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 ␣-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 was 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.

Surface displayed expression of a neutralizing epitope of spike protein from a Korean strain of porcine epidemic diarrhea virus

“…The preparation of cell-free extracts and the isolation of the cell wall fraction were conducted as previously described [38,39]. In order to prepare the cell-free extract, the cells were grown for three days, harvested, washed twice in extraction buffer (50 mM Tris-HCl, 2 mM EDTA), and homogenized three times in a bead beater (Biospec Products Inc., Bartlesville, OK, USA) for 1 min each, with 3 min intervals of icecooling.…”

“…The cell wall fraction recovered as precipitate after centrifugation was then washed three times in extraction buffer, and separated from the glass beads via standing. The fraction was then treated with α-1,3-glucanase in sodium acetate buffer (pH 5.2) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), in order to extract the K-COE from the cell wall [39]. A 150 mU amount of α-1,3glucanase per gram of wet cell wall was used, and the reaction was conducted for 4 h at 37 o C. PNGase F (New England Biolabs, Beverly, MA, USA) and jack bean β-mannosidase (Sigma) were used to deglycosylate the K-COE fusion construct, as described previously [40].…”

Cytochemical evaluation of localization and secretion of a heterologous enzyme displayed on yeast cell surface

“…A sec1 mutant accumulates vesicles during incubation at the restrictive temperature, 37³C [13]. A multicopy plasmid pGA11 and an integrative plasmid pIGA11 were constructed as described previously [1,3].…”

“…We established a method for displaying certain proteins on the cell surface of Saccharomyces cerevisiae by connecting a C-terminal half of K-agglutinin to the protein which was aimed at displaying on the cell surface [1^4]. In a previous study, a starch-utilizing S. cerevisiae strain was constructed by displaying a glucoamylase^K-agglutinin fusion protein on the cell wall with retaining glucoamylase activity [1]. The gene of Rhizopus oryzae glucoamylase, cleaving K-1,4-linked and K-1,6-linked glucose, was fused with the 3P-half of K-agglutinin gene.…”

Production of a recombinant lipase artificially localized on the Bacillus subtilis cell surface

“…Recently, cell surface engineering has been attracting a great deal of attention. In yeasts and bacteria, various antigenic determinants, heterologous enzymes, single-chain antibodies and polyhistidyl tags have been successfully displayed on the cell surface [18,19]. But a surface display for B. subtilis has not been developed yet.…”