Construction of light-driven artificial organelle

Light-driven artificial organelle was composed of two kinds of membrane proteins, bacteriorhodopsin (bR) and F-type ATP synthase (F o F 1 ). bR was isolated from a purple membrane of Halobacterium salinarum by ultra-centrifugation with sucrose density gradient (Fig. 1b and Supplementary Fig. 2). F o F 1 of Bacillus PS3 was purified as recombinant protein from Escherichia coli cells (Fig. 1b). The isolated bR were reconstructed as bR-embedding proteoliposomes (bR-PLs) for the measurement of light-dependent proton-pump activity. The size of bR-PLs were mostly 100–200 nm as diameter. We used phosphatidylcholine extract from soybean to form PLs which are stable in the reaction mixture of PURE system and also maintain the F o F 1 activity10. The formation of bR-PLs was carried out by reducing the detergent concentration in the mixture of lipids and purified protein according to the previous report19; however, we have found that only 25% bR were maintaining the proper membrane orientation (Supplementary Fig. 3C). To improve this ratio, we did some modifications in the preparation method by changing the timing of bR addition (Supplementary Fig. 3A), i.e., empty liposomes were first roughly preformed and, then, the purified bR was combined before completely removing the detergent. By this method, 70% bR was properly reconstructed in the PLs (Supplementary Fig. 3C). The improvement of the membrane orientation faithfully reflected into the proton-pump activity (Supplementary Fig. 3D). Since the efficiency of proton gradient generation directly affects the F o F 1 activity, we employed this optimized method for all of the following experiments.

During the light illumination, we observed a decrease of proton concentration at the outside of bR-PLs in proportion to bR concentration (Fig. 1c), suggesting that the protons were transported from the outside to inside of the bR-PL lumen (Supplementary Fig. 1A). In addition to the proton-pump activity, we also observed a rapid return of the proton concentration when the illumination ceased. This indicates proton leakage from the inside to outside of the bR-PL lumen. The proton leak was accelerated when the lateral fluidity of the bR-PL membranes was increased by temperature rise (Supplementary Fig. 4). For the sake of inhibiting the leak through the membrane, we added 30% cholesterol into the lipid composition of bR-PLs20, which resulted in 30% reduction of the proton leak (Supplementary Fig. 5). Thus, we kept this condition throughout the study.

Next, we estimated the membrane orientation of the reconstituted bR by evaluating the binding sensitivity of a histidine-tag, which elongated at the C-terminus of recombinant bR, to the Ni-NTA-conjugated magnet beads (Supplementary Fig. 6). If the reconstructed bR was keeping the working orientation, the C-terminus histidine-tag can bind to the magnet beads and be eluted in the elution fraction. The ratio of bR obtained in the elution fraction was normalized with the ratio of control experiment in which bR was monodispersed by dissolving the PLs with detergent (Triton). In the control experiment, 91% bR was collected in the elution fraction, although that should be 100% theoretically (Supplementary Fig. 6). Considering this result, we calculated that 86% bR was reconstructed in the working (outward C-terminus) orientation within the PL membrane; i.e., Elu. −Triton Elu. +Triton −1 100%. It should be noted that the opposite orienting bRs (inward C-terminus) pump protons from the inside to outside of the PLs. Thus, the net-working ratio of the reconstituted bR is calculated as 72% (Supplementary Table 1). Taking account of the bR membrane orientation, the initial reaction rate of bR was calculated as −2.87 ± 0.53 ΔpH min−1 nmol−1 or −0.11 ± 0.02 ΔpH min−1 mg−1, mean ± S.D. (Fig. 1c and Supplementary Table 1). On the other hand, the net-working ratio of the reconstituted F o F 1 was 65.1% after the normalization as with bR (Supplementary Fig. 7 and Supplementary Table 1), and the initial reaction rate was 128 ± 3.2 ATP nmol min−1 nmol−1 or 223 ± 6.1 ATP nmol min−1 mg−1 (Fig. 1d and Supplementary Table 1). The reverse function of F o F 1 , ATP-dependent proton-pump activity, was also detected (Supplementary Fig. 8), suggesting the full functionality of the reconstituted F o F 1 -PLs.

To construct artificial organelle, we assembled purified bR and F o F 1 to form bRF o F 1 -PLs. We prepared PLs in various proportion of bR against F o F 1 and illuminated with visible light passing a 500 nm long-pass filter. The amount of produced ATP was measured by means of luciferin and luciferase. The highest ATP photosynthesis was obtained in the case of 176 µM bR and 1 µM F o F 1 . This means that approximately 0.6 × 106 ATP was produced by a single bRF o F 1 -PL within 4 h of illumination (Fig. 1e). The maximum turnover number for ATP synthesis in the initial 5 min was 8.3 ± 0.3 s−1 in the case of 176 µM bR and 1 µM F o F 1 . This was almost double compared to the previous report18. Here, in a single PL, 3560 of the working bRs drive 18 F o F 1 (Supplementary Table 1). In all cases, we used 10 mM NaN 3 to inhibit the reverse (ATPase) activity of F o F 1 21. We found that the ATP production plateaued when the illumination was higher than 10 mW per cm2 (Supplementary Fig. 9).

The same reaction was also performed inside GUVs in which about 1.1 × 104 bRF o F 1 -PLs are contained in a 10 µm diameter GUV. After 6 h of illumination, we observed photosynthesized ATP from the inside of the GUVs (Fig. 1f), where 1.8 mM ATP was produced in a single GUV (Supplementary Table 1). This represents that 4.6 µmol ATP was produced per mg ATP synthase. The efficiency of ATP production in GUVs was roughly one-third that of the in vitro system, perhaps caused by lower light intensity inside a GUV. Since our artificial organelle can produce ATP inside GUV at the comparable concentration as a real living cell, we proceeded to design and construct the photosynthetic artificial cell system that synthesize protein by light.

Light-driven protein synthesis inside the artificial cell

We performed green fluorescent protein (GFP) synthesis inside GUVs by means of the photosynthesized ATP to demonstrate that the constructed artificial organelle works in an artificial cell system. For this purpose, we combined bRF o F 1 -PLs with the PURE system which is a cell-free protein synthesis system. The PURE system is reconstituted from purified translation factors6, and therefore we can customize the component factors suited for the designed artificial system. The PURE system was modified as shown in Supplementary Table 2 to allow the photosynthesized ATP be specifically used for the aminoacylation of tRNA (Supplementary Fig. 10), and supplied with a mRNA encoding GFP together with bRF o F 1 -PLs and NaN 3 . NaN 3 did not inhibit protein synthesis at concentrations below 50 mM (Supplementary Fig. 11). The prepared reaction mixture was encapsulated inside GUVs, and illuminated to induce protein synthesis. A large majority of the GUV population appeared in a range of 10–20 µm as diameter (n = 200) (Supplementary Fig. 12). After 6 h, we observed the fluorescence of internally synthesized GFP by confocal microscopy (Fig. 2a). This GFP synthesis was also confirmed in vitro (without GUVs) (Fig. 2b and Supplementary Fig. 13) and synchronized with timing of the ATP photosynthesis (Supplementary Fig. 14). These results indicate that GFP synthesis inside the GUVs was driven by the photosynthesized ATP (Supplementary Fig. 1B). We found that 50–60% of the GUVs emitted more fluorescence than the nonilluminated control GUVs (Fig. 2c) by flow-cytometry analysis. We also found a certain percentage of GUVs were not showing fluorescent even when illuminated. Although the definitive cause is not unclear, it has been reported that the encapsulation efficiency of PLs is affected by the size of PLs; i.e., less than 35% GUVs can encapsulate the PLs when their size are over 200 nm (diameter)18. Additionally, we cannot deny the possibility that inactivity of the internal artificial organelle by the fusion of bRF o F 1 -PLs and GUV membranes is limiting the successful artificial cell formation.

Fig. 2 Protein synthesis inside giant unilamellar vesicle (GUV) driven by light. Green fluorescent protein (GFP) was synthesized from its messenger RNA (mRNA) (a–e) or DNA (f–h) inside light illuminated GUV (a, c, e–h) or in vitro (b, d). GFP was synthesized inside GUV (a) or in vitro (the PURE system) (b) in which the photosynthesized adenosine triphosphate (ATP) was consumed for the aminoacylation of transfer RNA (tRNA). The insets in a, e and g indicate plot profile of green and red colors on the thin yellow line. c Flow-cytometric analysis of the GUVs of a. The illuminated GUVs are shown as green, whereas the GUVs incubated in the dark are shown as black. The X- and Y-axes represent the fluorescent intensity and the area of forward scattering, respectively. d GFP synthesis coupled with guanosine 5’-triphosphate (GTP) generation. GFP was synthesized in the PURE system with or without nucleoside-diphosphate kinase (NDK), GTP, guanosine diphosphate (GDP) and adenosine 5’-diphosphate (ADP). e The same reactions as in lanes 4 and 6 of d were performed inside GUVs as indicated as NDK+ and NDK−, respectively. f GFP synthesis from its DNA. A gene of whole GFP was introduced in the PURE system with or without bRF o F 1 -PLs, T7 RNA polymerase (T7RNAP), ATP and light. g A small part of GFP (GFP11: 15 amino acids) was synthesized from its encoding DNA inside GUVs containing T7RNAP, another large part of GFP (GFP1-10) purified form E. coli cells, and the PURE system lacking NDK. h The same GUVs of g were analyzed by flow-cytometer as in d. The synthesized GFP in b, d, and f were labeled with [35S]methionine. Scale bar: 10 µm. Source data are provided as a Source Data file Full size image

Next, we omitted GTP from the reaction mixture but introduced GDP and nucleoside-diphosphate kinases (NDKs), which allows the photosynthesized ATP to be consumed for synthesis of GTP that is a direct energy source of translation (Supplementary Fig. 10). The results showed that synthesized GFP was clearly detected by the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis when adenosine 5’-diphosphate (ADP), GDP and NDK were added (Fig. 2d, lane 4). We performed the same reaction inside GUVs and observed the fluorescence emission from the GUV lumen (Fig. 2e), suggesting that the photosynthesized energy was consumed not only for aminoacylation of tRNAs but also directly for translation inside GUVs.

In real cells, ATP is consumed not only as energy but also as a substrate of transcription. To build up this, we performed a transcription-and-translation coupled reaction in the artificial photosynthetic cell system. When T7 RNA polymerase and template DNA coding-GFP were introduced into the PURE system, photosynthesis of GFP was clearly detected by SDS-PAGE analysis (Fig. 2f). However, we could not detect significant fluorescence by microscopy observation and flow-cytometer analysis when we performed inside GUVs. This is because the synthesized GFP level was lower than the detection limit. To overcome this problem, we applied the split-GFP method developed by Cabantous et al.22, i.e., GFP is split into two parts: a small peptide (GFP11) and another large partner protein (GFP1-10). The fluorescence of GFP1-10 was restored by incorporating GFP11 (Supplementary Fig. 15). Although the intensity was rather weak, we observed the emission of GFP fluorescence from the GUVs when GFP11 was photosynthesized from the template DNA (Fig. 2g). In this reaction, we encapsulated the PURE system modified for transcription-and-translation reaction (Supplementary Table 2), and the purified GFP1-10. The successful photosynthesis of GFP11 was also confirmed in an in vitro reaction (Supplementary Fig. 16). By flow-cytometry analysis, we found that about 15% of the total GUVs emitted significant fluorescence as a consequence of transcription and translation inside (Fig. 2h). These results show that the photosynthesized ATP was consumed both as the substrates for mRNA transcription and as the energy for protein translation, just as in real cells.

Self-production of the artificial organelle components

The two kinds of component proteins of the artificial organelle produced ATP, and the resulting ATP drove protein synthesis. To test whether our artificial photosynthetic cell system can synthesize the component proteins of its own artificial organelle, we tried to photosynthesize bR, as well as F o F 1 . In this reaction, we used the translation-only PURE system (see For mRNA start in Supplementary Table 2). We expected that the newly photosynthesized de novo bRs localize onto the bRF o F 1 -PL membrane and increase ATP photosynthesis activity of the artificial organelle as a consequence of activity enhancement in the proton gradient generation of the bRF o F 1 -PL (Fig. 3a and Supplementary Fig. 1E and F). The fluorescence of the synthesized bR, which fused with GFP (bR-GFP), was mostly homogeneously observed inside the GUV lumen but not on the GUV membrane (Fig. 3b), indicating that the synthesized bR-GFP localized onto the internal PL membrane. This directed membrane localization is controlled by means of cholesterol which inhibits spontaneous membrane integration of protein23. We added 40% (mol%) cholesterol in the lipid composition of GUV membrane but not in the internal PL membrane. When bR-GFP was synthesized inside GUVs containing liposomes, the same homogeneous fluorescent distribution was observed within the GUV lumen. In contrast, when the GUVs were not encapsulating liposomes, many larger-size puncta appeared, suggesting aggregation of the synthesized bR-GFP (Supplementary Fig. 17). These results imply that the bR-GFP synthesized in the GUVs (Fig. 3b) localized onto the internal PL membrane avoiding protein aggregation. The membrane localization of bR was further confirmed in vitro by flotation assay. When bR was synthesized in a standard PURE system in the presence of liposomes, we found that the synthesized bR appeared in the liposome fractions (Fig. 3c) after ultra-centrifugation with a sucrose cushion, whereas almost all bR appeared in the pellet fraction when liposomes were omitted. This result directly shows the membrane localization of the de novo bR onto the PL membrane. Additionally, the membrane localized bR showed the proton-pump activity in response to the duration of protein synthesis reaction (Fig. 3d). Here, 11, 61, 124 or 233 bRs per one liposome were synthesized at the time of 10, 30, 60 or 180 min reaction, respectively (Supplementary Fig. 18). These results lead us to conclude that the de novo photosynthesized bRs spontaneously localized onto the internal PL membrane and may have increased the proton-pump activity there.

Fig. 3 Self-constituting protein synthesis in artificial photosynthetic cells. a Schematics of self-constituting protein synthesis. The numbers indicate the order of reactions; ➀: adenosine triphosphate (ATP) synthesis, ➁: aminoacylation of transfer RNA (tRNA) by aminoacyl-tRNA synthetase (ARS), ➂: translation by ribosomes (Rbs), ➃: de novo bacteriorhodopsin (bR) synthesis, and ➄: de novo F o synthesis. b Light-induced bR-GFP synthesis in giant unilamellar vesicles (GUVs). Bar: 10 µm. c Membrane localization of bR. The bRs synthesized in the PURE system with or without liposomes were fractionated by ultra-centrifugation with sucrose cushion. The percentages of bR in each fraction (%Frac.) are indicated at the bottom of the gels. d Proton-pump activity of bR synthesized in the PURE system. The measurement was performed as in Fig. 1c. The reaction times of protein synthesis are indicated by different colors. The white and gray areas represent light ON and OFF, respectively. e Enhanced artificial organelle by de novo bR. Wild-type (bR wt ) or mutant (bR mut ) bRs were photosynthesized in the PURE system containing bRF o F 1 -PLs. The ATP concentrations at the time 2 h was measured and converted into ATP per proteoliposome (PL). The value of the de novo bR wt -containing PL was normalized to that of the de novo bR mut -containing PL. ***P < 0.001. f Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the de novo photosynthesized bR. g Light-driven ATP synthesis by PLs consist of cell-free synthesized F o . Wild-type (a wt ) or mutant (a mut ) a-subunit protein was synthesized together with b- and c-subunits in the PURE system containing purified F 1 and bR-PLs. The measured ATP concentrations were converted into the produced ATP per PL. h Enhanced artificial organelle by de novo F o . a wt or a mut was photosynthesized together with b- and c-subunits in the presence of purified F 1 and bRF o F 1 -PLs. The ATP concentrations at the time 3 h was measured and converted into ATP per PL. The value of the de novo a wt -containing PL was normalized by that of the de novo a mut -containing PL. **P < 0.01. i SDS-PAGE analysis of the de novo photosynthesized F o . P values were from two-side t-test. All experiments were performed at least three times and their means and S.D. are shown. Source data are provided as a Source Data file Full size image

If the de novo photosynthesized bRs are functionable on the PL membrane, the ATP production rate of PL should be enhanced according to the increase of the number of bR per PL. To confirm this, we measured ATP concentration in the PURE system reaction mixture during the photosynthesis of de novo bR (bR wt ). In this experiment, we used the PLs consisting of a low concentration bR (i.e. 5 µM bR) to emphasize the effects of the de novo photosynthesized bR. The effect of the de novo photosynthesized bR was determined by comparing to the control experiment synthesizing a mutant bR (bR mut ) which does not have any proton-pomp activity (Supplementary Fig. 19), and therefore the ATP production rate of the PLs containing bR mut is constant throughout the bR photosynthesis. In the case of bR wt photosynthesis, the ATP concentration was higher than that in the case of bR mut photosynthesizing in all three independent measurements (Supplementary Fig. 20), especially after 10 min reaction. This is consistent with the result of proton-pump activity of the bR synthesized in PURE system (Fig. 3d). Here, the difference in the ATP concentration between bR wt and bR mut photosynthesizing reactions represents the effect of de novo photosynthesized bR wt . The ATP concentration in the bR wt -photosynthesizing reaction was approximately 1.5-fold higher than that of bR mut (Supplementary Fig. 20). It should be noted that the obtained ATP concentration indicates the net of the photosynthesized ATP minus the consumed ATP for the protein synthesis. The synthesis rate of the bR wt and bR mut was adjusted to be the same by regulating the amount of template mRNA (Supplementary Fig. 21), and thus the ATP consumption rates were equal in both cases. To directly compare all three measurements, we normalized each obtained result with the ATP per PL value at the endpoint time (120 min) of the de novo bR mut -photosynthesizing reaction. The ratio of the increased artificial organelle activity is shown in Fig. 3e. We also confirmed the photosynthesized bR by SDS-PAGE analysis (Fig. 3f) in which the photosynthesis rate was 2.5 nmol ml−1 min−1. After 2 h of reaction, the number of working bRs per PL increased from 100 working bRs per PL (original) to 110 bRs per PL (after photosynthesis). These series of results indicate that the ATP production rate was enhanced during the photosynthesis of de novo bR wt because the ability of proton gradient generation was improved by increasing the number of functional bRs on a PL.