Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display.

Enzymes that degrade synthetic polymers have attracted intense interest for eco-friendly plastic recycling. However, because enzymes did not evolve for the cleavage of abiotic polymers, directed evolution strategies are needed to enhance activity for plastic degradation. Previous directed evolution efforts relied on polymer degradation assays that were limited to screening ∼104 mutants. Here, we report a high-throughput yeast surface display platform to rapidly evaluate >107 enzyme mutants for increased activity in cleaving synthetic polymers. In this platform, individual yeast cells display distinct mutants, and enzyme activity is detected by a change in fluorescence upon the cleavage of a synthetic probe resembling a polymer of interest. Highly active mutants are isolated by fluorescence activated cell sorting and identified through DNA sequencing. To demonstrate this platform, we performed directed evolution of a polyethylene terephthalate (PET)-depolymerizing enzyme, leaf and branch compost cutinase (LCC). We identified activity-boosting mutations that substantially increased the kinetics of degradation of solid PET films. Biochemical assays and molecular dynamics (MD) simulations of the most active variants suggest that the H218Y mutation improves the binding of the enzyme to PET. Overall, this evolution platform increases the screening throughput of polymer-degrading enzymes by 3 orders of magnitude and identifies mutations that enhance kinetics for depolymerizing solid substrates.

Switchable DNA Catalysts for Proximity Labeling at Sites of Protein-Protein Interactions.

Proximity labeling (PL) has emerged as a powerful approach to elucidate proteomes within a defined radius around a protein of interest (POI). In PL, a catalyst is attached to the POI and tags nearby endogenous proteins, which are then isolated by affinity purification and identified by mass spectrometry. Although existing PL methods have yielded numerous biological insights, proteomes with greater spatial resolution could be obtained if PL catalysts could be activated at more specific subcellular locations, such as sites where both the POI and a chemical stimulus are present or sites of protein-protein interactions (PPIs). Here, we report DNA-based switchable PL catalysts that are attached to a POI and become activated only when a secondary molecular trigger is present. The DNA catalysts consist of a photocatalyst and a spectral quencher tethered to a DNA oligomer. They are catalytically inactive by default but undergo a conformational change in response to a specific molecular trigger, thus activating PL. We designed a system in which the DNA catalyst becomes activated on living mammalian cells specifically at sites of Her2-Her3 heterodimers and c-Met homodimers, PPIs known to increase the invasion and growth of certain cancers. While this study employs a Ru(bpy)3-type complex for tagging proteins with biotin phenol, the switchable DNA catalyst design is compatible with diverse synthetic PL photocatalysts. Furthermore, the switchable DNA PL catalysts can be constructed from conformation-switching DNA aptamers that respond to small molecules, ions, and proteins, opening future opportunities for PL in highly specific subcellular locations.

Switchable DNA Photocatalysts for Radical Polymerization Controlled by Chemical Stimuli.

Polymerization catalysts that activate in response to specific chemical triggers offer spatial and temporal control over polymer synthesis, facilitating the development of responsive materials and custom polymer coatings. However, existing catalysts switch their activity through mechanisms that are not generalizable to chemically diverse stimuli. To approach the level of control exhibited in biological polymer synthesis, switchable polymerization catalysts need to be configurable for activation in response to diverse chemical stimuli. Here, we combine synthetic photocatalysts with conformation-switching DNA aptamers to create polymerization catalysts that respond to diverse chemical stimuli. We use the secondary structure of DNA to bring a photocatalyst and quencher dye into proximity, turning off photocatalysis. The DNA structure can be precisely designed to change conformation in response to a molecular trigger, moving the photocatalyst far from the quencher and activating photocatalysis. We show these photocatalysts can initiate free-radical polymerization to form bulk hydrogels in response to complementary DNA, a metal ion (Zn2+), or small molecules (glucose and hydrocortisone). We demonstrate the biocompatibility of these switchable photocatalysts by triggering their activation on the surface of yeast cells. Finally, we perform reversible-deactivation radical polymerization through photoinduced electron/energy transfer reversible addition-fragmentation chain-transfer in a dual-stimulus manner, in which catalytic activity is regulated reversibly by photoirradiation and the conformational state of the DNA catalyst. These results demonstrate that DNA conformational changes triggered by chemically diverse stimuli can regulate the activity of radical polymerization photocatalysts. This platform offers new capabilities in spatially and temporally controlled polymer synthesis, with potential applications in diagnostics, sensing, and environmentally responsive materials.