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.
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Baltz RH (2008) Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 8(5):557–563. https://doi.org/10.1016/j.coph.2008.04.008
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. https://doi.org/10.1038/nrg3244
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. https://doi.org/10.1093/nar/gkl887
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):185–193
D’Haeseleer P (2005) How does gene expression clustering work? Nat Biotechnol 23(12):1499–1501. https://doi.org/10.1038/nbt1205-1499
Edgar R, Domrachev M, Lash AE (2002) Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30(1):207–210
Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci 95(25):14863–14868
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. https://doi.org/10.1128/jb.00029-14
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. https://doi.org/10.1073/pnas.0337542100
Hindra MMJ, Jones SE, Elliot MA (2014) Complex intra-operonic dynamics mediated by a small RNA in Streptomyces coelicolor. PLoS One 9(1):e85856. https://doi.org/10.1371/journal.pone.0085856
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. https://doi.org/10.1073/pnas.0508647102
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. https://doi.org/10.1038/ncomms11605
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical streptomyces genetics. The John Innes Foundation, Norwich
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. https://doi.org/10.1128/jb.00470-08
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):1413–1430
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. https://doi.org/10.1128/aem.00546-13
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. https://doi.org/10.1186/s12934-015-0351-0
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. https://doi.org/10.1186/1471-2164-9-56
Lv Q, Cheng R, Shi T (2014) Regulatory network rewiring for secondary metabolism in Arabidopsis thaliana under various conditions. BMC Plant Biol 14:180. https://doi.org/10.1186/1471-2229-14-180
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. https://doi.org/10.1186/1471-2164-11-10
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):4238–4244
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. https://doi.org/10.1271/bbb.69.431
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. https://doi.org/10.1016/j.chembiol.2009.01.015
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. https://doi.org/10.1007/s10295-009-0591-2
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. https://doi.org/10.1016/j.bbagrm.2013.09.006
Poole K (2007) Efflux pumps as antimicrobial resistance mechanisms. Ann Med 39(3):162–176. https://doi.org/10.1080/07853890701195262
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. https://doi.org/10.1111/j.1365-2958.2009.06624.x
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. https://doi.org/10.1073/pnas.0931429100
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. https://doi.org/10.1007/s10529-005-2222-y
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. https://doi.org/10.1038/nmeth.1246
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. https://doi.org/10.1128/mBio.00191-12
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. https://doi.org/10.1007/s00253-011-3803-4
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):849–854
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).
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This article does not contain any studies with human participants or animals performed by any of the authors.
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Kim, M.W., Lee, BR., You, S. et al. 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). Appl Microbiol Biotechnol 102, 3243–3253 (2018). https://doi.org/10.1007/s00253-018-8838-3