Coacervates as enzymatic microreactors

Abstract

Compartmentalization, a key aspect of biochemical regulation, naturally occurs in cellular organelles, including biomolecular condensates formed through liquid-liquid phase separation (LLPS). Inspired by biological compartments, synthetic coacervates have emerged as versatile microreactors, which can provide customed environments for enzymatic reactions. In this review, we explore recent advances in coacervate-based microreactors, while emphasizing the mechanisms by which coacervates accelerate enzymatic reactions, namely by enhancing substrate and enzyme concentrations, stabilizing intermediates, and providing molecular crowding. We discuss diverse coacervate systems, including those based on synthetic polymers, peptides, and nucleic acids, and describe the selection of enzymatic model systems, as well as strategies for enzyme recruitment and their impact on reaction kinetics. Furthermore, we discuss the challenges in monitoring reactions within coacervates and review the currently available techniques including fluorescence techniques, chromatography, and NMR spectroscopy. Altogether, this review offers a comprehensive perspective on recent progress and challenges in the design of coacervate microreactors, and addresses their potential in biocatalysis, synthetic biology, and nanotechnology.

Fig. 1. Enzymes recruitment strategies to coacervate reactors. The three central strategies for the recruitment of enzymes to the coacervate dense phase include spontaneous diffusion of enzymes into the condensates (left panel), conjugation of enzymes to IDRs which actively form the coacervates (middle panel), and conjugation of enzymes to ligands which bind specific motifs that are conjugated to the coacervate building block. Enzyme 3D structure is based on PDB 1IA7 (cellulase).

Fig. 2. Schematic illustration of the four common building blocks of designed coacervate microreactors, including polypeptides/peptides, nucleic acid-based polymers, glycan-based polymers, and synthetic polymers.

Fig. 3. Types of coacervate microreactors. (a) Coacervate microreactors formed by ELP-b-PEG dense phase inside cytomimetic lipid compartments, which are formed in response to hyperosmotic stress. (b) Top: Chemical structure of a 14-mer cationic peptide building block. Bottom: The cationic peptide undergoes LLPS as a factor of ionic strength and pH to form simple coacervates that can recruit substrates and enzymes. (c) DNA-based coacervate reactors which are formed by sequence-controlled multiblock ssDNA polymers via rolling-circle amplification, leading to the formation of all-DNA coacervates with a liquid core and a crosslinked duplex shell. (d) Glycan-based coacervate reactors formed by mixing positively charged Q-Am and negatively charged Cm-Am and Ni-NTA-Am. His-tagged client proteins including phosphatase are recruited to the coacervate phase by Ni-His affinity. Client proteins are taken up based on their phosphorylation-dependent affinity and are released following the enzymatic dephosphorylation in the condensed phase. (a) was reproduced from ref. with permission from Wiley-VCH GmbH; (b) was reproduced from ref. with permission from Nature Publishing Group; (c) was reproduced from ref. with permission from Nature Publishing Group; (d) was reproduced from ref. with permission from Wiley-VCH GmbH.

Fig. 4. Acceleration of enzymatic reaction kinetics in coacervates. (a)–(c). Acceleration of hydrolysis by β-lactoglobulin coacervates. (a) Schematic illustration of β-lactoglobulin and PEG LLPS which results in the formation into liquid droplets. β-Lactoglobulin undergoes conformational changes in the coacervate phase which allows it to catalyze the hydrolysis of ester bonds. (b) and (c) Comparative analysis of β-lactoglobulin catalysis showing 4-nitrophenyl acetate hydrolysis rate (b) and k_cat (c). (d) and (e) Acceleration of NADH oxidation in coacervates that are formed by NADH oxidase conjugated to IDRs. (d) Coacervate reactors are formed by IDRs (or LCDs) derived from the DEAD-box proteins Dbp1, Laf1, and Ddx4, that have varying net charge. The IDRs are fused to the N-terminus of the enzyme, creating chimeric proteins with different net charges. (e) Reaction kinetics in coacervates with varying net charge, monitored by a decrease in NADH concentration. (a)–(c) were reproduced from ref. with permission from Royal Society of Chemistry and (d) and (e) were reproduced from ref. with permission from Nature publishing group.

Fig. 5. Inhibition of enzymatic reaction kinetics in coacervates. (a)–(c). HRP-catalyzed reactions are restricted in coacervates that are formed by amylose derivatives, surrounded by a terpolymer membrane structure (a). (b) Chemical structures of H₂O₂ and Amplex red used as substrates to form the fluorescent product resorufin. (c) Michaelis–Menten curve which shows the rate of the reaction catalyzed by free and compartmentalized HRP. (d)–(f). Restricted phosphatase activity in peptide coacervates. (d) Schematic illustration of two oppositely charged peptide building blocks (WGR:WGE). (e) Encapsulation efficiency (EE%) of fluorescently-labelled phosphatase in peptide coacervates with varying charge ratio, achieved by varying the stoichiometry of WGR:WGE. (f) Reaction kinetics of phosphatase-catalyzed hydrolysis in coacervates with varying charge ratios, monitored by fluorescence spectroscopy of the MU product. (a)–(c) were reproduced from ref. with permission from European Chemical Societies Publishing and (d)–(f) were reproduced from ref. with permission from Wiley-VCH GmbH.

Fig. 6. Acceleration vs. restriction of reaction kinetics in coacervates. (a)–(c) Inhibition of β-galactosidase activity in coacervates (a) that are formed by high charge-density polycations. (b) Michaelis–Menten kinetics plot of the reaction in coacervates that are formed by polycations with varying charge density. (c) CD spectroscopy of β-galactosidase in the different coacervates, showing secondary structure retention in the complex coacervates. (d), (e) Reaction acceleration/restriction by spatial organization of enzymatic cascade in a coacervate-in-coacervate multi-compartment system. (d) The reactions are restricted when GO_x, HRP, and catalase are confined within the same compartment (case I) and accelerated when GO_x and HRP are spatially separated from catalase (case II), as shown by bright-field and time-dependent fluorescence spectroscopy images upon glucose addition. The inner and outer coacervates were outlined with dashed blue and white circles, respectively. (e) Time profile of resorufin fluorescence following oxidation from Amplex Red by the cascade. (a)–(c) were reproduced from ref. with permission from the American Chemical Society and (d), (e) were reproduced from ref. with permission from the Royal Society of Chemistry.

Rif Harris

Nofar Berman

Ayala Lampel