Synthetic scaffolds for pathway enhancement

Abstract

Controlling local concentrations of reactants, intermediates, and enzymes in synthetic pathways is critical for achieving satisfactory productivity of any desired products. An emerging approach to exert control over local concentrations is the use of synthetic biomolecular scaffolds to co-localize key molecules of synthetic pathways. These scaffolds bring the key molecules into close proximity by recruiting pathway enzymes via ligand binding and/or physically sequestrating enzymes and metabolites into isolated compartments. Novel scaffolds made of proteins, nucleic acids, and micro-compartments with increasingly complex architecture have recently been explored and applied to a variety of pathways, with varying degrees of success. Despite these strides, precise assembly of synthetic scaffolds remains a difficult task, particularly in vivo, where interactions both intended and unexpected can lead to unpredictable results. Additionally, because heterologous enzymes often have lowered activities in their new hosts, an ideal scaffold should provide a flexible platform that can adapt to kinetic imbalances in different contexts. In this review, we discuss some of the notable advances in the creation of these synthetic scaffolds and highlight the current challenges in their application.

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

“…The use of engineered enzyme assembly has been shown to substantially improve the efficiency of cascade reactions (19)(20)(21)(22). We argue that this method is ideally suited for pulling formaldehyde toward F6P production because of the improved substrate channeling.…”

“…Multienzyme supramolecular complexes, which self-assemble into spatially defined architectures, have been shown to be a promising approach to improve the efficiency of cascade reactions (19)(20)(21)(22). This spatial organization provides more stable enzyme structures, facilitates substrate channeling between active sites, and prevents the buildup of toxic intermediates (23)(24)(25).…”

Engineering the Bacterial Microcompartment Domain for Molecular Scaffolding Applications

“…Yet translating this technology to intracellular application has been partially constrained because the concentration of single-stranded nucleic acid building blocks and environmental properties important for nucleic acid folding (e.g., temperature, ions) are not easily manipulated in vivo (Pinheiro et al, 2011). While recent studies continue to advance the capability of nucleic acid assemblies achieved within the cell (Conrado et al, 2012; Myhrvold and Silver, 2015; Siu et al, 2015; Elbaz et al, 2016), proteins may offer another viable, naturally inspired solution. One early example of a synthetically designed scaffold was comprised of a string of protein–protein interaction domains that were used to recruit three cognate enzymes involved in the conversion of acetyl-CoA to mevalonate (Dueber et al, 2009).…”

Computation‐Guided Design of a Stimulus‐Responsive Multienzyme Supramolecular Assembly

“…Both phosphorylation‐ and photo‐crosslinking‐driven two‐enzyme systems showed improved reaction efficiency compared to non‐crosslinked enzyme mixture controls, and the three‐component assembly showed a further enhancement of product yields compared to the two‐enzyme assemblies. The magnitudes of increase in pathway efficacy were smaller compared to previous spatiallocalization‐based studies, which typically involve a reversible step so that colocalization has a large thermodynamic and kinetic boost . However, in contrast to colocalization with only spatial arrangement, the multistimulus‐responsive enzymeassembly approach provides temporal control and could be generally applicable to other pathway systems, including those with a reversible step.…”