P-31 NMR AND C-13 NMR-STUDIES OF RECOMBINANT SACCHAROMYCES-CEREVISIAE WITH ALTERED GLUCOSE PHOSPHORYLATION ACTIVITIES

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast. Intracellular metabolic state variables were determined by P-31 NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by C-13 NMR measurements under the same experimental conditions as used in the P-31 NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions. Only 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 drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been forseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.