Applied Microbiology and Biotechnology

, Volume 102, Issue 7, pp 3243–3253 | Cite as

Transcriptome analysis of wild-type and afsS deletion mutant strains identifies synergistic transcriptional regulator of afsS for a high antibiotic-producing strain of Streptomyces coelicolor A3(2)

  • Min Woo Kim
  • Bo-Rahm Lee
  • SungYong You
  • Eun-Jung Kim
  • Ji-Nu Kim
  • Eunjung Song
  • Yung-Hun Yang
  • Daehee Hwang
  • Byung-Gee Kim
Applied genetics and molecular biotechnology


Most secondary metabolism in Actinobacteria is controlled by multi-layered, gene-regulatory networks. These regulatory mechanisms are not easily identified due to their complexity. As a result, when a strong transcriptional regulator (TR) governs activation of biosynthetic pathways of target antibiotics such as actinorhodin (ACT), additional enhancement of the biosynthesis is difficult in combination with other TRs. To find out any “synergistic transcriptional regulators (sTRs)” that show an additive effect on the major, often strong, transcriptional regulator (mTR), here, we performed a clustering analysis using the transcriptome datasets of an mTR deletion mutant and wild-type strain. In the case of ACT biosynthesis in Streptomyces coelicolor, PhoU (SCO4228) and RsfA (SCO4677) were selected through the clustering analysis, using AfsS (SCO4425) as a model mTR, and experimentally validated their roles as sTRs. Furthermore, through analysis of synergistic effects, we were able to suggest a novel regulation mechanism and formulate a strategy to maximize the synergistic effect. In the case of the double TR mutant strain (ΔrsfA pIBR25::afsS), it was confirmed that the increase of cell mass was the major cause of the synergistic effect. Therefore, the strategy to increase the cell mass of double mutant was further attempted by optimizing the expression of efflux pump, which resulted in 2-fold increase in the cell mass and 24-fold increase in the production of ACT. This result is the highest ACT yield from S. coelicolor ever reported.


Combination of transcriptional regulators Clustering analysis Time-series transcriptome Actinorhodin Streptomyces coelicolor 



This research was supported by the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT and Future Planning (2016953757), and by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)(116139-03-1-SB010), and by the Institute for Basic Science (IBS-R13-G1).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_8838_MOESM1_ESM.pdf (615 kb)
ESM 1 (PDF 614 kb)


  1. Baltz RH (2008) Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 8(5):557–563. CrossRefPubMedGoogle Scholar
  2. Bar-Joseph Z, Gitter A, Simon I (2012) Studying and modelling dynamic biological processes using time-series gene expression data. Nat Rev Genet 13(8):552–564. CrossRefPubMedGoogle Scholar
  3. Barrett T, Troup DB, Wilhite SE, Ledoux P, Rudnev D, Evangelista C, Kim IF, Soboleva A, Tomashevsky M, Edgar R (2007) NCBI GEO: mining tens of millions of expression profiles—database and tools update. Nucleic Acids Res 35(Database issue):D760–D765. CrossRefPubMedGoogle Scholar
  4. Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19(2):185–193CrossRefPubMedGoogle Scholar
  5. D’Haeseleer P (2005) How does gene expression clustering work? Nat Biotechnol 23(12):1499–1501. CrossRefPubMedGoogle Scholar
  6. Edgar R, Domrachev M, Lash AE (2002) Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30(1):207–210CrossRefPubMedPubMedCentralGoogle Scholar
  7. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci 95(25):14863–14868CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gardner SG, Johns KD, Tanner R, McCleary WR (2014) The PhoU protein from Escherichia coli interacts with PhoR, PstB, and metals to form a phosphate-signaling complex at the membrane. J Bacteriol 196(9):1741–1752. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 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
  10. Hindra MMJ, Jones SE, Elliot MA (2014) Complex intra-operonic dynamics mediated by a small RNA in Streptomyces coelicolor. PLoS One 9(1):e85856. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hwang D, Rust AG, Ramsey S, Smith JJ, Leslie DM, Weston AD, de Atauri P, Aitchison JD, Hood L, Siegel AF, Bolouri H (2005) A data integration methodology for systems biology. Proc Natl Acad Sci U S A 102(48):17296–17301. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Jeong Y, Kim JN, Kim MW, Bucca G (2016) The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2). Nat Commun 7:11605. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical streptomyces genetics. The John Innes Foundation, NorwichGoogle Scholar
  14. Kim ES, Song JY, Kim DW, Chater KF, Lee KJ (2008) A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor. J Bacteriol 190(22):7559–7566. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Lee PC, Umeyama T, Horinouchi S (2002) afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2). Mol Microbiol 43(6):1413–1430CrossRefPubMedGoogle Scholar
  16. Lee HN, Kim JS, Kim P, Lee HS, Kim ES (2013) Repression of antibiotic downregulator WblA by AdpA in Streptomyces coelicolor. Appl Environ Microbiol 79(13):4159–4163. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Li S, Wang J, Li X, Yin S, Wang W, Yang K (2015) Genome-wide identification and evaluation of constitutive promoters in streptomycetes. Microb Cell Factories 14(1):172. CrossRefGoogle Scholar
  18. Lian W, Jayapal KP, Charaniya S, Mehra S, Glod F, Kyung YS, Sherman DH, Hu WS (2008) Genome-wide transcriptome analysis reveals that a pleiotropic antibiotic regulator, AfsS, modulates nutritional stress response in Streptomyces coelicolor A3(2). BMC Genomics 9:56. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lv Q, Cheng R, Shi T (2014) Regulatory network rewiring for secondary metabolism in Arabidopsis thaliana under various conditions. BMC Plant Biol 14:180. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Nieselt K, Battke F, Herbig A, Bruheim P, Wentzel A, Jakobsen ØM, Sletta H, Alam MT, Merlo ME, Moore J, Omara WAM, Morrissey ER, Juarez-Hermosillo MA, Rodríguez-García A, Nentwich M, Thomas L, Iqbal M, Legaie R, Gaze WH, Challis GL, Jansen RC, Dijkhuizen L, Rand DA, Wild DL, Bonin M, Reuther J, Wohlleben W, Smith MCM, Burroughs NJ, Martín JF, Hodgson DA, Takano E, Breitling R, Ellingsen TE, Wellington EMH (2010) The dynamic architecture of the metabolic switch in Streptomyces coelicolor. BMC Genomics 11:10–10. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Oganesyan V, Oganesyan N, Adams PD, Jancarik J, Yokota HA, Kim R, Kim S-H (2005) Crystal structure of the “PhoU-like” phosphate uptake regulator from Aquifex aeolicus. J Bacteriol 187(12):4238–4244CrossRefPubMedPubMedCentralGoogle Scholar
  22. Ohnishi Y, Yamazaki H, Kato J-Y, 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
  23. Okamoto S, Taguchi T, Ochi K, Ichinose K (2009) Biosynthesis of actinorhodin and related antibiotics: discovery of alternative routes for quinone formation encoded in the act gene cluster. Chem Biol 16(2):226–236. CrossRefPubMedGoogle Scholar
  24. Park SS, Yang YH, Song E, Kim EJ, Kim WS, Sohng JK, Lee HC, Liou KK, Kim BG (2009) Mass spectrometric screening of transcriptional regulators involved in antibiotic biosynthesis in Streptomyces coelicolor A3(2). J Ind Microbiol Biotechnol 36(8):1073–1083. CrossRefPubMedGoogle Scholar
  25. Patra B, Schluttenhofer C, Wu Y, Pattanaik S, Yuan L (2013) Transcriptional regulation of secondary metabolite biosynthesis in plants. Biochim Biophys Acta 1829(11):1236–1247. CrossRefPubMedGoogle Scholar
  26. Poole K (2007) Efflux pumps as antimicrobial resistance mechanisms. Ann Med 39(3):162–176. CrossRefPubMedGoogle Scholar
  27. 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
  28. Sola-Landa A, Moura RS, Martín JF (2003) The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans. Proc Natl Acad Sci U S A 100(10):6133–6138. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Thuy ML, Kharel MK, Lamichhane R, Lee HC, Suh JW, Liou K, Sohng JK (2005) Expression of 2-deoxy-scyllo-inosose synthase (kanA) from kanamycin gene cluster in Streptomyces lividans. Biotechnol Lett 27(7):465–470. CrossRefPubMedGoogle Scholar
  30. Valouev A, Johnson DS, Sundquist A, Medina C, Anton E, Batzoglou S, Myers RM, Sidow A (2008) Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat Methods 5(9):829–834. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Xu Y, Willems A, Au-Yeung C, Tahlan K, Nodwell JR (2012) A two-step mechanism for the activation of actinorhodin export and resistance in Streptomyces coelicolor. MBio 3(5):e00191–e00112. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Yang Y-H, Song E, Kim J-N, Lee B-R, Kim E-J, Park S-H, Kim W-S, Park H-Y, Jeon J-M, Rajesh T, Kim Y-G, Kim B-G (2012) Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor. Appl Microbiol Biotechnol 96(1):113–121. CrossRefPubMedGoogle Scholar
  33. Zhang L, Li WC, Zhao CH, Chater KF, Tao MF (2007) NsdB, a TPR-like-domain-containing protein negatively affecting production of antibiotics in Streptomyces coelicolor A3 (2). Wei Sheng Wu Xue Bao 47(5):849–854PubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Interdisciplinary Program for Biochemical EngineeringSeoul National UniversitySeoulSouth Korea
  2. 2.School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering InstituteSeoul National UniversitySeoulSouth Korea
  3. 3.Division of Cancer Biology and Therapeutics, Departments of Surgery & Biomedical SciencesCedars-Sinai Medical CenterLos AngelesUSA
  4. 4.Department of Microbial Engineering, College of EngineeringKonkuk UniversitySeoulSouth Korea
  5. 5.Department of New Biology and Center for Plant Aging ResearchInstitute for Basic Science, DGISTDaeguRepublic of Korea

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