Enzymatically reconfigurable liquid crystalline coacervate microdroplets as protocell models

Domain: physics
Published: 2025-11-26
Editorial outcome: major_revision

Abstract
Reconfigurable cell-sized compartments can act as artificial cellular models that dynamically respond to environmental stimuli, mimicking the adaptive behaviors of living cells. In this study, we present enzymatically active liquid crystalline (LC) coacervate microdroplets as a protocell model, which undergoes differentiation into helicoidal vesicles and eventually membranous protocells containing artificial organelles. The LC protocell microdroplets are developed through electrostatic complexation between a negatively charged polysaccharide and a cationic surfactant. We identify the amylase-mediated hydrolysis of polysaccharide as the molecular mechanism that is accounted for two-step structural changes of protocells, in which electrostatic complexation plays a crucial role. Notably, by integrating affinitive biomolecules into the LC microdroplets, we demonstrate the reconfiguration of protocells into yolk-shell coacervate vesicles that support biochemical reactions. Our findings illustrate the potential to build protocell models to mimic the differentiation behaviors of cellular materials and shed light to the understanding of the structural reconfiguration of protocells.

Introduction
The development of protocell models that display structural features and biomimetic functions of life is an important milestone in systems chemistry, soft matter bioengineering, and synthetic protobiology1,2. The protocells exhibit the features of morphological flexibility, dynamic responsiveness, and collective behaviors3–5, facilitating implementation of complex reconfiguration into high-order architectures6,7. Coacervate microdroplets formed by liquid-liquid phase separation (LLPS) have been recently developed as membrane-less protocell models which exhibit a wide range of life-like properties8,9, including compartmentalization10–13, enrichment of biomolecules14,15, regulation of enzymatic reactions16–19, and intercellular communication20–22. A less explored aspect in this field is the programmable reconfiguration of these protocell droplets that allow for transformation into higher-order structures under biologically relevant conditions23–25.

Recent advances have demonstrated the transformation process of membrane-less coacervates into coacervate vesicles via different mechanisms, including vacuolization26–30, membranization31–33, multi-phase separation34, and interface reorganization35–38. For instance, by taking the vacuolization mechanism, vacuolized protocells not only exhibit morphological changes, but are also endowed with functional promotion like biomolecular enrichment39–41, acceleration of biochemical reactions42,43, and enhanced interfacial stabilization44. Moreover, coacervate vesicles also enable to act as delivery vehicles for biopharmaceuticals, capable of preserving bioactivity and augmenting delivery efficacy45–47. However, a direct transformation from the membrane-less droplets into multicompartmentalized cell-like entities remains challenging48–50.

In this work, we demonstrate a helicoid pattern-featured vacuolization for developing organelle-containing coacervate vesicles. We construct liquid crystalline (LC) polysaccharide/double-tailed surfactant coacervates, realizing complex transformation into coacervate vesicles initiated by enzyme-mediated polysaccharide hydrolysis into oligosaccharide. The distinct hydrolysis rates result in LC coacervates experiencing intensive vacuolization step and coalescence step till reaching equilibrium. The resultant electrostatic interaction changes cause dissolution of LC structure which allows for the helicoid pattern within the intermediates. Alternatively, the charge screening or protonation also enables to initiate this complex transformation behavior. Significantly, the integration of interactive biomolecules enhances molecular interactions in the LC coacervates, resulting in transformation into yolk-shell coacervate vesicles capable of supporting enzymatic reactions. Our results present a typical reconfigurable coacervate protocells for implementing transformation into hierarchical compartments via complex vacuolization process and provide application potentials in dynamic life-like systems and bioengineering field.

Results
Construction of LC coacervate microdroplets
LC coacervate microdroplets were prepared with a negatively charged carboxymethylamylose (Cm-Am, 40 kDa, substitution degree of 1.2) and a cationic surfactant, didodecyldimethylammonium bromide (DDAB). Cm-Am can be hydrolyzed into fragments as catalyzed by amylase, involving the breakdown of the amylose polymers into low molecular weight oligosaccharides that bear ionic carboxymethyl groups, i.e., hydrolyzed Cm-Am (H-Cm-Am) (Fig. 1a, Supplementary Figs. S1 and S2). DDAB is a cationic surfactant, featuring a hydrophilic headgroup and two hydrophobic tails. The synergistic interactions of electrostatic attraction and the hydrophobic effect drove the complexation of Cm-Am and DDAB, ultimately resulting in the formation of LC coacervate microdroplets, which underwent complex structural transition in the presence of amylase (Fig. 1b). To better understand alterations in structures, we defined assemblies related to the transformation. Specifically, LC coacervate droplets were coacervates formed by LLPS with ordered liquid crystalline structure51,52, while helicoidal vesicles represented vacuolized LC coacervates featured by helicoidal inner patterns, coacervate vesicles were assemblies consisting of a coacervate rim and a water-filled cavity45, and yolk-shell coacervate vesicles (i.e., organelle-containing coacervate vesicles) were assemblies with a coacervate rim, a water-filled cavity, and an inner coacervate organelle. Coacervate microdroplets were isotropic coacervate droplets.

The formation of Cm-Am/DDAB LC coacervate microdroplets was observed across a broad concentration range, as illustrated by a phase diagram (Fig. 1c and Supplementary Fig. S3), exhibiting enhanced coacervation at higher concentrations. In this work, the LC microdroplets were primarily obtained at a total concentration of 10 mM with a monomer molar ratio of 1:1 (marked by a red triangle in Fig. 1c), wherein the LC coacervates were capable of exhibiting macroscopically visible phase separation in the Eppendorf tubes after centrifugation. These LC microdroplets displayed discrete spherical morphologies with high optical contrast, as well as liquid-like property, evidenced by spontaneous fusion behavior in the absence of external stimulus or special conditions (Supplementary Fig. S4 and Movie S1). The average diameter was measured to be 3.5 ± 1.0 μm, and the zeta potential was determined to be +3.9 ± 1.0 mV (Fig. 1d, e). Additionally, the LC microdroplets exhibited birefringent Maltese cross patterns with concentric retardation lines under polarized optical microscopy (POM) (Fig. 1f). This phenomenon disappeared after heating at 60 °C for 80 min (Supplementary Fig. S5), indicative of the system undergoing a phase transition at 60 °C. The LC coacervates exhibited an onion-like multilamellar structure without central water-filled lumen as characterized by cryogenic scanning electron microscopy (Supplementary Fig. S6). The ordered internal structure of the LC coacervates was further confirmed by small-angle X-ray scattering (SAXS), which revealed a sharp Bragg reflection peak at 0.177 Å−1, corresponding to a periodic molecular spacing of 3.55 nm (Fig. 1g and Supplementary Table 1). Considering the bilayer thickness of approximately 2.45 nm53, this spacing likely represented the periodic stacking of a DDAB bilayer and Cm-Am molecules.

LC coacervate microdroplets were able to sequestrate a variety of biomolecules. Experimentally, fluorescein isothiocyanate (FITC)-labeled dextran with relatively low molecular weights (i.e., 10k and 20 kDa) were uniformly distributed within the interior of the LC microdroplets (Supplementary Fig. S7). In contrast, FITC-dextran with a high molecular weight (i.e., 40k and 70 kDa) were predominantly absorbed onto the surface of the LC microdroplets, while FITC-dextran of 250 kDa was mostly excluded. The molecular weight-selective sequestration behavior of LC microdroplets was further demonstrated with a range of client compounds (Fig. 1h, i and Supplementary Fig. S8).

Amylase-induced LC microdroplet-to-coacervate vesicle transition
With the addition of amylase, the LC coacervate microdroplets underwent a two-step transition process toward coacervate vesicles with no other assemblies in the water-filled cavity. Initially, the LC microdroplets experienced intense vacuolization, resulting in the formation of an intermediate structure characterized by helicoidal inner patterns (225 s), followed by the second step, i.e., expansion of the assembly (375 s) caused by weakened multivalent electrostatic complexation and increased internal osmotic pressure during the hydrolysis of Cm-Am into small fragments (Fig. 2a, Supplementary Fig. S9, and Movie S2). The LC microdroplet with doped RITC-Cm-Am vacuolized into helicoidal vesicles as an intermediate, which underwent coalescence, ultimately giving rise to a hollow vesicle determined as a coacervate vesicle (Fig. 2b and Supplementary Movie S3). As a result, the helicoidal vesicles or coacervate vesicles both possessed thicker rims (i.e., 0.4 ± 0.2 or 0.3 ± 0.1 μm) and strong enrichment capability by compared to DDAB vesicles with thinner membranes (i.e., about 0.2 μm) and poor membrane permeability54, wherein the rim and membrane thickness were both measured by fluorescence microscopy and analyzed based on full width at half maximum of gray values in line scan profiles (Supplementary Figs. S10 and S11). Except for the differences in thickness of rims and vesicle membranes, we could also observe that coacervate vesicles were always hollow, whereas the vesicles sometimes possessed multilamellar structures. The cryogenic transmission electron microscopy (cryo-TEM) further confirmed the rim thickness of coacervate vesicles (i.e., about 120 nm) was much larger than membrane thickness in vesicles (i.e., about 6 nm) (Supplementary Fig. S12). In the reconfiguration process, the enzyme (RITC-amylase) was homogenously distributed (Fig. 2c). Notably, the LC coacervate microdroplet experienced a 1.6-fold increase in diameter, from 4.2 to 6.8 µm (Fig. 2d). This change was statistically supported by a comparison of the diameter of LC microdroplets (Fig. 1e, D of 3.5 ± 1.0 μm) and the resultant coacervate vesicles (Supplementary Fig. S13, D of 4.7 ± 2.0 μm), yielding a size ratio D /D of 1.3 ± 0.7. After reaching equilibrium, no obvious vacuolization or expansion was observed in the coacervate vesicle, even with the addition of extra amylase (Fig. 2e). Reconstructed three-dimensional (3D) images confirmed the occurrence of vacuolization and the appearance of helicoidal structure during the LC coacervate microdroplet-to-coacervate vesicle transition, accompanied by an obvious size expansion (Fig. 2f, g). Such reconfiguration process was also achieved in the LC coacervates with higher total concentrations (e.g., 15 or 20 mM) (Supplementary Fig. S14). Upon the addition of PBS buffer (10 mM, pH 6.9, containing 1 mM CaCl2), the Cm-Am/DDAB LC coacervates exhibited negligible changes. Furthermore, the helicoid patterns observed in the intermediate structures were predominantly left-handed, occurring in approximately 80% of cases (Fig. 2h). While the origin of mesoscale chirality remains unclear, we speculated that this phenomenon was related to the chirality of amylose (Supplementary Fig. S15), which possibly induced supramolecular chirality within the LC microdroplets55. Additionally, the vacuolization process was able to induce the rupture of coacervate vesicles, leading to their division (Supplementary Fig. S16). We statistically analyzed the diameters of the initial LC microdroplets (Dcoac) and the coacervate vesicles (Dvesicle), finding a linear correlation between them (Fig. 2i). A slope of 1.6 corresponding to the Dvesicle/Dcoac ratio was obtained, consistent with the diameter ratio (Fig. 2d). The formation of coacervate vesicles was likely maintained by a balance between the osmotic pressure and Laplace pressure within coacervate vesicles30.

We then examined the changes in populations of LC coacervate microdroplets, helicoidal structured vesicles, and coacervate vesicles after the addition of amylase (Fig. 2j). At 340 s, 90% of the LC coacervate microdroplet population (blue region 1) transformed into helicoidal structured vesicles (light blue region 2, 52% of the total populations), which was further reconfigured into coacervate vesicles (pink region 3, representing 38% of the total population). At 760 s, the proportion of coacervate vesicles increased to 83%, then remained stable thereafter. Meanwhile, the fractions of the initial LC microdroplets and helicoidal structured vesicles ultimately decreased to 7% and 10%, respectively, possibly due to the insufficient enzyme activity.

To gain further insights to the transition process, we investigated the impact of amylase concentration on the final microstructures of the Cm-Am/DDAB LC coacervate microdroplets. The 1.50 mg/mL amylase concentration, primarily used in this study, led to coacervate vesicles as the predominant final microstructure, accounting for 44% of the total population (Supplementary Fig. S17). In comparison, lower amylase concentrations (e.g., 0.15 and 0.75 mg/mL) primarily resulted in intact LC coacervate microdroplets (89% and 35% of the total population, respectively), while higher amylase concentrations (e.g., 2.25 and 3.00 mg/mL) predominantly led to multicompartmentalized droplets (51% and 47% of the total population, respectively), due to either insufficient or overly rapid hydrolysis of Cm-Am.

Possible mechanism of structural transition
To demonstrate the enzyme-mediated Cm-Am hydrolysis, we applied gel permeation chromatography (GPC) to monitor changes in the molecular weight of hydrolysis fragments (Fig. 3a). Upon the addition of amylase, the molecular weight decreased sharply from 40 to 5 kDa within 300 s, consistent with transformation of LC coacervates into helicoid-structured vesicles at vacuolization stage (225 s) (Fig. 2a) and the diameter of resulted assemblies reached a plateau at 300 s (Fig. 2d). Ultimately, the Cm-Am hydrolysis reached equilibrium accompanied with the generation of isotropic coacervate vesicles at coalescence stage (Supplementary Fig. S18). To further understand the effect of Cm-Am hydrolysis, we examined the internal ordering changes of assemblies by POM. The ordered LC droplets transformed into LC helicoid-structured vesicles at vacuolization stage (225 s), which was roughly consistent with rapid hydrolysis of Cm-Am within 300 s and eventually isotropic coacervate vesicles at coalescence stage (575 s) that was almost aligned with equilibrium hydrolysis of Cm-Am after 300 s (Fig. 3b and Supplementary Movie S4). We proposed the degree of Cm-Am hydrolysis determined the structural ordering of the assemblies: low-degree hydrolysis of Cm-Am electrostatically complexed with DDAB to form LC microdroplets, while high-degree hydrolysis led to the formation of isotropic coacervate vesicles (Fig. 3c). As proof of concept, we compared the structural ordering of coacervate microdroplets by using Cm-Am with varied degree of hydrolysis. H-Cm-Am 1 with 2 min hydrolysis (9 min elution) complexed with DDAB led to LC coacervate microdroplets (Fig. 3d, e). Whereas H-Cm-Am 2 with 12 h hydrolysis (10 min elution) gave rise to isotropic coacervate vesicles with DDAB (Fig. 3f). This difference was probably because long-chain Cm-Am hindered the mobility and rotation of DDAB, readily leading to LC structure, while short-chain fragments were unfavorable to be orderly organized with DDAB56,57.

To investigate the structural differences between the LC microdroplets and coacervate vesicles, we performed fluorescence recovery after photobleaching (FRAP) by using RITC-dextran (10k Da) as a fluorophore. After photobleaching by 569 nm laser, a rapid fluorescence recovery was observed, with recovery rate of 58% in LC microdroplets and 80% in coacervate vesicles within 500 s (Fig. 3g, h). Although the adjacent area available for replenishing the bleached region was smaller in coacervate vesicles, the recovery rate was faster compared to the LC microdroplets, indicating higher molecular diffusivity in the vesicular system. Specifically, the diffusion coefficient in coacervate vesicles (1.1 × 10−14 m²/s) was approximately eight times higher than in LC coacervate droplets (1.4 × 10−15 m²/s). This difference was attributed to the less ordered microenvironment within the coacervate vesicles, which facilitated faster molecular migration, whereas the liquid crystalline matrix of the microdroplets imposed restrictions on passive diffusion58.

We further investigated the transition from LC microdroplets to coacervate vesicles in mixed systems with varying Cm-Am/DDAB ratios. An LC phase was observed when the monomer molar ratio ranged from 8:1 to 1:4, with helicoidal vesicles noted during the transition (Supplementary Fig. S19). In contrast, when the monomer molar ratio exceeded 8:1 (e.g., 9:1) or fell below 1:4 (e.g., 1:5), isotropic structures were formed, and no helicoid patterns appeared in the intermediate structures. Therefore, we hypothesized that the highly ordered LC structure of the initial coacervate microdroplets was associated with the formation of helicoidal vesicles.

We next examined the structural transition in a variety of protocell microdroplets by varying the coacervate components. First, we replaced DDAB with dimethyldimyristylammonium bromide (DMAB) and observed the formation of ordered LC microdroplets (Fig. 4a) with a periodic spacing of 4.19 nm (Fig. 4b, c)59. The transition from LC microdroplet to coacervate vesicle was observed, which was accompanied by the formation of helicoidal vesicles as intermediates (Fig. 4d and Supplementary Movie S5). Likewise, carboxymethylated dextrin (Cm-dextrin, substitution degree of around 0.9) was used as a short-chain analog of Cm-Am (Fig. 4e and Supplementary Fig. S20), which upon interacting with DDAB, gave rise to LC microdroplets with a periodical spacing of 3.52 nm (Fig. 4f, g)60. The Cm-dextrin/DDAB LC coacervates transitioned into helicoidal structures and eventually coacervate vesicles (Fig. 4h and Supplementary Movie S6). By contrast, no helicoidal vesicles emerged in the structural transition of isotropic coacervates formed by Cm-Am and dodecyltrimethylammonium bromide (DTAB), myristyltrimethylammonium bromide (MTAB), or poly(allylamine hydrochloride) (PAH), wherein the coacervate vesicles were also ultimately generated (Fig. 4i–l and Supplementary Fig. S21)61. Therefore, the appearance of helicoid pattern was possibly attributed to Cm-Am-hydrolysis-induced frustration of lamellar structure in LC droplets, leading to the fusion and connections between disrupted phase62,63.

Finally, we examined the role of electrostatic interactions by adding sodium chloride to screen the charge attraction and hydrochloric acid to protonate carboxymethyl group of Cm-Am (Fig. 4m). In both cases, LC microdroplets transformed into helicoidal vesicles and eventually coacervate vesicles (Fig. 4n, Supplementary Fig. S22 and Movies S7, S8). A lower dose of sodium chloride (15 mM) or hydrochloric acid (1 mM) caused negligible changes in the LC microdroplets (Fig. 4o–q). In contrast, high concentrations of perturbation reagents (e.g., 150 mM of sodium chloride or 50 mM of hydrochloric acid) led to the direct dissociation of the LC microdroplets due to strong charge screening or protonation. This was also observed by the addition of both amylase and sodium chloride or hydrochloric acid (Supplementary Fig. S23).

Simultaneous formation of artificial organelles within protocell compartments
Interestingly, the incorporation of biomolecules into ordered LC coacervate microdroplets led to the formation of organelle-containing protocells, which were also referred to as yolk-shell coacervate vesicles (Fig. 5a). Compared to the coacervate vesicles obtained after the reconfiguration of LC coacervates without biomolecules, the yolk-shell coacervate vesicles featured a coacervate organelle in the water-filled cavity. To further interrogate the complex process, we separately loaded proteins and nucleic acids into the LC microdroplets, such as fluorescence-labeled bovine serum albumin (RITC-BSA), horseradish peroxidase (RITC-HRP), glucose oxidase (FITC-GOx), carboxytetramethylrhodamine-labeled single-stranded oligonucleotide (TAMRA-ssDNA, 99 nt), and TAMRA-dsDNA (25bp) (Fig. 5b, c, Supplementary Fig. S24 and Movies S9, S10). The resulting coacervates maintained liquid crystalline structures and predominantly transformed into coacervate vesicles with an internal coacervate organelle (i.e., yolk-shell coacervate vesicles) in the presence of amylase (Supplementary Figs. S25 and S26). The thickness of rims in yolk-shell coacervate vesicles was measured by fluorescence microscopy and analyzed based on full width at half maximum of gray values in line scan profiles, exhibiting thicker rims (i.e., 0.4 ± 0.1 μm) by compared to DDAB vesicle membranes (i.e., about 0.2 μm) (Supplementary Fig. S27). The yolk-shell coacervate vesicles also possessed a strong enrichment ability, in contrast to controllable permeability of vesicles. During the structural transition, the coacervate components were homogenously distributed throughout the assemblies (Fig. 5d and Supplementary Movie S11).

We hypothesized that the formation of organelle-containing coacervate vesicles was attributed to the enhanced association between interactive biomolecules and DDAB through strong hydrophobic and electrostatic interactions64,65. The multivalent complexation between biomolecules (e.g., proteins, nucleic acids), Cm-Am, and DDAB resulted in LC coacervate microphase that restricted the amylase-catalyzed digestion of Cm-Am, leaving the unreacted inner LC coacervates. In a control experiment, we introduced FITC-dextran (10k Da) into the LC coacervates and observed the generation of coacervate vesicles without internal organelles (Fig. 5e and Supplementary Movie S12). This is due to the weak interactions between FITC-dextran (10 kDa) and Cm-Am/DDAB LC coacervates. To evaluate the binding affinities between DDAB and biomolecules, we utilized isothermal titration calorimetry (ITC) to determine the dissociation constants (Kd), showing 1.57 ± 0.35 × 10−7, 1.00 ± 0.00 × 10−9, or 1.00 ± 0.00 × 10−3 M for the interactions of DDAB with BSA, ssDNA (99 nt), or dextran (10 kDa), respectively (Supplementary Fig. S28). It indicated significantly stronger affinities of DDAB with BSA (i.e., 7-fold) or ssDNA (i.e., 1060-fold) by compared to that with Cm-Am (i.e., 1.06 ± 0.28 × 10−6 M), while the interactions between dextran and DDAB could be neglected. To further support this, we evaluated the interaction strength in the initial LC coacervates, with and without the incorporation of biomolecules, using the critical salt concentration required to dissolve the droplets66. The resulting critical salt concentrations were 353, 429, and 188 mM for the LC droplets with incorporated HRP, ssDNA (99 nt), and dextran (10 kDa), corresponding to 3.6-, 4.4-, or 1.9-fold increases compared to 97 mM for LC droplets without biomolecules (Supplementary Fig. S29). To assess the interaction strength, we measured the diffusion coefficients of FITC-labeled poly(ethylenimine) (PEI) within these droplet systems67,68, which was determined to be 2.2 × 10−15, 1.8 × 10−15, and 3.5 × 10−15 m2/s in the LC microdroplets with incorporated HRP, ssDNA (99 nt), and dextran (10 kDa), respectively, corresponding to 1.9-, 2.3-, and 1.1-fold decrease in comparison to that of LC droplets without biomolecules (4.1 × 10−15 m2/s) (Fig. 5f and Supplementary Fig. S30). We therefore believed that molecular associations between guest biomolecules and Cm-Am/DDAB LC coacervates resulted into the formation of inner organelle-containing coacervate vesicles (Fig. 5g).

In the yolk-shell coacervate vesicles, after reaching equilibrium, we observed that the rim and inner organelle remained persistently attached without coalescing, even after the addition of a new batch of amylase, when the hydrolysis of Cm-Am reached equilibrium (Supplementary Figs. S31 and S32). We hypothesized that the composition in the rim and internalized organelle was different, i.e., the degree of hydrolysis of Cm-Am was higher in the rim than in the internal organelles. Specifically, the critical salt concentration in the rim and the organelles was determined to be 9.7 and 50 mM, respectively (Supplementary Fig. S33). Additionally, the diffusion coefficient of FITC-PEI within the rim was 1.4 × 10−14 m2/s, 1.8-fold higher than that in the organelle (i.e., 7.6 × 10−15 m2/s) (Fig. 5f), suggesting a molecularly more crowded environment inside the organelle.

The ability confining biochemical reactions within organelle-containing coacervate vesicles was crucial for the functionality of artificial cells. To investigate this, we performed a hammerhead ribozyme (HH-min)-mediated RNA catalysis reaction within the organelle-embedded coacervate vesicles69. In this system, the cleavage of a fluorescence resonance energy transfer (FRET)-substrate strand catalyzed by HH-min increased the distance between the fluorophore (6-carboxyfluorescein) and Black Hole quencher 1 (BHQ1), resulting in an increase of green fluorescence intensity (Fig. 5h, i). Significantly, the LC coacervates with homogenously integrated cyanine 5 labeled-HH-min (Cy5-HH-min) and carboxyfluorescein-labeled substrate (FAM-substrate) were capable of transforming to yolk-shell coacervate vesicles initiated by amylase (Supplementary Fig. S34), while a progressive increase of green fluorescence intensity from HH-min-mediated reaction was observed in the rim and inner artificial organelle (Fig. 5j and Supplementary Movie S13), confirming the support of enzymatic reactions by the system. Moreover, the RNA catalysis rates in yolk-shell coacervate vesicles were faster than that in the initial LC microdroplets (Fig. 5k, l), attributing to the higher molecular diffusivity in the isotropic yolk-shell coacervate vesicles, which were confirmed by the disappearance of birefringence phenomenon in the POM image and Bragg reflection peak in the SAXS profile (Supplementary Figs. S35 and S36). Meanwhile, the reaction rate in the rim was slightly faster than that in the organelle.

Methods
Preparation of LC or isotropic coacervate microdroplets
Typically, the pH of Cm-Am solution (10 mM) and DDAB solution (10 mM) were adjusted to 8 before mixing. Cm-Am/DDAB LC coacervates were prepared by directly mixing the aforementioned aqueous solutions of Cm-Am (25 μL, 10 mM, pH 8) and DDAB (25 μL, 10 mM, pH 8), following vortexing for 5 s. The pH was then checked and adjusted as necessary to ensure it remained at 8. Other coacervate suspensions were prepared following the similar procedure except for using other surfactants or polyelectrolytes under varied total concentrations and monomer molar ratios, such as Cm-Am/DTAB isotropic coacervate suspension (50 mM, 1:1), Cm-Am/MTAB isotropic microdroplet dispersion (50 mM, 1:1), Cm-Am/DMAB LC coacervate dispersion (5 mM, 1:1), Cm-dextrin/DDAB LC microdroplet suspension (10 mM, 1:1), and Cm-Am/PAH isotropic coacervate suspension (50 mM, 2:1).

Turbidity diagram
The turbidity diagram was measured by mixing different concentrations of Cm-Am (5, 10, 20, 40, and 50 mM, pH 8) and DDAB (10, 20, 30, and 40 mM, pH 8) solutions with different monomer molar ratios. The turbidity of these samples was recorded by a plate reader (CLARIOstar plus, BMG Labtech) at an excitation wavelength of 600 nm, where no absorbance for coacervate components. The coacervation behaviors of the samples were investigated on an optical microscope after incubation for 5 h. Measurements were repeated three times.

Sequestration properties of Cm-Am/DDAB LC coacervate microdroplets
Typically, Cm-Am/DDAB LC coacervate microdroplets (100 μL) were prepared in Eppendorf tubes using the procedure described above. Then, Hoechst (1 μL, 5 μM, in water), HPTS (1 μL, 20 μM, in water), Rh6G (1 μL, 50 μM, in water), NBD-PE (1 μL, 20 μg/mL, in ethanol), FITC-dextran (10, 20, 40, 70, and 250 kDa, 1 μL, 1 mg/mL, in water), RITC-DEAE-dextran (20k Da, 1 μL, 1 mg/mL, in water), RITC-PDDA (1 μL, 0.5 mg/mL, in water), TAMRA-dsDNA (1012 bp, 1 μL, 2 mg/mL, in 10 mM of Tris-HCl buffer with containing 1 mM of EDTA, pH 8), or RITC-Cm-Am (1 μL, 1 mg/mL, in water) was added to the Cm-Am/DDAB LC microdroplets suspension, respectively.

Enzyme-mediated reconfiguration from coacervate microdroplets to coacervate vesicles
Typically, 30 µL of Cm-Am/DDAB LC coacervate microdroplets prepared as the above procedure were added to a sample cell with a glass coverslip, settling for ca. 10 min. Then, 5.3 µL of amylase (10 mg/mL in 10 mM PBS buffer containing 1 mM CaCl2, pH 6.9) was added into the coacervate-containing sample cell. The reconfiguration process was monitored by confocal laser scanning microscopy (CLSM).

The amylase-mediated transformation of LC coacervates with higher concentrations was explored following the above procedures except using 15 mM or 20 mM of Cm-Am/DDAB LC coacervate microdroplets (30 µL, monomer molar ratio of 1:1). The amylase-mediated reconfiguration behaviors for other LC or isotropic coacervate microdroplets were investigated following similar procedures except for using different doses of amylase (10 mg/mL). Specifically, 3 µL of amylase (10 mg/mL) was added to Cm-Am/DTAB isotropic coacervate microdroplets (30 µL, total concentration of 50 mM, monomer molar ratio of 1:1), Cm-Am/MTAB isotropic microdroplets (30 µL, total concentration of 50 mM, monomer molar ratio of 1:1), or Cm-Am/DMAB LC coacervate droplets (30 µL, total concentration of 5 mM, monomer molar ratio of 1:1). One microliter of amylase (10 mg/mL) was added to Cm-dextrin/DDAB LC microdroplets (30 µL, total concentration of 10 mM, monomer molar ratio of 1:1). Two microliter of amylase (10 mg/mL) was added to Cm-Am/PAH isotropic microdroplets (30 µL, total concentration of 10 mM, monomer molar ratio of 2:1).

The effects of PBS buffer on LC coacervates were also investigated by adding 5.3 µL of PBS buffer (10 mM, pH 6.9, containing 1 mM CaCl2) to 30 µL of Cm-Am/DDAB LC coacervates (total concentration: 10 mM; monomer molar ratio: 1:1). All experiments were independently repeated at least three times, yielding consistent results.

Preparation of H-Cm-Am/DDAB LC coacervate microdroplets or coacervate vesicles
Typically, 300 μL of Cm-Am solution (10 mM) was priorly hydrolyzed by amylase (100 μL, 10 mg/mL in 10 mM PBS buffer containing 1 mM CaCl2, pH 6.9) for 2 min, after which the mixture was heated under 90 °C for 40 min to denature amylase. Then, the mixture was centrifugated, discarding the precipitation of amylase. Afterwards, H-Cm-Am 1 were obtained. The H-Cm-Am 1/DDAB LC coacervate microdroplets were prepared by mixing the aforementioned H-Cm-Am 1 (10 mM) and DDAB solution (10 mM) in a monomer molar ratio of 1:1. Likewise, H-Cm-Am 2/DDAB isotropic coacervate vesicles (total concentration of 10 mM, monomer molar ratio of 1:1) were prepared following the above procedure except the used H-Cm-Am 2 (10 mM) was obtained after prior hydrolysis of Cm-Am (10 mM) for 12 h.

Transformation from LC coacervate microdroplets to isotropic coacervate vesicles induced by sodium chloride or hydrochloric acid
Typically, 30 µL of Cm-Am/DDAB LC coacervate suspension prepared as the above procedure was added to a sample cell with a glass coverslip, settling for ca. 10 min. Then, 3 µL of sodium chloride with a concentration of 800 mM was added, and the reconfiguration process was recorded by CLSM. Similarly, the dynamic behaviors of Cm-Am/DDAB LC coacervate microdroplets with adding 3 µL of 150 mM or 1500 mM of sodium chloride were performed following the above procedures.

The hydrochloric acid-mediated dynamic behaviors were achieved following the above procedures except that 3 µL of hydrochloric acid solution with a concentration of 10, 100, and 500 mM was added, respectively.

The dynamic behaviors of LC coacervates initiated by mixture of amylase and high concentrations of sodium chloride or hydrochloric acid were achieved following the above procedure except the addition of 5 µL of amylase solution (10 mg/mL) with containing sodium chloride (955 mM) or hydrochloric acid (355 mM). All experiments were repeated at least three times independently, yielding consistent results.

Diagram of LC coacervate microdroplets with the addition of sodium chloride or hydrochloric acid
Typically, 30 µL of Cm-Am/DDAB LC coacervate suspension prepared as the above procedure was added to a sample cell with a glass coverslip, settling for ca. 10 min. Then, different doses of sodium chloride (1500 mM) were added into the LC coacervates, respectively, and the changes of LC coacervates were monitored by CLSM. When the assemblies almost remained unchanged, we set a period of 30 min for salt concentration equilibration and the then the ultimate structures were recorded. Similarly, the effects of different concentrations of hydrochloric acid on LC coacervates were also explored following the above procedures except using varied doses of hydrochloric acid (500 mM).

Reconfiguration of LC coacervate microdroplets integrated with biomacromolecules into organelle-containing coacervate vesicles
Typically, 5 µL of RITC-BSA (3 mg/mL, in 10 mM of PBS buffer, pH 8) was added to 50 µL of Cm-Am/DDAB LC coacervate suspension (total concentration of 10 mM, monomer molar ratio of 1:1, pH 8), followed by a 10-min incubation to establish partitioning equilibrium. The structural change of the sample was recorded by CLSM after adding 5.3 µL of amylase (10 mg/mL).

Likewise, the structural changes of Cm-Am/DDAB LC coacervate microdroplets with the incorporation of RITC-HRP (5 µL, 5 mg/mL, in 10 mM of PBS buffer, pH 8), RITC-GOx (5 µL, 5 mg/mL, in 10 mM of PBS buffer, pH 8), TAMRA-ssDNA (99 nt, 2 µL, 25 μM, in 10 mM of Tris-HCl buffer with containing 1 mM of EDTA, pH 8), TAMRA-dsDNA (25 bp, 1 µL, 50 μM, in 10 mM of Tris-HCl buffer with containing 1 mM of EDTA, pH 8), and FITC-dextran (10 kDa, 5 µL, 5 mg/mL, in water), Cy5-HH-min (0.5 µL, 50 μM, in water), and FAM-substrate (0.5 µL, 50 μM, in water) was recorded following the above procedure. All experiments were independently repeated at least three times, yielding consistent results.

The ribozyme-mediated catalysis within LC coacervate microdroplets and organelle-containing coacervate vesicle
Typically, 0.5 µL of hammerhead ribozyme derived from satellite RNA of tobacco ringspot virus (HH-min, 50 μM) was added into 30 µL of Cm-Am/DDAB LC coacervate suspension (total concentration of 10 mM, monomer molar ratio of 1:1, pH 8). Thirty microliters of HH-min-loaded Cm-Am/DDAB LC coacervate suspension was added to the sample cell, followed by settling for ca. 10 min on the coverslip before imaging. After adding amylase (5.3 µL, 10 mg/mL) for the reconfiguration process from LC coacervate microdroplets to yolk-shell coacervate vesicles, 0.5 µL of FRET-substrate (20 μM) was added to initiate the HH-min-mediated cleavage of the FRET-substrate strand. The enhancement of green fluorescence intensity was monitored by CLSM, which was associated with the increase in the distance between FAM and Black Hole quencher 1 (BHQ1). In a control experiment, the same condition was provided as mentioned above except none of amylase was added into Cm-Am/DDAB LC microdroplets. The data were obtained from three independent experiments.

Statistics and reproducibility. No statistical method was used to predetermine sample size. No data were excluded from the analysis. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. All representative CLSM images were obtained from three independent experiments with similar results. Statistical parameters including the definitions and exact value of n (e.g., total number of measured samples) were reported in the figures and corresponding figure legends. Error bars displayed on graphs represent the mean ± standard deviation (s.d.) from at least three independent experiments, as indicated in the corresponding figure legends. CLSM images were analyzed by ImageJ software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Recommendation: minor revisions needed\nThe content of the manuscript entitled “Enzymatically reconfigurable liquid crystalline coacervate microdroplets as protocell models” describes a liquid crystalline (LC) microdroplet-based protocell system that transitions into hierarchical coacervate vesicles through electrostatic and hydrophobic interactions between a polysaccharide and a cationic surfactant. The addition of amylase triggers structural changes, leading to the formation of helicoid-structured and coacervate vesicles capable of embedding biomolecules. These findings provide a protocell model that mimics cellular compartmentalization (artificial organelles), offering insights into cell-like structures and functions.\nThis work clearly meets the standards expected by both the research community and the journal, demonstrating significant value. The hypothesis is well-supported by experimental data and a detailed experimental description. However, some explanations are either missing or insufficiently addressed. Therefore, I recommend minor revisions before publication.\nKeywords\nThe word enzyme should appear in keywords\nResults and discussion\nThe molecular weight of carboxymethyl amylose is described is in the supplementary material but would be useful in the main text. It would help the reader to understand some process.\nTransformation from coacervate microdroplets to coacervate vesicles induced by sodium chloride or hydrochloric acid.\nTransformations are observed without the presence of the amylase enzyme; this aspect should be discussed further. What happens if both aspects are combined (amylase enzyme and high salt concentrations)?\nExperimental Descriptions\nAqueous Solution and pH Control (pH 8): The pH of coacervate microdroplets is 8. How was this pH value adjusted and is maintained?\nIncubation Period: Biomolecules are added after coacervate microdroplet formation. Is an incubation period required before proceeding with further steps?\nControl Experiment with Buffer and CaCl2: Since amylase is introduced along with a buffer and CaCl2, have you tested a control solution without the enzyme? If so, does it cause any reconfiguration of the coacervate microdroplets?\nMedium for Biomacromolecules: The text discusses the transformation of biomacromolecule-integrated coacervate microdroplets into organelle-containing coacervate vesicles but only mentions stock biomolecule concentrations. Could you specify the medium used (e.g., water, buffer) for these stock solutions?", "reviewer_2": "The manuscript “Enzymatically reconfigurable liquid crystalline coacervate microdroplets as protocell models” by L. Jia, C. Zhou, and Y. Qiao presents an investigation into coacervate droplets composed of negatively charged carboxymethyl amylose (Cm-Am) and a cationic surfactant (didodecyldimethylammonium bromide, DDAB) that display liquid crystalline lamellar phases. The study reports that enzymatic hydrolysis of Cm-Am by amylase induces morphological transitions from liquid crystalline coacervates to vacuolized droplets with helicoidal patterns and subsequently to coacervate vesicles. Additionally, in the presence of guest biomolecules (proteins or nucleic acids) interacting with the coacervate components, a transition to coacervate-containing vesicles is observed, which is suggested as a model of organelle-containing protocell. Both liquid crystalline coacervates and yolk-shell vesicles are demonstrated to be compatible with hammerhead ribozyme catalysis.\nWhile the manuscript is well written and covers an interesting topic, it lacks sufficient novelty and originality to meet the publication standards of Nature Communications. The reported system is closely related to previously published works by the same group and others (e.g., Mu W., et al., Nat. Chem., 2024, 16, 158-167). The main novelty, the transition from coacervate droplets to vesicles, has also been reported by others (e.g., Choi H., et al., Adv. Sci., 2024, 11, 2305978; Wen P., et al., Nat. Chem., 2025). Moreover, the manuscript does not provide new mechanistic insights or broaden the generality of the observed phenomena.\nConsidering these points, I recommend that the manuscript not be accepted for publication in Nature Communications. Below are key points that should be addressed before submission to another journal.\nMajor Comments:\n1. Origin of Life Claims: The assertion that this work contributes to the understanding of the origin of life is not well supported. The investigated system is highly artificial, and the morphological transitions rely on enzymatic catalysis, which is not prebiotic. The generalization of the observed phenomena to other prebiotic systems is also limited. Therefore, references to the origin of life (lines 27, 43 and 67) should be removed.\n2. Phase Diagram Characterization: Figure 1c shows a phase diagram based solely on turbidity measurements, which cannot differentiate between liquid crystalline droplets, isotropic droplets, or vesicles. Microscopy observations should be included to better characterize the phase diagram. Specifically, it would be helpful to investigate morphological transitions as a function of the Cm-Am/DDAB ratio. Supplementary Figure S11 shows different morphologies outside the phase diagram range reported in Figure 1c. Expanding the phase diagram to include up to 20 mM Cm-Am and DDAB and characterizing the droplets using polarized optical microscopy, without amylase, would be necessary.\n3. Critical Salt Concentration Measurements: The description of critical salt concentration measurements is unclear (SI lines 224–238). The reported procedure results in NaCl concentrations of 429, 353, 188, and 97 mM, which do not align with the data presented in Figure 4o or the experiments in Figure S17. Please, clarify how the phase diagram in Figure 4o was obtained, the waiting time for salt concentration equilibration, and the definition of critical salt concentration.\n4. Structural Characterization of Coacervate Vesicles: The structural properties of the coacervate vesicle phase and the internal organelle are not sufficiently investigated. Some images (Figures 3f, 4j, and S11) indicate birefringence, suggesting that the vesicle phase may be liquid crystalline. However, the schematic diagrams in Figures 1b and 3c depict vesicles and internal coacervates as solutions of micelles, poly-anions, and guest biomolecules, which may not accurately represent the observed phases. Further investigation using polarized microscopy would provide more insight into the structural organization of both compartments. The intriguing helicoidal pattern transition is not structurally characterized or understood.\n5. Enzyme-Mediated Reconfiguration Procedure: The description of the enzyme-mediated reconfiguration (SI, lines 182– 187) is unclear regarding the tested conditions. Assuming an initial total charge concentration of 10 mM (5 mM from DDAB and 5 mM from Cm-Am), adding amylase (5.3 μL to 30 μL of coacervate solution) reduces the concentration to 8.5 mM, which, according to Figure 1c, would lead close to the single-phase region due to dilution. To confirm that the morphological transition is not solely due to dilution, the authors should test higher initial total charge concentrations (e.g., 15 mM or 20 mM) and provide negative controls.\n6. Confinement of Reactions in Organelle-Containing Vesicles: I agree with the sentence that “The ability confining endogenous metabolic reactions within organelle-containing coacervate vesicles was crucial for the functionality of artificial cells”. Anyway, for a sub-compartment to be really an organelle it would require that the reactions were confined in the organelle only. This is not convincingly demonstrated. The biomolecule partitioning between the inner organelle and vesicle rim appears similar, and hammerhead ribozyme catalysis occurs in both phases without significant differences. Thus, the conclusion that the system represents a protocell model capable of evolving complex compartments (lines 392–394) is not fully supported.\nMinor Comments:\n• Caption of Figure S11, S13 and other SI figures: The addition of amylase is often not mentioned. Please write it where needed.\n• Figure 4o: Clarify the meaning of “droplet concentration.” If it refers to total charge concentration, update the label accordingly.\n• Helicoidal Pattern Transition: Explain why the helicoidal pattern transition also occurs with Cm-dextrin, despite the disturbance of the liquid crystalline phase by short H-Cm-Am.\n• Biomolecule-Induced Transition: lines 296–306: Clarify whether coacervate droplets transition to isotropic or remain liquid crystalline upon biomolecule addition. Provide polarized microscopy images in addition to fluorescence images to assess the internal organelle’s phase (see Supplementary Figure S24).\n• Vacuolized Intermediates: Indicate whether the vacuolized intermediates shown in Figure S11 are birefringent.\nIn summary, while the manuscript presents an interesting study, the lack of sufficient novelty and mechanistic insights limits its suitability for Nature Communications. Addressing the points above would strengthen the manuscript for consideration in other journals.", "reviewer_3": "The manuscript titled “Enzymatically reconfigurable liquid crystalline coacervate microdroplets as protocell models” authored by Jia et al. describes the formation and transition of liquid crystalline coacervates to helicoidal vesicles and membranous protocells that contain organelles. The droplets are prepared from Cm-Am and DDAB an anionic polyelectrolyte and a cationic surfactant respectively and the transition is driven by amylase action on the Cm-Am polyelectrolyte. The manuscript is well presented and describes an enzymatically driven phase transition in compartments to form droplets of different structures. However, the authors should check the interpretation of the data and the claim that coacervate vesicles have been formed. The results suggest that Cm-Am/DDAB droplets are multilamellar vesicles and not coacervates or coacervate vesicles. Coacervates are typically amorphous and do not exhibit structure. The data further shows that the multilamellar vesicles transform upon addition with amylase to vesicles with an internal structure which could be interpreted as a coacervate or a surfactant droplet or smaller vesicle. If the context of the data was presented correctly and better descriptions (supported by scientific data) of the structures were given then this work could be suitable for publication. Whilst there have been examples previously of transitions between multilamellar vesicles to vesicles before (Langmuir, 2018, 34, 5, 2171-2179 amongst other papers), to the best of my knowledge this is the first example of a dynamic system i.e. where an enzyme drives the structural transition.\nPlease see below for specific comments about the manuscript:\n1. Please define the following terms: liquid crystalline coacervate droplets, helicoidal vesicles, coacervate vesicles and microdroplets. This will provide clarity into how the data for the droplet structures are interpreted. Please also keep the terminology consistent through the text\n2. The authors describe the Cm-Am/DDAB coacervate droplets as LC coacervate microdroplets. The data actually suggests that these are multilamellar vesicles given the maltese crosses. Thus indicating that they are not coacervates. Further, the data suggests that the addition of amylase transforms the multilamellar vesicle into a vesicle with internal compartment. With the helicoidal structure a metasable state that appears depending on the ratio of Cm-Am and DDAB. Some questions remain about the internal compartment. What is the internal structuration of this? How can they confirm that is a coacervate and not a surfactant droplet or a smaller vesicle.\n3. SAX data and EM should be used to can confirm the location of the Cm-Am within the bilayers via electron density mapping. As well as the presence of bilayers within the LC coacervate and the yolk shell system.\n4. Does DDAB have a CVC on it’s own what is the bilayer spacing and what is this in comparison to Cm-Am and DDAB vesicles?\n5. What leads to the size expansion after the addition of amylase and what does this tell you about the mechanism of structural change?\n6. Could the authors provide some more explanation of the significance of the FRAP data with regard to the organelle and the rim? How does the FRAP recovery compare to control experiments i.e. Cm-Am/ DDAB alone or fragmented Cm-Am and DDAB. Does this give some insight to the chemical components of rim and the organelle? Can you show where Cm-Am is located? What does SAXs show? Can this confirm bilayers? The data suggests that it is an onion vesicle which transforms to another vesicle?\n7. Provide reference papers to other droplets that form LC phases with coacervate forming components as well as literature which discusses multilamellar vesicle to vesicle transitions. It could be useful to look at Langmuir, 2018, 34, 5, 2171-2179 amongst other papers\n8. How can you confirm vacuolisation?\n9. Show images from whole populations to get an idea of how robust this process is.\n10. For the critical salt concentration in the organelle and rim, please can you provide some explanation as to how the critical salt concentration was obtained?\n11. Please explain interaction strength? This is not given by diffusion coefficient or by salt concentration. Interaction strength is usually Kd.\n12. HHmin-RNA is not a metabolic reaction.\n13. Where is the evidence that the reaction takes place outside of the vacuole? Showing the location of the substrate and RNA could be useful here as there is no evidence to prove this statement. An alternative explanation could be that reaction happens ourside of these regions and the products can diffuse into the yolk-shell... “‘Moreover, the RNA catalysis rates in yolk-shell to the higher molecular diffusivity in the isotropic yolk-shell coacervate vesicles (Supplementary Fig. S24). Meanwhile, the reaction rate in the rim was slightly faster than that”\n14. Please show the structures that might form if fragmented Cm-Am is mixed with DDAB. This could confirm that the change in structure is due to the dynamic effect of the enzyme.\n15. “This phenomenon disappeared after heating at 60 °C for 80 min (Supplementary Fig. S3), confirming the presence of a LC internal structure51.” This statement does not show that it has an LC internal structure just that the system can undergo a phase transition at 60 oC.\n16. Does the time frame for change in structure correlate to the rates of amylase degradation?\n17. Do the membranes on the helicoid structures show bilayers?\n18. Please amend this sentence as the diffusion coefficient is not attributed to the concentration of molecules but by the ability for the molecules to diffuse internally. “Additionally, the diffusion coefficient of FITC-PEI within the rim was 1.4 × 10-14 m2/s, 1.8-fold higher than that in the organelle (i.e.,7.6 × 10-15 m2/s) (Fig. 5f and Supplementary Fig. S21). This difference is likely due to the lower partitioning of biomolecules in the rim compared to the organelle, which was confirmed by the fluorescence intensity of these biomolecules (Supplementary Fig. S22).”"}, "author_response": "Responses to reviewers’ comments\nWe thank the reviewers for their constructive comments that have enabled us to improve the manuscript.\nAs detailed below, we have conducted additional experiments and responded to the comments point-by-point.\nResponses to Reviewer#1\nThe content of the manuscript entitled “Enzymatically reconfigurable liquid crystalline coacervate\nmicrodroplets as protocell models” describes a liquid crystalline (LC) microdroplet-based protocell system\nthat transitions into hierarchical coacervate vesicles through electrostatic and hydrophobic interactions\nbetween a polysaccharide and a cationic surfactant. The addition of amylase triggers structural changes,\nleading to the formation of helicoid-structured and coacervate vesicles capable of embedding biomolecules.\nThese findings provide a protocell model that mimics cellular compartmentalization (artificial organelles),\noffering insights into cell-like structures and functions.\nThis work clearly meets the standards expected by both the research community and the journal,\ndemonstrating significant value. The hypothesis is well-supported by experimental data and a detailed\nexperimental description. However, some explanations are either missing or insufficiently addressed.\nTherefore, I recommend minor revisions before publication.\nResponse: We thank the reviewer for these comments.\n1. The word enzyme should appear in keywords.\nResponse: We thank the reviewer for the comments. We have now included the word “enzyme” in the\nkeywords as suggested.\n2. The molecular weight of carboxymethyl amylose is described in the supplementary material but would\nbe useful in the main text. It would help the reader to understand some process.\nResponse: We thank the reviewer for the comments. We have added the molecular weight of carboxymethyl\namylose (40k Da) in the revised manuscript (Page 3, Paragraph 2).\n3. Transformation from coacervate microdroplets to coacervate vesicles induced by sodium chloride or\nhydrochloric acid. Transformations are observed without the presence of the amylase enzyme; this aspect\nshould be discussed further. What happens if both aspects are combined (amylase enzyme and high salt\nconcentrations)?\nResponse: We thank the reviewer for the suggestion. We supplemented experiments by simultaneously\nadding amylase and sodium chloride or hydrochloric acid, which led to the rapid dissociation of the LC\ncoacervates. The corresponding discussion and data are now included in the manuscript (Page 10, Paragraph\n4) and SI (Page 7, Paragraph 5; new Figure S19).\nRevised text, Page 10, Paragraph 4\nIn contrast, high concentrations of perturbation reagents (e.g., 150 mM of sodium chloride or 50 mM of\nhydrochloric acid) led to the direct dissociation of the LC microdroplets due to strong charge screening or\nprotonation. This was also observed by the addition of both amylase and sodium chloride or hydrochloric\nacid (Supplementary Fig. S19).\nRevised method, Supplementary Information, Page 7, Paragraph 5\nR1\nThe hydrochloric acid-mediated dynamic behaviours were achieved following the above procedures\nexcept that 3 µL of hydrochloric acid solution with a concentration of 10 mM, 100 mM, and 500 mM was\nadded, respectively.\nThe dynamic behaviours of LC coacervates initiated by mixture of amylase and high concentrations\nof sodium chloride or hydrochloric acid were achieved following the above procedures except the addition\nof 5 µL of amylase solution (10 mg/mL) with containing sodium chloride (955 mM) or hydrochloric acid\n(355 mM).\nNew Figure S19\nSupplementary Figure S19. (a,b) Time-dependent optical microscopy images showed the disassembly of\nCm-Am/DDAB LC coacervate microdroplets (total concentration of 10 mM, monomer molar ratio of 1:1)\nupon the simultaneous addition of amylase (1.5 mg/mL) and sodium chloride (150 mM) (a) or hydrochloric\nacid (50 mM) (b). Scale bars, 5 1um.\n4. Aqueous Solution and pH Control (pH 8): The pH of coacervate microdroplets is 8. How was this pH\nvalue adjusted and is maintained?\nResponse: We thank the reviewer for the comments. The pH of the aqueous solutions used for the\npreparation of LC or isotropic coacervate microdroplets was adjusted to 8 prior to mixing. These solutions\nincluded Cm-Am (10 mM or 50 mM), DDAB (10 mM), DTAB (50 mM), MTAB (50 mM), DMAB (5\nmM), Cm-dextrin (10 mM), and PAH (50 mM). The adjusted solutions were then mixed to form LC or\nisotropic coacervate microdroplets, and the pH was maintained at 8 throughout. We have revised the\ncorresponding text in the SI (Page 5, Paragraph 4).\nRevised method, Supplementary Information, Page 5, Paragraph 4\nPreparation of LC or isotropic coacervate microdroplets. Typically , the pH of Cm-Am solution (10 mM)\nand DDAB solution (10 mM) were adjusted to 8 before mixing. Cm-Am/DDAB LC coacervates were\nprepared by directly mixing the aforementioned aqueous solutions of Cm-Am (25 1uL, 10 mM, pH 8) and\nDDAB (25 1uL, 10 mM, pH 8), following vortexing for 5 s. The pH was then checked, and adjusted as\nnecessary to ensure it remained at 8.\n5. Incubation Period: Biomolecules are added after coacervate microdroplet formation. Is an incubation\nperiod required before proceeding with further steps?\nResponse: We thank the reviewer for the comments. The biomolecules were incubated with LC coacervate\nR2\nmicrodroplets for 10 min before confocal microscopy image. We have revised the corresponding text in the\nSI (Page 8, Paragraph 2).\nRevised method, Supplementary Information, Page 8, Paragraph 2\nReconfiguration of LC coacervate microdroplets integrated with biomacromolecules into organelle-\ncontaining coacervate vesicles. Typically, 5 µL of RITC-BSA (3 mg/mL, in 10 mM of PBS buffer, pH 8) was\nadded to 50 µL of Cm-Am/DDAB LC coacervate suspension (total concentration of 10 mM, monomer\nmolar ratio of 1:1, pH 8), followed by a 10-min incubation to establish partitioning equilibrium.\n6. Control Experiment with Buffer and CaCl2: Since amylase is introduced along with a buffer and CaCl2,\nhave you tested a control solution without the enzyme? If so, does it cause any reconfiguration of the\ncoacervate microdroplets?\nResponse: We thank the reviewer for the constructive comments. We performed a control experiment by\nadding PBS buffer (10 mM, pH 6.9, containing 1 mM of CaCl ) without amylase and did not observe any\n2\nstructural changes of the LC coacervate microdroplets. We have added the results in the manuscript (Page\n5, Paragraph 2).\nRevised text, Page 5, Paragraph 2\nUpon the addition of PBS buffer (10 mM, pH 6.9, containing 1 mM CaCl2), the Cm-Am/DDAB LC\ncoacervates exhibited negligible changes. Furthermore, the helicoid patterns observed in the intermediate\nstructures were predominantly left-handed, occurring in approximately 80% of cases (Fig. 2h).\nRevised method, Supplementary Information, Page 7, Paragraph 1\nThe effects of PBS buffer on the LC coacervates were also investigated by adding 5.3 µL of PBS buffer\n(10 mM, pH 6.9, containing 1 mM CaCl2) to 30 µL of Cm-Am/DDAB LC coacervates (total concentration:\n10 mM; monomer molar ratio: 1:1). All experiments were independently repeated at least three times,\nyielding consistent results.\n7. Medium for Biomacromolecules: The text discusses the transformation of biomacromolecule-integrated\ncoacervate microdroplets into organelle-containing coacervate vesicles but only mentions stock\nbiomolecule concentrations. Could you specify the medium used (e.g., water, buffer) for these stock\nsolutions?\nResponse: We thank the reviewer for the suggestion. We have included details as suggested in the SI (Page\n7, Paragraph 2; Page 8, Paragraph 2).\nRevised method, Supplementary Information, Page 7, Paragraph 2\nSequestration properties of Cm-Am/DDAB LC coacervate microdroplets. Typically, Cm-Am/DDAB LC\ncoacervate microdroplets (100 1uL) were prepared in Eppendorf tubes using the procedure described above.\nThen, Hoechst (1 1uL, 5 1uM, in water), HPTS (1 1uL, 20 1uM, in water), Rh6G (1 1uL, 50 1uM, in water),\nNBD-PE (1 1uL, 20 1ug/mL, in ethanol), FITC-dextran (10k Da, 20k Da, 40k Da, 70k Da, and 250k Da, 1 1uL,\n1 mg/mL, in water), RITC-DEAE-dextran (20k Da, 1 1uL, 1 mg/mL, in water), RITC-PDDA (1 1uL, 0.5 mg/mL,\nin water), TAMRA-dsDNA (1012 bp, 1 1uL, 2 mg/mL, in 10 mM of Tris-HCl buffer with containing 1mM of\nEDTA, pH 8), or RITC-Cm-Am (1 1uL, 1 mg/mL, in water) was added to the Cm-Am/DDAB LC microdroplet\nsuspension, respectively.\nR3\nRevised method, Supplementary Information, Page 8, Paragraph 2\nReconfiguration of LC coacervate microdroplets integrated with biomacromolecules into organelle-\ncontaining coacervate vesicles. Typically, 5 µL of RITC-BSA (3 mg/mL, in 10 mM of PBS buffer, pH 8) was\nadded to 50 µL of Cm-Am/DDAB LC coacervate suspension (total concentration of 10 mM, monomer\nmolar ratio of 1:1, pH 8), followed by a 10-min incubation to establish partitioning equilibrium. The\nstructural change of the sample was recorded by CLSM after adding 5.3 µL of amylase (10 mg/mL).\nLikewise, the structural changes of Cm-Am/DDAB LC coacervate microdroplets with the\nincorporation of RITC-HRP (5 µL, 5 mg/mL, in 10 mM of PBS buffer, pH 8), RITC-GOx (5 µL, 5 mg/mL,\nin 10 mM of PBS buffer, pH 8), TAMRA-ssDNA (99 nt, 2 µL, 25 1uM, in 10 mM of Tris-HCl buffer with\ncontaining 1mM of EDTA, pH 8), TAMRA-dsDNA (25 bp, 1 µL, 50 1uM, in 10 mM of Tris-HCl buffer with\ncontaining 1mM of EDTA, pH 8), FITC-dextran (10k Da, 5 µL, 5 mg/mL, in water), Cy5-HH-min (0.5 µL,\n50 1uM, in water), and FAM-substrate (0.5 µL, 50 1uM, in water) was recorded following the above\nprocedure. All experiments were independently repeated at least three times, yielding consistent similar\nresults.\nResponses to Reviewer #2\nThe manuscript “Enzymatically reconfigurable liquid crystalline coacervate microdroplets as protocell\nmodels” by L. Jia, C. Zhou, and Y. Qiao presents an investigation into coacervate droplets composed of\nnegatively charged carboxymethyl amylose (Cm-Am) and a cationic surfactant\n(didodecyldimethylammonium bromide, DDAB) that display liquid crystalline lamellar phases. The study\nreports that enzymatic hydrolysis of Cm-Am by amylase induces morphological transitions from liquid\ncrystalline coacervates to vacuolized droplets with helicoidal patterns and subsequently to coacervate\nvesicles. Additionally, in the presence of guest biomolecules (proteins or nucleic acids) interacting with the\ncoacervate components, a transition to coacervate-containing vesicles is observed, which is suggested as a\nmodel of organelle-containing protocell. Both liquid crystalline coacervates and yolk-shell vesicles are\ndemonstrated to be compatible with hammerhead ribozyme catalysis.\nWhile the manuscript is well written and covers an interesting topic, it lacks sufficient novelty and\noriginality to meet the publication standards of Nature Communications. The reported system is closely\nrelated to previously published works by the same group and others (e.g., Mu W., et al., Nat. Chem., 2024,\n16, 158-167). The main novelty, the transition from coacervate droplets to vesicles, has also been reported\nby others (e.g., Choi H., et al., Adv. Sci., 2024, 11, 2305978; Wen P., et al., Nat. Chem., 2025). Moreover,\nthe manuscript does not provide new mechanistic insights or broaden the generality of the observed\nphenomena.\nResponse: We thank the reviewer for the comments.\nWe thank the reviewer for their thoughtful evaluation and for acknowledging that the manuscript is\nwell written and addresses an interesting topic. We respectfully understand the concern regarding the\nnovelty and originality of our work.\nIn this study, we presented a system of liquid crystalline (LC) coacervate droplets that underwent a\ndistinct morphological transformation via a helicoid pattern-featured vacuolization process. This\ntransformation—from highly ordered LC droplets to less ordered coacervate vesicles—was triggered either\nby enzymatic hydrolysis of polysaccharides or by environmental stimuli such as salt or acid. We have\nemphasized in the revised manuscript that these LC coacervate droplets are structurally and mechanistically\ndistinct from conventional multilamellar vesicles.\nR4\nWe would also like to clarify that this work significantly differs from our previous publication (Nat.\nChem. 2024, 16, 158), where we developed protocell networks capable of facilitating localized biochemical\nreactions, directional molecular translocation, and topographical reconfiguration. That work did not involve or\nreport the unique structural transformation of LC coacervate droplets observed in the current study.\nMoreover, while previous studies referenced by the reviewer (e.g., Choi H. et al., Adv. Sci. 2024, 11,\n2305978; Wen P. et al., Nat. Chem. 2025) reported the transformation of coacervate microdroplets to\nvesicles via interfacial reorganization, the mechanism proposed in our study is fundamentally different. Our\nobservations revealed a helicoid-patterned vacuolization mechanism, which, to the best of our knowledge,\nhas not been previously described.\nIn response to the reviewer’s comment, we have revised the manuscript to more clearly highlight these\nnovel aspects, particularly in the introduction and discussion sections, to better distinguish our work from\nprior studies.\nTaken together, we believe these revisions and clarifications reinforce the novelty and significance of\nour study and demonstrate that it meets the publication standards of Nature Communications.\nMajor Comments:\n1. Origin of Life Claims: The assertion that this work contributes to the understanding of the origin of life is\nnot well supported. The investigated system is highly artificial, and the rely on enzymatic catalysis, which is\nnot prebiotic. The generalization of the observed phenomena to other prebiotic systems is also limited. This\nassertion that this work contributes to the understanding of the origin of life is not well supported. The\ninvestigated system is highly artificial, and the morphological transitions rely on enzymatic catalysis, which is\nnot prebiotic. The generalization of the observed phenomena to other prebiotic systems is also limited.\nTherefore, references to the origin of life (lines 27, 43 and 67) should be removed.\nResponse: We thank the reviewer for the suggestion. The references are now removed.\n2. Phase Diagram Characterization: ① Figure 1c shows a phase diagram based solely on turbidity measurements, which\ncannot differentiate between liquid crystalline droplets, isotropic droplets, or vesicles. Microscopy observations should be\nincluded to better characterize the phase diagram. ② Specifically, it would be helpful to investigate morphological transitions\nas a function of the Cm-Am/DDAB ratio. ③ Supplementary Figure S11 shows different morphologies outside the phase\ndiagram range reported in Figure 1c. Expanding the phase diagram to include up to 20 mM Cm-Am and DDAB and\ncharacterizing the droplets using polarized optical microscopy, without amylase, would be necessary.\nResponse: We thank the reviewer for the constructive comments.\n① Though the phase diagram in Figure 1c was presented by turbidity, the coacervation behaviours of each\nsample were assessed by optical microscopy. The detailed description has been provided in SI (Page 5,\nParagraph 5). Additionally, we supplemented fluorescence and polarized optical microscopy to differentiate\nliquid crystalline droplets, isotropic droplets, or vesicles in the phase diagram. The data have been added in\nSI (new Figure S2).\nRevised text, Page 3, Paragraph 3\nThe formation of Cm-Am/DDAB LC coacervate microdroplets was observed across a broad concentration\nrange, as illustrated by a phase diagram (Fig. 1c and Supplementary Fig. S2), exhibiting enhanced\ncoacervation at higher concentrations.\nNew Figure S2\nR5\nSupplementary Figure S2. (a,b) Polarized (top) and fluorescence microscopy images (bottom) showing\nisotropic and LC coacervates with total concentrations of 10 mM (a) or 20 mM (b) were formed under\nvaried Cm-Am/DDAB monomer molar ratio. Specifically, the isotropic coacervates with total\nconcentration of 10 mM (doped with NBD-PE) were formed under the monomer molar ratio higher than\n2:1 or lower than 1:3 and LC coacervates were generated under the monomer molar ratio ranged from 1:1\nto 1:2 (a), while the isotropic coacervates with total concentration of 20 mM were formed under the\nmonomer molar ratio higher than 8:1 or lower than 1:4 and LC coacervates were generated under the\nmonomer molar ratio ranged from 3:1 to 1:2 (b). Scale bars, 5 μm.\n② As shown in Supplementary Figure S15, the Cm-Am/DDAB ratio affects the morphological\ntransformation. At monomer molar ratios of Cm-Am/DDAB ranging from 8:1 to 1:4, the transformation\nfollowed a characteristic process involving helicoid pattern formation. By contrast, when the molar ratio\nexceeded 8:1 or fell below 1:4, the helicoid pattern was no longer observed. This result has been discussed\nin the main text (Page 10, Paragraph 2).\n③ As suggested by the reviewer, we performed the experiments with increased concentrations of Cm-\nAm/DDAB LC coacervates. The data have been included in the main text (Figure 1c) and SI (new Figure\nS2).\nRevised method, Supplementary Information, Page 5, Paragraph 5\nTurbidity diagram. The turbidity diagram was measured by mixing different concentrations of Cm-Am (5\nmM, 10 mM, 20 mM, 40 mM, 50 mM, pH 8) and DDAB (10 mM, 20 mM, 30 mM, 40 mM, pH 8) solutions\nwith different monomer molar ratios.\nRevised Figure 1c\nR6\n3. Critical Salt Concentration Measurements: ① The description of critical salt concentration\nmeasurements is unclear (SI lines 224–238). ② The reported procedure results in NaCl concentrations of\n429, 353, 188, and 97 mM, which do not align with the data presented in Figure 4o or the experiments in\nFigure S17. ③ Please, clarify how the phase diagram in Figure 4o was obtained, the waiting time for salt\nconcentration equilibration, and the definition of critical salt concentration.\nResponse: We thank the reviewer for the comment.\n① The description of critical salt concentration measurements has been revised to improve clarity and\nreadability in SI (Page 8, Paragraph 4).\nRevised method, Supplementary Information, Page 8, Paragraph 4\nCritical salt concentration of LC coacervate microdroplets. To evaluate the stability of the LC coacervate\nmicrodroplets, defined amounts of sodium chloride were added, and the ability to maintain coacervate\nformation was assessed using optical microscopy. An increase in salt concentration led to the dissolution\nof the LC coacervates. The minimum NaCl concentration at which complete dissolution occurred was\ndefined as the critical salt concentration. Typically, 50 µL of Cm-Am/DDAB LC coacervate\nmicrodroplets loaded with HRP (5 µL, 5 mg/mL) were obtained following the above procedures. Then, 30 µL of the LC\nmicrodroplets was added to a sample cell with a glass coverslip, settling for ca. 10 min. Subsequently, 4 µL\nof sodium chloride with a concentration of 3 M was added and the disassembly process was recorded by\nconfocal laser scanning microscopy.\nSimilarly, 50 µL of the Cm-Am/DDAB LC microdroplets loaded with ssDNA (99 nt, 2 µL, 50 μM) or\ndextran (10k Da, 5 µL, 5 mg/mL) were prepared following the abovementioned method. To a solution of\nR7\nthe LC coacervate suspension (30 µL), varied amounts (5 µL, 2 µL, or 1 µL) of sodium chloride (3 M) was\nadded and the formation of LC coacervate microdroplets was examined by confocal laser scanning\nmicroscopy.\nThe critical salt concentrations for both the rims and the inner organelles were determined by\ngradually adding sodium chloride to suspensions of yolk-shell coacervate vesicles. The concentration at\nwhich only the rims disassembled while the internal organelles remained intact was defined as the critical\nsalt concentration of the rims. In contrast, the concentration at which both rims and organelles were\ndissolved was defined as the critical salt concentration of the organelles.\nSimilarly, the critical salt concentrations of the rims and the inner organelles were measured by\ngradually adding sodium chloride to the yolk-shell coacervate vesicle suspensions, wherein the\nconcentration of sodium chloride for only disassembling rims with concomitant maintenance of intact\norganelles was recorded as critical salt concentration of rims, and that for dissolving both rims and\norganelles was recorded as critical salt concentration of organelle. Specifically, 1 µL of sodium chloride\n(300 mM) were added into 30 µL of yolk-shell coacervate vesicles which were obtained after the\nreconfiguration of HRP-integrated Cm-Am/DDAB LC microdroplets. After 30 min of salt concentration\nequilibration period, 5 µL of sodium chloride (300 mM) was continuously added into the yolk-shell\ncoacervate vesicles. The disassembly process was recorded by confocal laser scanning microscopy.\n② Figure 4o showed the effects of sodium chloride on LC coacervates, in which biomolecules (i.e., HRP,\nssDNA, and dextran) were not present. By contrast, Figure S25 revealed the critical amount of salt required\nfor disassembling LC coacervates, in which biomolecules (i.e., HRP, ssDNA, and dextran) were loaded.\nWe have accordingly revised the main text (Page 12, Paragraph 2; Page 13, Paragraph 2) and SI (Figures\nS25, S29).\nRevised text, Page 12, Paragraph 2\nThe resulting critical salt concentrations were 353 mM, 429 mM, and 188 mM for the LC droplets with\nincorporated HRP, ssDNA (99 nt), and dextran (10k Da), corresponding to 3.6-, 4.4-, or 1.9-fold increases\ncompared to 97 mM of that for the LC droplets without biomolecules (Supplementary Fig. S17).\nRevised text, Page 13, Paragraph 2\nSpecifically, the critical salt concentration in the rim and the organelles was determined to be 9.7 mM and\n50 mM, respectively.\nRevised Figure S25\nR8\nSupplementary Figure S25. The critical salt concentration of initial Cm-Am/DDAB LC coacervate\nmicrodroplets with or without biomolecules. (a-d) Time series of optical microscopy images showing the\ncritical salt concentration of the LC coacervates (total concentration of 10 mM, monomer molar ratio of\n1:1) with incorporated HRP (a), ssDNA (99 nt) (b), or dextran (10k Da) (c) was 353 mM, 429 mM, or 188\nmM of sodium chloride, respectively, while that of the LC droplets without biomolecules was 97 mM of\nsodium chloride (d). Scale bars, 5 1um.\nRevised Supplementary Figure S29\nSupplementary Figure S29. (a,b) Disassembly of yolk-shell coacervate vesicles with the addition of NaCl: (a)\n9.7 mM; (b) 50 mM. The critical salt concentration of the rim was 9.7 mM of sodium chloride (a), while\nthat of the inner organelle was 50 mM of sodium chloride (b). (c,d) Fluorescence recovery photobleaching:\n(c) FITC-PEI on the rim and (d) FITC-PEI on the internalized organelle. HRP (0.5 mg/mL) was loaded in\nCm-Am/DDAB LC coacervates (total concentration of 10 mM, monomer molar ratio of 1:1). Scale bars, 5\n1um.\n③ We have added the experimental methods used to obtain the diagrams in Figure 4o and Figure S18,\nincluding the equilibration time for salt concentration (30 minutes) and the definition of the critical salt\nconcentration, defined as the minimum amount of salt required to dissolve the LC coacervates.\nNew method, Supplementary Information, Page 7, Paragraph 6\nDiagram of LC coacervate microdroplets with the addition of sodium chloride or hydrochloric acid.\nTypically, 30 µL of Cm-Am/DDAB LC coacervate suspension prepared as the above procedure was added\nto a sample cell with a glass coverslip, settling for ca. 10 min. Then, different doses of sodium chloride\n(1500 mM) were added into the LC coacervates, respectively, and the changes of LC coacervates were\nmonitored by confocal laser scanning microscopy. When the assemblies almost remained unchanged, we\nset a period of 30 min for salt concentration equilibration and then the ultimate structures were recorded.\nSimilarly, the effects of different concentrations of hydrochloric acid on LC coacervates were also explored\nfollowing the above procedures except using varied doses of hydrochloric acid (500 mM).\n4. Structural Characterization of Coacervate Vesicles: ① the structural properties of the coacervate vesicle\nphase and the internal organelle are not sufficiently investigated. Some images (Figures 3f, 4j, and S11)\nindicate birefringence, suggesting that the vesicle phase may be liquid crystalline. However, the schematic\ndiagrams in Figures 1b and 3c depict vesicles and internal coacervates as solutions of micelles, poly-anions,\nand guest biomolecules, which may not accurately represent the observed phases. Further investigation using\npolarized microscopy would provide more insight into the structural organization of\nR9\nboth compartments. ② The intriguing helicoidal pattern transition is not structurally characterized or\nunderstood.\nResponse: We thank the reviewer for the constructive comments.\n① The polarized microscopy images in Figures 3f and 4j indicated isotropic features of H-Cm-Am 2/DDAB\ncoacervate vesicles and Cm-Am/DTAB coacervates since the observed ring-like optical contrast was\nattributed to thin-film interference or slight refractive index mismatch at the assembly interfaces rather than\nbirefringence (Nat. Commun., 2023, 14, 2606; Curr. Opin. Colloid Interface Sci., 2021, 56, 101500). In\nFigure S15, the coacervates formed at Cm-Am/DDAB monomer molar ratios of 9:1, 8:1, 1:4, or 1:5 exhibited\nisotropic properties, whilst that formed at Cm-Am/DDAB monomer molar ratios of 3:1 or 1:2 displayed liquid\ncrystalline structures. As suggested, we characterized the coacervate vesicle and the internal organelle of\nyolk-shell coacervate vesicle under polarized optical microscopy, both exhibiting isotropic properties. We\nhave added the data in Figures S14 and S31. As reported in the references (Front. Soft Matter, 2022, 2,\n991375; Sci. Rep., 2022, 12, 12676; ACS Appl. Mater. Interfaces, 2022, 14, 31, 36277), the liquid crystalline\nassemblies generally exhibited a characteristic texture with a cross and multiple concentric rings, wherein\nlong-range orientational order existed between anisotropic molecules. In contrast, the isotropic assemblies\ngenerally lacked an internal texture and presented optical contrast at the edge due to curvature-enhanced\ninterference or scattering effects, in which short-range orientational order appeared between isotropic\nmolecules. Therefore, in Figures 1b and 3c, the isotropic coacervate vesicles or yolk-shell coacervate vesicles\nwere depicted as disordered assembly of micelles, poly-anions, and guest biomolecules. The results have been\ndemonstrated in the main text (Page 8, Paragraph 2).\nRevised text, Page 8, Paragraph 2\nUpon the addition of amylase, the molecular weight decreased sharply from 40k Da to 5k Da within 300 s,\nconsistent with transformation of LC coacervates into helicoid-structured vesicles at vacuolization stage\n(225 s) (Fig. 2a) and the diameter of resulted assemblies reached a plateau at 300 s (Fig. 2d). Ultimately,\nthe Cm-Am hydrolysis reached equilibrium accompanied with the generation of isotropic coacervate\nvesicles at coalescence stage (Supplementary Fig. S14).\nNew Figure S14\nSupplementary Figure S14. Fluorescence (left) and polarized optical microscopy images (right) revealed\nthe formation of isotropic coacervate vesicles (doped NBD-PE) after the addition of amylase (1.5 mg/mL)\nto Cm-Am/DDAB LC coacervates (total concentration of 10 mM, monomer molar ratio of 1:1). Scale bar,\n5 μm.\nRevised Figure S31\nR10\nSupplementary Figure S31. Optical (left) and polarized microscopy images (right) of an H-Cm-Am/DDAB\nyolk-shell coacervate vesicle obtained after the reconfiguration of HRP-integrated Cm-Am/DDAB LC\ncoacervate microdroplet (total concentration of 10 mM, monomer molar ratio of 1:1) displayed the\ndisappearance of birefringence phenomenon. It indicated isotropic structures in the rim and included\nartificial organelle within the yolk-shell coacervate vesicle. Scale bar, 5 1um.\n®\nThe helicoidal pattern transformation has been characterized by polarized optical microscopy as shown\nin Fig. 3b. Specifically, the liquid crystalline droplets at vacuolization stage firstly transformed into helicoid-\nstructured vesicles which displayed ordered liquid crystalline structures, in which low-degree hydrolysis of\nCm-Am occurred. The helicoid-structured vesicles at coalescence stage continuously transformed into\ncoacervate vesicles which exhibited isotropic structures. The lack of LC feature is due to the high-degree\nhydrolysis of Cm-Am that weakens the charge complexation. These findings have been demonstrated in the\nmanuscript (Page 8, Paragraph 2).\n5. Enzyme-Mediated Reconfiguration Procedure: ① The description of the enzyme-mediated\nreconfiguration (SI, lines 182–187) is unclear regarding the tested conditions. ® Assuming an initial total\ncharge concentration of 10 mM (5 mM from DDAB and 5 mM from Cm-Am), adding amylase (5.3 1uL to\n30 1uL of coacervate solution) reduces the concentration to 8.5 mM, which, according to Figure 1c, would\nlead close to the single-phase region due to dilution. ③ To confirm that the morphological transition is not\nsolely due to dilution, the authors should test higher initial total charge concentrations (e.g., 15 mM or 20\nmM) and provide negative controls.\nResponse: We thank the reviewer for the constructive comments.\n①\nWe have clarified the tested conditions for the enzyme-mediated reconfiguration to in SI (Page 6,\nParagraph 4).\nRevised method, Supplementary Information, Page 6, Paragraph 4\nThe amylase-mediated transformation of LC coacervates with higher concentrations was explored\nfollowing the above procedures except using 15 mM or 20 mM of Cm-Am/DDAB LC coacervate microdroplets\n(30 µL, monomer molar ratio of 1:1). The amylase-mediated reconfiguration behaviours for other LC or\nisotropic coacervate microdroplets were investigated following similar procedures except using different doses\nof amylase (10 mg/mL). Specifically, 3 µL of amylase (10 mg/mL) was added to Cm-Am/DTAB isotropic\ncoacervate microdroplets (30 µL, total concentration of 50 mM, monomer molar ratio of 1:1), Cm-Am/MTAB\nisotropic microdroplets (30 µL, total concentration of 50 mM, monomer molar ratio of 1:1), or Cm-Am/DMAB\nLC coacervate droplets (30 µL, total concentration of 5 mM, monomer molar\nratio of 1:1). 1 µL of amylase (10 mg/mL) was added to Cm-dextrin/DDAB LC microdroplets (30 µL, total\nconcentration of 10 mM, monomer molar ratio of 1:1). 2 µL of amylase (10 mg/mL) was added to Cm-\nAm/PAH isotropic microdroplets (30 µL, total concentration of 10 mM, monomer molar ratio of 2:1).\n®\nUnder total coacervate concentration of 8.5 mM (i.e., 30 μL of coacervates with total concentration of\nR11\n10 mM diluted by 5.3 μL of amylase), the optical density of coacervate system was measured to be 0.94 as\nshown in Figure 1c. This high OD indicated the formation of coacervate microdroplets.\nRevised caption, Figure 1\n(c) Phase diagram illustrating the formation of microdroplets at varying concentrations of Cm-Am and\nDDAB. The coacervation region as determined by optical microscope.\n③ As suggested, we studied the LC coacervates with total concentrations of 15 mM or 20 mM and\nobserved structural transition from LC coacervates to coacervate vesicles with the addition of amylase. We\nhave added the data and corresponding discussion to the manuscript (Page 5, Paragraph 2) and SI (Page 6,\nParagraph 4; new Figure S10).\nRevised text, Page 5, Paragraph 2\nReconstructed three-dimensional (3D) images confirmed the occurrence of vacuolization and the appearance\nof helicoidal structure during the LC coacervate microdroplet-to-coacervate vesicle transition, accompanied\nby an obvious size expansion (Fig. 2f,g). Such reconfiguration process was also achieved in\nthe LC coacervates with higher total concentrations (e.g., 15 mM or 20 mM) (Supplementary Fig. S10).\nUpon the addition of PBS buffer (10 mM, pH 6.9, containing 1 mM of CaCl ), the Cm-Am/DDAB LC\n2\ncoacervates exhibited negligible changes. Furthermore, the helicoid patterns observed in the intermediate\nstructures were predominantly left-handed, occurring in approximately 80% of cases (Fig. 2h).\nNew Figure S10\nSupplementary Figure S10. (a,b) Time sequence of fluorescence microscopy images revealing the\nmorphological transformation from LC coacervate microdroplets to coacervate vesicles. The total\nconcentrations of Cm-Am/DDAB LC coacervates were 15 mM (a) and 20 mM (b), respectively. Scale bars,\n5 μm.\n6. Confinement of Reactions in Organelle-Containing Vesicles: I agree with the sentence that “The ability\nconfining endogenous metabolic reactions within organelle-containing coacervate vesicles was crucial for the\nfunctionality of artificial cells”. Anyway, for a sub-compartment to be really an organelle it would require that\nthe reactions were confined in the organelle only. This is not convincingly demonstrated. The biomolecule\npartitioning between the inner organelle and vesicle rim appears similar, and hammerhead ribozyme catalysis\noccurs in both phases without significant differences. Thus, the conclusion that the system represents a\nprotocell model capable of evolving complex compartments (lines 392–394) is not fully\nR12\nsupported.\nResponse: We thank the reviewer for the constructive comments. We have revised the text in Conclusion\n(Page 15, Paragraph 2).\nRevised text, Page 15, Paragraph 2\nImportantly, the incorporation of biomolecules, such as proteins and nucleic acids, facilitated the\nmorphological transformation into coacervate vesicles embedding a coacervate droplet which structurally\nresembled artificial organelles capable of supporting biochemical reactions.\nMinor Comments:\n1. Caption of Figure S11, S13 and other SI figures: The addition of amylase is often not mentioned. Please\nwrite it where needed.\nResponse: We thank the reviewer for the comments. We have included the information of amylase addition\nin the main text (captions of Figure 3-5) and SI (captions of Figure S7, S12, S15, S17, S30).\nRevised caption, Figure 3\n(b) Time series of POM images showing the structural transition from LC coacervates to isotropic\ncoacervate vesicles initiated by amylase (1.5 mg/mL).\nRevised caption, Figure 4\nFigure 4. Structural transition of LC microdroplets under varied conditions. (a-l) Schematic\nrepresentations (a,e,i), POM images (b,f,j), SAXS profiles (c,g,k), and time-lapse optical (d,h) or fluorescence\nimages (l) depicting Cm-Am/DMAB (a-d) or Cm-dextrin/DDAB LC microdroplets (e-h) transformed into\ncoacervate vesicles via helicoid pattern-featured vacuolization in the presence of amylase (0.9 or 0.3 mg/mL),\nwhile isotropic Cm-Am/DTAB microdroplets (doped with RITC-Cm-Am) (i-l) reconfigured into coacervate\nvesicles without helicoid inner patterns after adding amylase (0.9 mg/mL).\nRevised caption, Figure 5\nFigure 5. LC coacervate microdroplets with integrated biomolecules reconfigured into isotropic yolk-shell\ncoacervate vesicles. (a) Scheme showed the transformation of LC coacervate microdroplets with integrated\nproteins or nucleic acids into organelle-containing coacervate vesicles induced by the hydrolysis of Cm-Am.\n(b,c) Time-lapse fluorescence images displaying LC microdroplets with the incorporation of RITC-BSA (0.3\nmg/mL) (b) or TAMRA-ssDNA (99 nt, 1.0 μM) (c) reconfigured into yolk-shell coacervate vesicles initiated\nby amylase (0.9 mg/mL). (d,e) Time sequence of fluorescence images exhibited homogenous distribution of\nRITC-Cm-Am during the reconfiguration of HRP-integrated LC microdroplets (d), while the LC\nmicrodroplets with integrated FITC-dextran (10k Da, 0.5 mg/mL) transformed into coacervate vesicles in\nthe presence of amylase (0.9 mg/mL) (e).\nRevised caption, Figure S7\nSupplementary Figure S7. (a,b) CLSM images showing Cm-Am/DDAB LC coacervate microdroplets (total\nconcentration of 10 mM, monomer molar ratio of 1:1, doped NBD-PE) vacuolized into intermediates with\nhelicoid inner patterns (a) and ultimately transformed into H-Cm-Am/DDAB coacervate vesicles initiated\nby amylase (1.5 mg/mL) (b). Scale bars, 5\nm.\nRevised caption, Figure S12\nR13\nSupplementary Figure S12. Time sequence of optical (top) and fluorescence microscopy images (bottom)\ndisplaying a Cm-Am/DDAB LC coacervate microdroplet (total concentration of 10 mM, monomer molar\nratio of 1:1, doped NBD-PE) vacuolized into a helicoid pattern-containing intermediate after the addition\nof amylase (1.5 mg/mL), followed by the rupture of coacervate vesicle due to the sharp decrease in size.\nScale bar, 5 1um.\nRevised caption, Figure S15\nSupplementary Figure S15. (a-f) Time-dependent polarized optical and fluorescence microscopy images\nshowing disordered isotropic Cm-Am/DDAB coacervate microdroplets (total concentration of 20 mM, doped\nwith HPTS) prepared at Cm-Am/DDAB monomer molar ratio of 9:1, 8:1, 1:4, and 1:5 transformed into\ncoacervate vesicles without the emergence of helicoid patterns in the vacuolized intermediates after adding\namylase (a,b,e,f), while ordered LC microdroplets generated under Cm-Am/DDAB monomer molar ratio of\n3:1 and 1:2 reconfigured into coacervate vesicles via helicoid pattern-featured vacuolization with\nbirefringence in the presence of amylase (1.5 mg/mL) (c,d). Scale bars, 5 1um.\nRevised caption, Figure S17\nSupplementary Figure S17. (a-f) Schematic illustrations (a,d), polarized optical microscopy images (b,e),\nand time-lapse fluorescence microscopy images (c,f) showing disordered isotropic Cm-Am/MTAB\ncoacervate microdroplets (total concentration of 50 mM, monomer molar ratio of 1:1, doped with RITC-\nCm-Am) (a-c) or Cm-Am/PAH microdroplets (total concentration of 50 mM, monomer molar ratio of 2:1)\n(d-f) transformed into coacervate vesicles without experience of helicoid pattern-featured vacuolization\nprocess in the presence of amylase (0.9 or 0.6 mg/mL). Scale bars, 5 m.\nRevised caption, Figure S30\nSupplementary Figure S30. (a,b) Time-lapse fluorescence microscopy images displaying Cy5-HH-min (0.8\n1uM) (a) and FAM-substrate (0.8 1uM) (b) were both homogenously distributed in Cm-Am/DDAB LC\ncoacervate microdroplets and the resultant LC coacervates were capable of transforming to organelle-\ncontaining coacervate vesicles via helicoid pattern-featured vacuolization process initiated by amylase (0.9\nmg/mL). Scale bars, 5 1um.\n2. Figure 4o: Clarify the meaning of “droplet concentration.” If it refers to total charge concentration,\nupdate the label accordingly.\nResponse: We thank the reviewer for the comments. We have revised the label in Figure 4o and S18.\nRevised Figure 4\nR14\nRevised Figure S18\n3. Helicoidal Pattern Transition: Explain why the helicoidal pattern transition also occurs with Cm-dextrin,\ndespite the disturbance of the liquid crystalline phase by short H-Cm-Am.\nResponse: We thank the reviewer for the comments. DDAB/Cm-dextrin (Mw = 10k Da) can form liquid\ncrystalline coacervate microdroplets which underwent helicoidal pattern transformation in the presence of\namylase as shown in Figure 4h. As evidenced by GPC analysis and POM images in Figure 3a,b, the liquid\ncrystalline phase disappeared when the molecular weight of short H-Cm-Am was lower than 5k Da. This\ndiscussion has been included in the manuscript (Page 8, Paragraph 2) as shown in the responses to Q4 of\nmajor comments.\nR15\n4. Biomolecule-Induced Transition: lines 296–306: Clarify whether coacervate droplets transition to isotropic\nor remain liquid crystalline upon biomolecule addition. Provide polarized microscopy images in addition to\nfluorescence images to assess the internal organelle’s phase (see Supplementary Figure S24). Response: We\nthank the reviewer for the suggestion. The coacervates maintained LC feature with the addition of\nbiomolecules. We also supplemented polarized microscopy images for coacervates with integration of\nbiomolecules, which exhibited liquid crystalline structures. The internal organelle in the yolk-shell coacervate\nvesicles displayed isotropic structure as shown in the response to Q4 of major comments. The corresponding\ndiscussion and data have been added in the manuscript (Page 12, Paragraph 1; Page 13, Paragraph 3) and SI\n(new Figure S21).\nRevised text, Page 12, Paragraph 1\nThe resulting coacervates maintained liquid crystalline structures and predominantly transformed into\ncoacervate vesicles with an internal coacervate organelle (i.e., yolk-shell coacervate vesicles) in the\npresence of amylase (Supplementary Figs.S21,S22), wherein the yolk-shell coacervate vesicles possessed\nthicker rims (i.e., 0.4 ± 0.1 1um) by compared to thinner membranes of multilamellar vesicles (i.e., several\nnanometres) and the strong enrichment ability in contrast to controllable permeability of vesicles\n(Supplementary Fig.S23).\nNew Figure S21\nSupplementary Figure S21. Fluorescence (top) and polarized microscopy images (bottom) indicating Cm-\nAm/DDAB coacervates (total concentration of 10 mM, monomer molar ratio of 1:1) remained LC\nstructures upon the integration of RITC-BSA (a), TAMRA-ssDNA (b), or FITC-dextran (c). Scale bars, 5\n1um.\n5. Vacuolized Intermediates: Indicate whether the vacuolized intermediates shown in Figure S11 are\nbirefringent.\nResponse: We thank the reviewer for the comments. No birefringence was observed in the vacuolized\nintermediates for Cm-Am/DDAB systems when the monomer molar ratio exceeded 8:1 (e.g., 9:1) or fell\nbelow 1:4 (e.g., 1:5). By contrast, birefringence was observed in the vacuolized intermediates when Cm-\nAm/DDAB molar ratios were between 8:1 to 1:4. We have added new data in Figure S15.\nRevised Figure S15\nR16\nSupplementary Figure S15. (a-f) Time-dependent polarized (top) and fluorescence microscopy images\n(bottom) showing disordered isotropic Cm-Am/DDAB coacervate microdroplets (total concentration of 20\nmM, doped with HPTS) prepared at Cm-Am/DDAB monomer molar ratio of 9:1, 8:1, 1:4, and 1:5\ntransformed into coacervate vesicles without the emergence of helicoid patterns in the vacuolized\nintermediates after adding amylase (1.5 mg/mL) (a,b,e,f), while ordered LC microdroplets generated under\nCm-Am/DDAB monomer molar ratio of 3:1 and 1:2 reconfigured into coacervate vesicles via helicoid\npattern-featured vacuolization with birefringence in the presence of amylase (1.5 mg/mL) (c,d). Scale bars, 5 μm.\n6. In summary, while the manuscript presents an interesting study, the lack of sufficient novelty and\nmechanistic insights limits its suitability for Nature Communications. Addressing the points above would\nstrengthen the manuscript for consideration in other journals.\nResponse: We thank the reviewer for their time and thoughtful evaluation of our manuscript. We appreciate the\nrecognition of the interest and relevance of our study. While we understood the concerns regarding the level of\nnovelty and mechanistic insight, we respectfully noted that our work presented new findings on protocell\ndroplets formed from LC coacervates, which exhibited complex and dynamic structural transformations. To our\nknowledge, this behaviour has not been systematically explored in previous literatures. In response to the\nreviewer’s comments, we have made substantial revisions to the manuscript to more clearly articulate the\nconceptual advances and to strengthen the mechanistic interpretation of our results. We believe these\nimprovements better convey the significance of our findings and provide a clearer\nR17\nframework for understanding the underlying processes.\nResponses to Reviewer #3\nThe manuscript titled “Enzymatically reconfigurable liquid crystalline coacervate microdroplets as\nprotocell models” authored by Jia et al. describes the formation and transition of liquid crystalline\ncoacervates to helicoidal vesicles and membranous protocells that contain organelles. The droplets are\nprepared from Cm-Am and DDAB an anionic polyelectrolyte and a cationic surfactant respectively and the\ntransition is driven by amylase action on the Cm-Am polyelectrolyte. The manuscript is well presented and\ndescribes an enzymatically driven phase transition in compartments to form droplets of different structures.\n① However, the authors should check the interpretation of the data and the claim that coacervate vesicles\nhave been formed. The results suggest that Cm-Am/DDAB droplets are multilamellar vesicles and not\ncoacervates or coacervate vesicles. Coacervates are typically amorphous and do not exhibit structure. ②\nThe data further shows that the multilamellar vesicles transform upon addition with amylase to vesicles with\nan internal structure which could be interpreted as a coacervate or a surfactant droplet or smaller vesicle. If\nthe context of the data was presented correctly and better descriptions (supported by scientific data) of the\nstructures were given then this work could be suitable for publication. ③ Whilst there have been examples\npreviously of transitions between multilamellar vesicles to vesicles before (Langmuir, 2018, 34, 5, 2171-2179\namongst other papers), to the best of my knowledge this is the first example of a dynamic system i.e. where an\nenzyme drives the structural transition.\nResponse: We thank the reviewer for these comments.\n① Based on the following analysis, we proposed that Cm-Am/DDAB droplets were LC coacervates and\ntransformed into coacervate vesicles with the addition of amylase.\na) Multilamellar vesicles were spherical structures composed of multiple concentric phospholipid\nbilayers, resembling an onion-like architecture. Our droplets did not exhibit a hollow structure with an\naqueous inner phase. When we added hydrophobic fluorophobic (e.g., NBD-PE), the whole droplets were\nstained (Figure S6), suggesting hydrophobic domains were evenly distributed. By contrast, the inner pool\nof multilamellar vesicles was not supposed to encapsulate hydrophobic fluorophores. b) Although\ncoacervates were mostly amorphous, ordered LC coacervates have been reported in the references (e.g.,\nNat. Commun., 2023, 14, 2606; ACS Nano, 2020, 14, 15071; Nat. Chem., 2024, 16, 158) which were all\ncited in the main text (Page 2, Paragraph 2; Page 3, Paragraph 2; Page 9, Paragraph 1). These systems were\ntypically formed using mixed surfactants, surfactant/salt, or surfactant/DNA combinations, where\nhydrophobic interactions played a central role in coacervate formation. This represented a key distinction\nfrom complex coacervates, which were driven solely by electrostatic attractions. c) A key feature of\ncoacervates is their ability to efficiently sequester biomolecules such as proteins (Figure 5b, c, e, and S20).\nIn contrast, the uptake of proteins across the hydrophobic bilayers of multilamellar vesicles was relatively\nslow.\nSecond, coacervate vesicles were obtained after vacuolization of coacervates, displaying a fluidic layer\nas evidenced by FRAP experiment (Figure 3h) and a water-filled cavity. The liquid-like layer was thick\nwithout a multilamellar structure (new Figure S8) and was able to sequestrate biomolecules (Figure 5b, c, e,\nand S20). We also added the corresponding data in the manuscript (Page 5, Paragraph 2).\nRevised text, Page 5, Paragraph 2\nThe LC microdroplet with doped RITC-Cm-Am vacuolized into helicoidal vesicle as an intermediate with\nR18\nthick rim (i.e., about 0.4 ± 0.2 1um), which underwent coalescence, ultimately giving rise to a hollow vesicle\ndetermined as a coacervate vesicle due to the thicker rim (i.e., 0.3 ± 0.1 1um) in comparison to thinner\nmembranes in multilamellar vesicles (i.e., several nanometres) and strong enrichment capability unlike\ncontrollable permeability of vesicles (Fig. 2b, Supplementary Fig. S8, and Movie S3)54.\nNew Figure S8\nSupplementary Figure S8. (a,b) Statistical analysis of thickness for rims in helicoidal vesicles (a) or\ncoacervate vesicles (b) showed an average thickness of 0.4 ± 0.2 1um or 0.3 ± 0.1 1um, respectively. Number\nof helicoidal vesicles or coacervate vesicles measured, n = 200.\n② As evidenced by fluorescence tracking experiment in the main text (Page 12, Paragraph 1 and 2),\nthe internal structure within yolk-shell coacervate vesicles was composed of Cm-Am fragments, DDAB,\nand biomolecules, similar to the composition in the rim which was coacervate vesicle (Figure 5b-d). While the\ninternal organelles also exhibited molecularly crowded interior, liquid-like property as confirmed by FRAP\nexperiment, and strong enrichment ability (Figure 5b-f). Thus, the organelles were determined as\ncoacervates.\n③ As pointed out by the reviewer, previous studies demonstrated a structural transition from multilamellar\nto unilamellar vesicles upon peptide incorporation, which altered the membrane thickness and elasticity of\nthe multilamellar structures. In contrast, our work revealed a dynamic complex system that transformed\nfrom LC coacervate droplets to coacervate vesicles.\n1. Please define the following terms: liquid crystalline coacervate droplets, helicoidal vesicles, coacervate\nvesicles and microdroplets. This will provide clarity into how the data for the droplet structures are\ninterpreted. Please also keep the terminology consistent through the text.\nResponse: We thank the reviewer for the comments. Liquid crystalline coacervate droplets were the\ncoacervate droplets combined with ordered liquid crystalline structure and intrinsic liquid-like property of\ncoacervates formed by LLPS (Curr. Opin. Colloid Interface Sci., 2021, 56, 101500). Helicoidal vesicles in\nthis work were vacuolized coacervates featured with helicoidal inner patterns, which were generated during\nthe vacuolization of liquid crystalline coacervate droplets. Coacervate vesicles were assemblies with a liquid\nlayer and a water-filled cavity (Nat. Chem., 2025, 17, 279), which was obtained after the reconfiguration of\nLC or isotropic coacervate droplets. Coacervate microdroplets were isotropic coacervate droplets appeared\nin this work. We have kept the terminology consistent through the text. To provide a clear description, we\nadded relevant definitions in the main text (Page 3, Paragraph 2).\nRevised text, Page 3, Paragraph 2\nThe synergistic interactions of electrostatic attraction and the hydrophobic effect drove the complexation of\nCm-Am and DDAB, ultimately resulting in the formation of LC coacervate microdroplets which\nR19\nunderwent complex structural transition in the presence of amylase (Fig. 1b). To better understand\nalterations in structures, we defined assemblies related to the transformation. Specifically, LC coacervate\ndroplets possessed ordered LC structure and intrinsic liquid property of coacervates formed by LLPS51,52,\nwhile helicoidal vesicles represented vacuolized LC coacervates featured by helicoidal inner patterns,\na co n a d c ervate vesicles were assemblies with a liquid layer and a water-filled cavity45. Coacervate\nmicrodroplets were isotropic coacervate droplets.\n2. ① The authors describe the Cm-Am/DDAB coacervate droplets as LC coacervate microdroplets. The\ndata actually suggests that these are multilamellar vesicles given the maltese crosses. Thus, indicating that\nthey are not coacervates. ② Further, the data suggests that the addition of amylase transforms the\nmultilamellar vesicle into a vesicle with internal compartment. With the helicoidal structure a metastable\nstate that appears depending on the ratio of Cm-Am and DDAB. Some questions remain about the internal\ncompartment. What is the internal structuration of this? How can they confirm that is a coacervate and not\na surfactant droplet or a smaller vesicle.\nResponse: We thank the reviewer for the suggestion.\n① We acknowledge that LC coacervate droplets exhibited multilamellar features; they lacked a distinct\ninner aqueous core. No clear segregation of hydrophobic and hydrophilic domains was observed, as both\nhydrophobic and hydrophilic fluorophores were uniformly distributed throughout the droplets (Figure 1h,\nS5, and S6). Unlike multilamellar vesicles, these LC droplets readily underwent fusion, suggesting a more\ndynamic and less compartmentalized structure (Figure S3). Moreover, biomolecules such as proteins were\nable to efficiently penetrate and distribute evenly within the LC droplets (Figure 5b, c, e, and S20). In\ncontrast, the diffusion of hydrophilic biomolecules into multilamellar vesicles was hindered by the presence\nof hydrophobic lipid bilayers, which acted as a barrier to entry. Liquid crystalline coacervate microdroplets\nhave been reported in the references (e.g., Nat. Commun., 2023, 14, 2606; ACS Nano, 2020, 14, 15071;\nNat. Chem., 2024, 16, 158).\n② As demonstrated in the main text (Page 12, Paragraph 1), we proposed that LC coacervates transformed\ninto coacervate vesicles with an inner coacervate. The inner coacervates were derived from the original LC\ncoacervate droplets. We have added a new discussion to the manuscript (Page 12, Paragraph 1) and the\ncorresponding data to SI (new Figure S23).\nAs evidenced by fluorescence tracking experiments in main text (Page 12, Paragraph 1 and 2), the\ninternal compartments of yolk-shell coacervate vesicles were composed of Cm-Am fragments, DDAB,\nand biomolecules, similar to the compositions in the rims which were coacervate vesicles (Figure 5b-d).\nThe internal organelle also revealed molecularly crowded interior, liquid-like property as evidenced by FRAP\nexperiment, and strong enrichment ability (Figure 5b-f). Thus, the internal compartment was determined as\na coacervate.\nRevised text, Page 12, Paragraph 1\nThe resulting coacervates maintained liquid crystalline structures and predominantly transformed into\ncoacervate vesicles with an internal coacervate organelle (i.e., yolk-shell coacervate vesicles) in the\npresence of amylase (Supplementary Figs. S21,S22), wherein the yolk-shell coacervate vesicles possessed\nthicker rims (0.4 ± 0.1 μm) by compared to thinner membranes of multilamellar vesicles (i.e., several\nnanometres) and the strong enrichment ability in contrast to controllable permeability of vesicles\n(Supplementary Fig.S23).\nR20\nNew Figure S23\nSupplementary Figure S23. Statistical analysis of thickness for rims in yolk-shell coacervate vesicles\nshowed an average thickness of 0.4 ± 0.1 μm. Number of samples measured, n = 200.\n3. SAX data and EM should be used to confirm the location of the Cm-Am within the bilayers via electron\ndensity mapping. As well as the presence of bilayers within the LC coacervate and the yolk shell system\nResponse: We thank the reviewer for the constructive suggestion. As shown in the manuscript (Page 3,\nParagraph 3), SAXS profile of Cm-Am/DDAB LC coacervates (Figure 1g) showed the periodic molecular\nspacing was 3.55 nm which was slightly larger than the bilayer thickness of DDAB (i.e., approximately 2.45\nnm). This suggested that the periodic layer consisted of DDAB and Cm-Am, in which Cm-Am might locate\nbetween adjacent DDAB bilayers (Figure 1b). We also supplemented SAXS profile of yolk-shell coacervate\nvesicles, showing the disappearance of SAXS peaks. We have added a discussion in the manuscript (Page\n13, Paragraph 3) and the data in SI (Page 11, Paragraph 4; new Figure S32).\nAdditionally, according to cryo-TEM imaging (Figure RL1), the multilamellar structures of LC\ncoacervates were not clearly resolved due to the exceedingly thick of coacervate phase (i.e., several\nmicrometres) which was beyond electron penetration depth limit of cryo-TEM (i.e., less than a micrometre)\n(Curr. Opin. Struct. Biol., 2016, 37, 81). The coacervate vesicles with about 0.3-μm-thick rims (Figure S8)\nwere resolved by cryo-TEM, displaying uniform electron density contrast without multiple-bilayer structures.\nWe failed to obtain electron density mapping analysis because it cannot distinguish the location of Cm-Am in\nthe LC coacervates and yolk-shell coacervate vesicles due to the similar elemental compositions (i.e., carbon,\nhydrogen, and oxygen elements) of Cm-Am with DDAB and ice.\nAs evidenced by fluorescence tracking experiments in the main text (Page 4, Paragraph 2; Page 12,\nParagraph 1), Cm-Am was homogenously distributed in the LC coacervates and yolk-shell coacervate\nvesicles, while the multiple bilayers were not observed under fluorescence microscopy due to the limit of\nresolution.\nRevised text, Page 13, Paragraph 3\nMoreover, the RNA catalysis rates in yolk-shell coacervate vesicles were faster than that in the initial LC\nmicrodroplets (Fig. 5k,l), attributing to the higher molecular diffusivity in the isotropic yolk-shell coacervate\nvesicles. The lack of LC feature was confirmed by the disappearance of birefringence\nphenomenon in POM image and Bragg reflection peak in SAXS profile (Supplementary Figs. S31,S32).\nRevised method, Page 11, Paragraph 4\nSmall angle X-ray scattering (SAXS). SAXS experiments were performed at room temperature on Ganesha\nsystem 380 (SAXSLAB, U.S) equipped with a multilayer focused copper K radiation as the X-ray 381 source\na\n(Genix 3D Cu ULD) and a semiconductor with silver behenate for the small angle region and LaB6 for the\nwide angle region, respectively. Cm-Am/DDAB, Cm-Am/DMAB, Cm-dextrin/DDAB, and Cm-Am/DTAB\ncoacervate suspension prepared following above procedures were centrifuged at 9390 × g for\nR21\n10 min to remove the water phase and collect the coacervate phase for SAXS analysis.\nBSA-integrated Cm-Am/DDAB LC coacervates prepared following above procedures were incubated\nwith amylase for 1 h, leading to the generation of yolk-shell coacervate vesicles, followed by centrifugation\nat 9390 × g for 10 min to remove the water phase and collect the coacervate phase for SAXS analysis.\nNew Figure S32\nSupplementary Figure S32. SAXS profile of yolk-shell coacervate vesicles displayed no obvious Bragg\nreflection peak, indicating the disordered internal structures due to amylase-mediated disruption of\nperiodical stacking between Cm-Am and DDAB. The yolk-shell coacervate vesicles used here were\nobtained after the reconfiguration of BSA-integrated LC coacervates (total concentration of 10 mM,\nmonomer molar ratio of 1:1).\nThis data shows the presence of a small bragg peak.\nFigure RL1. (a,b) Cryo-TEM images of LC coacervates (a) and coacervate vesicles (b) both showing\nuniform electron density contrast without multiple-bilayer structures. Scale bars, 500 nm.\nIt is surprising that the LC coacervates show no bilayers. Can the samples be sliced to reduce the thickness\nof the sample?\n4. Does DDAB have a CVC on it’s own what is the bilayer spacing and what is this in comparison to Cm-\nAm and DDAB vesicles?\nResponse: We thank the reviewer for the comment. The CVC of DDAB was about 0.7 mM (Colloid Surf.\nB, 2003, 32, 365; Colloids Surf. A 2015, 480, 253). The corresponding bilayer spacing of DDAB vesicles\nwas about 2.45 nm (J. Phys. II France, 1993, 3, 487), which was slightly smaller than that of Cm-\nAm/DDAB LC coacervate microdroplets (i.e., 3.55 nm) as shown in Figure 1g, likely due to the periodic\nstacking of a DDAB bilayer and Cm-Am molecules in the LC coacervates as shown in Figure 1b. This\nresult has been provided in the main text (Page 3, Paragraph 3).\n5. What leads to the size expansion after the addition of amylase and what does this tell you about the\nmechanism of structural change?\nResponse: We thank the reviewer for the comment. Two factors may contribute to this result. First, the\nhydrolysis of Cm-Am into smaller fragments weakened the multivalent electrostatic complexation, leading to\nlooser packing of the lipid layers. Second, the hydrolysis products, being small and osmotically active,\nincreased the internal osmotic pressure, driving water influx and causing the multilayers to swell. We\nR22\nclarified the corresponding explanation in the manuscript (Page 5, Paragraph 2)\nRevised text, Page 5, Paragraph 2\nInitially, the LC microdroplets experienced intense vacuolization, resulting in the formation of an\nintermediate structure characterized by helicoidal inner patterns (225 s), followed by the second step, i.e.,\nexpansion of the assembly (375 s) caused by weakened the multivalent electrostatic complexation and\nincreased internal osmotic pressure during the hydrolysis of Cm-Am into small fragments (Fig. 2a,\nSupplementary Fig. S7, and Movie S2).\n0. ① Could the authors provide some more explanation of the significance of the FRAP data with\nregard to the organelle and the rim? ② How does the FRAP recovery compare to control experiments\ni.e. Cm-Am/DDAB alone or fragmented Cm-Am and DDAB. Does this give some insight to the chemical\ncomponents of rim and the organelle? ③ Can you show where Cm-Am is located? What does SAXs show?\nCan this confirm bilayers? The data suggests that it is an onion vesicle which transforms to another\nvesicle? Response: We thank the reviewer for the comment.\n①\nThe FRAP data reflected lager diffusion coefficient in the rim than that in the internal organelle. This\nresult suggested a less viscous microenvironment in the rims. We have included a discussion in the main\ntext (Page 13, Paragraph 2).\nRevised text, Page 13, Paragraph 2\nAdditionally, the diffusion coefficient of FITC-PEI within the rim was 1.4 × 10-14 m2/s, 1.8-fold higher than that\nin the organelle (i.e., 7.6 × 10-15 m2/s) (Fig. 5f), suggesting a molecularly more crowded environment\ninside the organelle.\n®\nAs demonstrated in the main text (Page 8, Paragraph 2), the fragmented Cm-Am (i.e., H-Cm-Am 2) and\nDDAB could form isotropic coacervate vesicles (Figure 3f). Thus, the diffusion coefficient of H-Cm-Am\n2/DDAB coacervate vesicles might be larger than that of Cm-Am/DDAB LC coacervates since the liquid\ncrystalline matrix of the microdroplets could impose the restrictions on passive diffusion, which was similar to\nthe FRAP experiments for the assemblies before and after transformation of LC coacervates (Figure 3h).\nThe diffusion coefficients were dependent on viscosity of the medium, which was related to the\ncomponents of medium (Soft Matter, 2020, 16, 114; J. Phys. Chem. B, 2005, 109, 6485; Macromolecules,\n2015, 48, 8608). Therefore, the differences in diffusion coefficients could roughly reflect that the components\nof assemblies were distinct, while it cannot determine the specific compositions. It was not reliable to propose\nthe composition of rim and the organelle according to diffusion coefficients. In contrast, we could infer the\nstructures of assemblies based on the known compositions and diffusion coefficients.\n®\nCm-Am was homogenously distributed in the rim and internal organelle of yolk-shell coacervate vesicles\n(Figure 5d). Due to the resolution limit of fluorescence microscopy, the specific molecular arrangements\ncannot be observed. The SAXS profile of yolk-shell coacervate vesicles showed the absence of Bragg\nreflection peak, indicating the disappearance of ordered structures. Thus, we proposed that LC coacervates\ntransformed into isotropic coacervate vesicles. The data have been shown in Q3 and also added in new Figure\nS32. Our work showed the transformation from an ordered liquid crystalline coacervates to yolk-shell\ncoacervate vesicles rather than that from onion vesicles to another vesicles.\n6. Provide reference papers to other droplets that form LC phases with coacervate forming components as\nR23\nwell as literature which discusses multilamellar vesicle to vesicle transitions. It could be useful to look at\nLangmuir, 2018, 34, 5, 2171-2179 amongst other papers.\nResponse: We thank the reviewer for the suggestion. We have cited the relevant references as suggested by\nthe reviewers, including Nat. Commun., 2023, 14, 2606; ACS Nano, 2020, 14, 15071; Nat. Chem., 2024, 16,\n158 as mentioned in the main text (Page 2, Paragraph 2; Page 3, Paragraph 2; Page 9, Paragraph 1). The\nfollowing literatures discussed multilamellar vesicle to vesicle transitions, namely, J. Phys.: Conf. Ser., 2011,\n272, 012011; J. Mol. Liq., 2019, 290, 111245; J. Phys. Chem. B, 2010, 114, 8056. The work mentioned by\nthe reviewer (Langmuir, 2018) was cited in the main text (Page 5, Paragraph 2).\n8. How can you confirm vacuolisation?\nResponse: We thank the reviewer for the comment. We confirmed vacuolisation by fluorescence\nmicroscopy images (Figure 2b, f) and movies (Movies S1, S2) of Cm-Am/DDAB LC coacervates\ntransformation process. To this end, RITC-Cm-Am and NBD-PE were added to localize Cm-Am and\nDDAB, respectively. We have provided a discussion in the main text (Page 5, Paragraph 2).\nRevised text, Page 5, Paragraph 2\nReconstructed three-dimensional (3D) images confirmed the occurrence of vacuolization and the\nappearance of helicoidal structure during the LC coacervate microdroplet-to-coacervate vesicle\ntransition, accompanied by an obvious size expansion (Fig. 2f,g).\n9. Show images from whole populations to get an idea of how robust this process is.\nResponse: We thank the reviewer for the constructive comment. We utilized large-scale microscopy\nimages and movies to represent the whole populations (Figure 3b, Figure 4d, h, n, Figure 5b, c, d, e, and\nFigure S20, Movies S2, S4-S10, S12). We also performed statistical analysis of the population profiles of\nthe final microstructures formed after the transformation of the entire Cm-Am/DDAB LC coacervate\nmicrodroplet population, with or without the integration of biomolecules (Figure S13, S22).\n3. For the critical salt concentration in the organelle and rim, please can you provide some explanation\nas to how the critical salt concentration was obtained?\nResponse: We thank the reviewer for the constructive comment. The critical salt concentrations of rims\nwere determined as the threshold concentration of sodium chloride that can disassemble the rims but not\nthe organelles. The critical salt concentrations of organelles were determined as the concentration of\nsodium chloride that can disassemble both rims and organelles. We have added this information in Methods\n(Page 9, Paragraph 3).\nRevised method, Supporting information, Page 9, Paragraph 3\nThe critical salt concentrations for both the rims and the inner organelles were determined by\ngradually adding sodium chloride to suspensions of yolk-shell coacervate vesicles. The concentration at\nwhich only the rims disassembled while the internal organelles remained intact was defined as the critical\nsalt concentration of the rims. In contrast, the concentration at which both rims and organelles were\ndissolved was defined as the critical salt concentration of the organelles.\nSimilarly, the critical salt concentrations of the rims and the inner organelles were measured by\ngradually adding sodium chloride to the yolk-shell coacervate vesicle suspensions, wherein the\nconcentration of sodium chloride for only disassembling rims with concomitant maintenance of intact\norganelles was recorded as critical salt concentration of rims, and that for dissolving both rims and\nR24\norganelles was recorded as critical salt concentration of organelle. Specifically, 1 µL of sodium chloride\n(300 mM) were added into 30 µL of yolk-shell coacervate vesicles which were obtained after the\nreconfiguration of HRP-integrated microdroplets. After 30 min of salt concentration equilibration period,\n5 µL of sodium chloride (300 mM) was continuously added into the yolk-shell coacervate vesicles.\n11. Please explain interaction strength? This is not given by diffusion coefficient or by salt concentration.\nInteraction strength is usually K .\nd\nResponse: We thank the reviewer for the constructive suggestion. Interaction strength was a measure for\nthe intensity of interactions between molecules. We utilized isothermal titration calorimetry (ITC) to\ndetermine the binding affinities of DDAB with BSA, ssDNA (99 nt), or dextran (10k Da), showing the\ndissociation constants (K ) of 1.57 ± 0.35 × 10-7 M, 1.00 ± 0.00 × 10-9 M, 1.00 ± 0.00 × 10-3 M, respectively.\nd\nIt indicated stronger affinities of DDAB with BSA (i.e., 7-fold) or ssDNA (i.e., 1060-fold) by compared to\nthat with Cm-Am (i.e., 1.06 ± 0.28 × 10-6 M), while the interactions between dextran and DDAB could be\nneglected. Thus, the integration of BSA and ssDNA into LC coacervates could enhance the interactions.\nWe have added the data and corresponding discussion in the manuscript (Page 12, Paragraph 2) and SI\n(Page 13, Paragraph 1; new Figure S24).\nRevised text, Page 12, Paragraph 2\nThis is due to the weak interactions between FITC-dextran (10k Da) and Cm-Am/DDAB LC coacervates.\nTo evaluate the binding affinities between DDAB and biomolecules, we utilized isothermal titration\ncalorimetry (ITC) to determine the dissociation constants (K ), showing 1.57 ± 0.35 × 10-7 M, 1.00 ± 0.00\nd\n× 10-9 M, or 1.00 ± 0.00 × 10-3 M for the interactions of DDAB with BSA, ssDNA (99 nt), or dextran (10k\nDa), respectively (Supplementary Fig. S18+1). It indicated significantly stronger affinities of DDAB with\nBSA (i.e., 7-fold) or ssDNA (i.e., 1060-fold) by compared to that with Cm-Am (i.e., 1.06 ± 0.28 × 10-6 M),\nwhile the interactions between dextran and DDAB could be neglected.\nRevised method, Supplementary Information, Page 13, Paragraph 1\nIsothermal titration calorimetry (ITC) assay. Typically, ITC measurements were performed on a low-\nvolume nano ITC instrument (AFFINITY ITC LV) at 25 ℃. All solutions were prepared in 50 mM of Tris-\nHCl buffer (pH 8). For the interactions between BSA and DDAB, BSA (300 1uL, 50 1uM) was loaded in a\nsyringe and DDAB (400 1uL, 1 mM) was added into sample cell, followed by 2.5 1uL/injection and total 20\ninjections of the titration with concomitant 200 s intervals between injections. Similarly, the titrations of\nssDNA, dextran, or Cm-Am to DDAB were performed following the above procedures, except using ssDNA\n(99 nt, 300 1uL, 20 1uM) to titrate DDAB (400 1uL, 0.2 mM), dextran (10k Da, 300 1uL, 200 1uM) to titrate\nDDAB (400 1uL, 1 mM), or Cm-Am (300 1uL, 120 1uM) to titrate DDAB (400 1uL, 0.3 mM). For each\nexperiment, a control was performed by titrating Tris-HCl buffer (50 mM, pH 8). The data were analysed\nby Launch NanoAnalyze software. All experiments were performed in triplicate. The dissociation constants\n(K ) for exceedingly strong (bigger than 1.00 × 10-9 M) or weak (smaller than 1.00 × 10-3 M) affinities\nd\ninteraction were may beyond the detection limit of the instrument (i.e., ranging from 1.00 × 10-9 M to 1.00\n× 10-3 M), but the measured K values still can be used to obtain relative binding affinities in comparison\nd\nwith other systems.\nNew Figure S24\nR25\nSupplementary Figure S24. (a-d) ITC raw data (top panel) and isotherm (bottom panel) for the titrations\nof BSA (a), ssDNA (99 nt) (b), dextran (10k Da) (c), or Cm-Am (d) to DDAB, showing K of 1.57 ± 0.35 ×\nd\n10-7 M, 1.00 ± 0.00 × 10-9 M, 1.00 ± 0.00 × 10-3 M, or 1.06 ± 0.28 × 10-6 M, respectively.\n12. HHmin-RNA is not a metabolic reaction.\nResponse: We thank the reviewer for the constructive comments. We have replaced 'metabolic reactions'\nby 'biochemical reactions' or 'enzymatic reactions' in the manuscript (Abstract; Introduction; Page 13,\nParagraph 3).\nRevised Abstract\nNotably, by integrating affinitive biomolecules into the LC microdroplets, we demonstrate the\nreconfiguration of protocell into yolk-shell coacervate vesicles that support biochemical reactions.\nRevised Introduction\nSignificantly, the integration of interactive biomolecules enhances molecular interactions in the LC\ncoacervates, resulting transformation into yolk-shell coacervate vesicles capable of supporting enzymatic\nreactions.\nRevised text, Page 13, Paragraph 3\nThe ability confining biochemical reactions within organelle-containing coacervate vesicles was crucial\nfor the functionality of artificial cells.\n13. Where is the evidence that the reaction takes place outside of the vacuole? Showing the location of the\nsubstrate and RNA could be useful here as there is no evidence to prove this statement. An alternative\nexplanation could be that reaction happens outside of these regions and the products can diffuse into the\nyolk-shell... “M‘oreover, the RNA catalysis rates in yolk-shell to the higher molecular diffusivity in the\nisotropic yolk-shell coacervate vesicles (Supplementary Fig. S24). Meanwhile, the reaction rate in the rim\nwas slightly faster than that”\nResponse: We thank the reviewer for the constructive comments. To determine the location of HH-min-\nmediated RNA catalysis reaction, we added FAM-labelled substrate and Cy5-labelled HH-min into Cm-\nR26\nAm/DDAB LC coacervates prior to the addition of amylase. It showed that these RNA were enriched by\nthe LC coacervate microdroplets. Upon the addition of amylase to initiate structural changes, these RNA\nwere found to locate inside the coacervate vesicles. We have added the discussion in the main text (Page\n13, Paragraph 3) and data in the SI (Figure S30).\nRevised text, Page 13, Paragraph 3\nIn this system, the cleavage of a fluorescence resonance energy transfer (FRET)-substrate strand catalyzed by\nHH-min increased the distance between the fluorophore (6-carboxyfluorescein) and Black Hole quencher 1\n(BHQ1), resulting in an increase in green fluorescence intensity (Fig. 5h,i). Significantly, the\nLC coacervates with homogenously integrated cyanine 5 labelled-HH-min (Cy5-HH-min) and\ncarboxyfluorescein-labelled substrate (FAM-substrate) were capable of transforming to yolk-shell\ncoacervate vesicles initiated by amylase, while the HH-min-mediated reaction indeed led to a progressive\nincrease of green fluorescence intensity both in the rim and inner artificial organelle (Fig. 5j,\nSupplementary Fig. S30, and Movie S13), confirming the enzymatic reactions were supported by yolk-shell\ncoacervate vesicles.\nRevised method, Supplementary Information, Page 4, Paragraph 2\nFluorescent dyes. Hoechst 33258 (Hoechst, ≥98%, Sigma), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium\nsalt (HPTS, ≥96%, Sigma), nitro benzoxadiazol-phosphoethanolamine (NBD-PE, >99%, Avanti), rhodamine\nB isothiocyanate (RITC, ≥70%, Sigma), fluorescein isothiocyanate (FITC, ≥90%, Sigma), rhodamine 6G\n(Rh6G, 95%, Sigma), 1-pyrenebutyric acid N-hydroxysuccinimide ester (Py, 95%, Sigma), RITC-labelled\npoly(diallyldimethylammonium chloride) (RITC-PDDA, 100−200k Da, labelling ratio = 10 mol%, kindly\nprovided by E. Wischerhoff and A. Laschewsky, Fraunhofer Institute for Applied Polymer Research, Potsdam,\nGermany), FITC-dextran (10k Da, 20k Da, 40k Da, 250k Da, 0.003-0.020 mol FITC per mol glucose, 70k Da,\nFITC:glucose = 1:250, Sigma), RITC-dextran (10k Da, 0.002-0.01 mol RITC per mol glucose, Sigma), cyanine\n5-labelled HH-min (Cy5-HH-min, Sangon Biotech), and carboxyfluorescein-\nlabelled substrate (FAM-substrate, Sangon Biotech) were all purchased and used without further\npurification.\nRevised Figure S30\nSupplementary Figure S30. (a,b)Time-lapse fluorescence microscopy images displaying Cy5-HH-min (0.8\n1uM) (a) and FAM-substrate (0.8 1uM) (b) were both homogenously distributed in Cm-Am/DDAB LC\ncoacervate microdroplets and the resultant LC coacervates were capable of transforming to organelle-\ncontaining coacervate vesicles via helicoid pattern-featured vacuolization process initiated by amylase (0.9\nR27\nmg/mL). Scale bars, 5 μm.\n14. Please show the structures that might form if fragmented Cm-Am is mixed with DDAB. This could\nconfirm that the change in structure is due to the dynamic effect of the enzyme.\nResponse: We thank the reviewer for the constructive comments. H-Cm-Am 1 obtained from 2-min\nhydrolysis of Cm-Am formed liquid crystalline coacervate microdroplets with DDAB (Fig. 3e). While H-\nCm-Am 2 obtained from 12-h hydrolysis of Cm-Am generated isotropic coacervate vesicles with DDAB\n(Fig. 3f). This suggests that the structural changes were attributed to the dynamic effect of the enzyme.\nThese results have been included in the manuscript (Page 8, Paragraph 2).\n15. “This phenomenon disappeared after heating at 60 °C for 80 min (Supplementary Fig. S3),\nconfirming the presence of a LC internal structure51.” This statement does not show that it has an LC\ninternal structure just that the system can undergo a phase transition at 60 °C.\nResponse: We thank the reviewer for the suggestion. To improve clarity, we have revised the statement in\nthe manuscript (Page 3, Paragraph 3).\nRevised text, Page 3, Paragraph 3\nThis phenomenon disappeared after heating at 60 °C for 80 min (Supplementary Fig. S4), indicative of the\nsystem undergoing a phase transition at 60 °C.\n16. Does the time frame for change in structure correlate to the rates of amylase degradation?\nResponse: We thank the reviewer for the comments. Upon the addition of amylase, LC coacervates\ntransformed to helicoid-structured vesicles at vacuolization stage (225 s) (Figure 2a) and the diameter of\nresulted assemblies reached a plateau at 300 s (Figure 2d), which was consistent with the rapid hydrolysis of\nCm-Am within 300 s (Figure 3a). Ultimately, the helicoid-structured vesicles at coalescence stage\nreconfigured into isotropic coacervate vesicles when the Cm-Am hydrolysis reached equilibrium. We have\nadded corresponding discussion in the manuscript (Page 8, Paragraph 2). In addition, the transformation time\nframe of some coacervates recorded by optical microscopy might not be highly aligned with amylase\ndegradation rate, which was probably related to the addition location of amylase.\nRevised text, Page 8, Paragraph 2\nUpon the addition of amylase, the molecular weight decreased sharply from 40k Da to 5k Da within 300 s,\nconsistent with transformation of LC coacervates into helicoid-structured vesicles at vacuolization stage\n(225 s) (Fig. 2a) and the diameter of resulted assemblies reached a plateau at 300 s (Fig. 2d). Ultimately,\nthe Cm-Am hydrolysis reached equilibrium accompanied with the generation of isotropic coacervate\nvesicles at coalescence stage (Supplementary Fig. S14).\n17. Do the membranes on the helicoid structures show bilayers?\nResponse: We thank the reviewer for the comments. The helicoidal vesicles exhibited thick rims (i.e., 0.4\n± 0.2 μm) without multiple bilayers as shown in cryo-TEM images (Figure RL1). We have added a\ndiscussion in the manuscript (Page 5, Paragraph 2) and the data in SI (new Figure S8) as responding to the\nsummary comments of reviewer#3.\n18. Please amend this sentence as the diffusion coefficient is not attributed to the concentration of molecules but\nby the ability for the molecules to diffuse internally. “Additionally, the diffusion coefficient of FITC-\nR28\nPEI within the rim was 1.4 × 10-14 m2/s, 1.8-fold higher than that in the organelle (i.e.,7.6 × 10-15 m2/s)\n(Fig. 5f and Supplementary Fig. S21). This difference is likely due to the lower partitioning of biomolecules\nin the rim compared to the organelle, which was confirmed by the fluorescence intensity of these\nbiomolecules (Supplementary Fig. S22).”\nResponse: We thank the reviewer for the comments. We have revised the sentence in the main text (Page\n13, Paragraph 2).\nRevised text, Page 13, Paragraph 2\nAdditionally, the diffusion coefficient of FITC-PEI within the rim was 1.4 × 10-14 m2/s, 1.8-fold higher than that\nin the organelle (i.e.,7.6 × 10-15 m2/s) (Fig. 5f), suggesting a molecularly more crowded environment\ninside the organelle.\nR29"}, {"round": 2, "reviewer_comments": {"reviewer_2": "My concerns have been addressed in the authors' rebuttal and in the revised manuscript. I recommend the publication of this manuscript.", "reviewer_3": "Please see attached document.\nPlease note that Reviewer #3 assessed authors’ responses to Reviewer #1 and considers them addressed."}, "author_response": "Responses to reviewers’ comments\nWe thank the reviewers for their constructive comments that have enabled us to improve the manuscript.\nAs detailed below, we have conducted additional experiments and responded to the comments point-by-\npoint.\nResponses to Reviewer #3\nThe authors have undertaken a point by-point rebuttal to the comments on the manuscript with additional\nexperiments and explanations where requested. However, there still remains some changes that will be\nrequired. These are summarised below with specific responses to the reproduced verbatim authors rebuttal\nin particular places.\nResponse: We thank the reviewer for these comments.\n1. The description of the different structures is still unclear. Whilst the additional experiments have\nprovided more insight into the nature of the systems these are not reflected in their descriptions. In addition,\nthere yolk-shell coacervate vesicle is not introduced during the descriptions. I understand that the yolk-\nshell coacervate vesicle is the same as the coacervate vesicle. Under this premise there is a discrepancy in\nthe description. In one place they say that the coacervate vesicle has a liquid layer with a water core.\nHowever, they show that the water core is dissolves with salt. Further comments are provided below. It is\nimportant that this is correctly and consistently defined based on the data and previous studies to provide\nsufficient support based in the data for the phenomena they are describing.\nResponse: We thank the reviewer for the comments. We revised all definitions of the assemblies mentioned\nin the manuscript to improve clarity, and added the definition of yolk-shell coacervate vesicles to the main\ntext (Page 3, Paragraph 2).\nRevised text, Page 3, Paragraph 2\nSpecifically, LC coacervate droplets were coacervates formed by LLPS with ordered liquid crystalline\nstructure51,52, while helicoidal vesicles represented vacuolized LC coacervates featured by helicoidal inner\npatterns, coacervate vesicles were assemblies consisting of a coacervate rim and a water-filled cavity45,\nand yolk-shell coacervate vesicles (i.e., organelle-containing coacervate vesicles) were assemblies with a\ncoacervate rim, a water-filled cavity, and an inner coacervate organelle. Coacervate microdroplets were\nisotropic coacervate droplets.\nRevised text, Page 5, Paragraph 2\nWith the addition of amylase, the LC coacervate microdroplets underwent a two-step transition process\ntoward coacervate vesicles with no other assemblies in the water-filled cavity.\nRevised text, Page 12, Paragraph 2\nInterestingly, the incorporation of biomolecules into ordered LC coacervate microdroplets led to the\nformation of organelle-containing protocells which were also referred to as yolk-shell coacervate vesicles\n(Fig. 5a). Compared to the coacervate vesicles obtained after the reconfiguration of LC coacervates\nwithout biomolecule integration. The yolk-shell coacervate vesicles featured a coacervate organelle in the\nwater-filled cavity.\n2. The text needs to be checked carefully as often times the new text additions can be modified to increase\nR1\nclarity.\nResponse: We thank the reviewer for the constructive suggestion. We have carefully reviewed and revised\nthe text to increase clarity.\nDirect responses to the rebuttal are given in BOLD:\n1. Please define the following terms: liquid crystalline coacervate droplets, helicoidal vesicles, coacervate\nvesicles and microdroplets. This will provide clarity into how the data for the droplet structures are\ninterpreted. Please also keep the terminology consistent through the text.\nResponse: We thank the reviewer for the comments. Liquid crystalline coacervate droplets were the\ncoacervate droplets combined with ordered liquid crystalline structure and intrinsic liquid-like property of\ncoacervates formed by LLPS (Curr. Opin. Colloid Interface Sci., 2021, 56, 101500). Helicoidal vesicles in\nthis work were vacuolized coacervates featured with helicoidal inner patterns, which were generated during\nthe vacuolization of liquid crystalline coacervate droplets. Coacervate vesicles were assemblies with a liquid\nlayer and a water-filled cavity (Nat. Chem., 2025, 17, 279), which was obtained after the reconfiguration of\nLC or isotropic coacervate droplets. Coacervate microdroplets were isotropic coacervate droplets appeared\nin this work. We have kept the terminology consistent through the text. To provide a clear description, we\nadded relevant definitions in the main text (Page 3, Paragraph 2).\nRevised text, Page 3, Paragraph 2\nThe synergistic interactions of electrostatic attraction and the hydrophobic effect drove the complexation of\nCm-Am and DDAB, ultimately resulting in the formation of LC coacervate microdroplets which\nR19\nunderwent complex structural transition in the presence of amylase (Fig. 1b). To better understand\nalterations in structures, we defined assemblies related to the transformation. Specifically, LC coacervate\ndroplets possessed ordered LC structure and intrinsic liquid property of coacervates formed by LLPS51,52,\nwhile helicoidal vesicles represented vacuolized LC coacervates featured by helicoidal inner patterns,\naconadc ervate vesicles were assemblies with a liquid layer and a water-filled cavity45. Coacervate\nmicrodroplets were isotropic coacervate droplets.\n① As stated above, please ensure consistency in your definitions and provide full explanations. Here you do\nnot introduce yolk-shell coacervate vesicles but discuss them extensively in the second half of the manuscript.\n② The description of the coacervate vesicles is also different to your data. Here it is defined as “Coacervate\nvesicles were assemblies with a liquid layer and a water-filled cavity” but your data shows that the core\ndisassembles with salt which does not correlate to a water filled cavity. However, I think I now understand what\nyou mean by a coacervate vesicle. It is a core shell structure with two different phases in the core and the\noutside which was not immediately apparent by the term that was used for it. ③ More clarity in the description\nof the LC coacervate should be provide “ordered liquid crystalline structure and intrinsic liquid-like\nproperty of coacervates formed by LLPS”. Liquid crystals are liquid by definition, is this the same/different\nliquid as the liquid formed by LLPS.\nResponse: We thank the reviewer for the comments.\n① We checked all definitions in the manuscript to ensure consistency and added the definition of yolk-\nshell coacervate vesicle to the main text (Page 3, Paragraph 2).\nRevised text, Page 3, Paragraph 2\nSpecifically, LC coacervate droplets were coacervates formed by LLPS with ordered liquid crystalline\nR2\nstructure51,52, while helicoidal vesicles represented vacuolized LC coacervates featured by helicoidal inner\npatterns, coacervate vesicles were assemblies consisting of a coacervate rim and a water-filled cavity45,\nand yolk-shell coacervate vesicles (i.e., organelle-containing coacervate vesicles) were assemblies with a\ncoacervate rim, a water-filled cavity, and an inner coacervate organelle. Coacervate microdroplets were\nisotropic coacervate droplets.\n② The main distinction between coacervate vesicles and yolk-shell coacervate vesicles lies in the presence\nor absence of a coacervate organelle within the water-filled cavity. Coacervate vesicles lacking an inner\norganelle were obtained after the transformation of LC coacervates without biomolecule integration. In\ncontrast, the incorporation of biomolecules promoted their association with coacervate components,\npotentially restricting amylase-catalyzed Cm-Am hydrolysis. This, in turn, led to the formation of an inner\ncoacervate organelle during the reconfiguration of LC coacervates.\n③ We agreed that the liquid crystals were liquid by definition. We have revised the description in the\nmanuscript accordingly (Page 3, Paragraph 2).\nRevised text, Page 3, Paragraph 2\nSpecifically, LC coacervate droplets were coacervates formed by LLPS with ordered liquid crystalline\nstructure51,52, while helicoidal vesicles represented vacuolized LC coacervates featured by helicoidal inner\npatterns, coacervate vesicles were assemblies consisting of a coacervate rim and a water-filled cavity45,\nand yolk-shell coacervate vesicles (i.e., organelle-containing coacervate vesicles) were assemblies with a\ncoacervate rim, a water-filled cavity, and an inner coacervate organelle. Coacervate microdroplets were\nisotropic coacervate droplets.\n2. ① The authors describe the Cm-Am/DDAB coacervate droplets as LC coacervate microdroplets. The\ndata actually suggests that these are multilamellar vesicles given the maltese crosses. Thus, indicating that\nthey are not coacervates. ② Further, the data suggests that the addition of amylase transforms the\nmultilamellar vesicle into a vesicle with internal compartment. With the helicoidal structure a metastable\nstate that appears depending on the ratio of Cm-Am and DDAB. Some questions remain about the internal\ncompartment. What is the internal structuration of this? How can they confirm that is a coacervate and not\na surfactant droplet or a smaller vesicle.\nResponse: We thank the reviewer for the suggestion. ① We acknowledge that LC coacervate droplets\nexhibited multilamellar features; they lacked a distinct inner aqueous core. No clear segregation of\nhydrophobic and hydrophilic domains was observed, as both hydrophobic and hydrophilic fluorophores were\nuniformly distributed throughout the droplets (Figure 1h, S5, and S6). Unlike multilamellar vesicles, these\nLC droplets readily underwent fusion, suggesting a more dynamic and less compartmentalized structure\n(Figure S3). Moreover, biomolecules such as proteins were able to efficiently penetrate and distribute evenly\nwithin the LC droplets (Figure 5b, c, e, and S20). In contrast, the diffusion of hydrophilic biomolecules into\nmultilamellar vesicles was hindered by the presence of hydrophobic lipid bilayers, which acted as a barrier to\nentry. Liquid crystalline coacervate microdroplets have been reported in the references (e.g., Nat. Commun.,\n2023, 14, 2606; ACS Nano, 2020, 14, 15071; Nat. Chem., 2024, 16, 158).\n② As demonstrated in the main text (Page 12, Paragraph 1), we proposed that LC coacervates transformed\ninto coacervate vesicles with an inner coacervate. The inner coacervates were derived from the original LC\ncoacervate droplets. We have added a new discussion to the manuscript (Page 12, Paragraph 1) and the\nR3\ncorresponding data to SI (new Figure S23).\nAs evidenced by fluorescence tracking experiments in main text (Page 12, Paragraph 1 and 2), the\ninternal compartments of yolk-shell coacervate vesicles were composed of Cm-Am fragments, DDAB,\nand biomolecules, similar to the compositions in the rims which were coacervate vesicles (Figure 5b-d).\nThe internal organelle also revealed molecularly crowded interior, liquid-like property as evidenced by FRAP\nexperiment, and strong enrichment ability (Figure 5b-f). Thus, the internal compartment was determined\nas a coacervate.\nIt could be possible for multilamellar vesicles to fuse under the correct conditions, in addition, if the\nbilayers are leaky (which could be possible if molecules/ polymers are interrelated within the bilayers)\nthen this can allow the penetration of molecules from the outside of the MLV to the inside. Again, some\nadditional clarification for the coacervate part of the LC coacervate should be provided.\nResponse: We thank the reviewer for the comments. Firstly, fusion of MLV typically required the\nintroduction of external factors, such as adding polymers for membrane dehydration or osmotic pressure\nchanges, introducing cations for headgroup charge screening, changing pH or temperature for regulation of\nlipid arrangements (ACS Nano 2017, 11, 1273; Nat. Struct. Mol. Biol., 2008, 15, 675). In contrast, the\nfusion of LC coacervate microdroplets occurred spontaneously without the external stimulus or special\nconditions. Secondly, after centrifugation, MLV showed no macroscopically visible phase separation in the\nEppendorf tubes, whereas LC coacervate droplets exhibited two distinct phases that can be clearly observed by\nnaked eyes. Thirdly, the penetration of molecules through leakiness in bilayers of MLV led to a lack of\nselectivity in molecular enrichment. Perhaps all of molecules can be encapsuled in MLV. Conversely, LC\ncoacervates could selectively sequester macromolecules based on molecular weight (e.g., FITC-dextran in\nFigure S6; other client macromolecules in Figure S7), in which this selective enrichment ability was prevalently\nobserved in coacervates formed by LLPS. We added relevant discussion in the manuscript (Page 3,\nParagraph 3)\nRevised text, Page 3, Paragraph 3\nIn this work, the LC microdroplets were primarily obtained at a total concentration of 10 mM with a monomer\nmolar ratio of 1:1 (marked by a red triangle in Fig. 1c), wherein the LC coacervates were capable of exhibiting\nmacroscopically visible phase separation in the Eppendorf tubes after centrifugation. These LC microdroplets\ndisplayed discrete spherical morphologies with high optical contrast, as well as liquid-like property,\nevidenced by spontaneous fusion behaviour in the absence of external stimulus or special conditions\n(Supplementary Fig. S3 and Movie S1).\nRevised text, Page 12, Paragraph 1\nThe resulting coacervates maintained liquid crystalline structures and predominantly transformed into\ncoacervate vesicles with an internal coacervate organelle (i.e., yolk-shell coacervate vesicles) in the\npresence of amylase (Supplementary Figs. S21,S22), wherein the yolk-shell coacervate vesicles possessed\nthicker rims (0.4 ± 0.1 μm) by compared to thinner membranes of multilamellar vesicles (i.e., several\nnanometres) and the strong enrichment ability in contrast to controllable permeability of vesicles\n(Supplementary Fig.S23).\nNew Figure S23\nR4\nSupplementary Figure S23. Statistical analysis of thickness for rims in yolk-shell coacervate vesicles\nshowed an average thickness of 0.4 + 0.1 1um. Number of samples measured, n = 200.\nWhere does this data come from? Electron microscopy or optical microscopy. Examples of the images\nwhere these were taken from should be shown to determine how reliable this data is given then optical\nmicroscopy data would not be the best way to obtain the rim thickness.\nResponse: We thank the reviewer for the comments. The thickness of rims in yolk-shell coacervate vesicles\nwere measured by fluorescence microscopy and determined based on the full width at half maximum of gray\nvalue in linescan profiles. We added two example images in Figure S26 to show the reliability, as well as\nproviding two example images of helicoidal vesicles or coacervate vesicles in Figure S9. We compared the rim\nthickness of coacervate vesicles (i.e., about 300 nm) with that of DDAB vesicle membranes (i.e., approximately\n200 nm) measured also by fluorescence microscopy (Figure S10), revealing bigger thickness. Except for the\ndifferences in thickness of rims and vesicle membranes, we could also observe that coacervate vesicles were\nalways hollow, whereas the vesicles sometimes possessed multilamellar structures.\nDue to the rim thickness was close to the resolution limit of fluorescence microscopy, we further used\ncryo-TEM which showed coacervate vesicles have much thicker rims (i.e., about 120 nm) by compared to\nDDAB vesicle membranes (i.e., around 6 nm) (Figure S11). These results indicated coacervate vesicles and\nvesicles were two different assemblies.\nThe rim thickness of coacervate vesicles reported in other studies were also measured by fluorescence\nmicroscopy (Angew. Chem. Int. Ed., 2022, 61, e202202302; Angew. Chem. Int. Ed. 2024, 63, e202407472).\nWe added data and corresponding discussion in the manuscript (Page 5, Paragraph 2; Page 12, Paragraph\n2) and SI (Page 13, Paragraph 2; Figure S9, S10, S11, and S26).\nRevised text, Page 5, Paragraph 2\nThe LC microdroplet with doped RITC-Cm-Am vacuolized into helicoidal vesicles as an intermediate which\nunderwent coalescence, ultimately giving rise to a hollow vesicle determined as a coacervate vesicle (Fig.\n2b and Supplementary Movie S3). As a result, the helicoidal vesicles or coacervate vesicles both possessed\nthicker rims (i.e., 0.4 + 0.2 1um or 0.3 + 0.1 1um) and strong enrichment capability by compared to DDAB\nvesicles with thinner membrane (i.e., about 0.2 1um) and poor membrane permeability54, wherein the rim\nand membrane thickness were both measured by fluorescence microscopy and analysed based on full width\nat half maximum of gray values in linescan profiles (Supplementary Fig. S9,S10). Except for the\ndifferences in thickness of rims and vesicle membranes, we could also observe that coacervate vesicles were\nalways hollow, whereas the vesicles sometimes possessed multilamellar structures. The cryogenic transmission\nelectron microscopy (cryo-TEM) further confirmed the rim thickness of coacervate vesicles (i.e., about 120\nnm) was much larger than membrane thickness in vesicles (i.e., about 6 nm) (Supplementary Fig. S11). In\nthe reconfiguration process, the enzyme (RITC-amylase) was homogenously distributed (Fig. 2c).\nRevised text, Page 12, Paragraph 2\nR5\nThe resulting coacervates maintained liquid crystalline structures and predominantly transformed into\ncoacervate vesicles with an internal coacervate organelle (i.e., yolk-shell coacervate vesicles) in the presence\nof amylase (Supplementary Figs. S24,S25). The thickness of rims in yolk-shell coacervate vesicles\nwas measured by fluorescence microscopy and analysed based on full width at half maximum of gray values\nin linescan profiles, exhibiting thicker rims (i.e., 0.4 ± 0.1 1um) by compared to DDAB vesicle membranes\n(i.e., about 0.2 1um) (Supplementary Fig. S26). The yolk-shell coacervate vesicles also possessed a strong\nenrichment ability, in contrast to controllable permeability of vesicles.\nRevised method, Page 13, Paragraph 2\nCryogenic transmission electron microscopy (cryo-TEM). Typically, 2 1uL aliquot of coacervate vesicles\nobtained after the incubation of Cm-Am/DDAB coacervates (30 1uL, total concentration of 20 mM, monomer\nmolar ratio of 1:1) with amylase (5.3 1uL) for 30 min were applied to holey carbon films (2 1um hole diameter)\ncovered 300 mesh grids (R2/1 batch of GiG, CN), which were hydrophilized by plasma process in advance.\nThe supernatant fluid was sucked up with Whatman 1 filter paper for 1.5 s and then the film was quickly\nput into liquid ethane at -165 ℃ using a LEICA EM GP plunger (LEICA, DE). The frozen samples were\ntransferred into a Themis 300 transmission electron microscope (Thermo Fisher Scientific, USA) using a\nGatan 626 cryo-transfer holder (Gatan, USA) and observed with an accelerating voltage of 120 kV.\nSimilarly, the characterization of DDAB vesicles by cryo-TEM was also following the above\nprocedures, except 1 mM of DDAB vesicle dispersion (pH 8) were used.\nRevised Figure S9\nSupplementary Figure S9. (a-d) Fluorescence microscopy images (left) and corresponding linescan\nR6\nprofiles (right) of helicoidal vesicles (doped NBD-PE) (a) or coacervate vesicles (c) and statistical analysis\nof thickness for rims in helicoidal vesicles (b) or coacervate vesicles (d) showed an average thickness of 0.4\n+ 0.2 1um or 0.3 + 0.1 1um, respectively. Number of helicoidal vesicles or coacervate vesicles measured, n\n= 200. The thickness of rims was determined by full width at half maximum of gray values in linescan\nprofiles. Scale bars, 5 1um.\nNew Figure S10\nSupplementary Figure S10. (a,b) Fluorescence microscopy images (a) and corresponding linescan\nprofiles (b) of DDAB vesicles (5 mM, doped NBD-PE) displayed the membrane thickness of around 0.2 1um.\nThe thickness of membranes was determined by full width at half maximum of gray values in linescan\nprofiles. Scale bar, 5 1um.\nNew Figure S11\nSupplementary Figure S11. (a,b) Cryo-TEM images of coacervate vesicles (a) and DDAB vesicles (b)\nshowing the thickness of rim in the coacervate vesicles was about 120 nm and the thickness of vesicle\nmembrane was around 6 nm. Scale bars, 200 nm.\nRevised Figure S26\nR7\nSupplementary Figure S26. (a,b) Fluorescence microscopy images (left) and corresponding linescan\nprofiles (right) of yolk-shell coacervate vesicles (doped RITC-HRP) (a) and statistical analysis of thickness for\nrims in yolk-shell coacervate vesicles (b) showed an average thickness of 0.4 ± 0.1 1um. Number of samples\nmeasured, n = 200. The thickness of rims was determined by full width at half maximum of gray\nvalues in linescan profiles. Scale bars, 5 1um.\n3. SAX data and EM should be used to confirm the location of the Cm-Am within the bilayers via electron\ndensity mapping. As well as the presence of bilayers within the LC coacervate and the yolk shell system.\nResponse: We thank the reviewer for the constructive suggestion. As shown in the manuscript (Page 3,\nParagraph 3), SAXS profile of Cm-Am/DDAB LC coacervates (Figure 1g) showed the periodic molecular\nspacing was 3.55 nm which was slightly larger than the bilayer thickness of DDAB (i.e., approximately 2.45\nnm). This suggested that the periodic layer consisted of DDAB and Cm-Am, in which Cm-Am might locate\nbetween adjacent DDAB bilayers (Figure 1b). We also supplemented SAXS profile of yolk-shell coacervate\nvesicles, showing the disappearance of SAXS peaks. We have added a discussion in the manuscript (Page\n13, Paragraph 3) and the data in SI (Page 11, Paragraph 4; new Figure S32).\nAdditionally, according to cryo-TEM imaging (Figure RL1), the multilamellar structures of LC\ncoacervates were not clearly resolved due to the exceedingly thick of coacervate phase (i.e., several\nmicrometres) which was beyond electron penetration depth limit of cryo-TEM (i.e., less than a micrometre)\n(Curr. Opin. Struct. Biol., 2016, 37, 81). The coacervate vesicles with about 0.3-μm-thick rims (Figure S8)\nwere resolved by cryo-TEM, displaying uniform electron density contrast without multiple-bilayer structures.\nWe failed to obtain electron density mapping analysis because it cannot distinguish the location of Cm-Am in\nthe LC coacervates and yolk-shell coacervate vesicles due to the similar elemental compositions (i.e., carbon,\nhydrogen, and oxygen elements) of Cm-Am with DDAB and ice.\nAs evidenced by fluorescence tracking experiments in the main text (Page 4, Paragraph 2; Page 12,\nParagraph 1), Cm-Am was homogenously distributed in the LC coacervates and yolk-shell coacervate\nvesicles, while the multiple bilayers were not observed under fluorescence microscopy due to the limit of\nresolution.\nRevised text, Page 13, Paragraph 3\nMoreover, the RNA catalysis rates in yolk-shell coacervate vesicles were faster than that in the initial LC\nmicrodroplets (Fig. 5k,l), attributing to the higher molecular diffusivity in the isotropic yolk-shell coacervate\nvesicles. The lack of LC feature was confirmed by the disappearance of birefringence\nphenomenon in POM image and Bragg reflection peak in SAXS profile (Supplementary Figs. S31,S32).\nR8\nRevised method, Page 11, Paragraph 4\nSmall angle X-ray scattering (SAXS). SAXS experiments were performed at room temperature on Ganesha\nsystem 380 (SAXSLAB, U.S) equipped with a multilayer focused copper Ka radiation as the X-ray 381 source\n(Genix 3D Cu ULD) and a semiconductor with silver behenate for the small angle region and LaB6 for the\nwide angle region, respectively. Cm-Am/DDAB, Cm-Am/DMAB, Cm-dextrin/DDAB, and Cm-Am/DTAB\ncoacervate suspension prepared following above procedures were centrifuged at 9390 × g for 10 min to\nremove the water phase and collect the coacervate phase for SAXS analysis.\nBSA-integrated Cm-Am/DDAB LC coacervates prepared following above procedures were incubated\nwith amylase for 1 h, leading to the generation of yolk-shell coacervate vesicles, followed by centrifugation\nat 9390 × g for 10 min to remove the water phase and collect the coacervate phase for SAXS analysis.\nNew Figure S32\nSupplementary Figure S32. SAXS profile of yolk-shell coacervate vesicles displayed no obvious Bragg\nreflection peak, indicating the disordered internal structures due to amylase-mediated disruption of\nperiodical stacking between Cm-Am and DDAB. The yolk-shell coacervate vesicles used here were\nobtained after the reconfiguration of BSA-integrated LC coacervates (total concentration of 10 mM,\nmonomer molar ratio of 1:1).\nThis data shows the presence of a small bragg peak.\nResponse: We thank the reviewer for the comments. The small Bragg reflection peak of yolk-shell\ncoacervate vesicles appeared at 0.177 Å−1, consistent with that of LC Cm-Am/DDAB coacervates (Figure\n1g). This may be due to the relatively short hydrolysis time (1 h), which was insufficient for complete\nhydrolysis, leaving some coacervates only partially transformed. To clarify whether bilayers were present\nin the yolk-shell coacervate vesicles, we extended the hydrolysis time from 1 h to 3 h during sample\npreparation. In this case, no Bragg reflection peaks were observed in the SAXS profile, indicating that the\nyolk-shell coacervate vesicles did not contain bilayers. We added data into SI (Page 11, Paragraph 5; Figure\nS35)\nRevised method, Page 11, Paragraph 5\nBSA-integrated Cm-Am/DDAB LC coacervates prepared following above procedures were incubated\nwith amylase for 1 h or 3 h, leading to the generation of yolk-shell coacervate vesicles, followed by\ncentrifugation at 9390 × g for 10 min to remove the water phase and collect the coacervate phase for SAXS\nanalysis.\nRevised Figure S35\nR9\nSupplementary Figure S35. (a,b) SAXS profile of yolk-shell coacervate vesicles displayed the gradual\ndisappearance of Bragg reflection peaks at 0.177 Å−1 due to the hydrolysis of BSA-integrated LC\ncoacervates (total concentration of 10 mM, monomer molar ratio of 1:1) for 1 h (a) or 3 h (b). It indicated\nthe disordered internal structures due to amylase-mediated disruption of periodical stacking between Cm-\nAm and DDAB.\nFigure RL1. (a,b) Cryo-TEM images of LC coacervates (a) and coacervate vesicles (b) both showing\nuniform electron density contrast without multiple-bilayer structures. Scale bars, 500 nm.\nIt is surprising that the LC coacervates show no bilayers. Can the samples be sliced to reduce the thickness\nof the sample?\nResponse: We thank the reviewer for the comments. We utilized cryo-SEM to characterize the internal\nstructures of fractured LC coacervate samples, revealing an onion-like multilamellar structure without a\ncentral water-filled lumen. The corresponding data and discussion have been added to the manuscript (Page\n4, Paragraph 1) and SI (Page 13, Paragraph 3; new Figure S5).\nRevised text, Page 4, Paragraph 1\nThis phenomenon disappeared after heating at 60 °C for 80 min (Supplementary Fig. S4), indicative of the\nsystem undergoing a phase transition at 60 °C. The LC coacervates exhibited an onion-like multilamellar\nstructure without central water-filled lumen as characterized by cryogenic scanning electron microscopy\n(cryo-SEM) (Supplementary Fig. S5). The ordered internal structure of the LC coacervates was further\nconfirmed by small angle X-ray scattering (SAXS), which revealed a sharp Bragg reflection peak at 0.177\nÅ−1, corresponding to a periodic molecular spacing of 3.55 nm (Fig. 1g).\nRevised method, Page 13, Paragraph 4\nCryogenic scanning electron microscopy (cryo-SEM). Cryo-SEM was performed on a FE-SEM (Hitachi\nRegulus 8230 or ZEISS Crossbeam 550) at an operating voltage of 5 kV or 2 kV. Liquid nitrogen was used\nR10\nin the cryogenic system. Cm-Am/DDAB coacervate microdroplet suspension (total concentration of 20 mM,\nmonomer molar ratio of 1:1) was placed on the sample holder between two rivets and was rapidly frozen\nin liquid nitrogen for 3 minutes. Then, the sample was transferred to the preparation chamber. The\nfractured surface was sublimed at -90 °C for 20 min to remove condensed ice, after which the samples were\nsputtered with tungsten at a current of 5 mA for 1 min. During imaging, the sample temperature was\nmaintained at -140 °C.\nNew Figure S5\nSupplementary Figure S5. (a-c) Cryo-SEM image of LC Cm-Am/DDAB coacervates (total concentration\nof 20 mM, monomer molar ratio of 1:1) revealed onion-like multilamellar microstructures. Scale bars,\n1 μm.\n4. Where is the evidence that the reaction takes place outside of the vacuole? Showing the location of the\nsubstrate and RNA could be useful here as there is no evidence to prove this statement. An alternative\nexplanation could be that reaction happens outside of these regions and the products can diffuse into the\nyolk-shell... “M‘oreover, the RNA catalysis rates in yolk-shell to the higher molecular diffusivity in the\nisotropic yolk-shell coacervate vesicles (Supplementary Fig. S24). Meanwhile, the reaction rate in the rim\nwas slightly faster than that”.\nResponse: We thank the reviewer for the constructive comments. To determine the location of HH-min-\nmediated RNA catalysis reaction, we added FAM-labelled substrate and Cy5-labelled HH-min into Cm-\nAm/DDAB LC coacervates prior to the addition of amylase. It showed that these RNA were enriched by\nthe LC coacervate microdroplets. Upon the addition of amylase to initiate structural changes, these RNA\nwere found to locate inside the coacervate vesicles. We have added the discussion in the main text (Page\n13, Paragraph 3) and data in the SI (Figure S30).\nThis data still does not conclusively show that the reaction takes place inside. It is possible that the inside\nthe droplet there is no reaction and the reaction only takes place outside of the droplet.\nResponse: We thank the reviewer for the comments. As shown by the fluorescence labelling experiment\n(Figure S33), both Cy5-HH-min and the FAM-labelled substrate were sequestered within the coacervates,\nwhile negligible fluorescence was detected in the surrounding medium. This indicates that the HH-min-\nmediated reaction could occur only inside the LC coacervate droplets.\nR11\nResponses to reviewers’ comments\nWe thank the reviewers for their constructive comments that have enabled us to improve the manuscript.\nAs detailed below, we have conducted additional experiments and responded to the comments point-by-\npoint.\nResponses to Reviewer #3\nThe authors have addressed all of the comments. In principle the work is now suitable for publication with one\nchange. Care should be taken in the interpretation of the location of the RNA based reaction based on\nfluorescence. The authors should show that the reaction really does take place in the compartment. One way\nto do this is to demonstrate that there is no reaction outside of the compartment by analysis of the supernatant.\nEither showing that no reaction takes place there or that there is no RNA based on experiments which do not\ninvolve fluorescence. Alternatively, the authors can choose to soften the statement that the reactions are taking\nplace inside of the droplet.\nResponse: We thank the reviewer for these comments. We revised the corresponding text to soften the\nstatement (Page 10, Paragraph 2).\nRevised text, Page 10, Paragraph 2\nIn this system, the cleavage of a fluorescence resonance energy transfer (FRET)-substrate strand catalyzed\nby HH-min increased the distance between the fluorophore (6-carboxyfluorescein) and Black Hole quencher\n1 (BHQ1), resulting in an increase of green fluorescence intensity (Fig. 5h,i). Significantly, the LC\ncoacervates with homogenously integrated cyanine 5 labelled-HH-min (Cy5-HH-min) and\ncarboxyfluorescein-labelled substrate (FAM-substrate) were capable of transforming to yolk-shell\ncoacervate vesicles initiated by amylase (Supplementary Fig. S34), while a progressive increase of green\nfluorescence intensity from HH-min-mediated reaction was observed both in the rim and inner artificial\norganelle (Fig. 5j and Supplementary Movie S13), confirming the support of enzymatic reactions by the\nsystem.\nR1"}]

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