yano S, Yamaguchi T, Takeda K, Okuda T (1977) Macrolide antibiotics M-4365 produced by micromonospora I. Taxonomy, production, isolation, characterization and properties. J Antibiot 30:443–449PubMedGoogle Scholar
  6. Gaisser S, Reather J, Wirtz G, Kellenberger L, Staunton J, Leadlay P (2000) A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea. Mol Microbiol 36:391–401CrossRefPubMedGoogle Scholar
  7. Hong JSJ, Park SH, Choi CY, Sohng JK, Yoon YJ (2004) New olivosyl derivatives of methymycin/pikromycin from an engineered strain of Streptomyces venezuelae. FEMS Microbiol Lett 238:291–399PubMedCrossRefGoogle Scholar
  8. Jung WS, Kim ES, Kang HY, Choi CY, Sherman DH, Yoon YJ (2003) Site-directed mutagenesis on putative macrolactone ring size determinant in the hybrid pikromycin–tylosin polyketide synthase. J Microbiol Biotechnol 13:823–827Google Scholar
  9. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hoopwood DA (2000) Practical Streptomyces genetics. John Innes Centre, NorwichGoogle Scholar
  10. Lee SK, Basnet DB, Hong JSJ, Jung WS, Choi CY, Lee HC, Sohng JK, Ryu KG, Kim DJ, Ahn JS, Kim BS, Oh HC, Sherman DH, Yoon YJ (2005) Structural diversification of macrolactones by substrate-flexible cytochrome P450 monooxygenase. Adv Synth Catal 347:1369–1378CrossRefGoogle Scholar
  11. Martin CJ, Timoney MC, Sheridan RM, Kendrew SG, Wilkinson B, Staunton J, Leadlay PF (2003) Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase. Org Biomol Chem 1:4144–4147CrossRefPubMedGoogle Scholar
  12. McDaniel R, Ebert-Khosla S, Hopwood DA, Khosla C (1993) Engineered biosynthesis of novel polyketides. Science 262:1546–1550PubMedGoogle Scholar
  13. McDaniel R, Ebert-Khosla S, Hopwood DA, Khosla C (1995) Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 375:549–554CrossRefPubMedGoogle Scholar
  14. O’Hagan D (1991) The polyketide metabolites. Ellis Horwood, ChichesterGoogle Scholar
  15. Omura S, Ikeda H, Matsubara H, Sadakane N (1980) Hybrid biosynthesis and absolute configuration of macrolide antibiotic M-4365 G1. J Antibiot 33:1570–1572PubMedGoogle Scholar
  16. Pfeifer BA, Khosla C (2001) Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev 65:106–118CrossRefPubMedGoogle Scholar
  17. Reeves CD, Ward SL, Revill WP, Suzuki H, Marcus M, Petrakovsky OV, Marquez S, Fu H, Dong SD, Katz L (2004) Production of hybrid 16-membered macrolides by expressing combinations of polyketide synthase genes in engineered Streptomyces fradiae hosts. Chem Biol 11:1465–1472CrossRefPubMedGoogle Scholar
  18. Rodriguez E, Hu Z, Ou S, Volchegursky Y, Hutchinson CR, McDaniel R (2003) Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains. J Ind Microbiol Biotechnol 30:480–488CrossRefPubMedGoogle Scholar
  19. Rodriguez E, Ward S, Fu H, Revil WP, McDaniel R, Katz L (2004) Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae. Appl Microbiol Biotechnol 66:85–91CrossRefPubMedGoogle Scholar
  20. Rowe CJ, Cortes J, Gaisser S, Staunton J, Leadlay PF (1998) Construction of new vectors for high level expression in actinomycetes. Gene 216:215–223CrossRefPubMedGoogle Scholar
  21. Rude MA, Khosla C (2004) Engineered biosynthesis of polyketides in heterologous hosts. Chem Eng Sci 59:4693–4701CrossRefGoogle Scholar
  22. Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  23. Stassi DL, Kakavas SJ, Reynolds KA, Gunawardana G, Swanson S, Zeidner D, Jackson M, Liu H, Buko A, Katz L (1998) Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering. Proc Natl Acad Sci U S A 95:7305–7309CrossRefPubMedGoogle Scholar
  24. Stratigopoulos G, Cundliffe E (2002) Expression analysis of the tylosin–biosynthetic gene cluster: pivotal regulatory role of the tylQ product. Chem Biol 9:71–78CrossRefPubMedGoogle Scholar
  25. Wenzel SC, Muller R (2005) Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. Curr Opin Biotechnol 16:1–13CrossRefGoogle Scholar
  26. Wilson DJ, Xue Y, Reynolds KA, Sherman DH (2001) Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J Bacteriol 183:3468–3475CrossRefPubMedGoogle Scholar
  27. Xue Y, Sherman DH (2000) Alternative modular polyketide synthase expression controls macrolactone structure. Nature 403:571–575CrossRefPubMedGoogle Scholar
  28. Xue Y, Sherman DH (2001) Biosynthesis and combinatorial biosynthesis of pikromycin-related macrolides in Streptomyces venezuelae. Metab Eng 3:15–26CrossRefPubMedGoogle Scholar
  29. Xue Y, Zhao L, Liu HW, Sherman DH (1998a) A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity. Proc Natl Acad Sci U S A 95:12111–12116CrossRefPubMedGoogle Scholar
  30. Xue Y, Wilson D, Zhao L, Liu H, Sherman DH (1998b) Hydroxylation of macrolactone YC-17 and narbomycin in mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae. Chem Biol 5:661–667CrossRefPubMedGoogle Scholar
  31. Xue Q, Ashley G, Hutchinson CR, Santi DV (1999) A multiplasmid approach to preparing large libraries of polyketides. Proc Natl Acad Sci U S A 96:11740–11745CrossRefPubMedGoogle Scholar
  32. Yamase H, Zhao L, Liu HW (2000) Engineering a hybrid sugar biosynthetic pathway: production of l-rhamnose and its implication on dihydrostreptose biosynthesis. J Am Chem Soc 122:12397–12398CrossRefGoogle Scholar
  33. Yoon YJ, Beck JB, Kim BS, Kang HY, Reynolds KA, Sherman DH (2002) Generation of multiple bioactive macrolides by hybrid modular polyketide synthases in Streptomyces venezuelae. Chem Biol 9:203–214CrossRefPubMedGoogle Scholar
  34. Yu TW, Muller R, Muller M, Zhang X, Draeger G, Kim CG, Leistner E, Floss HG (2001) Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, starter unit of rifamycin biosynthesis in Amycolatopsis mediterranei S699. J Biol Chem 276:12546–12555CrossRefPubMedGoogle Scholar
  35. Zhao L, Sherman DH, Liu HW (1998a) Biosynthesis of desosamine: construction of a new methymycin/neomethymycin analogue by deletion of a desosamine biosynthetic gene. J Am Chem Soc 120:10256–10257CrossRefGoogle Scholar
  36. Zhao L, Que NLS, Xue Y, Sherman DH, Liu HW (1998b) Mechanistic studies of desosamine biosynthesis: C-4 deoxygenation precedes C-3 transamination. J Am Chem Soc 120:12159–12160CrossRefGoogle Scholar
  37. Zhao L, Ahlert J, Xue YQ, Thorson JS, Sherman DH, Liu HW (1999) Engineering a methymycin/pikromycin–calicheamicin hybrid: construction of two new macrolides carrying a designed sugar moiety. J Am Chem Soc 121:9881–9882CrossRefGoogle Scholar
  38. Zhao L, Borisova SA, Yeung SM, Liu HW (2001) Study of C-4 deoxygenation in the biosynthesis of desosamine: evidence implicating a novel mechanism. J Am Chem Soc 123:7909–7910CrossRefPubMedGoogle Scholar
  39. Ziermann R, Betlach MC (1999) Recombinant polyketide synthesis in Streptomyces: engineering of improved host strain. Biotechniques 26:106–110PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Won Seok Jung
    • 1
  • Sang Kil Lee
    • 2
  • Jay Sung Joong Hong
    • 1
  • Sung Ryeol Park
    • 3
  • Soon Jeong Jeong
    • 3
  • Ah Reum Han
    • 1
  • Jae Kyung Sohng
    • 4
  • Byung Gee Kim
    • 1
    • 2
  • Cha Yong Choi
    • 1
    • 2
  • David H. Sherman
    • 5
  • Yeo Joon Yoon
    • 3
  1. 1.Interdisciplinary Program of Biochemical Engineering and BiotechnologySeoul National UniversitySeoulSouth Korea
  2. 2.School of Chemical and Biological EngineeringSeoul National UniversitySeoulSouth Korea
  3. 3.Division of Nano Sciences, Department of ChemistryEwha Woman’s UniversitySeoulSouth Korea
  4. 4.Institute of Biomolecule Reconstruction, Department of Pharmaceutical EngineeringSun Moon UniversityChungnamSouth Korea
  5. 5.Life Sciences Institute, Department of Medicinal ChemistryUniversity of MichiganAnn ArborUSA

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