Phosphorus-31 NMR spectroscopy in metabolomics and carbon-13 tracer studies of yeast Saccharomyces cerevisiae

TY - THES

T1 - Phosphorus-31 NMR spectroscopy in metabolomics and carbon-13 tracer studies of yeast Saccharomyces cerevisiae

T2 - Master's thesis

AU - Jouhten, Paula

N1 - CA: BEL diplomityö Helsinki University of Technology. Department of Chemical Technology 149 p. + app. 2 p.

PY - 2004

Y1 - 2004

N2 - In the literature survey part of this study, metabolic engineering of the carbon metabolism of yeast Saccharomyces cerevisiae for efficient production of bioethanol from hexose- and pentose-containing feeds was discussed. Also the basics of NMR spectroscopy, one- and two-dimensional methods and the special characteristics of 31P NMR were introduced. Then the value of metabolomics and analysis of the metabolic fluxes in characterisation of the metabolism of improved strains was discussed. Also the current status of research and the methods used in the fields of metabolomics, systems biology and analysis of the metabolic fluxes were presented. The experimental part of this study began with one-dimensional 31P NMR measurements of yeast extracts. Then the pH dependence behaviour of the phosphorus-31 chemical shifts of the sugar phosphates, the intermediates of glycolysis and pentose phosphate pathway, was examined. Optimal conditions in terms of pH and temperature for one-dimensional 31P NMR measurement of the sugar phosphates were determined. A low temperature was optimal for phosphorus-31 signal lineshapes and a neutral pH gave the best resolution. One-dimensional 1H NMR, two-dimensional DQF-COSY and TOCSY measurements were made to study the proton chemical shifts and the signal patterns of the spinsystems in the sugar phosphates. A two-dimensional heteronuclear NMR method, 1H-31P HSQC-TOCSY, that correlates the phophorus-31 chemical shifts with proton chemical shifts of complete proton spinsystems was developed for determination of positional fractional carbon-13 enrichments in sugar phosphates produced in carbon-13 tracer experiments for analysis of metabolic fluxes. Adiabatic mixing was introduced to the TOCSY spinlock for better lineshapes and compatibility with Varian's nanoprobe. The adiabatic 1H-31HSQC-TOCSY called 1H-31P HSQC-AD-TOCSY was used for measurement of phosphorus-containing compounds in yeast metabolite extract. To improve the resolution in the narrow sugar phosphate phosphorus-31 frequency range, a selective inversion of sugar phosphates with two 180o inversion pulses was introduced. The selective 1H-31P HSQC-AD-TOCSY was used in selective measurement of sugar phosphates in complex yeast metabolite extract samples. Finally, carbon-13 tracer experiments with S. cerevisiae strains containing a xylose pathway were made with [2-13C]-D-glucose as a carbon source were made. The construction of the first strain had become unstable and it grew poorly on medium lacking amino acids. The residual [2-13C]-D-glucose was purified from the supernatant and another experiment was performed with the purified [2-13C]-D-glucose and with another clone of the S. cerevisiae strain. Perchloric acid quenching and extraction were performed. However, none of the studied sugar phosphates was identified in the NMR experiments of the last labelled metabolite extract.

AB - In the literature survey part of this study, metabolic engineering of the carbon metabolism of yeast Saccharomyces cerevisiae for efficient production of bioethanol from hexose- and pentose-containing feeds was discussed. Also the basics of NMR spectroscopy, one- and two-dimensional methods and the special characteristics of 31P NMR were introduced. Then the value of metabolomics and analysis of the metabolic fluxes in characterisation of the metabolism of improved strains was discussed. Also the current status of research and the methods used in the fields of metabolomics, systems biology and analysis of the metabolic fluxes were presented. The experimental part of this study began with one-dimensional 31P NMR measurements of yeast extracts. Then the pH dependence behaviour of the phosphorus-31 chemical shifts of the sugar phosphates, the intermediates of glycolysis and pentose phosphate pathway, was examined. Optimal conditions in terms of pH and temperature for one-dimensional 31P NMR measurement of the sugar phosphates were determined. A low temperature was optimal for phosphorus-31 signal lineshapes and a neutral pH gave the best resolution. One-dimensional 1H NMR, two-dimensional DQF-COSY and TOCSY measurements were made to study the proton chemical shifts and the signal patterns of the spinsystems in the sugar phosphates. A two-dimensional heteronuclear NMR method, 1H-31P HSQC-TOCSY, that correlates the phophorus-31 chemical shifts with proton chemical shifts of complete proton spinsystems was developed for determination of positional fractional carbon-13 enrichments in sugar phosphates produced in carbon-13 tracer experiments for analysis of metabolic fluxes. Adiabatic mixing was introduced to the TOCSY spinlock for better lineshapes and compatibility with Varian's nanoprobe. The adiabatic 1H-31HSQC-TOCSY called 1H-31P HSQC-AD-TOCSY was used for measurement of phosphorus-containing compounds in yeast metabolite extract. To improve the resolution in the narrow sugar phosphate phosphorus-31 frequency range, a selective inversion of sugar phosphates with two 180o inversion pulses was introduced. The selective 1H-31P HSQC-AD-TOCSY was used in selective measurement of sugar phosphates in complex yeast metabolite extract samples. Finally, carbon-13 tracer experiments with S. cerevisiae strains containing a xylose pathway were made with [2-13C]-D-glucose as a carbon source were made. The construction of the first strain had become unstable and it grew poorly on medium lacking amino acids. The residual [2-13C]-D-glucose was purified from the supernatant and another experiment was performed with the purified [2-13C]-D-glucose and with another clone of the S. cerevisiae strain. Perchloric acid quenching and extraction were performed. However, none of the studied sugar phosphates was identified in the NMR experiments of the last labelled metabolite extract.

M3 - Master's thesis

PB - Helsinki University of Technology

CY - Espoo

ER -