CFN Virtual Colloquium

Amongst the suite of technologies being developed for synthetic biology, cell-free approaches are becoming ever more prominent as they offer many advantages to address confounding issues associated with cellular systems including especially toxicity and off-pathway affects. Making these systems as simple and efficient as possible will be key to their success. From a purely minimalist perspective, multistep biosynthetic systems (i.e. enzymatic cascades) only require enzymes, their cofactors, and the substrate, however, such approaches typically suffers from reaction diffusion limitations and long-term enzyme instability. We are attempting to address these latter issues in pursuit of efficient minimalist synthetic systems by using nanoparticles (NPs) to both stabilize the enzymes and allow them to form nanoclusters that access channeled catalysis. Using semiconductor quantum dots (QDs) and the enzymes from saccharification and oxidative glycolysis as a prototypical system, we have developed methods that allow the enzymes to self-assemble with the QDs into catalytic nanoclusters. Within these systems, catalytic flux is improved by several orders of magnitude and detailed analysis along with numerical simulations show that this arises from both enzymatic stabilization and channeling phenomena. Incorporation of non-spherical QDs and optimization of relative enzymatic ratios (and catalytic rates) have also boosted efficiency dramatically. Examples of systems utilizing 10-14 enzymatic steps will be presented along with analysis of cluster formation and their channeling processes. The potential of this approach will be discussed in comparison to other scaffolded-type reconstituted enzymatic cascades including those assembled on DNA nanostructures along with how they can be further improved.