Applied Microbiology and Biotechnology

, Volume 104, Issue 4, pp 1647–1660 | Cite as

Construction and application of a “superplasmid” for enhanced production of antibiotics

  • Qin Liu
  • Qin Lin
  • Xinying Li
  • Muhammad Ali
  • Jing HeEmail author
Applied genetics and molecular biotechnology


More than two-third of known antibiotics are produced by actinomycetes of the genus Streptomyces. Unfortunately, the production rate from Streptomyces natural antibiotic is extremely slow and thus cannot satisfy industrial demand. In this study, the production of antibiotics by Streptomyces is enhanced by a “superplasmid” which including global regulatory factors afsR, cyclic adenosine receptor protein (CRP), RNA polymerase beta subunits (rpoB) with point mutation and acetyl coenzyme A carboxylase gene (accA2BE), these elements are controlled by the PermE* promoter and then transfer into Streptomyces coelicolor M145, Streptomyces mutabilis TRM45540, Streptomyces hygroscopicus XM201, and Streptomyces hygroscopicus ATCC29253 by conjugation to generate exconjugants. NMR, HPLC, and LC–MS analyses revealed that the superplasmid led to the overproduction of actinorhodin (101.90%), undecylprodigiosin (181.60%) in S. coelicolor M145:: pLQ003, of rapamycin (110%), hygrocin A (163.4%) in S. hygroscopicus ATCC29253:: pLQ003, and of actinomycin D (11.78%) in S. mutabilis TRM45540:: pLQ003, and also to the downregulation of geldanamycin in S. hygroscopicus XM201, but we found that mutant strains in mutant strains of S. hygroscopicus XM201 with regulatory factors inserted showed several peaks that were not found in wild-type strains. The results of the present work indicated that the regulator net working in Streptomyces was not uniform, the superplasmid we constructed possibly caused this overproduction and downregulation in different Streptomyces.


Streptomyces Endogenous antibiotic Superplasmid Regulation 



This work is supported by the National Natural Science Foundation of China (31870089), the Natural Science Foundation for Distinguished Young Scholars of Hubei Province of China (No. 2018CFA069), the fundamental Research Funds for the Central Universities (No. 2662018PY053).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approved

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

Supplementary material

253_2019_10283_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1268 kb)


  1. Bachmann B, Van Lanen SG, Baltz RH (2014) Microbial genome mining for accelerated natural products discovery: is a renaissance in the making. J Ind Microbiol Biotechnol 41(2):75–84Google Scholar
  2. Baltz RH (2014) Spontaneous and induced mutations to rifampicin, streptomycin and spectinomycin resistances in actinomycetes: mutagenic mechanisms and applications for strain improvement. J Antibiot (Tokyo) 67(9):19–24Google Scholar
  3. Bhatia SK, Lee BR, Sathiyanarayanan G, Song HS, Kim J, Jeon JM, Kim JH, Park SH, Yu JH, Park K, Yang YH (2016a) Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source. Bioresour Technol 217:1–9Google Scholar
  4. Bhatia SK, Lee BR, Sathiyanarayanan G, Song HS, Kim J, Jeon JM, Yoon JJ, Ahn J, Park K, Yang YH (2016b) Biomass-derived molecules modulate the behavior of Streptomyces coelicolor for antibiotic production. Chem Soc Rev 46(23):7176–7190Google Scholar
  5. Botas A, Perez-Redondo R, Rodriguez-Garcia A, Alvarez-Alvarez R, Yague P, Manteca A, Liras P (2018) ArgR of Streptomyces coelicolor is a pleiotropic transcriptional regulator: effect on the transcriptome, antibiotic production, and differentiation in liquid cultures. Front Microbiol 9:361PubMedPubMedCentralGoogle Scholar
  6. Caballero JL, Malpartida F, Hopwood DA (1991) Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture. Mol Gen Genet 228(3):372–380PubMedGoogle Scholar
  7. Caixia Lai JX, Tozawa Y, Okamoto-Hosoya Y, Yao X, Ochi K (2002) Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans. Microbiology 148:3365–3373PubMedGoogle Scholar
  8. Chattopadhyay R, Parrack P (2006) Cyclic AMP-dependent functional forms of cyclic AMP receptor protein from Vibrio cholerae. Arch Biochem Biophys 447(1):1–6Google Scholar
  9. Chaudhary AK, Singh B, Maharjan S, Jha AK, Kim BG, Sohng JK (2014) Switching antibiotics production on and off in actinomycetes by an IclR family transcriptional regulator from Streptomyces peucetius ATCC 27952. J Microbiol Biotechnol 24(8):65–72Google Scholar
  10. Chen K, Zhang D, Liu S, Wang NS, Wang M, Du G, Chen J (2013) Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application. Appl Microbiol Biotechnol 97(17):1–9Google Scholar
  11. Dai S, Ouyang Y, Wang G, Li X (2011) Streptomyces autolyticus JX-47 large-insert bacterial artificial chromosome library construction and identification of clones covering geldanamycin biosynthesis gene cluster. Curr Microbiol 63(1):68–74PubMedGoogle Scholar
  12. Dang L, Liu J, Wang C, Liu H, Wen J (2017) Enhancement of rapamycin production by metabolic engineering in Streptomyces hygroscopicus based on genome-scale metabolic model. J Ind Microbiol Biotechnol 44(2):259–270PubMedGoogle Scholar
  13. Derouaux A, Halici S, Nothaft H, Neutelings T, Moutzourelis G, Dusart J, Titgemeyer F, Rigali S (2004) Deletion of a cyclic AMP receptor protein homologue dminishes germination and affects morphological development of Streptomyces coelicolor. J Bacteriol 186(6):1893–1897PubMedPubMedCentralGoogle Scholar
  14. Donovan GT, Norton JP, Bower JM, Mulvey MA (2013) Adenylate cyclase and the cyclic AMP receptor protein modulate stress resistance and virulence capacity of uropathogenic Escherichia coli. Infect Immun 81(1):49–58Google Scholar
  15. Espert SM, Elsinghorst EA, Munson GP (2011) The tib adherence locus of enterotoxigenic Escherichia coli is regulated by cyclic AMP receptor protein. J Bacteriol 193(6):69–76Google Scholar
  16. 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):3–9Google Scholar
  17. Floriano B, Bibb M (1996) afsR is a pleiotropic but conditionally requiredregulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol Microbiol 21(2):385–396PubMedGoogle Scholar
  18. Fujii T, Gramajo HC, Takano E, Bibb MJ (1996) redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. J Bacteriol 178(11):2–5Google Scholar
  19. Fujimoto N, Toyama A, Takeuchi H (2002) Binding modes of cyclic AMP and environments of tryptophan residues in 1:1 and 1:2 complexes of cyclic AMP receptor protein and cyclic AMP. Biopolymers 67(3):86–96Google Scholar
  20. Fussenegger M, Morris RP, Fux C, Rimann M, von Stockar B, Thompson CJ, Bailey JE (2000) Streptogramin-based gene regulation systems for mammalian cells. Nat Biotechnol 18(11):3–8Google Scholar
  21. Gao C, Hindra MD, Yin C, Elliot MA (2012a) Crp is a global regulator of antibiotic production in Streptomyces. MBio 3(6)Google Scholar
  22. Gao Z, Li F, Wu G, Zhu Y, Yu T, Yu S (2012b) Roles of hinge region, loops 3 and 4 in the activation of Escherichia coli cyclic AMP receptor protein. Int J Biol Macromol 50(1):1–6PubMedGoogle Scholar
  23. Geng H, Liu H, Liu J, Wang C, Wen J (2017) Insights into the metabolic mechanism of rapamycin overproduction in the shikimate-resistant Streptomyces hygroscopicus strain UV-II using comparative metabolomics. World J Microbiol Biotechnol 33(6):101PubMedGoogle Scholar
  24. He W, Lei J, Liu Y, Wang Y (2008) The LuxR family members GdmRI and GdmRII are positive regulators of geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Arch Microbiol 189(5):1–10Google Scholar
  25. Hong Y-S, Lee D, Kim W, Jeong J-K, Kim C-G, Sohng JK, Lee J-H, Paik S-G, Lee JJ (2004) Inactivation of the carbamoyltransferase gene refines post-polyketide synthase modification steps in the biosynthesis of the antitumor agent geldanamycin. Am Chem Soc 126:11142–11143Google Scholar
  26. Horinouchi S (2003) AfsR as an integrator of signals that are sensed by multiple serine/threonine kinases in Streptomyces coelicolor A3(2). J Ind Microbiol Biotechnol 30(8):2–7Google Scholar
  27. Hu Zeng ZD, Wen S, Sun Y, Xu W, He Z, Zhai G, Liu Y (2015) Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system. Appl Microbiol Biotechnol:10575–10585PubMedGoogle Scholar
  28. Hu H, Zhang Q, Ochi K (2002) Activation of antibiotic biosynthesis by specified mutations in the rpoB gene (encoding the RNA polymerase subunit) of Streptomyces lividans. J Bacteriol 184(14):3984–3991PubMedPubMedCentralGoogle Scholar
  29. Jiang M, Yin M, Wu S, Han X, Ji K, Wen M, Lu T (2017) GdmRIII, a TetR family transcriptional regulator, controls geldanamycin and elaiophylin biosynthesis in Streptomyces autolyticus CGMCC0516. Sci Rep 7(1):4803PubMedPubMedCentralGoogle Scholar
  30. Kang SG, Jin W, Bibb M, Lee KJ (1998) Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture. FEMS Microbiol Lett 168(2):1–6Google Scholar
  31. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical streptomyces genetics. John Innes Foundation, pp 93–95Google Scholar
  32. Kim W (2010) Identification of three positive regulators in the geldanamycin PKS gene cluster of Streptomyces hygroscopicus JCM4427. Int J Microbiol Biotechnol 20(11):1484–1490Google Scholar
  33. Kim YJ, Song JY, Moon MH, Smith CP, Hong SK, Chang YK (2007) pH shock induces overexpression of regulatory and biosynthetic genes for actinorhodin productionin Streptomyces coelicolor A3(2). Appl Microbiol Biotechnol 76(5):19–30Google Scholar
  34. Kim MJ, Nihira T, Choi S-U (2012) Cloning and characterization of afsR homologue regulatory gene from Streptomyces acidiscabies ATCC 49003. J Korean Soc Appl Biol Chem 55(5):663–668Google Scholar
  35. Kung DW, Griffith DA, Esler WP, Vajdos FF, Mathiowetz AM, Doran SD, Amor PA, Bagley SW, Banks T, Cabral S, Ford K, Garcia-Irizarry CN, Landis MS, Loomis K, McPherson K, Niosi M, Rockwell KL, Rose C, Smith AC, Southers JA, Tapley S, Tu M, Valentine JJ (2015) Discovery of spirocyclic-diamine inhibitors of mammalian acetyl CoA-carboxylase. Bioorg Med Chem Lett 25(22):2–6Google Scholar
  36. Kuscer E, Coates N, Challis I, Gregory M, Wilkinson B, Sheridan R, Petkovic H (2007) Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. J Bacteriol 189(13):56–63Google Scholar
  37. Li S, Wang H, Li Y, Deng J, Lu C, Shen Y, Shen Y (2014) Biosynthesis of hygrocins, antitumor naphthoquinone ansamycins produced by Streptomycessp. LZ35. ChemBioChem 15(1):94–102PubMedGoogle Scholar
  38. Little R Jr, Bremer H (1983) Physiological characterizaton of Escherichia coli rpoB mutants with abnormal control of ribosome synthesis. mSystems:1162–1170Google Scholar
  39. Liu WH, Klapper A (2017) Afsrs synthesis with the extended euclidean rational approximation algorithm. Adv Math Commun 11(1):139–150Google Scholar
  40. Liu G, Chater KF, Chandra G, Niu G, Tan H (2013a) Molecular regulation of antibiotic biosynthesis in streptomyces. Microbiol Mol Biol Rev 77(1):12–43Google Scholar
  41. Liu Z, Zhao X, Bai F (2013b) Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering. Appl Microbiol Biotechnol 97(10):1–8Google Scholar
  42. Long W, Fang B, Ignaszak A, Wu Z, Wang YJ, Wilkinson D (2017) Biomass-derived nanostructured carbons and their composites as anode materials for lithium ion batteries. Chem Soc Rev 46(23):7176–7190PubMedGoogle Scholar
  43. Loomba R, Kayali Z, Noureddin M, Ruane P, Lawitz E, Gitlin N, Bennett M, Harting E, McColgan BJ, Myers RP, Subramanian M, McHutchison JG, Middleton MS, Sirlin CB, Lai M, Charlton MR, Harrison SA (2017) Acetyl-coA carboxylase (ACC) inhibitor GS-0976 leads to significant improvements in MRI-PDFF in a phase 2, randomized, placebo-controlled trial of patients with NASH. Hepatology 66(6):1260a–1261aGoogle Scholar
  44. López-García M, Yagüe P, González-Quiñónez N, Rioseras B, Manteca A (2018) The SCO4117 ECF sigma factor pleiotropically controls secondary metabolism and morphogenesis in. Front Microbiol 9:312PubMedPubMedCentralGoogle Scholar
  45. Luo J, Hong Y, Lu Y, Qiu S, Chaganty BK, Zhang L, Wang X, Li Q, Fan Z (2017) Acetyl-CoA carboxylase rewires cancer metabolism to allow cancer cells to survive inhibition of the Warburg effect by cetuximab. Cancer Lett 384:39–49PubMedGoogle Scholar
  46. Maharjan S, Oh T-J, Lee HC, Sohng JK (2008) Heterologous expression of metK1-sp and afsR-sp in Streptomyces venezuelae for the production of pikromycin. Biotechnol Lett 30(9):1621–1626PubMedGoogle Scholar
  47. Maharjan S, Park JW, Yoon YJ, Lee HC, Sohng JK (2010) Metabolic engineering of Streptomyces venezuelae for malonyl-CoA biosynthesis to enhance heterologous production of polyketides. Biotechnol Lett 32(2):77–82Google Scholar
  48. Martin JF, Sola-Landa A, Santos-Beneit F, Fernandez-Martinez LT, Prieto C, Rodriguez-Garcia A (2011) Cross-talk of global nutritional regulators in the control of primary and secondary metabolism in Streptomyces. Microb Biotechnol 4(2):165–174PubMedPubMedCentralGoogle Scholar
  49. 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 (Tokyo) 70(5):534–541Google Scholar
  50. Martin-Martin S, Rodriguez-Garcia A, Santos-Beneit F, Franco-Dominguez E, Sola-Landa A, Martin JF (2017) Self-control of the PHO regulon: the PhoP-dependent protein PhoU controls negatively expression of genes of PHO regulon in Streptomyces coelicolor. J Antibiot (Tokyo):1–10Google Scholar
  51. Maughan H, Galeano B, Nicholson WL (2004) Novel rpoB mutations conferring rifampin resistance on Bacillus subtilis: global effects on growth, competence, sporulation, and germination. J Bacteriol 186(8):2481–2486PubMedPubMedCentralGoogle Scholar
  52. McKenzie N, Nodwell J (2007) Phosphorylated AbsA2 negatively regulates antibiotic production in Streptomyces coelicolor through interactions with pathway-specific regulatory gene promoters. J Bacteriol 189(14):84–92Google Scholar
  53. Niu G, Chater KF, Tian Y, Zhang J, Tan H (2016) Specialised metabolites regulating antibiotic biosynthesis in Streptomyces spp. FEMS Microbiol Rev 40(4):54–73Google Scholar
  54. Ochi K, Hosaka T (2013) New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters. Appl Microbiol Biotechnol 97(1):87–98PubMedGoogle Scholar
  55. Parajuli N, Viet HT, Ishida K, Tong HT, Lee HC, Liou K, Sohng JK (2005) Identification and characterization of the afsR homologue regulatory gene from Streptomyces peucetius ATCC 27952. Res Microbiol 156(5–6):7–12Google Scholar
  56. Paudel S, Lee HC, Kim BS, Sohng JK (2011) Enhancement of pradimicin production in Actinomadura hibisca P157-2 by metabolic engineering. Microbiol Res 167(1):2–9Google Scholar
  57. Piette A, Derouaux A, Gerkens P, Noens EE, Mazzucchelli G, Vion S, Koerten HK, Titgemeyer F, De Pauw E, Leprince P, van Wezel GP, Galleni M, Rigali S (2005) From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant. J Proteome Res 4(5):699–708Google Scholar
  58. Praveen V, Tripathi CKM, Bihari V, Srivastava SC (2008) Production of actinomycin-d by the mutant of a new isolate of Streptomyces sindenensis. Braz J Microbiol 39:689–692PubMedPubMedCentralGoogle Scholar
  59. Queiroz Sousa MFV, Lopes CE, Pereira JN (2001) A chemically defined medium for production of actinomycin D by Streptomyces parvulus. Braz Arch Biol Technol 44(3):227–231Google Scholar
  60. Rascher A, Hu Z, Viswanathan N, Schirmer A, Reid R, Nierman WC, Lewis M, Hutchinson CR (2003) Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602. FEMS Microbiol Immunol 218(2):223–230Google Scholar
  61. Ryu YG, Butler MJ, Chater KF, Lee KJ (2006) Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor. Appl Environ Microbiol 72(11):7132–7139PubMedPubMedCentralGoogle Scholar
  62. Santos-Beneit F, Rodriguez-Garcia A, Martin JF (2011) Complex transcriptional control of the antibiotic regulator afsS in Streptomyces: PhoP and AfsR are overlapping, competitive activators. J Bacteriol 193(9):42–51Google Scholar
  63. Sawai R, Suzuki A, Takano Y, Lee PC, Horinouchi S (2004) Phosphorylation of AfsR by multiple serine/threonine kinases in Streptomyces coelicolor A3(2). Gene 334:53–61PubMedGoogle Scholar
  64. Shi J, Pan J, Liu L, Yang D, Lu S, Zhu X, Shen B, Duan Y, Huang Y (2016) Titer improvement and pilot-scale production of platensimycin from Streptomyces platensis SB12026. J Ind Microbiol Biotechnol 43(7):27–35Google Scholar
  65. Sidney Farber GDA, Evans A, Mitus A (1960) Clinical studies of actinomycin D with special reference to Wilms’ tumor in children. J Urol 168:2560–2562Google Scholar
  66. Talà ADF, Gallo G, Pinatel E, Calcagnile M, Testini M, Fico D, Rizzo D, Sutera A, Renzone G, Scaloni A (2018) Pirin: a novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces. Metab Eng 48(undefined):254-268PubMedGoogle Scholar
  67. Tomono A, Shimazu T, Inoue H, Nagasawa H, Yoshida M, Ohnishi Y, Horinouchi S (2006) Self-activation of serine/threonine knase AfsK on autophosphorylation at threonine-168. J Antibiot 59(2):117–123PubMedGoogle Scholar
  68. Wang W, Wang H, Hu H, Peng H, Zhang X (2015) Overexpression of afsR and optimization of metal chloride to improve lomofungin production in Streptomyces lomondensis S015. J Microbiol Biotechnol 25(5):672–680PubMedGoogle Scholar
  69. Wang H, Zhao G, Ding X (2017) Morphology engineering of Streptomyces coelicolor M145 by sub-inhibitory concentrations of antibiotics. Sci Rep 7(1):13226PubMedPubMedCentralGoogle Scholar
  70. Wei J, Zhang Y, Yu TY, Sadre-Bazzaz K, Rudolph MJ, Amodeo GA, Symington LS, Walz T, Tong L (2016) A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation. Cell Discov 2:16044PubMedPubMedCentralGoogle Scholar
  71. Wu C-Z (2012) New geldanamycin analogs from Streptomyces hygroscopicus. J Microbiol Biotechnol 22(11):1478–1481PubMedGoogle Scholar
  72. Yan H, Bao F, Zhao L, Yu Y, Tang J, Zhou X (2015) Cyclic AMP (cAMP) receptor protein-cAMP complex regulates heparosan production in Escherichia coli strain nissle 1917. Appl Environ Microbiol 81(22):7687–7696PubMedPubMedCentralGoogle Scholar
  73. Yin S, Wang X, Shi M, Yuan F, Wang H, Jia X, Yuan F, Sun J, Liu T, Yang K, Zhang Y, Fan K, Li Z (2017) Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus. Sci China Life Sci 60(9):992–999PubMedGoogle Scholar
  74. Yoo Y, Hwang J, Shin H, Cui H, Lee J, Yoon Y (2015) Characterization of negative regulatory genes for the biosynthesis of rapamycin in Streptomyces rapamycinicus and its application for improved production. J Ind Microbiol Biotechnol 42(1):25–35Google Scholar
  75. Zhu H, Sandiford SK, van Wezel GP (2014) Triggers and cues that activate antibiotic production by actinomycetes. J Ind Microbiol Biotechnol 41(2):371–386PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  1. 1.National Key Laboratory of Agricultural Microbiology, College of Life Science and TechnologyHuazhong Agricultural UniversityWuhanChina
  2. 2.Biotechnology Program, Department of Environmental SciencesCOMSATS University IslamabadAbbottabadPakistan

Personalised recommendations