Membraneless Compartmentalization Facilitates Enzymatic Cascade Reactions and Reduces Substrate Inhibition

Abstract

Living cells possess membraneless organelles formed by liquid-liquid phase separation. With the aim of better understanding the general functions of membraneless microcompartments, this paper constructs acellular multicompartment reaction systems using an aqueous multiphase system. Membraneless coacervate droplets are placed within a molecularly crowded environment, where a larger dextran (DEX) droplet is submerged in a polyethylene glycol (PEG) solution. The coacervate droplets are capable of sequestering reagents and enzymes with a long retention time, and demonstrate multistep cascading reactions through the liquid-liquid interfaces. The ability to change phase dynamics is also demonstrated through salt-mediated dissolution of coacervate droplets, which leads to the release and mixing of separately sequestered reagents and enzymes. Finally, as phase-separated materials in membraneless organelles are often substrates and substrate analogues for the enzymes sequestered or excluded in the organelles, this paper explores the interaction between DEX and dextranase, an enzyme that hydrolyzes DEX. The results reveal that dextranase suffers from substrate inhibition when partitioned directly in a DEX phase but that this inhibition can be mitigated and reactions greatly accelerated by compartmentalization of dextranase inside a coacervate droplet that is adjacent to, but phase-separated from, the DEX phase. The insight that compartmentalization of enzymes can accelerate reactions by mitigating substrate inhibition is particularly novel and is an example where artificial membraneless organelle-like systems may provide new insights into physiological cell functions.

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…The different local environments experienced by biomolecules (e.g.,i nt erms of viscosity, dielectric constant, pH, …) can affect their activity.I na ddition, the differential sequestration of enzymes,s ubstrates, intermediates and products within the droplets can result in decreasedr eactivity (e.g.,i ft he droplets sequester an enzyme but exclude its substrate). [81] Overall, these studies indicate that the extentso fp artitioning of biomolecules into one or the other phase produced by LLPS can strongly affect the rates of biochemical reactions. [80] The phenomenon known as substrate inhibition has also been reported as ap lausible cause of reduced enzyme reactivity:i n one study dextranase was shown to be inactivated when sequestered in dextran-rich droplets, due to the high local concentration of substrate dextranm acromolecules.…”

“…Osmotic pressure differences across semipermeable membranes have been exploited to induce changes in the local concentrations of encapsulated phase-separating species and therefore to induce phase separation. Dextranase, for instance, has been used to hydrolyse dextran into smaller oligosaccharides and hence to trigger the dissolution of dextran-rich droplets suspended in aP EG-rich phase [81] or embedded within ah ydrogel, [123] resulting in the gradualr eleaseo fs equestered proteins. [78b] Similarly,m ultistep osmotic shocks applied to lipid vesicles containing cell lysate and PEG were shown to induce lysate phase separation due to liposome shrinkage, resulting in localised protein expression.…”

“…[156] This hierarchicalo rganisation might allow the spatial orchestration of multistep reactions and has recently been observed in stress granules, [157] nuclear speckles [158] and the nucleolus, [159] where it has been attributedt o balanced surfacet ensionsb etween coexisting liquid phases [159] and/or differences in effective solvationv olumes. [81] In comparison, in an otable work, elastin-like polypeptides were designed to spontaneously produce complex organisation upon thermally driven phase separation, including nano-or microdroplets-within-droplet microstructures and multilayered droplets. Dynamic substructures (vacuoles) have been observed within PLys/oligonucleotide coacervate microdroplets subjected to ac ontinuous electric field.…”

Embracing Heterogeneity and Disorder

“…Conveniently, this type of LLPS and stable molecule partitioning phenomena can be produced outside the cell using readily available aqueous solutions made from common polymers such as poly(ethylene glycol) (PEG) and dextran (DEX) in place of IDPs and nucleic acids (Figure A). Since our first applications in highly‐parallel gene delivery and knockdown (Figure ), we have expanded the microfluidic use of aqueous two phase systems (ATPS) to stem cell patterning, construction of physically segregated but chemically communicating bacteria communities, cross‐reaction free immunoassays, and preparation of membrane‐less organelle mimetic reaction systems …”

“…Since our first applications in highlyparallel gene delivery and knockdown [13] (Figure 5), we have expanded the microfluidic use of aqueous two phase systems (ATPS) to stem cell patterning, [14] construction of physically segregated but chemically communicating bacteria communities, [15] cross-reaction free immunoassays, [16] and preparation of membrane-less organelle mimetic reaction systems. [17] Beyond the mainly technological use of the ATPSs that result from the LLPS of PEG and DEX solutions as is performed in my lab and as briefly described above, there have been significant advances in understanding of LLPS of protein and nucleic acid solutions in the cell beyond formation of nucleoli. There is even a category of disease referred to as intrinsically disordered protein diseases where abnormal intracellular LLPS formation may be a contributing factor.…”