title: Construction of a starch-utilizing yeast by cell surface engineering.
abstract: We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase 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 alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid 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 glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Murai
authors.forename: M
authors.initials: M
authors.lastname: Ueda
authors.forename: M
authors.initials: M
authors.lastname: Yamamura
authors.forename: H
authors.initials: H
authors.lastname: Atomi
authors.forename: Y
authors.initials: Y
authors.lastname: Shibasaki
authors.forename: N
authors.initials: N
authors.lastname: Kamasawa
authors.forename: M
authors.initials: M
authors.lastname: Osumi
authors.forename: T
authors.initials: T
authors.lastname: Amachi
authors.forename: A
authors.initials: A
authors.lastname: Tanaka
mesh_terms: D002462:Cell Membrane / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013213:Starch / Q000378:metabolism*
chemical_list: D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase