Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Three trehalose synthetic pathways in the acarbose-producing Actinoplanes sp. SN223/29 and evidence for the TreY role in biosynthesis of component C


In this study, three trehalose gene clusters, treX-Y-Z, tpS1, and treS, of the acarbose-producing strain, Actinoplanes sp. SN223/29, have been identified. In particular, five trehalose synthetic genes were sequenced and characterized in detail. They were cloned and expressed in Escherichia coli BL21(DE3)pLysS using the His-tag vector pET19b. The recombinant proteins were purified by Ni2+-nitrilotriacetic acid agarose affinity chromatography, and their functions were characterized biochemically. Both the maltooligosyltrehalose synthase (TreY–TreZ) pathway and the trehalose synthase (TreS) pathway have maximum activity at 40°C and at pH 7.5 and 7.0, respectively, in 100-mM phosphate buffer. Meanwhile, the trehalose-6-phosphate synthase (TpS1) showed maximum activity at 35°C and at pH 7.5 in 100 mM Tris–HCl. As a cofactor candidate, Mg2+ enhanced the activities of all three trehalose synthetic reactions significantly. TreY produced component C from acarbose by its proposed isomerase activity, but TreS did not. This study suggests that the mutation of treY can improve acarbose production by repressing component C production. Based on the data obtained in this study, a model for component C production in Actinoplanes sp. SN 223/29 is proposed.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Austin CA (1996) Screening cDNA libraries by hybridization with double-stranded DNA probes and oligonucleotides. Methods in Mol Biol 69:147–154

  2. Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G (2006) Insights on the evolution of trehalose biosynthesis. BMC Evol Biol 6:109–123

  3. Bell W, Sun W, Hohmann S, Wera S, Reinders A, De Virgilio C, Wiemken A, Thevelein JM (1998) Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex. J Biol Chem 273:33311–33319

  4. Bergmeyer HU (1974) Methoden der enzymatischen Analyse, Band I und II. Verlag Chemie GmbH, Weinheim

  5. Birnboim HC, Doly J (1979) A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

  6. Blattner FR, Plunkett GI, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner FD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474

  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  8. Brunkhorst C, Schneider E (2005) Characterization of maltose and maltotriose transport in the acarbose-producing bacterium Actinoplanes sp. Res Microbiol 156:851–857

  9. Cardoso FS, Castro RF, Borges N, Santos H (2007) Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response. Microbiology 153:270–280

  10. Choi BT, Shin CS (2003) Reduced formation of byproduct component C in acarbose fermentation by Actinoplanes sp. CKD485–16. Biotechnol Prog 19:1677–1682

  11. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE, Tekaia F, Badcock K, Basham D, Brown DT Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544

  12. Crüger A, Piepersberg W, Sistler J (1998) Acarbose biosynthesis genes from Actinoplanes sp., process for the isolation thereof and the use thereof. United States Patent 5753501

  13. De Smet KAL, Weston A, Brown IN, Young DB, Robertson BD (2000) Three pathways for trehalose biosynthesis in mycobacteria. Microbiology 146:199–208

  14. Di Lernia I, Morana A, Ottombrino A, Fusco S, Ross IM, De Rosa M (1998) Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch. Extremophiles 2:409–416

  15. Frommer W, Puls W, Schaefer D, Schmidt D (1975) Glucoside-hydrolase enzyme inhibitors. US Patent 3,876,766

  16. Goeke K, Drepper A, Pape H (1996) Formation of acarbose phosphate by a cell-free extract from the acarbose producer Actinoplanes sp. J Antibiot 49:661–663

  17. Gueguen Y, Rolland JL, Schroeck S, Flament D, Defretin S, Saniez MH, Dietrich J (2001) Characterization of the maltooligosyl trehalose synthase from the thermophilic archaeon Sulfolobus acidocaldarius. FEMS Microbiol Lett 194:201–206

  18. Hattori S, Yamamoto K, Teshima S, Kawamura Y (1998) Method and reagent for detecting a ligand in a sample. US Patent 5,952,171

  19. Hemker M, Stratmann A, Goeke K, Schroder W, Lenz J, Piepersberg W, Pape H (2001) Identification, cloning, expression, and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes sp. strain SE50. J Bacteriol 183:4484–4492

  20. Jarling M, Cauvet T, Grundmeier M, Kuhnert K, Pape H (2004) Isolation of mak1 from Actinoplanes missouriensis and evidence that Pep2 from Streptomyces coelicolor is a maltokinase. J Basic Microb 44:360–373

  21. Jensen PR, Williams PG, Oh D-C, Zeigler L, Fenical W (2007) Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Appl Environ Microbiol 73:1146–1152

  22. Kaasen I, Falkenberg P, Styrvold OB, Strom AR (1992) Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR). J Bacteriol 174:889–898

  23. Kim YH, Kwon TK, Park S, Seo HS, Cheong JJ, Kim CH, Kim JK, Lee JS, Choi YD (2000) Trehalose synthesis by sequential reactions of recombinant maltooligosyl-trehalose synthase and maltooligosyltrehalose trehalohydrolase from Brevibacterium helvolum. Appl Environ Microbiol 66:4620–4624

  24. Kitamoto Y, Osaki N, Tanaka H, Sasaki H, Mori N (2000) Purification and properties of a-glucose 1-phosphate-forming trehalose phosphorylase from basidiomycete, Pleurotus ostreatus. Mycoscience 41:607–613

  25. Klein W, Ehmann U, Boos W (1991) The repression of trehalose transport and metabolism in Escherichia coli by high osmolarity is mediated by trehalose-6-phosphate phosphatase. Res Microbiol 142:359–371

  26. Kobayashi K, Kato M, Miura Y, Kettoku M, Komeda T, Iwamatsu A (1996) Gene cloning and expression of new trehalose-producing enzymes from the hyperthermophilic archaeum Sulfolobus solfataricus KMI. Biosci Biotech Biochem 60:1882–1885

  27. Lee JS, Wittchen KD, Stahl C, Strey J, Meinhardt F (2001) Cloning, expression, and carbon catabolite repression of the bamM gene encoding beta-amylase of Bacillus megaterium DSM319. Appl Microbiol Biotechnol 56:205–211

  28. Li L, Bannantine JP, Zhang Q, Amonsin Q, May BJ, Alt D, Banerji N, Kanjilal S, Kapur S (2005) The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci U S A 102:12344–12349

  29. Maruta K, Kubota M, Fukuda S, Kurimoto M (2000) Cloning and nucleotide sequence of a gene encoding a glycogen debranching enzyme in the trehalose operon from Arthrobacter sp. Q36. Biochim Biophys Acta 1476:377–381

  30. Maruta K, Hattori K, Nakada T, Kubota M, Sugimoto T, Kurimoto M (1996) Cloning and sequencing of trehalose biosynthesis genes from Rhizobium sp. M-11. Biosci Biotechnol Biochem 60:717–720

  31. Ohguchi M, Kubota N, Wada T, Yoshinaga K, Uritani M (1997) Purification and properties of trehalose-synthesizing enzyme from Pseudomonas sp. F1. J Ferment Bioeng 84:358–360

  32. Pan YT, Koroth-Edavana V, Jourdian WJ, Edmondson R, Carroll JD, Pastuszak I, Elbein AD (2004) Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme. Eur J Biochem 271:4259–4269

  33. Redenbach M, Kieser HM, Denapaite D, Eichner A, Cullum J, Kinashi H, Hopwood DA (1996) A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21:77–96

  34. Saito K, Yamazaki H, Ohnishi Y, Fujimoto S, Takahashi E, Horinouchi S (1998) Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli. Appl Microbiol Biotechnol 50:193–198

  35. Sambrook J, Frisch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd editionnd edn. Cold Spring Harbor Laboratory, New York

  36. Sanger F, Nickler S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Nat Acad Sci 74:5463–5467

  37. Savchenko A, Vieille C, Kang S, Zeikus JG (2002) Pyrococcus furiosus alpha-amylase is stabilized by calcium and zinc. Biochemistry 41:6193–6201

  38. Schwarz A, Goedl C, Minani A, Nidetzky B (2007) Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of a,a-trehalose. J Biotechnol 129:140–150

  39. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

  40. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FSL, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GKS, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock REW, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964

  41. Stratmann A, Mahmud T, Lee S, Distler J, Floss HG, Piepersberg W (1999) The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose. J Biol Chem 274:10889–10896

  42. Streeter JG, Bhagwat A (1999) Biosynthesis of trehalose from malto-oligosaccharides in Rhizobia. Can J Microbiol 45:716–721

  43. Tsusaki K, Nishimoto T, Nakata T, Kubota M, Chaen H, Fukuta S, Kurimoto M (1996) Cloning and sequencing of trehalose synthase gene from Pimelobacter sp. R48. Biochim Biophys Acta 1290:1–3

  44. Tsusaki K, Nishimoto T, Nakada T, Kubota M, Chaen H, Fukuda S, Sugimoto T, Kurimoto M (1997) Cloning and sequencing of trehalose synthase gene from Thermus aquaticus ATCC33923. Biochim Biophys Acta 1334:28–32

  45. Vandercammen A, Francois J, Hers HG (1989) Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae. Eur J Biochem 182:613–620

  46. Wehmeier UF, Piepersberg W (2004) Biotechnology and molecular biology of the a-glucosidase inhibitor acarbose. Appl Microbiol Biotechnol 63:613–625

Download references


The authors thank Bayer AG (Wuppertal, Germany) for providing the strain Actinoplanes sp. SN223/29 and Professor Piepersberg from the University of Wuppertal (Germany) for the λ-EMBL3 phage library of Actinoplanes sp. SN223/29. This work was supported by a grant (Code # 20050301034449) from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

Author information

Correspondence to Joo-Won Suh.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, J., Hai, T., Pape, H. et al. Three trehalose synthetic pathways in the acarbose-producing Actinoplanes sp. SN223/29 and evidence for the TreY role in biosynthesis of component C. Appl Microbiol Biotechnol 80, 767 (2008).

Download citation


  • Actinoplanes sp.
  • TreX-Y-Z
  • TpS1
  • TreS
  • Trehalose biosynthesis
  • Acarbose
  • Component C