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Metabolomics

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

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

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

Abstract

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.

Keywords

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 

Notes

Acknowledgments

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)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Toshiaki Fukui
    • 1
  • 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

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