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Applied Microbiology and Biotechnology

, Volume 97, Issue 24, pp 10469–10477 | Cite as

Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus

  • Lingjuan Jiang
  • Junhong Wei
  • Lei Li
  • Guoqing NiuEmail author
  • Huarong TanEmail author
Applied genetics and molecular biotechnology

Abstract

Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus. It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics.

Keywords

Engineering Precursor Gougerotin Gene cluster Streptomyces graminearus 

Notes

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31171202 and 31270110) and the Ministry of Science and Technology of China (grant nos. 2012CB721103 and 2013CB734001). We would like to thank Dr. Bertolt Gust (University of Tübingen, Tübingen, Germany) for providing the PCR targeting system.

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 116:43–49PubMedCrossRefGoogle Scholar
  2. Combes P, Till R, Bee S, Smith MC (2002) The Streptomyces genome contains multiple pseudo-attB sites for the ϕC31-encoded site-specific recombination system. J Bacteriol 184:5746–5752PubMedCrossRefGoogle Scholar
  3. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645PubMedCrossRefGoogle Scholar
  4. Du D, Zhu Y, Wei J, Tian Y, Niu G, Tan H (2013) Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters. Appl Microbiol Biotechnol 97:6383–6396PubMedCrossRefGoogle Scholar
  5. Fierro F, Barredo JL, Díez B, Gutierrez S, Fernández FJ, Martín JF (1995) The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci U S A 92:6200–6204PubMedCrossRefGoogle Scholar
  6. Gregory MA, Till R, Smith MC (2003) Integration site for Streptomyces phage ϕBT1 and development of site-specific integrating vectors. J Bacteriol 185:5320–5323PubMedCrossRefGoogle Scholar
  7. Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100:1541–1546PubMedCrossRefGoogle Scholar
  8. Haneishi T, Arai M, Kitano N, Yamamoto S (1974) Aspiculamycin, a new cytosine nucleoside antibiotic. 3. Biological activities, in vitro and in vivo. J Antibiot 27:339–342PubMedCrossRefGoogle Scholar
  9. Kaysser L, Wemakor E, Siebenberg S, Salas JA, Sohng JK, Kammerer B, Gust B (2010) Formation and attachment of the deoxysugar moiety and assembly of the gene cluster for caprazamycin biosynthesis. Appl Environ Microbiol 76:4008–4018PubMedCrossRefGoogle Scholar
  10. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, Norwich, United KingdomGoogle Scholar
  11. Kondo F, Kitano N, Domon H, Arai M, Haneishi T (1974) Aspiculamycin, a new cytosine nucleoside antibiotic. IV. Antimycoplasma activity of aspiculamycin in vitro and in vivo. J Antibiot 27:529–534PubMedCrossRefGoogle Scholar
  12. Lacal JC, Vázquez D, Fernandez-Sousa JM, Carrasco L (1980) Antibiotics that specifically block translation in virus-infected cells. J Antibiot 33:441–446PubMedCrossRefGoogle Scholar
  13. Lee HN, Huang J, Im JH, Kim SH, Noh JH, Cohen SN, Kim ES (2010) Putative TetR family transcriptional regulator SCO1712 encodes an antibiotic downregulator in Streptomyces coelicolor. Appl Environ Microbiol 76:3039–3043PubMedCrossRefGoogle Scholar
  14. Li Y, Ling H, Li W, Tan H (2005) Improvement of nikkomycin production by enhanced copy of sanU and sanV in Streptomyces ansochromogenes and characterization of a novel glutamate mutase encoded by sanU and sanV. Metab Eng 7:165–173PubMedCrossRefGoogle Scholar
  15. Liao G, Li J, Li L, Yang H, Tian Y, Tan H (2009) Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes. Microb Cell Fact 8:61PubMedCrossRefGoogle Scholar
  16. Liao G, Li J, Li L, Yang H, Tian Y, Tan H (2010) Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production. Microb Cell Fact 9:6PubMedCrossRefGoogle Scholar
  17. Liu G, Tian Y, Yang H, Tan H (2005) A pathway-specific transcriptional regulatory gene for nikkomycin biosynthesis in Streptomyces ansochromogenes that also influences colony development. Mol Microbiol 55:1855–1866PubMedCrossRefGoogle Scholar
  18. Murakami T, Burian J, Yanai K, Bibb MJ, Thompson CJ (2011) A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor. Proc Natl Acad Sci U S A 108(38):16020–16025PubMedCrossRefGoogle Scholar
  19. Niu G, Li L, Wei J, Tan H (2013) Cloning, heterologous expression, and characterization of the gene cluster required for gougerotin biosynthesis. Chem Biol 20:34–44PubMedCrossRefGoogle Scholar
  20. Ostash B, Makitrinskyy R, Walker S, Fedorenko V (2009) Identification and characterization of Streptomyces ghanaensis ATCC14672 integration sites for three actinophage-based plasmids. Plasmid 61:171–175PubMedCrossRefGoogle Scholar
  21. Paget MS, Chamberlin L, Atrih A, Foster SJ, Buttner MJ (1999) Evidence that the extracytoplasmic function sigma factor σE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J Bacteriol 181:204–211PubMedGoogle Scholar
  22. Peschke U, Schmidt H, Zhang HZ, Piepersberg W (1995) Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78–11. Mol Microbiol 16:1137–1156PubMedCrossRefGoogle Scholar
  23. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  24. Sioud S, Aigle B, Karray-Rebai I, Smaoui S, Bejar S, Mellouli L (2009) Integrative gene cloning and expression system for Streptomyces sp. US 24 and Streptomyces sp. TN 58 bioactive molecule producing strains. J Biomed Biotechnol 2009:464986PubMedCrossRefGoogle Scholar
  25. Xia M, Huang D, Li S, Wen J, Jia X, Chen Y (2013) Enhanced FK506 production in Streptomyces tsukubaensis by rational feeding strategies based on comparative metabolic profiling analysis. Biotechnol Bioeng 110:2717–2730Google Scholar
  26. Yanai K, Murakami T, Bibb M (2006) Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus. Proc Natl Acad Sci U S A 103:9661–9666PubMedCrossRefGoogle Scholar
  27. Yu L, Cao N, Wang L, Xiao C, Guo M, Chu J, Zhuang Y, Zhang S (2012) Oxytetracycline biosynthesis improvement in Streptomyces rimosus following duplication of minimal PKS genes. Enzyme Microb Technol 50:318–324PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

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