The archaeal domain of life encloses microorganisms with extraordinary diverse and even unique metabolic capabilities including organotrophic, lithotrophic and phototrophic pathways evolved to survive harsh environmental conditions (Schäfer et al., 1999).

Phototrophic growth appears to be an exclusive prerogative found in halophilic archaea. Usually, they are aerobic chemoheterotrophs consuming preformed organic substances (mainly amino acids) as source of energy and carbon. However, due to low O₂ solubility in brine ponds, species like Halobacterium salinarium, use sunlight as supplementary energy fuel (Hartmann et al., 1980; Schäfer et al., 1999; Falb et al., 2008).

The photophosphorylation mechanism of this archaeon differs from others, relying on a single pigment-protein called bacteriorhodopsin (BR) (Figure 1A, in purple). BR is the smallest light-dependent proton pump known in nature and each protein monomer engages one retinal chromophore as light-harvesting group (a derivative of beta-carotene) (Henderson et al., 1990; Kimura et al., 1997). In the bacterial membrane BR is clustered into hexamers forming two-dimensional hexagonal patches of purple colour due to the presence of the retinal chromophores (Blaurock and Stoeckenius, 1971; Henderson and Unwin, 1975; Essen et al., 1998). Upon light excitation, the retinal group undergoes a conformational change. The original state is regained thanks to the outward movement of protons across the microbial membrane thus generating a proton potential. The pH gradient is used in the end to drive ATP synthesis by ATP synthase (Figure 1A, in yellow) (Lozier et al., 1975).

This photochemical energy conversion mechanism is considered unrelated to other forms of photosynthesis for two main reasons: (i) Halobacteria photochemistry does not require chlorophylls being instead carotenoid-based (ii) redox reactions are not involved in the photocycle since a direct ion transfer occurs upon interaction between light and the pigment-protein complex.

More recently, proteorhodopsin (PR), a retinal-containing integral membrane protein, functioning as a light-driven proton pump as well, was discovered in marine bacteria sharing high amino acid sequence similarity with archaeal BRs (Béjà et al., 2000; Béjà et al., 2001).

R. sphaeroides 2.4.1 is a Gram-negative, rod-shaped, purple non-sulphur bacterium with a broad range of metabolic capabilities (Woese et al., 1984; Ferguson et al., 1987; Woese, 1987). This organism populates habitats with low-light illumination, namely, ≤10% of full sunlight (Woronowicz and Niederman, 2010; Blankenship, 2014) and experiences the transition between chemotrophic to phototropic growth in response to decreasing of oxygen tension (O₂ ≤ 3%) (Altamura et al., 2018a). Its photosynthetic apparatus has served as a model for anoxygenic photosynthesis and has been extensively studied over the last 60 years (Sener et al., 2010; Cartron et al., 2014; Sener et al., 2016; Hitchcock et al., 2017). The primary components are, in order of energy utilization: light-harvesting complexes I (LH1) and II (LH2), Reaction Center (RC), ubiquinol: cytochrome c—oxidoreductase (bc1), ATP synthase (ATPsyn). Bacterial photosynthesis starts with Light Harvesting Complexes that absorb incident light and transfer the energy to the Reaction Center (Figure 1B, in blue). RC catalyses together with cytochrome bc1 (Figure 1B, in red), a cyclic redox reaction involving two couples of electron shuttles: periplasmic cytochrome c2 in its reduced (cyt²⁺) and oxidised (cyt³⁺) form (Figure 1B, pink and light pink, respectively), and quinone/quinol (Q/QH₂) molecules from the membrane pool (Figure 1B, in white). During the photo-redox cycle, protons are translocated from the cytoplasm to the periplasm according to the following stoichiometry: Q H 2 + 2 H i n + + 2 c y t 3 + → 2 h υ Q + 4 H o u t + + 2 c y t 2 + where in and out indicate the cytoplasmic and the periplasmic side of the membrane, respectively. A pH gradient is formed across the membrane and this proton-motive force is then exploited by the ATPsyn (Figure 1B, in yellow) to sustain the phosphorylation of ADP molecules into ATP.

In photoautotroph eukaryotes, the photosynthetic process occurs in specialized intracytoplasmic organelles named chloroplasts. In addition to the outer double-membrane layer (envelope), they also have an inner system of interconnected membrane-surrounded sacs, called thylakoids, where the photophosphorylation machinery is localised. The organelle aqueous lumen is named stroma and houses the enzymes for metabolic pathways occurring in the organelle most notably starch metabolism and CO₂-fixing Calvin-Benson cycle for photosynthetic carbon assimilation (Staehelin and Paolillo, 2020).

The main components of the photophosphorylation system are 4 multiprotein complexes (i) photosystem II (PSII, Figure 1C, in magenta), (ii) cytochrome b ₆ f (Figure 1C, in violet), (iii) photosystem I (PSI, Figure 1C, in green), and (iv) ATP synthase (Figure 1C, in yellow). Light-Harvesting Complexes are also associated with both photosystems, enhancing the light-absorption capabilities of their reaction centres (P680 and P700 for PSII and PSI, respectively).

All these complexes act synergically to generate a light-powered electron flux from a high-redox potential couple (H₂O/O₂ E_m = 800 mV) to a low-redox potential couple (NAD(P)H/NAD(P)⁺, E_m = −300 mV) (Figure 1C, red dashed line). The process is associated with the generation of a transmembrane proton gradient, driving the photophosphorylation of ADP in ATP molecules. The produced energy-rich compounds, NADPH and ATP, are in turn used to sustain the CO₂-fixing process.

Upon light absorption PSII transfers one electron of the special pair of chlorophylls P680 to a plastoquinone molecule (PQ, Figure 1C, in white) of the membrane pool. The water splitting, catalysed in the proximity of the lumen, drives the electron to the photo-oxidized P680. Once two electrons have been transferred in tandem, PQ is reduced to plastoquinol (PQH₂) with the uptake of protons from the stroma. PQH₂ delivers electrons to cytochrome b ₆ f that oxidizes plastoquinol and uses the gained electrons to reduce the electron-shuttle protein plastocyanin (PC, Figure 1C, in orange) contextually pumping protons across the thylakoid membranes. PC delivers the electrons to the photo-oxidized PSI which preliminarily has reduced ferredoxin (Fdx, Figure 1C, in pink), an iron-sulfur protein associated with the photosystem from the stromatic side of the membrane. Finally, the electrons of the Fdx are passed to NADP⁺ by the Ferredoxin-NADP⁺ oxidoreductase (FNR, Figure 1C, in brown), synthesizing NADPH molecules. The pH gradient, accumulated during the electron transfer pathway, is the proton motive force driving the synthesis of ATP molecules in the stroma. In Spinacia oleracea chloroplasts, a complete rotational catalysis of ATPsyn requires 14 H⁺ and brings to the production of 3 ATP molecules (Hahn et al., 2018).

Considering that a proton gradient of approximately 12 H⁺ is produced for every O₂ molecule evolved (Furbank and Badger, 1983), the overall stoichiometric equation of the process is: 8   p h o t o n s + 2 H 2 O + 2   N A D P + + 2.57   A D P + 2.57   P i → O 2 + 2   N A D P H + 2.57   A T P and describes the unidirectional electron transfer from H₂O to NADP⁺. The production of energy-rich molecules must be strictly balanced by their consumption to avoid an over-production of highly reactive chemical species leading to photodamage (Kramer and Evans, 2011).

Starting from the end of the last century, transmembrane proteins have been extensively studied by reconstitution into nano-sized lipid vesicles as simplified membrane models, obtaining proteoliposomes (Rigaud et al., 1995). This allowed to explore the characteristics of the proteins avoiding the complexity of the native membrane and the interference with other cellular components or side reactions. Particular attention was paid to energy-transducing membrane proteins, such as BR and FOF1-ATP synthases. When BR is reconstituted alone into phospholipid vesicles is capable of acidifying the liposome internal milieu upon illumination (van Dijck and van Dam, 1982). The case of the co-reconstitution of BR hexamers from Halobacterium halobium and FOF1-ATPsyn from bovine mitochondria in liposome is the first example of light energy transducer lipid compartments reported in 1974 by Racker and Stoeckenius (Racker and Stoeckenius, 1974). It was proven that these proteoliposomes could photo-synthesize ATP from ADP and inorganic phosphate under light irradiation. Although the observed rate of ATP synthesis was only 0.1% of the oxidative phosphorylation rate found in mitochondria, proteoliposomes embedding BR and ATP synthase became an ideal model to study the mechanism of energy coupling. A subsequent work pointed out that ATP synthesis yield is influenced by the protein purification method, the co-reconstitution procedure, and the type of phospholipids used which affects the protein distribution among liposomes (Van Der Bend et al., 1984).

Indeed, bacteriorhodopsin in monomeric form performs much better than in form of purple membrane patches being more homogeneously distributed among liposomes. This results in a 6.2 times higher ATP production rate (Wagner et al., 1987).

In the following years, several authors reported the possibility to reconstitute FOF1-ATP synthase complex along with monomeric bacteriorhodopsin in proteoliposomes, as well. A comparison among examples of these semi-synthetic photoactive organelles is reported in Otrin et al., 2019, where the type of compartment and the light transducing efficiency are also listed.

Later in 1997, Moore and co-workers replaced BR with an artificial reaction centre prepared by linking a synthetic tetraarylporphyrin to both a naphthoquinone moiety and a norbornene system with a carboxylic group and a carotenoid polyene (Steinberg-Yfrach et al., 1997), subsequently coupled with ATPsyn in an artificial photosynthetic membrane (Steinberg-Yfrach et al., 1998).

More recently, mimicking the bacterial photosynthetic apparatus, the Rection Center extracted from the R26 mutant of R. sphaeroides has been reconstituted in the GUV membrane with the 90% of physiological orientation. These giant proteoliposomes generate a transmembrane proton gradient under continuous light irradiation by increasing the internal pH with a rate of 0.061 ± 0.004 pH units per minute and exhibit a retained photo-efficiency of 80% after 24 h (Altamura et al., 2017a).

Although the co-reconstitution of bc1 along with RC in the GUV membrane with a correct orientation is still a challenging task (Hardy et al., 2018), it has been also shown that the bacterial RC proteins can be coupled with the bc1 extracted from bovine heart mitochondria to obtain a more efficient hybrid system in comparison to the bacterial one (Altamura et al., 2017b; Altamura et al., 2021a). This, in principle, gives the chance to improve the model bacterial apparatus with the most efficient protein complexes extracted from mammalian cells.

Different classes of multi-compartment systems have been developed and proposed to mimic the architecture of living cells including liposomes within layer-by-layer capsules (capsosomes), liposome-in-liposome (vesosomes), polymersome-in-polymersome architectures and multisomes (Schoonen and van Hest, 2016). Herein we focus the attention exclusively on multi-compartment architecture based on giant lipid vesicles.

In 2018, the first example was realized with engineered switchable photosynthetic organelles (100 nm diameter) encapsulated in giant lipid vesicles (10–100 µm diameter) (Figure 2A). The synthetic organelles were acting as energy modules enabling the conversion of light energy into ATP molecules, in turn used to sustain an internalized anabolic reaction. In the nano-liposome membrane two complementary photoconverters were co-reconstituted: plant-derived photosystem II (PSII) and bacterial-derived proteorhodopsin (PR) along with ATPsyn. Upon white light illumination, the PSII and PR generated a proton motive force across the proteoliposome membrane that was sufficient to induce ATP synthesis outside the artificial organelles (Figure 2A) (Lee et al., 2018). The maximum turnover number of the ATP synthase was 4.3 ± 0.1 s⁻¹. The artificial organelles sustained ATP conversion for 3 days (half-maximum efficiency) at room temperature and for 1 month at 4°C. Optical independent activation of the two photoconverters allowed dynamic control of ATP synthesis since red light facilitates and green light impedes ATP synthesis (Figure 2A). This artificial system was able to sustain two ATP-dependent reactions powered by light, such as carbon fixation and actin polymerization, altering, with the latter, the vesicle morphology (Figure 2A). In the can be used to develop biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviours.

One year later, Berhanu and co-workers reported the co-encapsulation in GUVs of a cell-free protein synthesis system and proteoliposomes containing BR and ATP synthase. The photoproduced ATP sustained the synthesis of BR or constituent proteins of ATP synthase, that spontaneously integrated into the artificial photosynthetic organelles enhancing its photosynthetic activity (Figure 2B). The ATPsyn turnover number in the first 5 min of proteoliposome bulk activity resulted in 8.3 ± 0.3 s⁻¹, while when encapsulated in GUVs it reduces to one-third (Berhanu et al., 2019).

More recently, it has been shown that the entire photosynthetic apparatus of a living organism can be encapsulated into GUVs in form of chromatophores, natural nanometric proteoliposomes extracted from R. sphaeroides. Under specific growth conditions this bacterium forms cytoplasmic membrane invaginations in which the photosynthetic system is confined (Feniouk et al., 2002; Noble et al., 2018). After a single French press step, bacterial cells are broken inducing the spontaneous closure of the present invaginations into chromatophores. The nano-vesicles still trap the intact photosynthetic apparat in their membranes with the physiological orientation of all protein complexes. These organelles were encapsulated in GUVs along with a commercial kit for DNA transcription showing that the photo-produced ATP was sufficient to sustain the synthesis of mRNA molecules (Figure 2C). The estimated turnover of the ATPsyn was around 80 s⁻¹ (Altamura et al., 2021b).