Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces

Abstract

Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-speci®c polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been ampli®ed and used to construct gene fusions with the b-galactosidase gene. On the basis of b-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and ®lament formation conditions.

Characterisation of banana wine fermented with recombinant wine yeast strains

“…To minimize these disadvantages, an increasing spectrum of relatively expensive commercial enzyme preparations (proteases, pectinases, glucanases, xylanases and arabinofuranosidases) are frequently added to the grape must and wine.As an alternative to such preparations, which often contain unwanted contaminating or side activities,wine yeasts are being developed to secrete proteolytic and polysaccharolytic enzymes that would remove hazeforming proteins and filter-clogging polysaccharides respectively. To this end, the overexpression of several bacterial,fungal and yeast genes has resulted in proteolytic,pectinolytic,glucanolytic and xylanolytic wine yeast strains (Querol and Ramon 1996;Pretorius, 1997;Van Rensburg and Pretorius, 2000;Gognies et al, 2001;Laing and Pretorius, 1993;Pérez-Gonzélez et al, 1993;Van Rensburg et al, 1998). The results on turbidity in this study are in support of what the above researchers had noted and such means of improving processing efficiency in bananas should be exploited to greater heights.…”

“…As an alternative strategy to the addition of costly enzyme preparations that often contain unwanted contaminating or side activities, wine yeasts are being developed to secrete proteolytic and polysaccharolytic enzymes that would remove hazeforming proteins and filter-clogging polysaccharides during fermentation. To this end, the overexpression of several bacterial, fungal and yeast genes has resulted in proteolytic, pectinolytic, glucanolytic and xylanolytic wine yeast strains (Querol and Ramon 1996;Pretorius, 1997;Van Rensburg and Pretorius, 2000;Gognies et al, 2001;Laing and Pretorius, 1993;Pérez-Gonzélez et al, 1993;Van Rensburg et al, 1998). To comply with the ever-increasing demands of modern winemakers and consumers for the best quality wine at every price point, it is also inevitable that novel enzymes will be designed for specific purposes and then tailored through protein engineering technologies (Van Rensburg and Pretorius, 2000).…”

Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability

“…Removing or releasing the chromatin barrier over the STA2 and MUC1 promoters in response to specific nutritional signals could therefore result in the observed activation, as it would make the promoter accessible to Flo8p, Msn1p and Ste12p, as well as to other transcription factors. It was demonstrated recently that the transcription of the PGL1 gene is regulated by the same signalling elements that regulate the transcription of MUC1 in conditions conducive for filamentous growth (Madhani et al ., 1999; Gognies et al ., 2001). The PGL1 gene encodes an endopolygalacturonase that enables the yeast cell to hydrolyse pectin.…”

Use of the Saccharomyces cerevisiae endopolygalacturonase promoter to direct expression in Escherichia coli

“…The main difference in the 5 0 non-coding sequences of the two strains is the insertion of delta Ty2 transposon in the intergenic DAL5-PGL1 fragment, which regulates expression of the PGL1 ORF in S. cerevisiae [16]. It was suggested [22] that endoPG activity in different S. cerevisiae strains is modulated at the transcriptional level and not by the structural gene.…”

“…On the other hand, when the PGL1 ORF was isolated under its own promoter, endoPG activity was hardly detectable [16] by conventional methods [33,37]. To bypass this problem and to improve expression efficiency, the PGL1 ORF was placed under the control of a strong promoter [3,17,20].…”