Synergies in coupled hydrolysis and fermentation of cellulose using a Trichoderma reesei enzyme preparation and a recombinant Saccharomyces cerevisiae strain.

We describe a procedure by which filter paper is digested with a cellulolytic enzyme preparation, obtained from Trichoderma reesei cultivated under solid state fermentation conditions and then fermented by a recombinant Saccharomyces cerevisiae strain. The yeast strain produces a ß-glucosidase encoded by the BGL1 gene from Saccharomycopsis fibuligera that quantitatively and qualitatively complements the limitations that the Trichoderma enzyme complex shows for this particular activity. The supplemental ß-glucosidase activity fuels the progression of cellulose hydrolysis and fermentation by decreasing the inhibitory effects caused by the accumulation of cellobiose and glucose. Fermentation of filter paper by this procedure yields ethanol concentrations above 70 g/L.

Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α-Glucosidase.

The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.