WO2011030964A1

WO2011030964A1 - Composition for atp synthesis, mounted for gloeobacter rhodopsin and atp synthase to have orientation

Info

Publication number: WO2011030964A1
Application number: PCT/KR2009/006736
Authority: WO
Inventors: Kwang-Hwan Jung; Keon Ah Lee; Ah Reum Choi
Original assignee: Industry-University Cooperation Foundation Sogang University
Priority date: 2009-09-14
Filing date: 2009-11-27
Publication date: 2011-03-17
Also published as: KR101197129B1; KR20110028853A

Abstract

Disclosed herein is a composition for ATP synthesis, mounted for Gloeobacter rhodopsin (hereinafter, referred to as GR) and ATP synthase to have orientation. Specifically, disclosed herein is a method for synthesizing ATP, including 1) overexpressing GR to translocate protons (H+) from the inside to the outside of the membrane in E. coli, 2) grinding the cells to prepare an inverted membrane, 3) adding Pi and irradiating to form a proton membrane gradient by GR, and 4) synthesizing ATP through ATP synthase, a membrane protein of E. coli from the membrane gradient. The composition for ATP synthesis and the method for preparing ATP according to the present invention may stably provide a large amount of ATP for mass production of biomaterials, and the costs may be greatly reduced due to renewable use of the composition for ATP synthesis according to the present invention.

Description

COMPOSITION FOR ATP SYNTHESIS, MOUNTED FOR GLOEOBACTER RHODOPSIN AND ATP SYNTHASE TO HAVE ORIENTATION

The present disclosure relates to a composition for artificial ATP synthesis.

Living cells constantly perform work and thus require energy. Energy is used for various processes such as maintenance of highly organized structures, synthesis of cellular components, movement, generation of electrical currents, production of light, etc. ATP is one of the main energy sources among various metabolites. ATP is the chemical link between catabolism and anabolism, and one of the factors which constitute the energy flow in a cell. ATP is exergonically converted into ADP and Pi or AMP and PPi, which is coupled to endergonic conversions of many other substrates. When ATP is hydrolyzed, a phosphoryl, pyrophosphoryl, or adenylyl group from ATP is transferred to the substrate. By these 'group transfer' reactions, ATP provides the energy for anabolic reactions. In ATP synthesis of bacteria, mitochondria, and chloroplast, protons move through the F₁F₀ ATP synthase for each ATP molecule produced when an electrochemical gradient of protons (ΔH+)[or sometimes Na+ ions(ΔNa+)] is generated across the membrane (Capaldi RA & Aggeler R, Trends Biochem. Sci. 27:154-160, 2002; Dimroth P, Biochim. Biophys. Acta 1318:11-51, 1997; Neumann S et al., J. Bacteriol. 180:3312-3316, 1998; Reidlinger J & Muller V, Eur. J. Biochem. 223:275-283, 1994; Yoshida M et al., Nat. Rev. Mol. Cell. Biol. 2:669-677, 2001). The F₁F₀ ATP synthase consists of F1 and F0, membrane-related peripheral portion and integral membrane component. The F₁ and F₀ consist of subunits of α₃β₃γδεand ab₂C_10-14, respectively. Transfer of ions through F₀ is coupled via this molecular machine to synthesis of ATP by F₁. For biochemical studies on ATP synthase machinery from various organisms, ATP synthase was isolated from a facultative thermoalkaliphilic Bacillus species by one study group (Olsson K et al., J. Bacteriol. 185:461-465, 2003; Peddie CJ et al., J. Bacteriol. 181:3172-3177, 1999), and the purified enzyme was reconstituted into proteoliposomes for ion transport studies, which showed that the ATP synthase utilizes protons as coupling ions for ATP synthesis (Gregory M et al., J. Bacteriol. 185 (15): 4442-4449, 2003).

Upon formation of a proton gradient across the membrane as a pre-step for ATP synthesis, microorganisms utilize the respiratory process using the electron transport system in cell membrane while some of microorganisms obtain aids from a membrane protein called rhodopsin. Rhodopsin is a photoactive retinylidene protein embedded in the membrane and these photoreceptor proteins are well distributed among a Kingdom of animal, fungi, protista, and bacteria. Rhodopsin was first isolated from the eyes of cattle in the 1930s. The protein is responsible for vision in the eyes of animals (Spudich JL, Trends Microbiol. 14(11): 480-487, 2006), while for microorganisms, it is not only used to pump hydrogen or chlorine ions or move cells but also is involved in phototactic responses (Spudich JL & Jung KH, In Handbook of Photosensory Receptors, ed. (2005) W.R. Griggs and J.L. Spudich, pp. 1-24). Among rhodopsins from microorganisms, proteorhodopsin (hereinafter, referred to as PR), discovered in a marine γ-proteobacterium of the SAR86 group, and worked as a light-driven proton pump (Beja O et al., Science 289:1902-1906, 2000; Beja O et al., Nature. 411:786-789, 2001). Until now, more than 2000 different PR genes have been identified in various oceans around the world, and it has been found that an estimated 13% of microorganisms in the photic zone as well as more than 50% of all marine organism communities found in maritime surface water are bacteria which contain the PR genes (Sabehi G et al., PLoS Biol. 3:e273, 2005).

Gloeobacter violaceus is a unicellular cyanobacterium that has been isolated from calcareous rocks in Switzerland (Rippka R et al., Arch. Microbiol. 100:419-436, 1974). According to phylogenetic analysis based on the 16S rRNA sequence, it branched off from the main cyanobacterial tree at a very early stage, which suggests that it may retain some of the primitive properties of early cyanobacteria (Nelissen B et al., J Mol Evol. 42(2):194-200, 1996; Nelissen B et al., Mol Biol Evol. 12(6):1166-1173 1995). G. violaceus, which performs photosynthesis, lacks a thylakoid membrane. However, the bacteria include all the photosynthesis machinery and respiratory system in the cell membrane (Rippka R et al., Arch. Microbiol. 100:419-436, 1974). Results of sequence homology comparisons showed that a gene homologous with microbial rhodopsin was found in the G. violaceus genome, having proton pumping activity like PR. This rhodopsin was called Gloeobacter rhodopsin (hereinafter, referred to as GR).

ATP is a material which provides energy necessary for cells and life forms. Recently, while a large amount of ATP has been used for mass production of biomaterials, ATP is not a material that is renewable when once used and is very expensive to synthesize. However, if a composition for in-vivo ATP synthesis system is used, ATP may be constantly provided and its renewal is also possible.

As a result of these studies, the present inventors made an inverted membrane by grinding GR cells overexpressed in E. coli, constructed a composition for ATP synthesis by allowing a proton membrane gradient produced by the GR to be formed through ATP synthase in E. coli, and completed the present invention.

One object of the present invention is to provide a composition for ATP synthesis, including an inverted membrane vesicle, which is mounted for Gloeobacter rhodopsin (hereinafter, referred to as GR) and ATP synthase to have orientation.

Another object of the present invention is to provide a method for preparing the inverted membrane vesicle.

Still another object of the present invention is to provide a method for synthesizing ATP by utilizing the composition for ATP synthesis.

In order to achieve the objects, the present invention provides a composition for ATP synthesis, including an inverted membrane vesicle including ATP synthase oriented to have an extroversion so as to pump in protons and produce ATP outside the membrane by a concentration gradient of Gloeobacter rhodopsin (hereinafter, referred to as GR) oriented to have an introversion and the pumped protons.

The present invention also provides a method for preparing a composition for ATP synthesis, mounted for GR and ATP synthase to have orientation, including Steps:

1) preparing a recombinant expression vector operably linked to a polynucleotide encoding GR;

2) transforming a host cell with the recombinant expression vector in Step 1) to prepare a transformant;

3) incubating the transformant in Step 2) to induce an expression of GR;

4) isolating a protoplast from a transformant overexpressing the GR in Step 3); and

5) preparing an inverted membrane vesicle from the protoplast isolated in Step 4).

Furthermore, the present invention provides a method for synthesizing ATP by using the composition for ATP synthesis, including:

1) adding Pi (phosphoryl group) and ADP to the composition for ATP synthesis of the present invention; and

2)irradiating the composition.

The present invention also provides a use of an inverted membrane vesicle including ATP synthase oriented to have an extroversion so as to pump in protons and produce ATP outside the membrane by a concentration gradient of Gloeobacter rhodopsin (hereinafter, referred to as GR) oriented to have an introversion and the pumped protons in preparation of a composition for ATP synthesis.

The composition for ATP synthesis and the method for preparing ATP according to the present invention may stably provide a large amount of ATP for mass production of biomaterials, and the costs may be greatly reduced due to renewable use of the composition for ATP synthesis according to the present invention.

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic diagram illustrating the liposome reconstitution of Gloeobacter rhodopsin (hereinafter, referred to as GR) and ATP synthase.

FIG. 2 is a graph illustrating the stability of E. coli membrane in which GR is expressed.

Features and advantages of the present invention will be more clearly understood by the following detailed description of the present preferred embodiments by reference to the accompanying drawings. It is first noted that terms or words used herein should be construed as meanings or concepts corresponding with the technical sprit of the present invention, based on the principle that the inventor can appropriately define the concepts of the terms to best describe his own invention. Also, it should be understood that detailed descriptions of well-known functions and structures related to the present invention will be omitted so as not to unnecessarily obscure the important point of the present invention.

Hereinafter, the terms as used herein will be described in detail.

The term "expression vector" as used herein refers to a linear or circular DNA molecule which includes a fragment encoding a polypeptide of interest operably linked to additional fragments which provide for its transcription. Such an additional fragment includes sequences of a promoter and a termination codon. The expression vector also includes at least one origin of replication, at least one selection marker, a polyadenylation signal, etc. The expression vector is typically derived from plasmid or viral DNA, or may contain elements of both.

The term "operably linked" as used herein indicates that the fragments are arranged such that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through an encoding sequence to a termination codon.

Hereinafter, the present invention will be described in detail.

In a specific example of the present invention, a vector for overexpression of Gloeobacter rhodopsin (hereinafter, referred to as GR) was transformed into E. coli for overexpression. Protoplasts were obtained from the overexpressed cells, followed by French Press to obtain a membrane fraction of GR-overexpressed E. coli. The membrane fraction was resuspended in an extraction buffer to obtain an inverted vesicle-inverted membrane of GR-expressed E. coli. GR, which is a membrane protein to translocate protons (H+) from the inside to the outside of the membrane, may be inverted while being bound to the membrane of E. coli to form a proton membrane gradient by introduction of protons from the outside to the inside of the membrane as in FIG. 1. It is also possible to synthesize ATP through ATP synthase which is a membrane protein of E. coli.

In a specific example of the present invention, the composition for ATP synthesis, prepared by the method and having a form in FIG. 1, was subjected to Pi and light to produce ATP in an amount of approximately 3.68904E-12 mole to approximately 8.82043E-11 mole per mg of GR for 1 min (See Table 1). In addition, the time for which the amount of ATP produced by the composition for ATP synthesis was reduced by half (T_f) was 2.12 days and the amount of ATP produced was exponentially reduced within 10 days (See Table 2 and FIG. 2).

From these results, it was confirmed that the composition for ATP synthesis of the present invention was useful in ATP synthesis.

The present invention provides a composition for ATP synthesis, including an inverted membrane vesicle including ATP synthase oriented to have an extroversion so as to pump in protons and produce ATP outside the membrane by a concentration gradient of Gloeobacter rhodopsin (hereinafter, referred to as GR) oriented to have an introversion and the pumped protons.

The GR is a membrane protein to translocate protons (H⁺) from the inside to the outside of the membrane, is derived from Gloeobacter violaceus, and may be a protein available from NCBI accession No. BAC88139.

GR in the composition for ATP synthesis may form a proton membrane gradient by introducing protons from the outside to inside of the membrane. The ATP synthase may also translocate protons introduced at a high concentration in a membrane vesicle inverted with a membrane protein integrally incorporated into E. coli membrane to the outside of the membrane to synthesize ATP. The composition for ATP synthesis of the present invention produced ATP in an amount of approximately 3.68904E-12 mole to approximately 8.82043E-11 mole per mg of GR for 1 min (See Table 1).

The stability of the composition for ATP synthesis of the present is closely associated with that of GR and ATP synthase, and the result was indirectly confirmed by measuring the change in the amount of ATP produced (reduction in an amount of ATP produced) as time proceeded. The time for which the amount of ATP produced by the composition for ATP synthesis of the present invention was reduced by half (T_f) was 2.12 days and the amount of ATP produced was exponentially reduced within 10 days (See Tables 1 and 2 and FIG. 2).

The present invention also provides a method for preparing an inverted membrane vesicle including GR and ATP synthase, including Steps:

1) preparing a recombinant expression vector operably linked to a polynucleotide represented by SEQ ID NO. 1 encoding Gloeobacter rhodopsin (hereinafter, referred to as GR);

3) incubating the transformant in Step 2) to induce an expression of GR;

In the method, any cell applicable to preparation techniques of an inverted membrane vesicle may be used as the host cell in Step 2), and E. coli may be preferably used.

In the method, preparation techniques of an inverted membrane vesicle in Step 5) are characterized by pressure control for preparing an inverted membrane vesicle by using a French press when cells are ground and concentration of each material in a solution phase for protein stability. These preparation techniques are frequently used to study biochemical properties of proteins such as measurements of concentrations of ions or materials moving through proteins which are conventionally studied.

The present invention also provides a method for synthesizing ATP using the composition for ATP synthesis, including adding Pi to the composition for ATP synthesis of the present invention and irradiating the composition.

The composition for ATP synthesis of the present invention produced ATP in an amount of approximately 3.68904E-12 mole to approximately 8.82043E-11 mole per mg of GR for 1 min (See Table 1), the time for which the amount of ATP produced by the composition for ATP synthesis of the present invention was reduced by half (T_f) was 2.12 days, and the amount of ATP produced was exponentially reduced within 10 days (See Tables 1 and 2 and FIG. 2).

Preferably, an amount of the Pi added is approximately 1 pM to approximately 100 nM as a final concentration. However, it may be controlled according to the amount of the composition for ATP synthesis.

A visible light at a wavelength of at least above 440 nm is preferably irradiated.

ATP was synthesized while the amount of the composition for ATP synthesis was exponentially reduced within 10 days, and thus the composition is available for 0 to 10 days.

Furthermore, the present invention provides the use of an inverted membrane vesicle including ATP synthase oriented to have an extroversion so as to pump in protons and produce ATP outside the membrane by a concentration gradient of Gloeobacter rhodopsin (hereinafter, referred to as GR) oriented to have an introversion and the pumped protons in preparation of a composition for ATP synthesis.

It can be seen that GR in embodiments of the present invention formed a proton membrane gradient by introducing protons from the outside to inside of the membrane and the ATP synthase also translocated protons introduced at a high concentration in a membrane vesicle inverted with a membrane protein integrally incorporated into E. coli membrane to the outside of the membrane to synthesize ATP. It can be also seen that the composition for ATP synthesis stably provided a large amount of ATP. Therefore, it can be seen that an inverted membrane vesicle including GR and ATP synthase is useful as a component of a composition for ATP synthesis.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

However, the following examples are provided for illustrative purposes only, and the scope of the present invention should not be limited thereto in any manner.

<Example 1> Overexpression of GR in E. coli

<1-1> Preparation of GR-overexpressed E. coli

In order to overexpress Gloeobacter rhodopsin (hereinafter, referred to as GR) protein (SEQ ID NO. 2), a process for preparing a vector including a nucleic acid represented by SEQ ID No. 1 encoding GR was performed as follows. GR from NCBI accession NO. BAC88139 was amplified by PCR in genome DNA of Gloeobacter violaceous, and the degenerate primer set used is as follows: a forward primer represented by SEQ ID No. 3 (5'-ATGTTGATGACCGTATTTTCTTCTGC-3') and a reverse primer represented by SEQ ID No. 4 (5'-CTAGGAGATAAGACTGCCTCCCCG-3'). PCR for gene amplification was performed using pfu polymerase (Koschem, Korea) for 30 cycles (95℃/1 min, 54℃/1 min, and 72℃/2 min 30 sec). The gene product amplified by PCR with a 6-His tag was inserted downstream of the lacUV5 promoter of the pKJ900 vector, using XbaI, Not I restriction enzyme, and ligase (New English Lab, USA) (Kim et al., Biochim. Biophys. Acta 1777 (6): 504-513, 2008). The vector including an ampicillin resistance gene was transformed into an E. coli strain DH5α(UT5600, New England Biolabs, USA) and was selected in LB Agar containing 50 μM of ampicillin (USB Corp, USA). The selected transformant was inoculated in a 500 ㎖ LB medium supplemented with ampicillin (USB corp, USA) at 50 ㎍/㎖, to which 1 mM IPTG and 5 μM all-trans retinal (Sigma-Aldrich, USA) were added, and incubated in an incubator with stirring for 4 hours (30℃, 200 rpm). Centrifugation was performed at 5000 rpm for 15 min to obtain a pink E. coli pellet in which rhodopsin was expressed.

<1-2> Yield of protoplasts

Approximately 3 g of cells (wet weight) was resuspended in a solution containing 50 mM Tris-HCl (pH 8.2) and 0.5 mM PMSF, supplemented with 3.6 mg of lysozyme, and stirred at room temperature for 45 min. Subsequently, DNase I (2 mg) and MgCl₂ (final concentration 5 mM) were added to the solution, which was stirred at room temperature for 15 min. Centrifugation was performed at 4℃ and 180,000 rpm for 1 hour to obtain a protoplast.

<Example 2> Yield of fraction of GR-overexpressed E. coli membrane

The protoplast pellet obtained in <Example 1> was resuspended in 4 ㎖ of extraction buffer [50 mM potassium phosphate buffer (pH 7.8), 0.5 mM PMSF, 2 mM MgCl₂, and 1 mM DTT]. The protoplast was ground using a pre-cooled French Pressure Cell Press (SLM Aminco, USA) (2 passages, 20,000 Ib/in²). Cells which had not been ground were centrifuged at 8,000 rpm for 2 min and removed. Supernatant was collected and centrifuged at 180,000 rpm for 45 min to obtain a membrane. The membrane was resuspended in 4 ㎖ of extraction buffer to yield an inverted vesicle-inverted membrane. In this way, a GR-expressed E. coli membrane having a form as in FIG. 1 was prepared.

<Example 3> ATP Assay

<3-1> Test method

P_i (Sigma-Aldrich, USA) was added to a sample dissolved in 4 ㎖ of extraction buffer to prepare a solution at final concentration of 5 mM. Light was irradiated for 20 min using a cut-off filter (Sigma Koki SCH-50S-44Y, Japan) at = 440 nm for light adaptation. Simultaneously, P_i was added to a sample at the same concentration to obtain a solution at final concentration of 5 mM, followed by dark adaptation. 20 minutes later, a luminometer (ADENOSINE 5 -TRIPHOSPHATE (ATP) BIOLUMINESCENT ASSAY KIT, Sigma, USA) was used to measure the amount of ATP produced. The amount of ATP produced in a dark-adapted sample was subtracted from that in a light-adapted sample to correct the value.

<3-2> Measurement of the amount of ATP produced per mg of GR for 1 min

In order to measure the amount of GR, a UV-visible spectrophotometer (Shimadzu UV-2550, Japan) was used to measure an amount of absorption of the composition for ATP synthesis. The O.D. (optical density) value thus obtained from this was used to calculate the molarity of GR protein. The amount of ATP produced per mg of GR for 1 min was measured by the method in Example <3-1>. A luminometer was used to measure the total amount of ATP produced per assay for 20 min, a value obtained from the dark adaptation was subtracted to correct the value, and the value was divided by the concentration of GR. The calculation processes and results were summarized in Table 1.

As a result, ATP in an amount of approximately 3.68904E-12 mole to approximately 8.82043E-11 mole was produced as shown in Table 1.

Table 1

Sample	GR1	GR2	GR3	GR4	GR5	GR6
1) L20 (R.L.I)	12,093,888	11,357,044	14,673,190	16,602,056	10,936,660	13,545,794
2) D20 (R.L.I)	7,550,470	10,403,830	1,400,263	2,027,044	545,492	10,177,552
3) L-D (R.L.I)	4,543,418	953,214	13,272,927	14,575,012	10,391,168	3,368,242
4) L-D (moles ATP/assay)	1.50929E-10	2.73458E-11	4.8762E-10	5.40183E-10	3.73075E-10	1.0879E-10
5) L-D (moles ATP/ Sample 4 ㎖)	6.03717E-09	1.09383E-09	1.95048E-08	2.16073E-08	1.4923E-08	4.35159E-09
6) Molarity (㎎/㎖)	2.523127693	3.706362839	2.840809253	3.062110251	2.89445798	3.573410689
7) ㎎/4 ㎖	10.09251077	14.82545136	11.36323701	12.248441	11.57783192	14.29364276
8) moles ATP/min/㎎	2.99092E-11	3.68904E-12	8.58242E-11	8.82043E-11	6.44465E-11	1.52221E-11

1) L20 was measured by adding Pi to a GR sample and irradiating the sample for 20 min

2) D20 was measured by adding Pi to a GR sample and leaving the sample in the dark for 20 min

* R.L.I is an acronym for Relative Light Intensity, indicating a value of a relative luminescence intensity measured by a luminometer

3) L-D is a subtraction of D20 form L20

4) A value obtained from a pre-measured ATP calibration curve. The unit is moles ATP/assay

5) Because the volume of a final sample was 4 ㎖, the total amount of ATP produced from the 4 ㎖ volume was calculated

6) The amount of GR contained in 1 ㎖ of a sample

7) The amount of GR contained in the total volume of 4 ㎖

8) [5)/20 min]/7). The unit is moles ATP/min/㎎

<3-3> Measurement of the change in ATP production as time

When an E. coli membrane vesicle in which GR was expressed was stored at 4℃, it was confirmed whether a change in the amount of ATP production occurred as time proceeded. The same experiments as above were repeated 7 and 65 days from the day when three samples (GR3, GR4, and GR5) in FIG. 1 were first prepared. The samples were those including the best production efficiency of ATP. The determined values were recorded in Table 2, and mean values of the three samples were used to plot a change in the amount of ATP production as time proceeded.

Table 2

Moles/ATP/min/㎎	GR3	GR4	GR5	Mean value
Day
0	8.58E-11	8.82E-11	6.44E-11	7.95E-11
Day
7	1.05E-11	1.08E-11	7.93E-12	9.74E-12
Day 65	2.48E-12	2.02E-12	3.16E-12	2.55E-12

That is, each of the half-times (T_f) was calculated with respect to a mean value of the three values according to each time of measurement in FIG. 2. The mean value was fitted to an exponential decay by using the following Math Figure 1, and the results were recorded in a small graph in FIG. 2.

MathFigure 1

N_t: the amount remaining without being decayed after t hours;

N₀: the initial amount of the material which may be decayed;

λ: decay constant.

The y-axis of the small graph in FIG. 2 was transformed into the log scale, was shown in the big graph in FIG. 2, and the T_f values were calculated from the big graph by using the Math Figure 2.

MathFigure 2

As a result, it was determined as shown in FIG. 2 that the amount of ATP produced was exponentially reduced within 10 days from the day when the samples were prepared (Small graph). 10 days later, it was not exponentially reduced. In addition, the time for which the amount of ATP was reduced by half was 2.12 days from the calculation.

As observed above, the composition for ATP synthesis and the method for synthesizing ATP using the same according to the present invention may be useful in mass production of biomaterials through ATP supply.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

A composition for ATP synthesis, comprising an inverted membrane vesicle comprising ATP synthase oriented to have an extroversion so as to pump in protons and produce ATP outside the membrane by a concentration gradient of Gloeobacter rhodopsin (hereinafter, referred to as GR) oriented to have an introversion and the pumped protons.
The composition as set forth in claim 1, wherein the GR is derived from Gloeobacter violaceus.
A method for preparing a composition for ATP synthesis, mounted for GR and ATP synthase to have orientation, comprising Steps:

1) preparing a recombinant expression vector operably linked to a polynucleotide represented by SEQ ID NO. 1 encoding GR;

2) transforming a host cell with the recombinant expression vector in Step 1) to prepare a transformant;

3) incubating the transformant in Step 2) to induce an expression of GR;

4) isolating a protoplast from a transformant overexpressing the GR in Step 3); and

5) preparing an inverted membrane vesicle from the transformant isolated in Step 4).
The method as set forth in claim 3, wherein the host cell in Step 2) is E. coli.
A method for synthesizing ATP by using the composition for ATP synthesis, comprising steps:

adding Pi and ADP to the composition for ATP synthesis of claim 1, and irradiating the composition.
The method as set forth in claim 5, wherein an amount of the Pi added is approximately 1 pM to approximately 100 nM.
The method as set forth in claim 5, wherein a visible light at a wavelength of at least 440 nm is irradiated.
A use of an inverted membrane vesicle comprising ATP synthase oriented to have an extroversion so as to pump in protons and produce ATP outside the membrane by a concentration gradient of Gloeobacter rhodopsin (hereinafter, referred to as GR) oriented to have an introversion and the pumped protons in preparation of a composition for ATP synthesis.
The use as set forth in claim 8, wherein the GR is derived from Gloeobacter violaceus.