Green fluorescent protein in Saccharomyces cerevisiae: Real-time studies of the GAL1 promoter

Green fluorescent protein (GFP) was used to study the regulation of the gal actose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains. G FP was cloned into the pGAL110 vector and transformed into the yeast strain s. Time course studies comparing culture fluorescence intensity and GFP con centration were conducted along with on-line monitoring of GFP expression. Our results demonstrated that GFP fluorescence could be used as a quantifia ble on-line reporter gene in yeast strains. The effect of an integrated GAL 10p-GAL4 transcription cassette was investigated. Induction time studies sh owed that there was no significant difference in GFP expression level by ad ding galactose at different culture times. A wide range of galactose concen trations was used to study the initial galactose concentration effect on GF P expression kinetics. A minimum of 0.05 g/L galactose doubled the GFP fluo rescence signal as compared to the control, whereas 0.1 g/L gave the highes t specific GFP yield. A simple analytical model was proposed to describe GF P expression kinetics based on the experimental results. In addition, this GFP-based approach was shown to have potential use for high-throughput stud ies. The use of GFP as a generic tool provided important insights to the GA L expression system and has great potential for further process optimizatio n applications. (C) 2000 John Wiley & Sons, Inc.