Construction and analysis of recombinant glucanolytic brewer's yeast strains

Abstract

Four different types of endo-fl-l,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the eoll gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The eoll gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or A D H 1 locus of the yeast. Integration into the PGK1 or A D H 1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the eoH gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of fl-glucans in worts.

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…Several cellulase genes from various fungi have also been expressed in S. cerevisiae. Five endo-␤-1,4-glucanases (encoded by egl1, egl2, egl3, egl4, and egl5) and two cellobiohydrolases (CBHI and CBHII) from T. reesei were efficiently secreted into the culture medium by S. cerevisiae transformants (4,27,521,522,525,586,588,655,783,784). Similar results were observed when the endo-␤-1,4-glucanase gene from Trichoderma longibrachiatum was expressed in a wine yeast strain (528,529).…”

Induction of production and secretion β(1→4) glucanase withSaccharomyces cerevesiaeby replacing theMET10gene withegl1gene fromTrichoderma reesei

“…The reason maybe the MET10 promoter used is weaker than PGK and ADH1 promoter (Penttilä et al. 1987; Suihko et al. 1991), and few copies of egl1 gene integrated into MET10 locus in the recombinants genome.…”

Development of New Strains for the Food Industry

“…(Penttilä et al, 1987; Suihko et al, 1991). To avoid those problems, brewers' yeasts have been transformed with plasmids carrying the egl1 gene from Trichoderma reesei coding for an endoglucanase (Suihko et al, 1991). The cDNA of the egl1 gene was expressed in the brewers' yeast under the control of the regulatory signals of the PGK1 and the ADH1 yeast genes.…”

“…The cDNA of the egl1 gene was expressed in the brewers' yeast under the control of the regulatory signals of the PGK1 and the ADH1 yeast genes. Both, autonomous and integrative plasmids have been used (Suihko et al, 1991). The transformants possessing a copy of the egl1 gene and regulatory signals of the PGK1 integrated in the PGK1 locus were very stable, and the β‐glucanase activity secreted was enough to eliminate all the β‐glucans present in the wort (Suihko et al, 1991).…”

“…Both, autonomous and integrative plasmids have been used (Suihko et al, 1991). The transformants possessing a copy of the egl1 gene and regulatory signals of the PGK1 integrated in the PGK1 locus were very stable, and the β‐glucanase activity secreted was enough to eliminate all the β‐glucans present in the wort (Suihko et al, 1991). In addition, brewers' yeasts had the same organoleptic features as the control using the untransformed strain.…”