Fluorescence based assay ofGALsystem in yeastSaccharomyces cerevisiae

Abstract

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.

Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae

“…The amount and the specific productivity of the enzyme produced in the Dgal1 mutant were considerably lower compared with the wild type and Dgal80 strains. This is in contrast to the recent report [7] in which Dgal1 strain was superior to the Dgal80 strain in the expression level of GFP by GAL1 promoter. This discrepancy may be due in part to the fact that cultivation time of 24 h employed in their experiment was not long enough for expression of GAL1 promoter for which full derepression is effected only after complete exhaustion of glucose (see the following section).…”

“…The Dgal1 mutant strain lacks galactokinase, and so cannot utilize galactose; high level of protein expression is evident even at low galactose concentrations. A recent fluorescence-based study of the GAL system using green fluorescence protein (GFP) screened and evaluated the contribution of 28 selected gene deletions to protein expression from the GAL1 promoter [7].…”

The GAL induction response in yeasts with impaired galactokinase Gal1p activity

“…Interestingly, in the Gal1p knockout strain the inducer was also not consumed, but the level of GAL genes induction was significantly higher compared to induction in strains with impaired kinase function. Previously, it has been demonstrated that GAL genes expression is higher in gal1 deletion strain, presumably due to maintaining constant galactose concentration (Hovland et al 1989;Napp and Da Silva 1994;Kim et al 2004;Kang et al 2005;Š tagoj et al 2005). Data from this study clearly indicated that depletion of inducer is not the main limiting factor in GAL genetic switch.…”

“…Prior studies have demonstrated higher induction level of GAL genetic response in Gal1p null strain (Hovland et al 1989;Napp and Da Silva 1994;Š tagoj et al 2005). It was shown that maintaining constant level of inducer gives a significant higher expression of recombinant genes driven by a galactose-inducible promoters (Kim et al 2004;Kang et al 2005).…”

“…An asterix (*) indicates identical amino acid residues promoter directed expression of green fluorescent protein (GFP). Previous studies have shown that GFP fluorescence closely correlates with expression levels from galactose regulated promoters(Kruckenberg et al 1999;De Wulf et al 2000;Š tagoj et al 2005).…”