New developments in nucleic acid nanotechnology and protein scaffold designs have enabled unparalleled control over the spatial organization of synthetic multienzyme cascade reactions. One of the goals of these new technologies is to create nanostructured enzyme cascade reactions that promote substrate channeling along the cascade and, in doing so, enhance cascade catalysis. The concept of substrate channeling has a long and rich history in biochemistry and has established methods of evaluation and quantification. In this Perspective, we review the most common of these methods and discuss them in the context of engineered multienzyme systems and natural bifunctional enzymes with known mechanisms of substrate channeling. In addition, we use experimental data and the results of simulations of coupled-enzyme reactions to develop a set of preliminary design rules for engineering multienzyme nanostructures. The design rules address the limitations on interenzyme distance and active site orientation in substrate channeling and suggest designs for promoting enhanced catalysis, specifically, that enzyme orientation should minimize interenzyme distance and that at distances greater than 1 nm between active sites, significant channeling occurs only if diffusion of the intermediate is bounded through interactions with the surface or scaffold between active sites. This field is rapidly developing and promises to create many more new and exciting technologies.

Advances in DNA bionanotechnology have led to the ability to create structures with well-defined chemical and physical features at the nanoscale. Such nanostructures can be used to create spatially organized enzymatic cascades that promote substrate channeling and result in enhanced cascade kinetics. Here, we investigate the effects of substrate−scaffold interactions on the catalytic activity of an enzyme−DNA complex using horseradish peroxidase (HRP) and a nanoscale DNA scaffold with three addressable sites. Kinetic assays with a library of HRP substrates revealed that DNA scaffolding enhances HRP activity in a manner that is analogous to the Sabatier Principle. In this case, the binding of the substrate is to the scaffold and not to the catalyst, but the Sabatier trend holds: weak and strong binding substrates showed no enhancement in kinetics, whereas intermediately bound substrates result in >300% increase in enzyme activity.M etabolic pathways are often organized in multienzyme complexes that promote the efficient transport and processing of substrates along the pathway, resulting in enhanced pathway kinetics and high yields. 1,2 When engineering new and re-engineering existing pathways, such nanoscale organization and the associated kinetic benefits are often lost. A number of recent efforts to reproduce these kinetic benefits in engineered pathways, both in vitro and in vivo, have focused on enzyme colocalization with nucleic acid 3−7 and protein scaffolding, 8−10 where scaffold is defined as a biomolecular structure to which proteins can be attached at specific sites. As a material, DNA can be used to create precisely defined multidimensional shapes with molecular-level control over structural and chemical features. 11 These capabilities can be used to create multienzyme cascades (or polyvalent enzyme displays) with well-defined spatial organization. However, interactions between enzyme substrates and scaffolds, and their potential effectsbeneficial or detrimentalon enzyme kinetics have yet to be explored. Here, we investigate a potentially advantageous (or constraining) aspects of these designs, interactions between substrates and the DNA scaffold.On the basis of well-known interactions between small molecules and DNA (e.g., DNA stains used in electrophoresis and anticancer drugs 12,13 ) and DNA templating of aniline monomers for the enzyme-mediated synthesis of polyaniline nanowires, 14 we reasoned that interactions between DNA scaffolds and enzyme substrates may affect local substrate concentrations and alter the kinetics of enzymes assembled in enzyme-DNA nanostructures. To investigate this possibility, we used a model system of horseradish peroxidase (HRP) assembled on a nanoscale DNA triangle. We elected to use this system because HRP oxidizes a wide range of chemically distinct substrates, including phenol; charged and uncharged phenolics; and colorimetric substrates, such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB) that are commonly use...

Synthetic biology and metabolic engineering seek to re-engineer microbes into “living foundries” for the production of high value chemicals. Through a “design-build-test” cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.

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In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases), Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER) and Atf1 is known to localize to lipid droplets (LDs). The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24–41 and 508–525, respectively) are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala) and fruits species (C. melo and S. lycopersicum) showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices.