Applied Microbiology and Biotechnology

, Volume 102, Issue 17, pp 7541–7553 | Cite as

Identification and metabolomic analysis of chemical elicitors for tacrolimus accumulation in Streptomyces tsukubaensis

  • Cheng Wang
  • Di Huang
  • Shaoxiong Liang
Applied microbial and cell physiology


Tacrolimus is a widely used immunosuppressive agent in the treatment of various clinical diseases. However, the low fermentation yield seriously limits its further application. To stimulate tacrolimus synthesis, nine chemical elicitors of five groups were evaluated for their effects on tacrolimus accumulation in S. tsukubaensis. The results showed that sodium butyrate (SB), dimethylsulfoxide (DMSO), and LaCl3 could increase tacrolimus accumulation by more than 30%. Cumulative effects of different chemical elicitors exhibited that the highest tacrolimus yield was improved by 64.7% (303.60 mg/L) in DMSO and La treatment, compared to the control. To decipher possible response mechanism, a weighted correlation network analysis (WGCNA) based on metabolomics was employed and datasets showed 13 distinct metabolic modules and 16 hub metabolites were possibly related to the stimulatory roles of DMSO, La, SB, and their combination treatments. The pathway analysis further exhibited that central carbon metabolism, amino acid metabolism, and fatty acid metabolism showed significant differences in the above chemical elicitor treatments. Thereinto, the carboxylation of propionyl-CoA from isoleucine and methionine degradation was first confirmed to be the main source of methylmalonyl-CoA by RT-PCR analysis in DMSO and La treatment. By further strengthening of the supply of methylmalonyl-CoA precursor in DMSO and La treatment, the final tacrolimus yield could reach to 372.12 mg/L, 2.02-fold higher than the control. To our knowledge, this is the first study to unveil the potential mechanism of different chemical elicitor stresses in S. tsukubaensis based on metabolomics, and the established information provide valuable guidance for further improving tacrolimus production.


Tacrolimus Chemical elicitors Metabolomic network S. tsukubaensis 


Funding information

This work was financially supported by the Research Start-up Funds of Northwest A&F University (Z109021804).

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_9177_MOESM1_ESM.pdf (120 kb)
ESM 1 (PDF 120 kb)


  1. Ban YH, Shinde PB, Hwang JY, Song MC, Kim DH, Lim SK, Sohng JK, Yoon YJ (2013) Characterization of FK506 biosynthetic intermediates involved in post-PKS elaboration. J Nat Prod 76(6):1091–1098CrossRefPubMedGoogle Scholar
  2. Ban YH, Park SR, Yoon YJ (2016) The biosynthetic pathway of FK506 and its engineering: from past achievements to future prospects. J Ind Microbiol Biotechnol 43(2–3):389–400CrossRefPubMedGoogle Scholar
  3. Barreiro C, Martínez-Castro M (2014) Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506). Appl Microbiol Biotechnol 98(2):497–507CrossRefPubMedGoogle Scholar
  4. Butler A, Cundliffe E (2001) Influence of dimethylsulfoxide on tylosin production in Streptomyces fradiae. J Ind Microbiol Biotechnol 27(1):46–51CrossRefPubMedGoogle Scholar
  5. Chahinian H, Nini L, Boitard E, Dubès J-P, Comeau L-C, Sarda L (2002) Distinction between esterases and lipases: a kinetic study with vinyl esters and TAG. Lipids 37(7):653–662CrossRefPubMedGoogle Scholar
  6. Chan YA, Podevels AM, Kevany BM, Thomas MG (2009) Biosynthesis of polyketide synthase extender units. Nat Prod Rep 26(1):90–114CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chen G, Gui-Yang-Sheng W, Li X, WATERS B, DAVIES J (2000) Enhanced production of microbial metabolites in the presence of dimethyl sulfoxide. J Antibiot 53(10):1145–1153CrossRefPubMedGoogle Scholar
  8. Dotzlaf JE, Metzger LS, Foglesong MA (1984) Incorporation of amino acid-derived carbon into tylactone by Streptomyces fradiae GS14. Antimicrob Agents Chemother 25(2):216–220CrossRefPubMedPubMedCentralGoogle Scholar
  9. Du W, Huang D, Xia M, Wen J, Huang M (2014) Improved FK506 production by the precursors and product-tolerant mutant of Streptomyces tsukubaensis based on genome shuffling and dynamic fed-batch strategies. J Ind Microbiol Biotechnol 41(7):1131–1143CrossRefPubMedGoogle Scholar
  10. Easton JB, Houghton PJ (2004) Therapeutic potential of target of rapamycin inhibitors. Expert Opin Ther Targets 8(6):551–564CrossRefPubMedGoogle Scholar
  11. 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):101CrossRefPubMedGoogle Scholar
  12. Glatz A, Pilbat A-M, Németh GL, Vince-Kontár K, Jósvay K, Hunya Á, Udvardy A, Gombos I, Péter M, Balogh G (2016) Involvement of small heat shock proteins, trehalose, and lipids in the thermal stress management in Schizosaccharomyces pombe. Cell Stress Chaperones 21(2):327–338CrossRefPubMedGoogle Scholar
  13. Goranovič D, Blažič M, Magdevska V, Horvat J, Kuščer E, Polak T, Santos-Aberturas J, Martínez-Castro M, Barreiro C, Mrak P (2012) FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis. BMC Microbiol 12(1):1CrossRefGoogle Scholar
  14. Graziani EI (2009) Recent advances in the chemistry, biosynthesis and pharmacology of rapamycin analogs. Nat Prod Rep 26(5):602–609CrossRefPubMedGoogle Scholar
  15. Hounsa C-G, Brandt EV, Thevelein J, Hohmann S, Prior BA (1998) Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress. Microbiology-UK 144(3):671–680CrossRefGoogle Scholar
  16. Huang D, Li S, Xia M, Wen J, Jia X (2013a) Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement. Microb Cell Factories 12(1):52CrossRefGoogle Scholar
  17. Huang D, Xia M, Li S, Wen J, Jia X (2013b) Enhancement of FK506 production by engineering secondary pathways of Streptomyces tsukubaensis and exogenous feeding strategies. J Ind Microbiol Biotechnol 40(9):1023–1037CrossRefPubMedGoogle 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(1):53–61CrossRefPubMedGoogle Scholar
  19. Kim HS, Park YI (2007) Lipase activity and tacrolimus production in Streptomyces clavuligerus CKD 1119 mutant strains. J Microbiol Biotechnol 17(10):1638–1644PubMedGoogle Scholar
  20. 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 40(9):1249–1255CrossRefPubMedGoogle Scholar
  21. Martínez-Castro M, Salehi-Najafabadi Z, Romero F, Pérez-Sanchiz R, Fernández-Chimeno RI, Martín JF, Barreiro C (2013) Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’. Appl Microbiol Biotechnol 97(5):2139–2152CrossRefPubMedGoogle Scholar
  22. Mishra A, Verma S (2012) Optimization of process parameters for tacrolimus (FK506) production by new isolate of Streptomyces sp. using response surface methodology. J Biochem Technol 3:419–425Google Scholar
  23. Mo S, Ban Y-H, 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(12):1473–1482CrossRefPubMedGoogle Scholar
  24. Mo S, Yoo YJ, Ban YH, Lee S-K, Kim E, Suh J-W, 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(7):2249–2255CrossRefPubMedPubMedCentralGoogle Scholar
  25. Moore JM, Bradshaw E, Seipke RF, Hutchings MI, McArthur M (2011) Use and discovery of chemical elicitors that stimulate biosynthetic gene clusters in Streptomyces bacteria. Methods Enzymol 517:367–385CrossRefGoogle Scholar
  26. Ordóñez-Robles M, Rodríguez-García A, Martín 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):8091–8103CrossRefPubMedGoogle Scholar
  27. Pei G, Chen L, Zhang W (2017) Chapter nine-WGCNA application to proteomic and metabolomic data analysis. Methods Enzymol 585:135–158CrossRefPubMedGoogle Scholar
  28. Qi H, Zhao S, Fu H, Wen J, Jia X (2014) Enhancement of ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by combining resin HP20 addition and metabolic profiling analysis. J Ind Microbiol Biotechnol 41(9):1365–1374CrossRefPubMedGoogle Scholar
  29. Singh BP, Behera BK (2009) Regulation of tacrolimus production by altering primary source of carbons and amino acids. Lett Appl Microbiol 49:254–259CrossRefPubMedGoogle Scholar
  30. Strom A, Kaasen I (1993) Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression. Mol Microbiol 8(2):205–210CrossRefPubMedGoogle Scholar
  31. Su Y, Wang J, Shi M, Niu X, Yu X, Gao L, Zhang X, Chen L, Zhang W (2014) Metabolomic and network analysis of astaxanthin-producing Haematococcus pluvialis under various stress conditions. Bioresour Technol 170:522–529CrossRefPubMedGoogle Scholar
  32. Sui X, Niu X, Shi M, Pei G, Li J, Chen L, Wang J, Zhang W (2014) Metabolomic analysis reveals mechanism of antioxidant butylated hydroxyanisole on lipid accumulation in Crypthecodinium cohnii. J Agric Food Chem 62(51):12477–12484CrossRefPubMedGoogle Scholar
  33. Thomson A, Carroll P, McCauley J, Woo J, Abu-Elmagd K, Starzl T, Van Thiel D (1993) FK506-a novel immunosuppressant for treatment of autoimmune disease-rationale and preliminary clinical-experience at the University of Pittsburgh. Springer Semin Immunopathol 14(4):323–344CrossRefPubMedPubMedCentralGoogle Scholar
  34. Turło J, Gajzlerska W, Klimaszewska M, Król M, Dawidowski M, Gutkowska B (2012) Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis. Enzym Microb Technol 51(6):388–395CrossRefGoogle Scholar
  35. Wang J, Chen L, Tian X, Gao L, Niu X, Shi M, Zhang W (2013) Global metabolomic and network analysis of Escherichia coli responses to exogenous biofuels. J Proteome Res 12(11):5302–5312CrossRefPubMedGoogle Scholar
  36. Wang J, Zhang X, Shi M, Gao L, Niu X, Te R, Chen L, Zhang W (2014) Metabolomic analysis of the salt-sensitive mutants reveals changes in amino acid and fatty acid composition important to long-term salt stress in Synechocystis sp. PCC 6803. Funct Integr Genomics 14(2):431–440CrossRefPubMedGoogle Scholar
  37. Wang C, Liu J, Liu H, Liang S, Wen J (2017a) Combining metabolomics and network analysis to improve tacrolimus production in Streptomyces tsukubaensis using different exogenous feedings. J Ind Microbiol Biotechnol 44(11):1527–1540CrossRefPubMedGoogle Scholar
  38. Wang C, Liu J, Liu H, Wang J, Wen J (2017b) A genome-scale dynamic flux balance analysis model of Streptomyces tsukubaensis NRRL18488 to predict the targets for increasing FK506 production. Biochem Eng J 123:45–56CrossRefGoogle Scholar
  39. Wang J, Liu H, Huang D, Jin L, Wang C, Wen J (2017c) Comparative proteomic and metabolomic analysis of Streptomyces tsukubaensis reveals the metabolic mechanism of FK506 overproduction by feeding soybean oil. Appl Microbiol Biotechnol 101(6):2447–2465CrossRefPubMedGoogle Scholar
  40. Xia J, Wishart DS (2010) MetPA: a web-based metabolomics tool for pathway analysis and visualization. Bioinformatics 26(18):2342–2344CrossRefPubMedGoogle Scholar
  41. 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(10):2717–2730CrossRefPubMedGoogle Scholar
  42. Yoon V, Nodwell JR (2014) Activating secondary metabolism with stress and chemicals. J Ind Microbiol Biotechnol 41(2):415–424CrossRefPubMedGoogle Scholar
  43. Yu X, Niu X, Zhang X, Pei G, Liu J, Chen L, Zhang W (2015) Identification and mechanism analysis of chemical modulators enhancing astaxanthin accumulation in Haematococcus pluvialis. Algal Res 11:284–293CrossRefGoogle Scholar
  44. Zhang W, Li F, Nie L (2010) Integrating multiple ‘omics’ analysis for microbial biology: application and methodologies. Microbiology-SGM 156(2):287–301CrossRefGoogle Scholar
  45. Zhang XS, Luo HD, Tao Y, Wang YY, Jiang XH, Jiang H, Li YQ (2016) FkbN and Tcs7 are pathway-specific regulators of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis L19. J Ind Microbiol Biotechnol 43(12):1693–1703CrossRefPubMedGoogle Scholar
  46. Zhu H, Sandiford SK, van Wezel GP (2014) Triggers and cues that activate antibiotic production by actinomycetes. J Ind Microbiol Biotechnol 41(2):371–386CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Forestry Engineering, College of ForestryNorthwest A&F UniversityYanglingPeople’s Republic of China
  2. 2.TEDA Institute of Biological Sciences and Biotechnology, Tianjin Economic-Technological Development Area (TEDA)Nankai UniversityTianjinPeople’s Republic of China
  3. 3.Key Laboratory of Molecular Microbiology and TechnologyMinistry of EducationTianjinPeople’s Republic of China
  4. 4.Tianjin Key Laboratory of Microbial Functional GenomicsTianjinPeople’s Republic of China
  5. 5.SynBio Research Platform, Collaborative Innovation Center of Chemical Science and EngineeringNankai UniversityTianjinPeople’s Republic of China
  6. 6.SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and TechnologyTianjin UniversityTianjinPeople’s Republic of China

Personalised recommendations