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. 1997 Apr;63(4):1362–1366. doi: 10.1128/aem.63.4.1362-1366.1997

Construction of a starch-utilizing yeast by cell surface engineering.

T Murai 1, M Ueda 1, M Yamamura 1, H Atomi 1, Y Shibasaki 1, N Kamasawa 1, M Osumi 1, T Amachi 1, A Tanaka 1
PMCID: PMC168429  PMID: 9097432

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.

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Selected References

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  1. Cameron D. C., Tong I. T. Cellular and metabolic engineering. An overview. Appl Biochem Biotechnol. 1993 Jan-Feb;38(1-2):105–140. doi: 10.1007/BF02916416. [DOI] [PubMed] [Google Scholar]
  2. Chiswell D. J., McCafferty J. Phage antibodies: will new 'coliclonal' antibodies replace monoclonal antibodies? Trends Biotechnol. 1992 Mar;10(3):80–84. doi: 10.1016/0167-7799(92)90178-x. [DOI] [PubMed] [Google Scholar]
  3. Fleet G. H., Manners D. J. The enzymic degradation of an alkali-soluble glucan from the cell walls of Saccharomyces cerevisiae. J Gen Microbiol. 1977 Feb;98(2):315–327. doi: 10.1099/00221287-98-2-315. [DOI] [PubMed] [Google Scholar]
  4. Georgiou G., Poetschke H. L., Stathopoulos C., Francisco J. A. Practical applications of engineering gram-negative bacterial cell surfaces. Trends Biotechnol. 1993 Jan;11(1):6–10. doi: 10.1016/0167-7799(93)90068-K. [DOI] [PubMed] [Google Scholar]
  5. Kamasawa N., Ohtsuka I., Kamada Y., Ueda M., Tanaka A., Osumi M. Immunoelectron microscopic observation of the behaviors of peroxisomal enzymes inducibly synthesized in an n-alkane-utilizable yeast cell, Candida tropicalis. Cell Struct Funct. 1996 Apr;21(2):117–122. doi: 10.1247/csf.21.117. [DOI] [PubMed] [Google Scholar]
  6. Lipke P. N., Kurjan J. Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins. Microbiol Rev. 1992 Mar;56(1):180–194. doi: 10.1128/mr.56.1.180-194.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lipke P. N., Wojciechowicz D., Kurjan J. AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating. Mol Cell Biol. 1989 Aug;9(8):3155–3165. doi: 10.1128/mcb.9.8.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Manners D. J., Masson A. J., Patterson J. C., Björndal H., Lindberg B. The structure of a beta-(1--6)-D-glucan from yeast cell walls. Biochem J. 1973 Sep;135(1):31–36. doi: 10.1042/bj1350031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Manners D. J., Masson A. J., Patterson J. C. The structure of a beta-(1 leads to 3)-D-glucan from yeast cell walls. Biochem J. 1973 Sep;135(1):19–30. doi: 10.1042/bj1350019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sawai-Hatanaka H., Ashikari T., Tanaka Y., Asada Y., Nakayama T., Minakata H., Kunishima N., Fukuyama K., Yamada H., Shibano Y. Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase. Biosci Biotechnol Biochem. 1995 Jul;59(7):1221–1228. doi: 10.1271/bbb.59.1221. [DOI] [PubMed] [Google Scholar]
  11. Schreuder M. P., Brekelmans S., van den Ende H., Klis F. M. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast. 1993 Apr;9(4):399–409. doi: 10.1002/yea.320090410. [DOI] [PubMed] [Google Scholar]
  12. Tajima M., Nogi Y., Fukasawa T. Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast. 1985 Sep;1(1):67–77. doi: 10.1002/yea.320010108. [DOI] [PubMed] [Google Scholar]
  13. Tanaka T., Kita H., Murakami T., Narita K. Purification and amino acid sequence of mating factor from Saccharomyces cerevisiae. J Biochem. 1977 Dec;82(6):1681–1687. doi: 10.1093/oxfordjournals.jbchem.a131864. [DOI] [PubMed] [Google Scholar]
  14. Wojciechowicz D., Lu C. F., Kurjan J., Lipke P. N. Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily. Mol Cell Biol. 1993 Apr;13(4):2554–2563. doi: 10.1128/mcb.13.4.2554. [DOI] [PMC free article] [PubMed] [Google Scholar]

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