Goal:The research goal of this project is to develop a nanotechnology-based system that allows enzymatic cascades to be joined together as needed in a predesignated manner to biosynthesize designer products in a modular "plug-and-play" fashion. These self-assembled systems will be used to enzymatically convert gross or bulk agricultural commodity products (as epitomized by sugars such as glucose) into high-value designer biosynthetic products (as epitomized by complex essential vitamins) that can then be fed back into both the agricultural and commercial chemical sectors. Many of the latter products are currently sourced from wet-chemical production which is costly and has a large environmental footprint. The overarching goal is to develop minimalist technologies that allow bulk agricultural products to be used as substrates for direct biosynthesis of any desired product in a modular-designer manner. Current cell-based synthetic biology (SynBio) techniques cannot yet undertake the synthesis proposed here nor the target molecule diversity we aim to achieve.The technical merit of this project lies in the ability to create biosynthetic cascades consisting of NPs self-assembled with enzymes that can convert agriculturally-derived bulk substrates into high value products in a highly efficient manner that has minimal environmental impact without requiring cells in an economical manner. Moreover, this approach will be modular allowing for facile switching between different substrates and choice of final product just through the choice of enzymatic cascades that are assembled to the nanoparticles (NPs).The central hypothesis of this proposal is that minimalistic designer biosynthetic cascades can be assembled and function extremely efficiently by understanding and controlling the following: (1) allowing the enzymes constituting a particular synthetic cascade to self-assemble to NPs and form nanoclusters; (2) linking such clustered NP-enzyme systems to work together to produce a far more complex desired product in a single reaction; (3) exploring the scope of non-natural products that can be efficiently produced with different substrates; and that this will require (4) utilizing different nanoparticulate materials to optimize the structure and function of the nanoclusters; (5) utilizing kinetic modeling to optimize the ratio of each enzyme used; and (6) designing the systems such that the enzymatic cascades access channeling phenomena which is the most efficient form of multi-enzyme catalysis (for more information on channeling, see Preliminary Results). The proposal and hypothesis testing will be carried out with the following specific objectives.Objectives:Objective 1: Demonstrate that different individual enzymatic pathways can be self-assembled onto nanoparticles (NPs) to give rise to designer nanoclusters capable of highly efficient multistep biocatalysis.The working hypothesis of this aim is that the self-assembly of enzymes constituting a particular biocatalytic reaction pathway onto NPs will enhance the efficiency of the pathway, in part by allowing them to form nanoclusters by crosslinking since many of the enzymes are dimers and tetramers. Moreover, we will show that this is a modular approach by switching between different sets of sequentially-functional enzymes that constitute different cascaded reactions. Each designer NP-enzyme cascade that is capable of catalyzing different reactions will be referred to as a catalytic module to reflect this. The small size of the cluster coupled to the high density of enzymes in close proximity to each other will allow the enzymes to engage in channeled biocatalysis and efficiently convert substrate to final product. Kinetic simulation of overall reaction rates using the Michaelis-Menten (MM) model will allow for optimization of each enzyme's ratio and overall catalytic flux based on matching of individual rates and functional limitations. Moreover, screening of different NP materials with different sizes/shapes will allow for selection of a NP-scaffold that maximizes both cluster formation and kinetic flux.Objective 2: Demonstrate that multiple different modules (nanoclusters consisting of NPs self-assembled with the enzymes corresponding to a particular cascade) can be functionally linked together to take an initial substrate (representing a bulk agricultural product) and catalyze its biosynthesis through multiple cascaded steps into a desired agriculturally-relevant high value product.The working hypothesis of this aim is that different modules, each consisting of a separate NP-clustered enzymatic cascade, can be functionally linked together in a given reaction to produce a desired product that is the sum of all the reactions modules present. Modules will be linked together by creating either super-clustered structures or physically-linked structures. This will allow product from one clustered pathway to act as substrate for the next pathway. Moreover, this approach can tolerate enzymes from many different sources including those produced by both prokaryotic and eukaryotic expression systems without the need for extensive protein engineering. The latter is something that is not currently attainable with cell-based SynBio systems.Objective 3: Demonstrate NPs self-assembled with enzymes can catalyze the synthesis of non-natural designer products that cells cannot synthesize as the substrates/products used in this pathway would be toxic to them.The working hypothesis of this aim is that many enzymes can tolerate non-natural substrates when utilized in vitro and this should extend to focused biosynthetic cascades. Since almost all in vivo biocatalytic pathways are interconnected, introduction of a non-natural substrate analog into a living cell's metabolic pathways is invariably toxic. As long as an analog does not hinder enzyme-substrate recognition and/or binding and does not preclude the actual chemical catalysis, it should allow an in vitro constituted pathway to utilize it to create a non-natural final product.