Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface

• Published in: Applied and Environmental Microbiology Vol. 81; no. 1; pp. 59 - 66
• Main Authors: Bae, Jungu, Kuroda, Kouichi, Ueda, Mitsuyoshi
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 2015
• Subjects: beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Cellulase - genetics; Cellulase - metabolism; Cellulose; Cellulose - metabolism; Cellulose 1,4-beta-Cellobiosidase - genetics; Cellulose 1,4-beta-Cellobiosidase - metabolism; Enzymes; Enzymology and Protein Engineering; Membrane Proteins - metabolism; Microbiology; Polypeptides; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Yeast
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/aem.02864-14

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.