PubMed · 4844622
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J Tiwari, A Fraser, R Beckman. 1974. Genetical feedback repression. I. Single locus models.. https://doi.org/10.1016/0022-5193(74)90117-9
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The mutant IGC 40 x 1001 of the yeast Pichia anomala IGC 4380, which displays inverse diauxic growth in a medium with glucose and malic acid, was studied to elucidate the biochemical mechanisms underlying that behavior. Time course changes of enzyme activities during growth of the mutant in that mixture of substrates indicated that the gluconeogenic enzymes remained active during the first phase of diauxic growth, while glycolytic enzyme activities were significantly reduced. This reduction was essentially due to an alteration in the maximum velocity and not in substrate affinity. Malate, citrate, and adenosine triphosphate did not affect significantly the activities of the glucose phosphorylating enzymes in cell extracts of either the mutant or the wild strain. In P. anomala, unlike Saccharomyces cerevisiae, the fructose/glucose phosphorylating ratio was not associated with repression/derepression conditions.
Leuconostoc mesenteroides NRRL B-512F dextransucrase was rapidly and irreversibly inactivated by o-phthalaldehyde. The dextransucrase-o-phthalaldehyde adduct showed a characteristic fluorescence maxima at 417 nm when excited at 337 nm. These results were consistent with the isoindole derivative formation in which the sulfhydryl group of cysteine and epsilon-amino group of lysine participate in the reaction. The stoichiometric determinations gave one isoindole derivative per enzyme molecule upon complete inactivation by o-phthalaldehyde. The enzyme showed no inhibition on treatment with thiol specific reagents. This indicated that cysteine is present in close proximity of the lysine and is involved in the isoindole derivative formation but is not participating in the catalysis. These results established for the first time that one lysine residue present at the active site is required for the activity of dextransucrase.
The effects of residual catabolite repression and the importance of induction timing were determined for a temperature-sensitive (ts) GAL-regulated stable yeast expression system. The Saccharomyces cerevisiae strain employed carries a reg1 mutation inhibiting catabolite repression, and a ts mutation enabling induction of the regulated GAL promoters by a temperature shift to 35 degrees C. Despite the reg1 mutation and induction method, glucose depressed lacZ expression from a GAL1 promoter during batch culture. beta-Galactosidase specific activity was consistently lower at higher initial glucose concentrations in both SDC (semi-defined) and YPDa (complex) media; decreases of 18-36% were observed as glucose concentration was increased between 1, 3, 5, and 10 g l-1. However, the reductions in beta-galactosidase specific activity due to residual catabolite repression were more than balanced by substantial improvements in biomass yield at higher glucose levels. Therefore, productivity rose with increasing glucose concentration; in YPDa medium, increasing initial glucose from 1 to 10 g l-1 resulted in a 2.6-fold increase in beta-galactosidase volumetric activity. Due to the negative effects of shifting temperature to 35 degrees C, the trade-offs between optimum growth and a lengthy induction period were also evaluated. Delaying the time of induction reduced final specific activities but improved cell yield, and waiting 14 h into batch culture to induce lacZ expression provided modest 9-15% improvements in overall productivity.