Metabolic Engineering Applications of in vivo ³¹P and ¹³C NMR Studies of Saccharomyces cerevisiae

Citation

Shanks, Jacqueline Vanni (1989) Metabolic Engineering Applications of in vivo ³¹P and ¹³C NMR Studies of Saccharomyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/tcbh-r327. https://resolver.caltech.edu/CaltechETD:etd-01242007-080430

Abstract

Noninvasive, multicomponent measurements are required for characterization of the results from metabolically manipulated organisms. The consequences of genetic manipulation by mutation and by introduction of recombinant plasmids to Saccharomyces cerevisiae is explored by ³¹P and ¹³C nuclear magnetic resonance (NMR) spectroscopy.

The in vivo NMR measurements should be quantified and detailed as much as possible. With this intent, analysis techniques of the in vivo ³¹P NMR spectrum are developed. A systematic procedure is formulated for estimating the relative intracellular concentrations of the sugar phosphates in S. cerevisiae based upon the ³¹P NMR spectrum. In addition, in vivo correlations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, β-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6-phosphate are determined. Elucidation of the cytoplasmic and vacuolar components of inorganic phosphate in the ³¹P NMR spectrum of S. cerevisiae is another method of analysis that was developed. Concentrations and pH values for these components may be determined from this method. An in vivo correlation relating the inorganic phosphate chemical shift of the vacuole with the chemical shift of the resonance for pyrophosphate and the terminal phosphate of polyphosphate (PP₁) is established. Extension of these strategies to other cellular systems should be straightforward.

Transient measurements provided by ³¹P NMR are applied to reg1 mutant and standard strains. The reg1 mutation will allow the expression of a cloned gene on a plasmid under the control of a GAL promoter in the presence of glucose. Despite similar values of ATP and cytoplasmic pH, the transient profiles of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, and 3-phosphoglycerate were very different for the two strains during anaerobic catabolism of glucose. The glucose uptake step or hexokinase step appears to be altered in the reg1 strain. The reg1 strain also catabolized galactose faster than the standard strain. These results concur with the hypothesis that the reg1 product operates early in the regulatory circuitry for glucose repression.

³¹P and ¹³C NMR measurements are used to analyze the performance of recombinant strains in which the glucose phosphorylation step had been altered. The strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain that had the highest in vitro enzyme activity for glucose phosphorylation did not have the highest glucose consumption rate or ethanol production. The differences observed in in vivo utilization of the glucose phosphorylation capacity among the strains are insensitive to cytoplasmic pH and levels of cytoplasmic inorganic phosphate, sugar phosphates, and total ATP. Regulation of hexokinase PII by magnesium-free ATP appears to account for the differences observed with respect to the strain containing this enzyme.

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