Molecular Breeding of Arming Yeasts with Hydrolytic Enzymes by Cell Surface Engineering

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Novel yeast cells armed with biocatalysts - glucoamylase, -amylase, CM-cellulase, β-glucosidase, and lipase — were constructed by a cell surface engineering system of yeast Saccharomyces cerevisiae. These surface-engineered yeast cells were termed “Arming yeasts”. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half of yeast α-agglutinin. Glucoamylase was shown to be displayed on the cell surface of S. cerevisiae in its active form, anchored covalently to the cell wall. S. cerevisiae is unable to utilise starch, while the arming cells could grow on starch as the sole carbon source. For enhancement of the ability to directly ferment starchy materials by the arming yeast, a surface-engineered yeast cell displaying two amylolytic enzymes was constructed. The gene encoding R. oryzae glucoamylase with its own secretion signal peptide and a truncated fragment of the a-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast a-factor, respectively, were fused with the gene encoding the C-terminal half of the yeast ?-agglutinin.The arming cell co-displaying glucoamylase and a-amylase could grow faster on starch as the sole carbon source than the cell displaying only glucoamylase. Furthermore, a novel celluloseutilising yeast cell displaying cellulolytic enzymes in their active forms on the cell surface of S. cerevisiae was constructed by the cell surface engineering. An arming yeast co-displaying FI-carboxymethylcellulase (CM-cellulase), one of the endo-type cellulase, and (3-glucosidase from Aspergillus aculeatus was endowed with the ability of cellooligosaccharide assimilation, suggesting the possibility that the assimilation of cellulosic materials may be carried out by S. cerevisiae expressing heterologous cellulase genes on the cell surface. Furthermore, a yeast cell armed with R. oryzae lipase was also constructed. These idea will be open to all living cells and the technique will be able to endow them with novel abilities.KeywordsSole Carbon SourceMolecular BreedingCellulolytic EnzymeClostridium ThermocellumAmylolytic EnzymeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.