Self-assembled nanoparticle-enzyme clusters and substrate channeling – a developing system for de novo biosynthesis

J.C. Breger, E. Oh, M.H. Stewart, K. Susumu, S.A. Walper, S.A. Díaz, S.L. Hooe, M. Thakur, M.G. Ancona, I.L. Medintz, G.A. Ellis
US Naval Research Laboratory,
United States

Keywords: enzyme, quantum dot, cascade, synthetic biology


To combat the scourge of organic waste associated with traditional industrial chemical synthesis a new paradigm has emerged called Synthetic Biology. Scientists are trying to understand and harness how Nature performs 1000’s of different types of chemical reactions at the same time in a single cell with just a few begin starting materials such as salt, sugar, water, and light. Our approach is to incorporate a nanoscaffold to bind different enzymes, bringing them in close proximity to each other, stabilizing their tertiary structure and enabling the formed nanoclusters to undergo “channeling”. Channeling occurs when the product of one enzyme is in close proximity of the next enzyme and is effectively “handed off” before product/substrate has a chance to diffuse away, increasing the overall flux. Channeling is the most efficient form of multienzyme catalysis. Our preferred prototypical nanoparticles (NP) are semiconductor quantum dots (QDs) due to their small size. Enzymes that have been produced containing an N-terminal polyhistidine tail can be assembled onto the QD’s surface in a ratiometric fashion through metal-affinity coordination between the imidazole side chains of the histidine residues and the Zn+2 rich surface of the QD. These self-assembled nanosystems allow for a high concentration of localized enzyme to access channeling phenomena. Besides determining which enzymes are needed to achieve the synthesis of a desired product, much research needs to be performed to address such questions as the optimal ratio of each enzyme present, optimal number of enzymes per NP, the order of assembly and other factors that influence the overall flux. To address these questions, we utilized the seven enzymes associated with oxidative glycolysis to convert the agricultural feedstock glucose to 3-phosphoglycerate. This system is tractable with the ability to monitor NADH formation over time as well as each enzyme in the pathway can be individually characterized through absorbance or fluorescence based protocols. The apparent Michaelis-Menten kinetic characteristics of each enzyme free in solution or attached to a NP were determined and utilized to simulate the ratio of each enzyme to QD to achieve the maximum flux in nanoclusters. The NP shape, size, and concentration were also characterized to determine their influence on enzymatic nanocluster formation. By increasing the number of enzymatic nanoclusters formed while optimizing the ratio of individual enzymes in a cascade, we achieved an 800-fold increase in the 7-enzyme flux compared to enzymes free in solution. The degree of enzyme packing or enzyme proximity that can be achieved as a result of NPs size and shape can also double the amount of NADH produced compared to when attached to QDs. Insights from these experiments with the seven enzymes associated with glycolysis are now being applied to other systems to make useful products that cannot be achieved by cell-based synthetic systems.