Comparison of wild‐type and Reg 1 mutant saccharomyces cerevisiae metabolic levels during glucose and galactose metabolism using ³¹P NMR

Abstract

The reg 1 mutation will allow the expression of a cloned gene on a plasmid under the control of a GAL promoter in the presence of glucose. The metabolism of wild-type and reg l mutant strains was examined by in vivo (31)P nuclear magnetic resonance (NMR) spectroscopy. Transient profiles of glucose 6-phosphate, fructose 6-phosphate, fructose 1, 6-diphosphate, and 3-phosphoglycerate indicated that glucose was processed differently for the reg 1 strain despite similar cytoplasrnic pH values and ATP levels. Intracellular phosphate became depleted in the transition to quasi-steady state and limited glycolysis in the reg 1 strain. The glucose uptake step or hexokinase step appears to be altered in the reg 1 strain. The reg 1 strain utilized galactose faster than the wild-type strain under the conditions used for NMR analysis. These results are consistent with the hypothesis that the REG 1 product operates early in the regulatory circuitry for glucose repression. This study illustrates the usefulness of transient information provided by NMR in understanding changes in the metabolism of genetically manipulated organisms.

Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae

“…We measured the extracellular pH and observed that it ranged between 4.0-5.1 for all culture media and procedures during the reaction period. The intracellular pH of yeast was determined to be between 6.4 -6.6 by 31 P NMR during galactose utilization (Shanks and Bailey, 1990). Using Eq.…”

In vivo nuclear magnetic resonance studies of glycolytic kinetics inLactococcus lactis

“…Isotopic labeling experiments coupled to 13 C-nuclear magnetic resonance (NMR) have proven to be useful in determining metabolic fluxes (Marx et al, 1996;Ramos and Santos, 1996;Sauer et al, 1997;Tran-Dinh et al, 1996;Veiga da Cunha et al, 1992). Furthermore, the noninvasive characteristics of NMR can be used to obtain invaluable information on the magnitude of intracellular metabolite pools (Pereira et al, 1996;Shanks and Bailey, 1990). As early as 1978 Shulman and coworkers showed that it was possible to use 13 C-NMR to study cell physiology and, in particular, to monitor the time courses of glycolytic intermediates in cell suspensions of Saccharomyces cerevisiae and Escherichia coli (den Hollander et al, 1979;Ugurbil et al, 1978).…”

Dynamicin vivo31P nuclear magnetic resonance study ofSaccharomyces cerevisiae in glucose-limited chemostat culture during the aerobic-anaerobic shift

“…Also, sensitivity was better, as judged from the ADP+ATP peak at −5 ppm: 4 in den Hollander et al . (1981), 5.5 in Shanks and Bailey (1990), as compared to 10 in the present study (Figure 2) per 1 min of measuring time. The following aspects relating to our experimental set‐up are considered to be highly relevant for metabolic studies: (a) in vivo pools with high turnover rates and concentrations in the order of 1–10 µmol/g DW (e.g.…”

“…During this second stage, the original value of total polyphosphate was restored (Figure 3b, Table 1). An efflux of phosphate and hydrolysis of polyphosphate have also been reported after addition of glucose to derepressed cells (Gillies et al ., 1981; Shanks and Bailey, 1990). Furthermore, this efflux related to glucose addition can be inhibited by inhibitors of glycolysis.…”