Catabolite repression and induction time effects for a temperature-sensitive GAL-regulated yeast expression system.
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.
Napp
S J
SJ
Department of Chemical and Biochemical Engineering, University of California, Irvine 92717.
Da Silva
N A
NA
D004794:Enzyme Repression; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000494:pharmacology; D007763:Lac Operon; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis*
D001616:beta-Galactosidase; D005947:Glucose