, Volume 10, Issue 2, pp 190–202 | Cite as

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

  • Toshiaki FukuiEmail author
  • Kenta Chou
  • Kazuo Harada
  • Izumi Orita
  • Yasumune Nakayama
  • Takeshi Bamba
  • Satoshi Nakamura
  • Eiichiro Fukusaki
Original Article


This study describes metabolite profiles of Ralstonia eutropha H16 focusing on biosynthesis of polyhydroxyalkanoates (PHAs), bacterial polyesters attracted as biodegradable bio-based plastics. As CoA-thioesters are important intermediates in PHA biosynthesis, four kinds of acyl-CoAs with medium chain length were prepared and used to establish analytical conditions for capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE–ESI-MS/MS). Metabolites were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose and PHA production phase on octanoate, and subjected to stable isotope dilution-based comparative quantification by multiple reaction monitoring using CE–ESI-MS/MS and 13C-labeled metabolites prepared by extraction from R. eutropha mutant grown on U-13C6-glucose. This procedure allowed to quantify relative changes of 94 ionic metabolites including CoA-thioesters. Hexose-phosphates except for glucose 1-phosphate were decreased in the PHA production phase than in the growth phase, suggesting reduced flux of sugar degradation after the cell growth. Several intermediates in TCA cycle and gluconeogenesis were increased in the PHA production phase on octanoate. Interestingly, ribulose 1,5-bisphosphate were detected in all the samples examined, raising possibilities of CO2 fixation by Calvin–Benson–Bassham cycle in this bacterium even under heterotrophic growth conditions. Turnover of acyl moieties through β-oxidation was suggested to be active on fructose, as CoA-thioesters of C6 and C8 were detected in the fructose-grown cells. In addition, major metabolic pools in R. eutropha cells were estimated from the signal intensities. The results of the present study provided new insights into global metabolisms in PHA-producing R. eutropha.


Capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE-ESI-MS/MS) Multiple reaction monitoring Isotope dilution-based comparative quantification Polyhydroxyalkanoate Ralstonia eutropha Metabolite profiling 



This work was supported by KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas “Applied Genomics” from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Supplementary material

11306_2013_567_MOESM1_ESM.xlsx (50 kb)
Supplementary material 1 (XLSX 51 kb)
11306_2013_567_MOESM2_ESM.xlsx (47 kb)
Supplementary material 2 (XLSX 48 kb)
11306_2013_567_MOESM3_ESM.pdf (128 kb)
Supplementary material 3 (PDF 129 kb)


  1. Armando, J. W., Boghigian, B. A., & Pfeifer, B. A. (2012). LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity. Letters in Applied Microbiology, 54, 140–148.CrossRefPubMedGoogle Scholar
  2. Bowien, B., & Kusian, B. (2002). Genetics and control of CO2 assimilation in the chemoautotroph Ralstonia eutropha. Archives of Microbiology, 178, 85–93.CrossRefPubMedGoogle Scholar
  3. Brigham, C. J., Budde, C. F., Holder, J. W., Zeng, Q., Mahan, A. E., Rha, C., et al. (2010). Elucidation of β-oxidation pathways in Ralstonia eutropha H16 by examination of global gene expression. Journal of Bacteriology, 192, 5454–5464.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Brigham, C. J., Speth, D. R., Rha, C., & Sinskey, A. J. (2012). Whole-genome microarray and gene deletion studies reveal regulation of the polyhydroxyalkanoate production cycle by the stringent response in Ralstonia eutropha H16. Applied and Environmental Microbiology, 78, 8033–8044.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Budde, C. F., Mahan, A. E., Lu, J., Rha, C., & Sinskey, A. J. (2010). Roles of multiple acetoacetyl coenzyme A reductases in polyhydroxybutyrate biosynthesis in Ralstonia eutropha H16. Journal of Bacteriology, 192, 5319–5328.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Budde, C. F., Riedel, S. L., Willis, L. B., Rha, C., & Sinskey, A. J. (2011). Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains. Applied and Environmental Microbiology, 77, 2847–2854.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cramm, R. (2009). Genomic view of energy metabolism in Ralstonia eutropha H16. Journal of Molecular Microbiology and Biotechnology, 16, 38–52.CrossRefPubMedGoogle Scholar
  8. Dalluge, J. J., Gort, S., Hobson, R., Selifonova, O., Amore, F., & Gokarn, R. (2002). Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry. Analytical and Bioanalytical Chemistry, 374, 835–840.CrossRefPubMedGoogle Scholar
  9. Dube, G., Henrion, A., Ohlendorf, R., & Vidal, C. (2001). Application of the combination of isotope ratio monitoring with isotope dilution mass spectrometry to the determination of glucose in serum. Rapid Communications in Mass Spectrometry, 15, 1322–1326.CrossRefPubMedGoogle Scholar
  10. Fong, J. C., & Schulz, H. (1981). Short-chain and long-chain enoyl-CoA hydratases from pig heart muscle. Methods in Enzymology, 71, 390–398.CrossRefPubMedGoogle Scholar
  11. Friedrich, C. G., Friedrich, B., & Bowien, B. (1981). Formation of enzymes of autotrophic metabolism during heterotrophic growth of Alcaligenes eutrophus. Journal of General Microbiology, 16, 69–78.Google Scholar
  12. Fukui, T., & Doi, Y. (1997). Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae. Journal of Bacteriology, 179, 4821–4830.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Fukui, T., & Doi, Y. (1998). Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Applied Microbiology and Biotechnology, 49, 333–336.CrossRefPubMedGoogle Scholar
  14. Fukui, T., Shiomi, N., & Doi, Y. (1998). Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. Journal of Bacteriology, 180, 667–673.PubMedPubMedCentralGoogle Scholar
  15. Fukusaki, E., Harada, K., Bamba, T., & Kobayashi, A. (2005). An isotope effect on the comparative quantification of flavonoids by means of methylation-based stable isotope dilution coupled with capillary liquid chromatography/mass spectrometry. Journal of Bioscience and Bioengineering, 99, 75–77.CrossRefPubMedGoogle Scholar
  16. Harada, K., Fukusaki, E., & Kobayashi, A. (2006). Pressure-assisted capillary electrophoresis mass spectrometry using combination of polarity reversion and electroosmotic flow for metabolomics anion analysis. Journal of Bioscience and Bioengineering, 101, 403–409.CrossRefPubMedGoogle Scholar
  17. Harada, K., Ohyama, Y., Tabushi, T., Kobayashi, A., & Fukusaki, E. (2008). Quantitative analysis of anionic metabolites for Catharanthus roseus by capillary electrophoresis using sulfonated capillary coupled with electrospray ionization-tandem mass spectrometry. Journal of Bioscience and Bioengineering, 105, 249–260.CrossRefPubMedGoogle Scholar
  18. Haywood, G. W., Anderson, A. J., Chu, L., & Daws, E. A. (1988). The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus. FEMS Microbiology Letters, 52, 259–264.CrossRefGoogle Scholar
  19. Kaddor, C., & Steinbüchel, A. (2011). Effects of homologous phosphoenolpyruvate-carbohydrate phosphotransferase system proteins on carbohydrate uptake and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16. Applied and Environmental Microbiology, 77, 3582–3590.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kahar, P., Tsuge, T., Taguchi, K., & Doi, Y. (2004). High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polymer Degradation and Stability, 83, 79–86.CrossRefGoogle Scholar
  21. Kawashima, Y., Cheng, W., Mifune, J., Orita, I., Nakamura, S., & Fukui, T. (2012). Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha. Applied and Environmental Microbiology, 78, 493–502.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kim, J. K., Harada, K., Bamba, T., Fukusaki, E., & Kobayashi, A. (2005). Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo 15N-isotope enrichment. Bioscience, Biotechnology, and Biochemistry, 69, 1331–1340.CrossRefPubMedGoogle Scholar
  23. King, R., Bonfiglio, R., Fernandez-Metzler, C., Miller-Stein, C., & Olah, T. (2000). Mechanistic investigation of ionization suppression in electrospray ionization. Journal of the American Society for Mass Spectrometry, 11, 942–950.CrossRefPubMedGoogle Scholar
  24. Lenz, O., Ludwig, M., Schubert, T., Burstel, I., Ganskow, S., Goris, T., et al. (2010). H2 conversion in the presence of O2 as performed by the membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha. ChemPhysChem, 11, 1107–1119.CrossRefPubMedGoogle Scholar
  25. Lindenkamp, N., Peplinski, K., Volodina, E., Ehrenreich, A., & Steinbüchel, A. (2010). Impact of multiple β-ketothiolase deletion mutations in Ralstonia eutropha H16 on the composition of 3-mercaptopropionic acid-containing copolymers. Applied and Environmental Microbiology, 76, 5373–5382.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Madison, L. L., & Huisman, G. W. (1999). Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and Molecular Biology Reviews, 63, 21–53.PubMedPubMedCentralGoogle Scholar
  27. Magnes, C., Sinner, F. M., Regittnig, W., & Pieber, T. R. (2005). LC/MS/MS method for quantitative determination of long-chain fatty acyl-CoAs. Analytical Chemistry, 77, 2889–2894.CrossRefPubMedGoogle Scholar
  28. Magnes, C., Suppan, M., Pieber, T. R., Moustafa, T., Trauner, M., Haemmerle, G., et al. (2008). Validated comprehensive analytical method for quantification of coenzyme A activated compounds in biological tissues by online solid-phase extraction LC/MS/MS. Analytical Chemistry, 80, 5736–5742.CrossRefPubMedGoogle Scholar
  29. Matsusaki, H., Abe, H., Taguchi, K., Fukui, T., & Doi, Y. (2000). Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3. Applied Microbiology and Biotechnology, 53, 401–419.CrossRefPubMedGoogle Scholar
  30. Mifune, J., Nakamura, S., & Fukui, T. (2008). Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Canadian Journal of Chemistry, 86, 621–627.CrossRefGoogle Scholar
  31. Mifune, J., Nakamura, S., & Fukui, T. (2010). Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polymer Degradation and Stability, 95, 1305–1312.CrossRefGoogle Scholar
  32. Mooney, B. P. (2009). The second green revolution? Production of plant-based biodegradable plastics. The Biochemical Journal, 418, 219–232.CrossRefPubMedGoogle Scholar
  33. Müller, C., Schäfer, P., Störtzel, M., Vogt, S., & Weinmann, W. (2002). Ion suppression effects in liquid chromatography-electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries. Journal of Chromatography B, 773, 47–52.CrossRefGoogle Scholar
  34. Nomura, C. T., et al. (2005). Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109. Applied and Environmental Microbiology, 71, 4297–4306.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Peplinski, K., Ehrenreich, A., Döring, C., Bömeke, M., Reinecke, F., Hutmacher, C., et al. (2010). Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism. Microbiology, 156, 2136–2152.CrossRefPubMedGoogle Scholar
  36. Pohlmann, A., Fricke, W. F., Reinecke, F., Kusian, B., Liesegang, H., Cramm, R., et al. (2006). Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature Biotechnology, 24, 1257–1262.CrossRefPubMedGoogle Scholar
  37. Putri, S. P., Nakayama, Y., Matsuda, F., Uchikata, T., Kobayashi, S., Matsubara, A., et al. (2013a). Current metabolomics: Practical applications. Journal of Bioscience and Bioengineering, 115, 579–589.CrossRefPubMedGoogle Scholar
  38. Putri, S. P., Yamamoto, S., Tsugawa, H., & Fukusaki, E. (2013b). Current metabolomics: Technological advances. Journal of Bioscience and Bioengineering, 116, 9–16.CrossRefPubMedGoogle Scholar
  39. Rehm, B. H. (2003). Polyester synthases: natural catalysts for plastics. The Biochemical Journal, 376, 15–33.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Riedel, S. L., Bader, J., Brigham, C. J., Budde, C. F., Yusof, Z. A., Rha, C., et al. (2012). Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations. Biotechnology and Bioengineering, 109, 74–83.CrossRefPubMedGoogle Scholar
  41. Shimizu, R., Chou, K., Orita, I., Suzuki, Y., Nakamura, S., & Fukui, T. (2013). Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly(3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses. BMC microbiology, in press.Google Scholar
  42. Soga, T., Ohashi, Y., Ueno, Y., Naraoka, H., Tomita, M., & Nishioka, T. (2003). Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. Journal of Proteome Research, 2, 488–494.CrossRefPubMedGoogle Scholar
  43. Soga, T., Ueno, Y., Naraoka, H., Matsuda, K., Tomita, M., & Nishioka, T. (2002a). Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions. Analytical Chemistry, 74, 6224–6229.CrossRefPubMedGoogle Scholar
  44. Soga, T., Ueno, Y., Naraoka, H., Ohashi, Y., Tomita, M., & Nishioka, T. (2002b). Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. Analytical Chemistry, 74, 2233–2239.CrossRefPubMedGoogle Scholar
  45. Steinbüchel, A., & Lütke-Eversloh, T. (2003). Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochemical Engineering Journal, 16, 81–96.CrossRefGoogle Scholar
  46. Stitt, M., & Fernie, A. R. (2003). From measurements of metabolites to metabolomics: an ‘on the fly’ perspective illustrated by recent studies of carbon-nitrogen interactions. Current Opinion in Biotechnology, 14, 136–144.CrossRefPubMedGoogle Scholar
  47. Sudesh, K., Abe, H., & Doi, Y. (2000). Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Progress in Polymer Science, 25, 1503–1555.CrossRefGoogle Scholar
  48. Sudesh, K., Bhubalan, K., Chuah, J. A., Kek, Y. K., Kamilah, H., Sridewi, N., et al. (2011). Synthesis of polyhydroxyalkanoate from palm oil and some new applications. Applied Microbiology and Biotechnology, 89, 1373–1786.CrossRefPubMedGoogle Scholar
  49. Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211–221.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Timm, A., & Steinbuchel, A. (1992). Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1. European Journal of Biochemistry, 209, 15–30.CrossRefPubMedGoogle Scholar
  51. Tsuge, T., Taguchi, K., Seiichi, T., & Doi, Y. (2003). Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid β-oxidation. International Journal of Biological Macromolecules, 31, 195–205.CrossRefPubMedGoogle Scholar
  52. Valentin, H. E., & Steinbüchel, A. (1994). Application of enzymatically synthesized short-chain-length hydroxy fatty-acid coenzyme-A thioesters for assay of polyhydroxyalkanoic acid synthases. Applied Microbiology and Biotechnology, 40, 699–709.CrossRefGoogle Scholar
  53. Yang, S., Sadilek, M., Synovec, R. E., & Lidstrom, M. E. (2009). Liquid chromatography-tandem quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry measurement of targeted metabolites of Methylobacterium extorquens AM1 grown on two different carbon sources. Journal of Chromatography A, 1216, 3280–3289.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Toshiaki Fukui
    • 1
    Email author
  • Kenta Chou
    • 1
  • Kazuo Harada
    • 2
    • 3
  • Izumi Orita
    • 1
  • Yasumune Nakayama
    • 2
  • Takeshi Bamba
    • 2
  • Satoshi Nakamura
    • 1
  • Eiichiro Fukusaki
    • 2
  1. 1.Department of Bioengineering, Graduate School of Bioscience and BiotechnologyTokyo Institute of TechnologyYokohamaJapan
  2. 2.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuitaJapan
  3. 3.Applied Environmental Biology, Graduate School of Pharmaceutical SciencesOsaka UniversitySuitaJapan

Personalised recommendations