Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6581–6592 | Cite as

Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces

  • Shuai Luo
  • Xin-Ai Chen
  • Xu-Ming MaoEmail author
  • Yong-Quan LiEmail author
Applied genetics and molecular biotechnology


Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields. For proof of the concept, using an antibiotic daptomycin from Streptomyces roseosporus, we introduced Himar1-based random mutagenesis combined with a reporter-guided screening strategy to identify a transcriptional regulator PhaR, whose loss-of-function deletion led to about 2.68-fold increase of the gene cluster expression and approximately 6.14-fold or 43% increased daptomycin production in the flask fermentation or in the fed-batch fermentation, respectively. Further study showed that PhaR negatively regulates the expression of daptomycin biosynthetic gene cluster by direct binding to its promoter (dptEp). Moreover, phaR expression gradually drops down during fermentation, and PhaR is positively auto-regulated by directly binding to its own promoter, which results in positive feedback regulation to persistently reduce phaR expression. Meanwhile, the declining PhaR protein remove its repressive effects during daptomycin production. All these results support that our strategy would be a powerful method for genetic screening and rational engineering for the yield improvement of antibiotics, and could be potentially used widely in other Streptomyces species.


Transposon mutagenesis Streptomyces Antibiotic production Transcriptional regulators Regulatory mechanism 



The authors thank Professor Andriy Luzhetskyy (Saarland University, Germany) for kindly providing the plasmid pHTM.

Funding information

This work was financially supported by the Natural Science Foundation of China (31520103901, 31730002, 31470212) to Yong-Quan Li, and the Natural Science Foundation of China (31571284) to Xu-Ming Mao.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9103_MOESM1_ESM.pdf (5.2 mb)
ESM 1 (PDF 5298 kb)


  1. Allenby NEE, Laing E, Bucca G, Kierzek AM, Smith CP (2012) Diverse control of metabolism and other cellular processes in Streptomyces coelicolor by the PhoP transcription factor: genome-wide identification of in vivo targets. Nucleic Acids Res 40(19):9543–9556. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bierman M, Logan R, Obrien 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(1):43–49. CrossRefPubMedGoogle Scholar
  3. Bilyk B, Weber S, Myronovskyi M, Bilyk O, Petzke L, Luzhetskyy A (2013) In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1. Appl Microbiol Biotechnol 97(1):351–359. CrossRefPubMedGoogle Scholar
  4. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6):1513–1523CrossRefPubMedPubMedCentralGoogle Scholar
  5. Boeck LD, Fukuda DS, Abbott BJ, Debono M (1988) Deacylation of A21978c, an acidic lipopeptide antibiotic complex, by Actinoplanes utahensis. J Antibiot 41(8):1085–1092CrossRefPubMedGoogle Scholar
  6. Chen JW, Wu QH, Hawas UW, Wang H (2016) Genetic regulation and manipulation for natural product discovery. Appl Microbiol Biotechnol 100(7):2953–2965. CrossRefPubMedGoogle Scholar
  7. Cobb RE, Wang Y, Zhao H (2015) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol 4(6):723–728. CrossRefPubMedGoogle Scholar
  8. Craig NL (1997) Target site selection in transposition. Annu Rev Biochem 66:437–474. CrossRefPubMedGoogle Scholar
  9. Damasceno JD, Beverley SM, Tosi LR (2010) A transposon toolkit for gene transfer and mutagenesis in protozoan parasites. Genetica 138(3):301–311. CrossRefPubMedGoogle Scholar
  10. Demain AL (2014) Importance of microbial natural products and the need to revitalize their discovery. J Ind Microbiol Biotechnol 41(2):185–201. CrossRefPubMedGoogle Scholar
  11. Eisenstein BI, Oleson FB, Baltz RH (2010) Daptomycin: from the mountain to the clinic, with essential help from Francis Tally, MD. Clin Infect Dis 50:S10–S15. CrossRefPubMedGoogle Scholar
  12. Flett F, Mersinias V, Smith CP (1997) High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting Streptomycetes. FEMS Microbiol Lett 155(2):223–229. CrossRefPubMedGoogle Scholar
  13. 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(4):1541–1546. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Huang D, Jia XQ, Wen JP, Wang GY, Yu GH, Caiyin QG, Chen YL (2011) Metabolic flux analysis and principal nodes identification for daptomycin production improvement by Streptomyces roseosporus. Appl Biochem Biotech 165(7–8):1725–1739. CrossRefGoogle Scholar
  15. Huang D, Wen JP, Wang GY, Yu GH, Jia XQ, Chen YL (2012) In silico aided metabolic engineering of Streptomyces roseosporus for daptomycin yield improvement. Appl Microbiol Biotechnol 94(3):637–649. CrossRefPubMedGoogle Scholar
  16. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Centre, EnglandGoogle Scholar
  17. Lampe DJ, Churchill ME, Robertson HM (1996) A purified mariner transposase is sufficient to mediate transposition in vitro. EMBO J 15(19):5470–5479PubMedPubMedCentralCrossRefGoogle Scholar
  18. Maier TM, Pechous R, Casey M, Zahrt TC, Frank DW (2006) In vivo Himar1-based transposon mutagenesis of Francisella tularensis. Appl Environ Microbiol 72(3):1878–1885. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mao XM, Zhou Z, Hou XP, Guan WJ, Li YQ (2009) Reciprocal regulation between SigK and differentiation programs in Streptomyces coelicolor. J Bacteriol 191(21):6473–6481. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Mao XM, Sun ZH, Liang BR, Wang ZB, Feng WH, Huang FL, Li YQ (2013) Positive feedback regulation of stgR expression for secondary metabolism in Streptomyces coelicolor. J Bacteriol 195(9):2072–2078. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Mao XM, Luo S, Zhou RC, Wang F, Yu P, Sun N, Chen XX, Tang Y, Li YQ (2015) Transcriptional regulation of the daptomycin gene cluster in Streptomyces roseosporus by an autoregulator, AtrA. J Biol Chem 290(12):7992–8001. CrossRefPubMedGoogle Scholar
  22. Mao XM, Luo S, Li YQ (2017) Negative regulation of daptomycin production by DepR2, an ArsR-family transcriptional factor. J Ind Microbiol Biotechnol 44:1653–1658. CrossRefPubMedGoogle Scholar
  23. Martin JF, Rodriguez-Garcia A, Liras P (2017) The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis. J Antibiot 70(5):534–541. CrossRefPubMedGoogle Scholar
  24. McHenney MA, Baltz RH (1996) Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production. Microbiology 142(Pt 9):2363–2373. CrossRefPubMedGoogle Scholar
  25. Miao V, Coeffet-LeGal MF, Brian P, Brost R, Penn J, Whiting A, Martin S, Ford R, Parr I, Bouchard M, Silva CJ, Wrigley SK, Baltz RH (2005) Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry. Microbiology 151:1507–1523. CrossRefPubMedGoogle Scholar
  26. Nett M, Ikeda H, Moore BS (2009) Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat Prod Rep 26(11):1362–1384. CrossRefPubMedPubMedCentralGoogle Scholar
  27. O’rourke S, Wietzorrek A, Fowler K, Corre C, Challis GL, Chater KF (2009) Extracellular signalling, translational control, two repressors and an activator all contribute to the regulation of methylenomycin production in Streptomyces coelicolor. Mol Microbiol 71(3):763–778. CrossRefPubMedGoogle Scholar
  28. Ohnishi Y, Yamazaki H, Kato JY, Tomono A, Horinouchi S (2005) AdpA, a central transcriptional regulator in the A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces griseus. Biosci Biotechnol Biochem 69(3):431–439. CrossRefPubMedGoogle Scholar
  29. Ordonez-Robles M, Rodriguez-Garcia A, Martin JF (2016) Target genes of the Streptomyces tsukubaensis FkbN regulator include most of the tacrolimus biosynthesis genes, a phosphopantetheinyl transferase and other PKS genes. Appl Microbiol Biotechnol 100(18):8091–8103. CrossRefPubMedGoogle Scholar
  30. Ordonez-Robles M, Santos-Beneit F, Rodriguez-Garcia A, Martin JF (2017) Analysis of the Pho regulon in Streptomyces tsukubaensis. Microbiol Res 205:80–87. CrossRefPubMedGoogle Scholar
  31. Pan YY, Liu G, Yang HY, Tian YQ, Tan HR (2009) The pleiotropic regulator AdpA-L directly controls the pathway-specific activator of nikkomycin biosynthesis in Streptomyces ansochromogenes. Mol Microbiol 72(3):710–723. CrossRefPubMedGoogle Scholar
  32. Pickens LB, Tang Y, Chooi YH (2011) Metabolic engineering for the production of natural products. Annu Rev Chem Biomol Eng 2(1):211–236. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Robbel L, Marahiel MA (2010) Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery. J Biol Chem 285(36):27501–27508. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Romero-Rodriguez A, Robledo-Casados I, Sanchez S (2015) An overview on transcriptional regulators in Streptomyces. Biochim Biophys Acta 1849(8):1017–1039. CrossRefPubMedGoogle Scholar
  35. Santos-Beneit F, Rodriguez-Garcia A, Sola-Landa A, Martin JF (2009) Cross-talk between two global regulators in Streptomyces: PhoP and AfsR interact in the control of afsS, pstS and phoRP transcription. Mol Microbiol 72(1):53–68. CrossRefPubMedGoogle Scholar
  36. Sousa C, deLorenzo V, Cebolla A (1997) Modulation of gene expression through chromosomal positioning in Escherichia coli. Microbiology 143:2071–2078CrossRefPubMedGoogle Scholar
  37. Sun JH, Kelemen GH, Fernandez-Abalos JM, Bibb MJ (1999) Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2). Microbiology 145(9):2221–2227CrossRefPubMedGoogle Scholar
  38. Thompson A, Gasson MJ (2001) Location effects of a reporter gene on expression levels and on native protein synthesis in Lactococcus lactis and Saccharomyces cerevisiae. Appl Environ Microbiol 67(8):3434–3439. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Vaishnav P, Demain AL (2011) Unexpected applications of secondary metabolites. Biotechnol Adv 29(2):223–229. CrossRefPubMedGoogle Scholar
  40. Volff JN, Altenbuchner J (1997) High frequency transposition of the Tn5 derivative Tn5493 in Streptomyces lividans. Gene 194(1):81–86CrossRefPubMedGoogle Scholar
  41. Wang L, Zhao Y, Liu Q, Huang Y, Hu C, Liao G (2012) Improvement of A21978C production in Streptomyces roseosporus by reporter-guided rpsL mutation selection. J Appl Microbiol 112(6):1095–1101. CrossRefPubMedGoogle Scholar
  42. Wang F, Ren NN, Luo S, Chen XX, Mao XM, Li YQ (2014) DptR2, a DeoR-type auto-regulator, is required for daptomycin production in Streptomyces roseosporus. Gene 544(2):208–215. CrossRefPubMedGoogle Scholar
  43. Weaden J, Dyson P (1998) Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis. Microbiology 144(Pt 7):1963–1970. CrossRefPubMedGoogle Scholar
  44. Widenbrant EM, Kao CM (2007) Introduction of the foreign transposon Tn4560 in Streptomyces coelicolor leads to genetic instability near the native insertion sequence IS1649. J Bacteriol 189(24):9108–9116. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Yuan PH, Zhou RC, Chen XP, Luo S, Wang F, Mao XM, Li YQ (2016) DepR1, a TetR family transcriptional regulator, positively regulates daptomycin production in an industrial producer, Streptomyces roseosporus SW0702. Appl Environ Microbiol 82(6):1898–1905. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Zhang R, Zeng A, Fang P, Qin Z (2008) Characterization of replication and conjugation of Streptomyces circular plasmids pFP1 and pFP11 and their ability to propagate in linear mode with artificially attached telomeres. Appl Environ Microbiol 74(11):3368–3376. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Pharmaceutical BiotechnologyZhejiang UniversityHangzhouChina
  2. 2.Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic EngineeringHangzhouChina

Personalised recommendations