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 in duction timing were determined for a temperature-sensitive (ts) GAL-re gulated stable yeast expression system. The Saccharomyces cerevisiae s train employed carries a reg1 mutation inhibiting catabolite repressio n, 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 promo ter during batch culture. Beta-galactosidase specific activity was con sistently lower at higher initial glucose concentrations in both SDC ( semi-defined) and YPD(a) (complex) media; decreases of 18-36% were obs erved 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 subst antial improvements in biomass yield at higher glucose levels. Therefo re, productivity rose with increasing glucose concentration; in YPD(a) 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-o ffs between optimum growth and a lengthy induction period were also ev aluated. Delaying the time of induction reduced final specific activit ies but improved cell yield, and waiting 14 h into batch culture to in duce lacZ expression provided modest 9-15% improvements in overall pro ductivity.