Abstract
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.
Original language | English |
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Qualification | Master Degree |
Awarding Institution |
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Place of Publication | Espoo |
Publisher | |
Publication status | Published - 2004 |
MoE publication type | G2 Master's thesis, polytechnic Master's thesis |