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FkbN and Tcs7 are pathway-specific regulators of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis L19

  • Genetics and Molecular Biology of Industrial Organisms - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

FK506 (tacrolimus), which is produced by many Streptomyces strains, is clinically used as an immunosuppressive agent and for treatment of inflammatory skin diseases. Here, we identified that the FK506 biosynthetic gene cluster in an industrial FK506-producing strain Streptomyces tsukubaensis L19 is organized as eight transcription units. Two pathway-specific regulators, FkbN and Tcs7, involved in FK506 biosynthesis from S. tsukubaensis L19 were characterized in vivo and in vitro. FkbN activates the transcription of six transcription units in FK506 biosynthetic gene cluster, and Tcs7 activates the transcription of fkbN. In addition, the DNA-binding specificity of FkbN was determined. Finally, a high FK506-producing strain was constructed by overexpression of both fkbN and tcs7 in S. tsukubaensis L19, which improved FK506 production by 89 % compared to the parental strain.

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References

  1. Allison AC (2000) Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 47:63–83

    Article  CAS  PubMed  Google Scholar 

  2. Andexer JN, Kendrew SG, Nur-e-Alam M, Lazos O, Foster TA, Zimmermann AS, Warneck TD, Suthar D, Coates NJ, Koehn FE, Skotnicki JS, Carter GT, Gregory MA, Martin CJ, Moss SJ, Leadlay PF, Wilkinson B (2011) Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate. Proc Natl Acad Sci USA 108:4776–4781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Barreiro C, Martinez-Castro M (2014) Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506). Appl Microbiol Biotechnol 98:497–507

    Article  CAS  PubMed  Google Scholar 

  4. Chen D, Zhang L, Pang B, Chen J, Xu Z, Abe I, Liu W (2013) FK506 maturation involves a cytochrome p450 protein-catalyzed four-electron C-9 oxidation in parallel with a C-31 O-methylation. J Bacteriol 195:1931–1939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chen D, Zhang Q, Cen P, Xu Z, Liu W (2012) Improvement of FK506 production in Streptomyces tsukubaensis by genetic enhancement of the supply of unusual polyketide extender units via utilization of two distinct site-specific recombination systems. Appl Environ Microbiol 78:5093–5103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cui H, Ni X, Liu S, Wang J, Sun Z, Ren J, Su J, Chen G, Xia H (2016) Characterization of three positive regulators for tetramycin biosynthesis in Streptomyces ahygroscopicus. FEMS Microbiol Lett. doi:10.1093/femsle/fnw109

    Google Scholar 

  7. De Schrijver A, De Mot R (1999) A subfamily of MalT-related ATP-dependent regulators in the LuxR family. Microbiology 145(Pt 6):1287–1288

    Article  PubMed  Google Scholar 

  8. Jr Gatto GJ, Boyne MT 2nd, Kelleher NL, Walsh CT (2006) Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster. J Am Chem Soc 128:3838–3847

    Article  CAS  Google Scholar 

  9. Jr Gatto GJ, McLoughlin SM, Kelleher NL, Walsh CT (2005) Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme. Biochemistry 44:5993–6002

    Article  CAS  Google Scholar 

  10. Gomez C, Olano C, Mendez C, Salas JA (2012) Three pathway-specific regulators control streptolydigin biosynthesis in Streptomyces lydicus. Microbiology 158:2504–2514

    Article  CAS  PubMed  Google Scholar 

  11. Goranovic D, Blazic M, Magdevska V, Horvat J, Kuscer E, Polak T, Santos-Aberturas J, Martinez-Castro M, Barreiro C, Mrak P, Kopitar G, Kosec G, Fujs S, Martin JF, Petkovic H (2012) FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis. BMC Microbiol 12:238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Goranovic D, Kosec G, Mrak P, Fujs S, Horvat J, Kuscer E, Kopitar G, Petkovic H (2010) Origin of the allyl group in FK506 biosynthesis. J Biol Chem 285:14292–14300

    Article  CAS  PubMed  PubMed Central  Google 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 USA 100:1541–1546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Henikoff S, Haughn GW, Calvo JM, Wallace JC (1988) A large family of bacterial activator proteins. Proc Natl Acad Sci USA 85:6602–6606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Horbal L, Rebets Y, Rabyk M, Luzhetskyy A, Ostash B, Welle E, Nakamura T, Fedorenko V, Bechthold A (2010) Characterization and analysis of the regulatory network involved in control of lipomycin biosynthesis in Streptomyces aureofaciens Tu117. Appl Microbiol Biotechnol 85:1069–1079

    Article  CAS  PubMed  Google Scholar 

  16. Huang D, Li S, Xia M, Wen J, Jia X (2013) Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement. Microb Cell Fact 12:52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huang D, Xia M, Li S, Wen J, Jia X (2013) Enhancement of FK506 production by engineering secondary pathways of Streptomyces tsukubaensis and exogenous feeding strategies. J Ind Microbiol Biotechnol 40:1023–1037

    Article  CAS  PubMed  Google Scholar 

  18. Husain S, Singh N (2002) The impact of novel immunosuppressive agents on infections in organ transplant recipients and the interactions of these agents with antimicrobials. Clin Infect Dis 35:53–61

    Article  CAS  PubMed  Google Scholar 

  19. Jiang H, Wang YY, Guo YY, Shen JJ, Zhang XS, Luo HD, Ren NN, Jiang XH, Li YQ (2015) An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units. FEBS J 282:2527–2539

    Article  CAS  PubMed  Google Scholar 

  20. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical streptomyces genetics. The John Innes Foundation, Norwich

    Google Scholar 

  21. Kino T, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, Kohsaka M, Aoki H, Imanaka H (1987) FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot (Tokyo) 40:1249–1255

    Article  CAS  Google Scholar 

  22. Kitani S, Ikeda H, Sakamoto T, Noguchi S, Nihira T (2009) Characterization of a regulatory gene, aveR, for the biosynthesis of avermectin in Streptomyces avermitilis. Appl Microbiol Biotechnol 82:1089–1096

    Article  CAS  PubMed  Google Scholar 

  23. Kolling R, Lother H (1985) AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli. J Bacteriol 164:310–315

    CAS  PubMed  PubMed Central  Google Scholar 

  24. MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil T (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61–68

    Article  CAS  PubMed  Google Scholar 

  25. Mao XM, Zhou Z, Cheng LY, Hou XP, Guan WJ, Li YQ (2009) Involvement of SigT and RstA in the differentiation of Streptomyces coelicolor. FEBS Lett 583:3145–3150

    Article  CAS  PubMed  Google Scholar 

  26. Mo S, Ban YH, Park JW, Yoo YJ, Yoon YJ (2009) Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor. J Ind Microbiol Biotechnol 36:1473–1482

    Article  CAS  PubMed  Google Scholar 

  27. Mo S, Kim DH, Lee JH, Park JW, Basnet DB, Ban YH, Yoo YJ, Chen SW, Park SR, Choi EA, Kim E, Jin YY, Lee SK, Park JY, Liu Y, Lee MO, Lee KS, Kim SJ, Kim D, Park BC, Lee SG, Kwon HJ, Suh JW, Moore BS, Lim SK, Yoon YJ (2011) Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. J Am Chem Soc 133:976–985

    Article  CAS  PubMed  Google Scholar 

  28. Mo S, Yoo YJ, Ban YH, Lee SK, Kim E, Suh JW, Yoon YJ (2012) Roles of fkbN in positive regulation and tcs7 in negative regulation of FK506 biosynthesis in Streptomyces sp. strain KCTC 11604BP. Appl Environ Microbiol 78:2249–2255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mo X, Wang Z, Wang B, Ma J, Huang H, Tian X, Zhang S, Zhang C, Ju J (2011) Cloning and characterization of the biosynthetic gene cluster of the bacterial RNA polymerase inhibitor tirandamycin from marine-derived Streptomyces sp. SCSIO1666. Biochem Biophys Res Commun 406:341–347

    Article  CAS  PubMed  Google Scholar 

  30. Motamedi H, Cai SJ, Shafiee A, Elliston KO (1997) Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506. Eur J Biochem 244:74–80

    Article  CAS  PubMed  Google Scholar 

  31. Motamedi H, Shafiee A (1998) The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur J Biochem 256:528–534

    Article  CAS  PubMed  Google Scholar 

  32. Motamedi H, Shafiee A, Cai SJ, Streicher SL, Arison BH, Miller RR (1996) Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520. J Bacteriol 178:5243–5248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Perez-Redondo R, Rodriguez-Garcia A, Martin JF, Liras P (1998) The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene. Gene 211:311–321

    Article  CAS  PubMed  Google Scholar 

  34. Richet E, Raibaud O (1989) MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator. EMBO J 8:981–987

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Rodriguez M, Nunez LE, Brana AF, Mendez C, Salas JA, Blanco G (2008) Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya. Mol Microbiol 69:633–645

    Article  CAS  PubMed  Google Scholar 

  36. Scheu AK, Martinez E, Soliveri J, Malpartida F (1997) abaB, a putative regulator for secondary metabolism in Streptomyces. FEMS Microbiol Lett 147:29–36

    Article  CAS  PubMed  Google Scholar 

  37. Shafiee A, Motamedi H, Chen T (1994) Enzymology of FK-506 biosynthesis. Purification and characterization of 31-O-desmethylFK-506 O:methyltransferase from Streptomyces sp. MA6858. Eur J Biochem 225:755–764

    Article  CAS  PubMed  Google Scholar 

  38. Stuetz A, Baumann K, Grassberger M, Wolff K, Meingassner JG (2006) Discovery of topical calcineurin inhibitors and pharmacological profile of pimecrolimus. Int Arch Allergy Immunol 141:199–212

    Article  CAS  PubMed  Google Scholar 

  39. Sun J, 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(Pt 9):2221–2227

    Article  CAS  PubMed  Google Scholar 

  40. Wang YY, Bai LF, Ran XX, Jiang XH, Wu H, Zhang W, Jin MY, Li YQ, Jiang H (2015) Biochemical characterization of a malonyl-specific acyltransferase domain of FK506 biosynthetic polyketide synthase. Protein Pept Lett 22:2–7

    Article  CAS  PubMed  Google Scholar 

  41. Wang YY, Ran XX, Chen WB, Liu SP, Zhang XS, Guo YY, Jiang XH, Jiang H, Li YQ (2014) Characterization of type II thioesterases involved in natamycin biosynthesis in Streptomyces chattanoogensis L10. FEBS Lett 588:3259–3264

    Article  CAS  PubMed  Google Scholar 

  42. Wang YY, Zhang XS, Luo HD, Ren NN, Jiang XH, Jiang H, Li YQ (2016) Characterization of discrete phosphopantetheinyl transferases in Streptomyces tsukubaensis L19 unveils a complicate phosphopantetheinylation network. Sci Rep 6:24255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Willins DA, Ryan CW, Platko JV, Calvo JM (1991) Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine. J Biol Chem 266:10768–10774

    CAS  PubMed  Google Scholar 

  44. Wilson DJ, Xue Y, Reynolds KA, Sherman DH (2001) Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J Bacteriol 183:3468–3475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 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 110:2717–2730

    Article  CAS  PubMed  Google Scholar 

  46. Yang H, Wang L, Xie Z, Tian Y, Liu G, Tan H (2007) The tyrosine degradation gene hppD is transcriptionally activated by HpdA and repressed by HpdR in Streptomyces coelicolor, while hpdA is negatively autoregulated and repressed by HpdR. Mol Microbiol 65:1064–1077

    Article  CAS  PubMed  Google Scholar 

  47. Zhang Y, Wang L, Zhang S, Yang H, Tan H (2008) hmgA, transcriptionally activated by HpdA, influences the biosynthesis of actinorhodin in Streptomyces coelicolor. FEMS Microbiol Lett 280:219–225

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by Zhejiang Provincial Natural Science Foundation of China (LR16H300001), National Natural Science Foundation of China (31670008 and 31470212), National Basic Research Program of China (973 Program) (2012CB721005), and National Key Technologies R&D Program of China (2011BAD23B05-2).

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    Authors and Affiliations

    1. College of Life Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang, China

      Xiao-Sheng Zhang, Hong-Dou Luo, Yang Tao, Xin-Hang Jiang & Hui Jiang

    2. School of Life Sciences, Fudan University, Shanghai, 200438, China

      Yue-Yue Wang

    3. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang, China

      Yong-Quan Li

    4. Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolism Engineering, Hangzhou, 310058, Zhejiang, China

      Yong-Quan Li

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    1. Xiao-Sheng Zhang
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    Correspondence to Hui Jiang or Yong-Quan Li.

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    Xiao-Sheng Zhang and Hong-Dou Luo contributed equally.

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    Zhang, XS., Luo, HD., Tao, Y. et al. FkbN and Tcs7 are pathway-specific regulators of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis L19. J Ind Microbiol Biotechnol 43, 1693–1703 (2016). https://doi.org/10.1007/s10295-016-1849-0

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