title: Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface.
abstract: Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Jungu
authors.initials: J
authors.lastname: Bae
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Kuroda
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan miueda@kais.kyoto-u.ac.jp.
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Ueda
mesh_terms: D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase