Assembly of Multi-Enzyme Cascades Using Synthetic Scaffoldins Displayed On the Yeast Surface

Substrate channeling is an important process involved in the transfer of intermediates between multiple enzymes.  For most multi-enzyme cascades, enzymes are randomly distributed within the complexes. An optimal system with the appropriate stoichiometric ratio and the right ordering of enzymes will likely result in the highest reaction rate because of the more efficient utilization of the substrate. Cellulosome is a naturally occurring enzyme complex that has been shown to enhance the hydrolysis rate of cellulose by more than 10-fold.  The major component of the cellulosome is a structural scaffoldin that consists of repeating cohesin domains, which are docked individually with an enzyme tagged with the corresponding dockerin domain. Because the interaction of the cohesin and dockerin modules is species-specific, artificial “designer cellulosomes” have been created containing the cohesin/dockerin pairs from different species. In this talk, a multi-enzyme cascade was established by assembly three different enzymes onto a synthetic scaffoldin displayed on the yeast surface. The effect of enzyme ordering and spacing on the conversion of methanol to carbon dioxide will be discussed.