Applied Microbiology and Biotechnology

, Volume 99, Issue 22, pp 9577–9589 | Cite as

Increased riboflavin production by manipulation of inosine 5′-monophosphate dehydrogenase in Ashbya gossypii

  • Rubén M. Buey
  • Rodrigo Ledesma-Amaro
  • Mónica Balsera
  • José María de Pereda
  • José Luis Revuelta
Biotechnologically relevant enzymes and proteins

Abstract

Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii.

Keywords

Ashbya gossypii Metabolic engineering Riboflavin Inosine 5′-monophosphate dehydrogenase 

Abstract

Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Herein, we report the functional and structural characterization of the IMPDH enzyme from the industrial fungus Ashbya gossypii. Our data show that the overexpression of the IMPDH gene increases the metabolic flux through the guanine pathway and ultimately enhances 40 % riboflavin production with respect to the wild type. Also, IMPDH disruption results in a 100-fold increase of inosine excretion to the culture media. Our results contribute to the developing metabolic engineering toolbox aiming at improving the production of metabolites with biotechnological interest in A. gossypii.

Notes

Acknowledgments

This work was supported by grant BIO2014-56930-P from the Spanish Ministerio de Economía y Competitividad. Rubén M Buey is supported by a “Ramón y Cajal” contract from the Spanish Ministerio de Economía y Competitividad and by a Marie Curie Career Integration Grant (EB-SxIP; FP7-PEOPLE-2011-CIG-293831). We thank MD. Sánchez and S. Domínguez for excellent technical help and N. Skinner for correcting the manuscript. X-ray crystallography data were collected at BL13 (XALOC) beamline at ALBA Synchrotron Light Facility with the collaboration of ALBA staff.

Conflict of interest

RMB, RLA, and JLR declare conflict of interest due to issued and outstanding patent applications covering aspects of this work.

Supplementary material

253_2015_6710_MOESM1_ESM.pdf (3.4 mb)
ESM 1(PDF 3495 kb)

References

  1. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. doi:10.1107/S0907444909052925 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Allison AC, Eugui EM (2000) Mycophenolate mofetil and its mechanisms of action. Immunopharmacology 47(2–3):85–118CrossRefPubMedGoogle Scholar
  3. Alonso-García N, Inglés-Prieto A, Sonnenberg A, de Pereda JM (2009) Structure of the calx-β domain of the integrin β4 subunit: insights into function and cation-independent stability. Acta Crystallogr D Biol Crystallogr 65(Pt 8):858–871. doi:10.1107/S0907444909018745 CrossRefPubMedGoogle Scholar
  4. Bacher A, Eberhardt S, Richter G (1996) Biosynthesis of riboflavin. In: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington DC, pp. 657–664Google Scholar
  5. Bacher A, Eberhardt S, Fischer M, Kis K, Richter G (2000) Biosynthesis of vitamin B2 (riboflavin). Annu Rev Nutr 20:153–167. doi:10.1146/annurev.nutr.20.1.153 CrossRefPubMedGoogle Scholar
  6. Bateman A (1997) The structure of a domain common to archaebacteria and the homocystinuria disease protein. Trends Biochem Sci 22(1):12–13CrossRefPubMedGoogle Scholar
  7. Benson DA, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2014) GenBank. Nucleic Acids Res 42(Database issue):D32–D37. doi:10.1093/nar/gkt1030 CrossRefPubMedGoogle Scholar
  8. Braun-Sand SB, Peetz M (2010) Inosine monophosphate dehydrogenase as a target for antiviral, anticancer, antimicrobial and immunosuppressive therapeutics. Future Med Chem 2(1):81–92. doi:10.4155/fmc.09.147 CrossRefPubMedGoogle Scholar
  9. Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17(4):540–552CrossRefPubMedGoogle Scholar
  10. Chong CR, Qian DZ, Pan F, Wei Y, Pili R, Sullivan Jr DJ, Liu JO (2006) Identification of type 1 inosine monophosphate dehydrogenase as an antiangiogenic drug target. J Med Chem 49(9):2677–2680. doi:10.1021/jm051225t CrossRefPubMedGoogle Scholar
  11. Cossins EA, Chen L (1997) Folates and one-carbon metabolism in plants and fungi. Phytochemistry 45(3):437–452CrossRefPubMedGoogle Scholar
  12. Dietrich FS, Voegeli S, Brachat S, Lerch A, Gates K, Steiner S, Mohr C, Pohlmann R, Luedi P, Choi S, Wing RA, Flavier A, Gaffney TD, Philippsen P (2004) The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science 304(5668):304–307. doi:10.1126/science.1095781 CrossRefPubMedGoogle Scholar
  13. Dobie F, Berg A, Boitz J, Jardim A (2007) Kinetic characterization of inosine monophosphate dehydrogenase of Leishmania donovani. Mol Biochem Parasitol 152(1):11–21. doi:10.1016/j.molbiopara.2006.11.007 CrossRefPubMedGoogle Scholar
  14. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501. doi:10.1107/S0907444910007493 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Forster C, Santos MA, Ruffert S, Kramer R, Revuelta JL (1999) Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii. J Biol Chem 274(14):9442–9448CrossRefPubMedGoogle Scholar
  16. Gan L, Seyedsayamdost M, Shuto S, Matsuda A, Petsko G, Hedstrom L (2003) The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase. Biochemistry 42(4):857–863. doi:10.1021/bi0271401 CrossRefPubMedGoogle Scholar
  17. Gattiker A, Rischatsch R, Demougin P, Voegeli S, Dietrich FS, Philippsen P, Primig M (2007) Ashbya Genome Database 3.0: a cross-species genome and transcriptome browser for yeast biologists. BMC Genomics 8:9. doi:10.1186/1471-2164-8-9 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gilbert H, Lowe C, Drabble W (1979) Inosine 5′-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5′-monophosphate. Biochem J 183(3):481–494CrossRefPubMedPubMedCentralGoogle Scholar
  19. Gross SS, Levi R (1992) Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem 267(36):25722–25729PubMedGoogle Scholar
  20. Hedstrom L (2009) IMP dehydrogenase: structure, mechanism, and inhibition. Chem Rev 109(7):2903–2928. doi:10.1021/cr900021w CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hedstrom L, Liechti G, Goldberg JB, Gollapalli DR (2011) The antibiotic potential of prokaryotic IMP dehydrogenase inhibitors. Curr Med Chem 18(13):1909–1918CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hyle J, Shaw R, Reines D (2003) Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast. J Biol Chem 278(31):28470–28478. doi:10.1074/jbc.M303736200 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Jaroszewski L, Li Z, Cai XH, Weber C, Godzik A (2011) FFAS server: novel features and applications. Nucleic Acids Res 39 (Web Server issue):W38-44 doi:10.1093/nar/gkr441Google Scholar
  24. Jenks MH, Reines D (2005) Dissection of the molecular basis of mycophenolate resistance in Saccharomyces cerevisiae. Yeast 22(15):1181–1190. doi:10.1002/yea.1300 CrossRefPubMedGoogle Scholar
  25. Jiménez A, Santos MA, Pompejus M, Revuelta JL (2005) Metabolic engineering of the purine pathway for riboflavin production in Ashbya gossypii. Appl Environ Microbiol 71(10):5743–5751. doi:10.1128/AEM.71.10.5743-5751.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Jiménez A, Santos MA, Revuelta JL (2008) Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii. BMC Biotechnol 8:67. doi:10.1186/1472-6750-8-67 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Juanhuix J, Gil-Ortiz F, Cuni G, Colldelram C, Nicolas J, Lidon J, Boter E, Ruget C, Ferrer S, Benach J (2014) Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the ALBA synchrotron. J Synchrotron Radiat 21(Pt 4):679–689. doi:10.1107/S160057751400825X CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kabsch W (2010) Xds. Acta Crystallogr D Biol Crystallogr 66(Pt 2):125–132. doi:10.1107/S0907444909047337 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kato T, Park E (2012) Riboflavin production by Ashbya gossypii. Biotechnol Lett 34(4):611–618. doi:10.1007/s10529-011-0833-z CrossRefPubMedGoogle Scholar
  30. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. doi:10.1093/bioinformatics/btm404 CrossRefPubMedGoogle Scholar
  31. Ledesma-Amaro R, Jiménez A, Santos MA, Revuelta JL (2013) Biotechnological production of feed nucleotides by microbial strain improvement. Process Biochem 48:7CrossRefGoogle Scholar
  32. Ledesma-Amaro R, Santos MA, Jiménez A, Revuelta JL (2014a) Strain design of Ashbya gossypii for single-cell oil production. Appl Environ Microbiol 80(4):1237–1244. doi:10.1128/AEM.03560-13 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Ledesma-Amaro R, Santos MA, Jiménez A, Revuelta JL (2014b) Tuning single-cell oil production in Ashbya gossypii by engineering the elongation and desaturation systems. Biotechnol Bioeng 111(9):1782–1791. doi:10.1002/bit.25245 CrossRefPubMedGoogle Scholar
  34. Ledesma-Amaro R, Buey RM, Revuelta JL (2015) Increased production of inosine and guanosine by means of metabolic engineering of the purine pathway in Ashbya gossypii. Microb Cell Fact 14:58. doi:10.1186/s12934-015-0234-4 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Long H, Cameron S, Yu L, Rao Y (2006) De novo GMP synthesis is required for axon guidance in Drosophila. Genetics 172(3):1633–1642. doi:10.1534/genetics.105.042911 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Makowska-Grzyska M, Kim Y, Wu R, Wilton R, Gollapalli D, Wang X, Zhang R, Jedrzejczak R, Mack J, Maltseva N, Mulligan R, Binkowski T, Gornicki P, Kuhn M, Anderson W, Hedstrom L, Joachimiak A (2012) Bacillus anthracis inosine 5′-monophosphate dehydrogenase in action: the first bacterial series of structures of phosphate ion-, substrate-, and product-bound complexes. Biochemistry 51(31):6148–6163. doi:10.1021/bi300511w CrossRefPubMedGoogle Scholar
  37. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40(Pt 4):658–674. doi:10.1107/S0021889807021206 CrossRefPubMedPubMedCentralGoogle Scholar
  38. McPhillips C, Hyle J, Reines D (2004) Detection of the mycophenolate-inhibited form of IMP dehydrogenase in vivo. Proc Natl Acad Sci U S A 101(33):12171–12176. doi:10.1073/pnas.0403341101 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Morrow CA, Valkov E, Stamp A, Chow EW, Lee IR, Wronski A, Williams SJ, Hill JM, Djordjevic JT, Kappler U, Kobe B, Fraser JA (2012) De novo GTP biosynthesis is critical for virulence of the fungal pathogen Cryptococcus neoformans. PLoS Pathog 8(10):e1002957 doi:10.1371/journal.ppat.1002957
  40. Murray AW (1971) The biological significance of purine salvage. Annu Rev Biochem 40:811–826. doi:10.1146/annurev.bi.40.070171.004115 CrossRefPubMedGoogle Scholar
  41. Nagano N, Orengo CA, Thornton JM (2002) One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions. J Mol Biol 321(5):741–765CrossRefPubMedGoogle Scholar
  42. Nair V, Shu Q (2007) Inosine monophosphate dehydrogenase as a probe in antiviral drug discovery. Antivir Chem Chemother 18(5):245–258CrossRefPubMedGoogle Scholar
  43. Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12(4):357–358PubMedGoogle Scholar
  44. Park EY, Ito Y, Nariyama M, Sugimoto T, Lies D, Kato T (2011) The improvement of riboflavin production in Ashbya gossypii via disparity mutagenesis and DNA microarray analysis. Appl Microbiol Biotechnol 91(5):1315–1326. doi:10.1007/s00253-011-3325-0 CrossRefPubMedGoogle Scholar
  45. Pimkin M, Markham G (2008) The CBS subdomain of inosine 5′-monophosphate dehydrogenase regulates purine nucleotide turnover. Mol Microbiol 68(2):342–359. doi:10.1111/j.1365-2958.2008.06153.x CrossRefPubMedPubMedCentralGoogle Scholar
  46. Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr 59(Pt 7):1131–1137CrossRefPubMedGoogle Scholar
  47. Prosise GL, Wu JZ, Luecke H (2002) Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site. J Biol Chem 277(52):50654-50659 doi:10.1074/jbc.M208330200
  48. Rao V, Shepherd S, Owen R, Hunter W (2013) Structure of Pseudomonas aeruginosa inosine 5′-monophosphate dehydrogenase. Acta Crystallogr Sect F Struct Biol Cryst Commun 69(Pt 3):243–247. doi:10.1107/s1744309113002352 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Ratcliffe AJ (2006) Inosine 5′-monophosphate dehydrogenase inhibitors for the treatment of autoimmune diseases. Curr Opin Drug Discov Devel 9(5):595–605PubMedGoogle Scholar
  50. Revuelta JL, Buitrago MJ, Santos MA (1998) Riboflavin biosynthesis in fungi. Patent nr WO9526406 In., C12N15/52(edn: BASF AG (DE))Google Scholar
  51. Riera TV, Zheng L, Josephine HR, Min D, Yang W, Hedstrom L (2011) Allosteric activation via kinetic control: potassium accelerates a conformational change in IMP dehydrogenase. Biochemistry 50(39):8508–8518. doi:10.1021/bi200785s CrossRefPubMedPubMedCentralGoogle Scholar
  52. Stahmann K, Revuelta J, Seulberger H (2000) Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl Microbiol Biotechnol 53(5):509-516 doi:10.1007/s002530051649
  53. Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2:Unit 2.3 doi:10.1002/0471250953.bi0203s00Google Scholar
  54. Traut TW (1994) Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140(1):1-22Google Scholar
  55. Trzhtsinskaya BV, Abramova ND (1991) Imidazole-2-thiones: synthesis, structure and properties. Sulfur Reports 10:389–421CrossRefGoogle Scholar
  56. Umejiego NN, Gollapalli D, Sharling L, Volftsun A, Lu J, Benjamin NN, Stroupe AH, Riera TV, Striepen B, Hedstrom L (2008) Targeting a prokaryotic protein in a eukaryotic pathogen: identification of lead compounds against cryptosporidiosis. Chem Biol 15(1):70–77. doi:10.1016/j.chembiol.2007.12.010 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Vandamme EJ (1992) Production of vitamins, coenzymes and related biochemicals by biotechnological processes. J Chem Technol Biotechnol 53(4):313–327CrossRefPubMedGoogle Scholar
  58. Wasserstrom L, Lengeler KB, Walther A, Wendland J (2013) Molecular determinants of sporulation in Ashbya gossypii. Genetics 195(1):87–99. doi:10.1534/genetics.113.151019 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Wendland J, Ayad-Durieux Y, Knechtle P, Rebischung C, Philippsen P (2000) PCR-based gene targeting in the filamentous fungus Ashbya gossypii. Gene 242(1–2):381–391CrossRefPubMedGoogle Scholar
  60. Wendland J, Dunkler A, Walther A (2011) Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii. FEMS Yeast Res 11(5):418–429. doi:10.1111/j.1567-1364.2011.00732.x CrossRefPubMedGoogle Scholar
  61. Winn MD, Isupov MN, Murshudov GN (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr D Biol Crystallogr 57(Pt 1):122–133CrossRefPubMedGoogle Scholar
  62. Zalkin H, Dixon JE (1992) De novo purine nucleotide biosynthesis. Prog Nucleic Acid Res Mol Biol 42:259–287CrossRefPubMedGoogle Scholar
  63. Zhang R, Evans G, Rotella F, Westbrook E, Huberman E, Joachimiak A, Collart FR (1999a) Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes. Curr Med Chem 6(7):537–543PubMedGoogle Scholar
  64. Zhang R, Evans G, Rotella FJ, Westbrook EM, Beno D, Huberman E, Joachimiak A, Collart FR (1999b) Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase. Biochemistry 38(15):4691–4700. doi:10.1021/bi982858v CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Rubén M. Buey
    • 1
  • Rodrigo Ledesma-Amaro
  • Mónica Balsera
    • 2
  • José María de Pereda
    • 3
  • José Luis Revuelta
    • 1
  1. 1.Metabolic Engineering Group, Departamento de Microbiología y GenéticaUniversidad de SalamancaSalamancaSpain
  2. 2.Department Abiotic Stress, Instituto de Recursos Naturales y AgrobiologíaConsejo Superior de Investigaciones CientíficasSalamancaSpain
  3. 3.Instituto de Biología Celular y Molecular del Cáncer, Consejo Superior de Investigaciones CientíficasUniversidad de SalamancaSalamancaSpain

Personalised recommendations