Functional Display of Complex Cellulosomes on the Yeast Surface via Adaptive Assembly

A new adaptive strategy was developed for the ex vivo assembly Of a functional tetravalent designer cellulosoine on the yeast cell surface. The design is based on the use of (1) a surface-bound anchoring scaffoldin composed of two divergent cohesin domains, (2) two dockerin-tagged adaptor scaffoldins to amplify the number of enzyme loading sites based on the specific dockerin-cohesin interaction with the anchoring scaffoldin, and (3) two dockerin-tagged enzymatic subunits (the endoglucanse Gt and the beta-glucosidase Bglf) for cellulose hydrolysis. Cells displaying the tetravalent cellulosome on the surface exhibited a 4.2-fold enhancement in the hydrolysis of phosphoric, acid swollen cellulose (PASC) compared with free enzymes. More importantly, cells displaying the tetravalent celluosome also exhibited an similar to 2-fold increase, production compared with cells displaying a divalent cellulosome usins asimilar enzyme loading. These results clearly indicate the more crucial role of enzyme proximity than just simply increasing the enzyme loading on the overall creating artificial cellulosome structures. The unique feature of the anchoring and the adaptor scaffolding strategy to amplify the number of enzymatic subunits can be easily extended to more compex cellulosomal structures to achieve an even higher level of enzyme synergy.