Toshiyuki Murai

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

Abstract The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

Abstract We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

Abstract An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

Evaluation of the function of arming yeast displaying glucoamylase on its cell surface by direct fermentation of corn to ethanol

We have constructed a Saccharomyces cerevisiae strain displaying glucoamylase from Rhizopus oryzae on its cell surface, which could grow on soluble starch as the sole source of carbon and energy (Murai et al., Appl. Environ. Microbiol., 63, 1362–1366, 1997). This system was introduced into a yeast strain, G-1315, with a high fermentation ability, and the direct production of ethanol from corn was examined. When a transformant of strain G-1315 displaying glucoamylase on the cell surface was cultivated statically at 27°C for 7 d using ground raw corn as starchy material, ethanol was produced at a concentration of 2.34% (w/w). Addition of an α-amylase reagent (Termamil) to liquefy the ground corn prior to fermentation improved the ethanol production to 5.32% (w/w), which was comparable to that of transformants secreting glucoamylase. These results demonstrate that the “arming yeast” developed by a cell surface engineering technique and its further improvement is applicable to direct fermentation of starchy materials.

Establishment of a simple system to analyse the molecular interaction in the agglutination of Saccharomyces cerevisiae

Saccharomyces cerevisiae a‐agglutinin, which is involved in mating and covalently anchoring to the cell wall, consists of two components, Aga1p and Aga2p, whose syntheses are individually regulated. To facilitate the analysis of the protein–protein interaction on agglutination between a‐ and α‐agglutinins, the construction of a yeast strain (MATa) with the functional protein prepared by genetic fusion of Aga1p‐ and Aga2p‐encoding genes and by the expression system using the UPR–ICL promoter derived from the n‐alkane‐assimilating yeast, Candida tropicalis, which is functional under the condition of lower glucose concentration was tried and the agglutination ability of the constructed strain was evaluated with a yeast strain (MATa) which expressed AGα1 encoding α‐agglutinin under the control of the same promoter. The genes were integrated into the yeast chromosomes. Cell agglutination between both (MATa) strains was observed microscopically when these two strains were mix‐cultured to a glucose‐decreased concentration. The agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed Aga1p–Aga2p fusion protein was enoughly functional for the interaction with the Agα1 protein, and that this phenomenon occurred dependent on glucose concentration, but independent of the peptide pheromones secreted by the cells of the opposite mating types. Using this system, the role of two disulphide linkages between Aga1p and Aga2p on the binding activity between Aga2p and Aga1p was first evaluated. Under the treatment by the SH‐compound (dithiothreitol), in which Agα2p is easily released into the medium from the intact cell surface, the Aga1p and Aga2p fusion protein was a good tool to make clear the role of the disulphide linkages. As a result, the linkages had a significant effect on not only the assembly but also the binding activity. The novel and simple system described here may further facilitate the study of molecular interaction in agglutination. Copyright

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

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.

Strong expression of peroxisomal malate synthase genes from n-alkane-utilizable yeast Candida tropicalis in Saccharomyces cerevisiae

Expression of the genes encoding several peroxisomal enzymes of the n-alkane-utilizable yeast Candida tropicalis has been examined in Saccharomyces cerevisiae to find out a novel and foreign promoter functional in S. cerevisiae. Among these genes, two malate synthase genes, as well as the isocitrate lyase gene, were strongly expressed in acetate-grown cells of S. cerevisiae. The amounts of the recombinant malate synthases expressed in S. cerevisiae were as much as 30% of the total amount of soluble proteins in the cells. These results showed that the upstream regions (1.1 kbp for the MS-1 gene and 0.8 kbp for the MS-2 gene) of the malate synthase genes [UPR-MSs] from C. tropicalis contained strong promoters functional in S. cerevisiae and that the construction of novel gene expression systems using UPR-MSs might be possible.