Method for producing membrane vesicles containing a target substance

Abstract

To provide a novel method for producing a membrane vesicle in which a target substance is encapsulated.SOLUTION: Provided is a method for producing a membrane vesicle in which a target substance is encapsulated which includes a process of producing a membrane vesicle by inducing disruptive bacteriolysis in a gram-negative bacterium. The disruptive bacteriolysis can be induced by an expression of cell wall degrading enzyme in the gram-negative bacterium, and expression enhancement of the cell wall degrading enzyme can be induced by DNA damage stress load. The production method of the present invention may include a process of culturing gram-negative bacterium before inducing disruptive bacteriolysis.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing membrane vesicles in which a substance of interest is encapsulated. [Background technology]

[0002] Membrane vesicles (sometimes referred to as "membrane vesicles" or "MVs" in this specification) are attracting attention as a fundamental technology for vaccine development and drug delivery due to their immune-inducing properties and their tendency to accumulate in cancer cells.

[0003] For example, in vaccine development, a technique has been developed in which membrane vesicles are prepared from bacteria and used as an immunogenic composition (Patent Documents 1 and 2). However, due to its purpose, this technique is limited to preparing membrane vesicles from bacteria, and has not yet reached the stage of encapsulating a target substance in the membrane vesicle. In addition, in the development of drug delivery technology, exosomes, which control intercellular communication, have attracted attention in recent years due to their targeting properties, and their application to pharmaceuticals has been considered (Patent Document 3). However, the encapsulation of a target substance in exosomes is achieved by electroporation, freezing and thawing, ultrasonic treatment, saponification, etc., and there are naturally limitations to mass production and quality control as pharmaceuticals. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2014-520801 [Patent Document 2] Special Publication No. 2006-503822 [Patent Document 3] Special Publication No. 2019-535839 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel method for producing membrane vesicles encapsulating a target substance. [Means for solving the problem]

[0006] The present inventors have been conducting intensive research focusing on the phenomenon of membrane vesicle production by bursting bacteriolysis, and have found that membrane vesicles encapsulating a target substance can be produced by inducing bursting bacteriolysis in Gram-negative bacteria. The present invention is based on this finding.

[0007] According to the present invention, the following inventions are provided. [1] A method for producing membrane vesicles encapsulating a target substance, comprising: (A) producing membrane vesicles by inducing burst lysis in a gram-negative bacterium. [2] The method for producing a gram-negative bacterium according to the above item [1], wherein burst lysis is induced in the presence of a target substance on either or both the outside and / or the inside of the bacterium. [3] The method for production described in [1] or [2] above, in which burst lysis is induced by expression of a cell wall-degrading enzyme in Gram-negative bacteria and, optionally, an enzyme that perforates the inner membrane of Gram-negative bacteria. [4] The method for production described in [3] above, wherein the gram-negative bacteria have genetic modifications that induce expression of a cell wall-degrading enzyme and, optionally, an enzyme that perforates the inner membrane of the gram-negative bacteria. [5] The method for production described in [3] above, in which expression of a cell wall decomposing enzyme and, in some cases, an enzyme that perforates the inner membrane of Gram-negative bacteria is induced by DNA-damaging stress. [6] The method according to the above [5], wherein the DNA damaging stress is induced by mitomycin, a fluoroquinolone antibiotic, reactive oxygen species and / or nitric oxide. [7] A production method described in any of [3] to [6] above, in which the production amount of membrane vesicles is adjusted by adjusting the degree of expression of a cell wall-degrading enzyme and, optionally, an enzyme that perforates the inner membrane of Gram-negative bacteria. [8] The method according to any one of the above [3] to [7], wherein the cell wall decomposing enzyme is an endolysin. [9] The method according to any one of the above [3] to [8], wherein the enzyme that perforates the inner membrane of gram-negative bacteria is a holin.

[10] The production method according to any one of [1] to [9] above, further comprising a step (X) of culturing a gram-negative bacterium prior to the induction of explosive bacteriolysis.

[11] The method for producing a substance according to the above

[10] , comprising culturing a gram-negative bacterium in a medium containing the substance of interest.

[12] The method for producing the target substance described in

[10] above, in which the target substance is produced by gram-negative bacteria.

[13] (Y) The method according to any one of

[10] to

[12] above, which comprises a step of separating the produced membrane vesicles from the medium.

[14] The method according to

[13] above, wherein magnetic force is used in the separation step.

[15] A method for producing a culture medium according to any one of [1] to

[14] above, comprising repeating the steps of (X) culturing gram-negative bacteria, (A) producing membrane vesicles by bursting lysis, and (Y) isolating the membrane vesicles one or more times.

[16] The method according to any one of the above

[10] to

[15] , wherein the medium contains a magnesium salt at a concentration of 1 to 30 mM mass% in the medium.

[17] The method according to any one of

[10] to

[16] above, wherein the shaking conditions for the culture are 180 to 250 rpm.

[18] The method according to any one of

[10] to

[17] above, wherein the pH of the medium is 4 to 10.

[19] The method according to any one of

[10] to

[18] above, wherein the viscosity of the medium is 1 to 15 Pa s.

[20] A membrane vesicle in which a target substance is encapsulated, the components of the membrane vesicle being derived from cultured gram-negative bacteria.

[21] A membrane vesicle containing a target substance, produced by the production method described in any one of [1] to

[19] above.

[22] A method for encapsulating a target substance in a membrane vesicle derived from a gram-negative bacterium, the method comprising the step of inducing burst lysis in the gram-negative bacterium in the presence of the target substance.

[0008] The production method of the present invention is advantageous in that it can easily and efficiently produce membrane vesicles in which a target substance is encapsulated. The production method of the present invention is also advantageous in that it can encapsulate a target substance in membrane vesicles simultaneously with the production of membrane vesicles without a separate process for encapsulating the target substance in the membrane vesicles, and is also advantageous in that it can avoid inactivation or disruption of the target substance during encapsulation in the membrane vesicles. The production method of the present invention is also advantageous in that it can control the production amount of membrane vesicles by external stimuli, and can be mass-produced by scaling up the culture system or by continuous production. [Brief description of the drawings]

[0009] [Figure 1] Figure 1 shows the uptake of a target substance (calcein) into membrane vesicles by burst lysis. Figure 1A shows the results for calcein alone (control), and Figure 1B shows the results for burst lysis in the presence of calcein. [Diagram 2] FIG. 2 shows the amount of membrane vesicles produced per cell in each cycle of subculture. [Diagram 3] FIG. 3 shows the amount of membrane vesicles produced per cell in the absence (control) and presence of MgSO4 in the medium. [Figure 4] FIG. 4 shows the amount of membrane vesicles produced per cell under each shaking condition. [Diagram 5] FIG. 5 shows the amount of membrane vesicles produced per cell in the cases where Ficoll was added to the medium (control) and where Ficoll was not added. [Figure 6] FIG. 6 shows the amount of calcein encapsulated per membrane vesicle per cell (A) and the amount of membrane vesicle produced (B) under each pH condition. [Figure 7] FIG. 7 shows the amount of membrane vesicles recovered when membrane vesicles with iron encapsulated (Fe-MV) and without iron encapsulation (MV) (control) were recovered using a magnet. [Figure 8]Figure 8 shows a photograph of a membrane vesicle pellet encapsulating a target substance produced by bacteria. When a signal substance (C10-HSL) was added to the medium, the membrane vesicle pellet turned purple in color (dark gray in the figure) due to violacein (left photo), whereas when no signal substance was added (control), the membrane vesicle pellet did not turn purple (right photo). Description of the Invention

[0010] The present invention relates to a method for producing membrane vesicles in which a target substance is encapsulated. The production method of the present invention comprises (A) a step of producing membrane vesicles by inducing explosive cell lysis in Gram-negative bacteria (a step of producing membrane vesicles by explosive cell lysis). Here, "explosive cell lysis (ECL)" refers to a phenomenon in which all or a part of the bacteria in a population of Gram-negative bacteria explode and die through the action of cell wall-degrading enzymes in the bacteria.

[0011] Burstive bacteriolysis can be induced by a cell wall (peptidoglycan layer) decomposing enzyme, for example, by expressing a cell wall decomposing enzyme in bacteria. Examples of cell wall decomposing enzymes include endolysins derived from bacteriophages, autolysins derived from bacteria, peptidoglycan lytic enzymes derived from other bacteria such as bacteriocins, those derived from virulence factors or other antibacterial polypeptides (e.g., lysostaphin, ALE-1 lysin, mutanolysin, enterolysin), lysozyme, etc., and preferably endolysins.

[0012] The expression of the cell wall decomposition enzyme in the gram-negative bacterium can be induced by genetic modification that results in gene expression of the cell wall decomposition enzyme. Genetic modification can be performed according to known molecular biology techniques such as recombinant gene technology, and for example, as described in Turnbull L, et al., Nat Commun. 2016 Apr 14;7:11220., the cell wall decomposition enzyme can be expressed by incorporating a plasmid or expression vector encoding the cell wall decomposition enzyme into the gram-negative bacterium. The expression of the cell wall decomposition enzyme can be regulated by a control sequence such as an inducible promoter, and in the plasmid or expression vector, such a control sequence can be placed in the vicinity of the base sequence encoding the cell wall decomposition enzyme.

[0013] The expression of cell wall decomposition enzymes can also be induced by DNA-damaging stress. DNA-damaging stress can be applied to bacteria by adding a DNA replication inhibitor (e.g., mitomycin), a fluoroquinolone antibiotic (e.g., ciprofloxacin), active oxygen, nitric oxide, or the like to the medium. The amount of these substances added can be appropriately adjusted, and can be, for example, 0.001 to 0.1% by mass in the medium.

[0014] The bursting lysis can be induced more effectively by using an enzyme that perforates the inner membrane of gram-negative bacteria in addition to the cell wall decomposing enzyme. That is, by contacting the inner membrane of the bacteria with the enzyme that perforates the inner membrane of gram-negative bacteria, holes are opened in the intracellular membrane, promoting the contact between the cell wall decomposing enzyme and the cell wall, and bursting lysis can be induced more effectively. An example of the protein that perforates the inner membrane of gram-negative bacteria is phage-derived holin, and a preferred combination with the cell wall decomposing enzyme is holin-endolysin. The expression of the protein that perforates the inner membrane of gram-negative bacteria can be carried out by molecular biological techniques such as gene recombination techniques, similar to the expression of the cell wall decomposing enzyme.

[0015] In the present invention, the bacteria to be subjected to burst lysis are gram-negative bacteria, for example, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), E. coli ( Escherichia coli ), Acetic acid bacteria ( Acetobacter Aceti , Acetobacter pasteurianus , Acetobacter Liguefaciens , Acetobacter xylinum , Acetobacter Polyoxygenes , Acetobacter methanolicus , Gluconobacter oxydans , Gluconobacter cerinus , Gluconobacter hansenii , Frater aurantia ) and the like. Gram-negative bacteria are composed of an intracellular membrane, a peptidoglycan layer (cell wall), and an outer cell membrane in this order from the inside of the bacterial body, and it is considered that membrane vesicles consisting of the outer cell membrane (OMV; Outer Membrane Vesicle) or membrane vesicles consisting of the outer cell membrane and the intracellular membrane (OIMV; Outer Inner Membrane Vesicle) are formed by burst bacteriolysis. In addition, some of the above bacteria are used as foods or probiotic preparations (e.g., Nissle 1917 strain of Escherichia coli), and the membrane vesicles of the present invention can be derived from such bacteria from the viewpoint of safety.

[0016] The production method of the present invention may further include (X) a step of culturing gram-negative bacteria in a medium (a step of culturing gram-negative bacteria) prior to the induction of bursting bacteriolysis. The gram-negative bacteria may be cultured under normal culture conditions, but in terms of the amount of membrane vesicles produced, it is preferable to culture the bacteria under the conditions described below.

[0017] In the production method of the present invention, burst lysis is induced in a state in which a target substance is present either outside or inside or both of the Gram-negative bacteria, and the target substance can be encapsulated in the membrane vesicle at the same time as the formation of the membrane vesicle. When the target substance is present outside the Gram-negative bacteria, the bacteria can be cultured in a medium containing the target substance. The content of the target substance in the medium can be appropriately adjusted, and can be, for example, 0.001 to 10% by mass (preferably 0.01 to 5% by mass) in terms of the concentration in the medium.

[0018] When the target substance is present inside the gram-negative bacterium, the target substance can be produced by the gram-negative bacterium itself to be subjected to burst lysis and accumulated in the bacterium. The target substance may be produced by incorporating the gene of the target substance into the bacterium by a molecular biological technique such as gene recombination technology. The target substance can be produced by the gram-negative bacterium itself by a molecular biological technique by a known method.

[0019] The target substance to be encapsulated in the membrane vesicle is not particularly limited as long as it is a substance of a size that can be encapsulated in the membrane vesicle (for example, a diameter of 10 to 600 nm, 20 to 500 nm, or 30 to 400 nm). Examples of the target substance include low molecular weight compounds, proteins, peptides, amino acids, and nucleic acids that are active ingredients of pharmaceuticals. Pharmaceuticals also include biological preparations, and the active ingredients of biological preparations can be the target substance. Examples of the active ingredients of biological preparations include antibodies, antigens, interferons, interleukins, vaccines, hormones, enzymes related to blood coagulation, thrombolytic components, and immunoregulatory factors. Pharmaceuticals also include gene therapy preparations, and the active ingredients of gene therapy preparations can be the target substance. Examples of the active ingredients of gene therapy preparations include single-stranded or double-stranded DNA, iRNA, mRNA, siRNA, miRNA, and antisense RNA.

[0020] In the production method of the present invention, the production amount of membrane vesicles can be adjusted by adjusting the expression level of the cell wall decomposing enzyme and, in some cases, the expression level of the enzyme that perforates the inner membrane of the gram-negative bacteria. For example, by increasing the expression level of the enzyme, the production amount of membrane vesicles can be increased, and by decreasing the expression level of the enzyme, the production amount of membrane vesicles can be decreased.

[0021] The production method of the present invention may further include (Y) a step of separating the produced membrane vesicles from the medium (membrane vesicle separation step). The separation may be performed by a known method, for example, by removing the bacterial cells from the medium containing the bacterial cells using a centrifuge, and then filtering, purifying, or otherwise treating the supernatant, as necessary, to separate and recover the membrane vesicles. The supernatant may also be further purified by ultracentrifugation or density gradient centrifugation. The separation may also be performed by encapsulating the target substance and iron in the membrane vesicles during the bursting bacteriolysis, and then separating and recovering the membrane vesicles from the medium using magnetic force. Separation using magnetic force is advantageous in that it makes it easy to separate and recover the membrane vesicles. These methods for separating and recovering the membrane vesicles may be appropriately combined depending on the purpose of using the membrane vesicles.

[0022] In the production method of the present invention, one cycle is composed of (X) a culture step of gram-negative bacteria, (A) a membrane vesicle production step by bursting bacteriolysis, and (Y) a membrane vesicle separation step, and this cycle can be repeated one or more times to continuously prepare membrane vesicles. In other words, once the conditions for bursting bacteriolysis and culture conditions are set, the desired membrane vesicles can be prepared in large quantities by repeating steps (X), (A), and (Y) under those conditions. There is no limit to the number of repetitions, but the upper limit can be set to, for example, 50, 40, 30, 20, or 10 times.

[0023] In the production method of the present invention, Mg is added to the medium for culturing Gram-negative bacteria. 2+ (e.g., magnesium salts such as MgSO4, MgCl2, etc.) can be added. 2+ The amount of Mg added can be, for example, 1 to 30 mM (preferably 5 to 20 mM) as magnesium salt to the medium. 2+It is believed that the addition of can stabilize the cell membrane of gram-negative bacteria, which is advantageous in that it can increase the production amount of membrane vesicles.

[0024] In the production method of the present invention, the shaking conditions in the culture of gram-negative bacteria are preferably more than 100 rpm, more preferably 180 to 250 rpm. The shaking conditions can be appropriately adjusted in relation to the amount of oxygen supplied to the medium, but increasing the shaking speed is advantageous in that the amount of membrane vesicles produced increases.

[0025] In the production method of the present invention, the pH of the medium in the culture of gram-negative bacteria can be adjusted, for example, within the range of pH 4 to 10 (preferably 5 to 9). From the viewpoint of increasing the production amount of membrane vesicles, however, a higher pH (more alkaline), for example, pH 8 to 10 (preferably pH 8 to 9), is preferable. On the other hand, from the viewpoint of increasing the amount of the target substance encapsulated in the membrane vesicles, a lower pH (more acidic), for example, pH 4 to 6 (preferably pH 5 to 6), is preferable. In the production method of the present invention, the pH of the culture medium can be appropriately set depending on the application.

[0026] In the production method of the present invention, the viscosity of the medium for culturing gram-negative bacteria cells is preferably high from the viewpoint of increasing the amount of the target substance encapsulated in the membrane vesicles, and the viscosity of the culture medium can be, for example, 1 to 15 Pa·s (preferably 2 to 11 Pa·s). To increase the viscosity of the medium, for example, ficoll, alginic acid, carboxymethylcellulose, carboxyvinyl polymer, etc. can be added to the medium as a thickening agent. The content of these thickening agents in the medium can be 5 to 20% by mass for ficoll, 1 to 3.5% by mass for carboxymethylcellulose, and 0.05 to 0.3% by mass for carboxyvinyl polymer, in terms of the concentration in the medium. These thickening agents can be added alone or in any combination.

[0027] According to another aspect of the present invention, the present invention provides a membrane vesicle in which a target substance is encapsulated, the constituent of the membrane vesicle being derived from a gram-negative bacterium. The membrane vesicle of the present invention has a particle diameter of 10 to 600 nm, 20 to 500 nm, or 30 to 400 nm, and is configured in the form of either an outer membrane-inner membrane vesicle (OIMV) or an outer membrane vesicle (OMV). The membrane vesicle of the present invention can be produced according to the description of the production method of the present invention. The target substance to be encapsulated in the membrane vesicle of the present invention may be the same as those described in the production method of the present invention.

[0028] According to another aspect of the present invention, the present invention provides a composition comprising the membrane vesicles of the present invention. The composition of the present invention can be provided in the form of a pharmaceutical product (e.g., a pharmaceutical composition), a food product (e.g., a food composition), a cosmetic product (e.g., a cosmetic composition), etc., depending on the nature of the substance to be encapsulated in the membrane vesicles. The composition of the present invention can be produced by containing the membrane vesicles of the present invention. The target substance to be encapsulated in the membrane vesicles contained in the composition of the present invention can be the same as those described in the production method of the present invention.

[0029] According to another aspect of the present invention, the present invention further provides a method for encapsulating a target substance in a membrane vesicle derived from a gram-negative bacterium. The method for encapsulating a target substance of the present invention comprises a step of inducing burst lysis in a gram-negative bacterium in the presence of a target substance. The method for encapsulating a target substance of the present invention can be carried out according to the description of the production method of the present invention. The target substance to be encapsulated in the membrane vesicle of the present invention can be the same as those described in the production method of the present invention. EXAMPLES

[0030] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0031] Example 1: Production of membrane vesicles by burst lysis and encapsulation of target substances in the membrane vesicles In Example 1, it was shown that the presence of the target substance induced burst lysis in Gram-negative bacteria, and the target substance was encapsulated in membrane vesicles.

[0032] (1) Method A bacterial body The bacterial body is Pseudomonas aeruginosa ( Pseudomonas aeruginosa The strain PAO1 ΔprtN was prepared by introducing a plasmid (pJN105 hol_lys, obtained from Addgene) capable of controlling the expression of holin-endolysin. The P. aeruginosa strain PAO1 ΔprtN was prepared by disrupting prtN from PAO1 (available from the RIKEN BioResource Research Center (BRC No: JCM 14847)) according to the method described in Environ Microbiol. 2014 Sep;16(9):2927-38.

[0033] B. Bacterial culture and induction of burst lysis LB liquid medium (4 ml) was used, and gentamicin (Nacalai Tesque) was added at 50 μg / ml to prevent the plasmid from being lost. 600 = 0.01 and cultured at 37°C for 12 hours at 200 rpm. After 4 hours of culture, arabinose (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 0.1% to express holin-endolysin, induce burst lysis, and produce membrane vesicles. Just before adding arabinose, 1 mM calcein (Nacalai Tesque) was added to the medium as a target substance.

[0034] C. Separation, purification, and recovery of membrane vesicles The culture solution was centrifuged (4°C, 30 minutes, 7,000 x g) to pellet the cells. The supernatant after centrifugation was filtered through a 0.45 μm pore size filter to remove the cells, and the sample was then ultracentrifuged (4°C, 1 hour, 150,000 x g). The pellet produced by ultracentrifugation was suspended in PBS. The resulting membrane vesicles were then washed by ultracentrifugation and resuspending, and this washing procedure was repeated twice. Next, the membrane vesicle pellet produced by ultracentrifugation was suspended in 45% iodixanol (AXIS-SHEILD), and 40, 35, 30, 25, 20, 15, and 10% iodixanol were layered on top, and density gradient centrifugation was performed (4°C, 3 hours, 100,000 x g, total volume 3 ml). The membrane vesicles were purified and collected by this density gradient centrifugation.

[0035] D. Quantification of membrane vesicles The collected membrane vesicles were quantified by measuring the fluorescence intensity of FM4-64 (Invitrogen) as a membrane lipid stain using a fluorescence plate reader (Varioscan flash fluorometer, Thermo Scientific). The amount of membrane vesicles produced per cell was calculated as RFU (relative fluorescence unit) (FM1-43 fluorescence intensity / OD 600 The presence of the encapsulated substance was confirmed by quantifying the fluorescence intensity of calcein using a fluorescence plate reader (Varioscan flash fluorometer, Thermo Scientific).

[0036] (2) Results The results are shown in Figure 1. Calcein indicates the presence of the substance to be encapsulated, and FM4-64 indicates the presence of membrane vesicles. In Figure 1A, in the sample containing only calcein (control), a calcein peak was observed at around fraction numbers 27 to 30 (area enclosed in a dashed square). On the other hand, in Figure 1B, in the sample in which calcein was encapsulated in membrane vesicles, the FM4-64 peak and the calcein peak were observed to coincide at around fraction numbers 8 to 16 (area enclosed in a dashed square), confirming that calcein was encapsulated in membrane vesicles due to burst lysis.

[0037] Example 2: Effect of subculture on membrane vesicle production In Example 2, the effect of subculture on the production of membrane vesicles by burst lysis was examined.

[0038] (1) Method A bacterial body The bacterial cells described in Example 1(1)A were used.

[0039] B. Bacterial culture and induction of burst lysis The procedure was the same as described in Example 1(1)(i), except that calcein was not added.

[0040] C. Subculture The bacteria from the culture in which burst lysis was induced were inoculated into a new medium, and the cycle of inducing burst lysis was repeated three times. Specifically, one cycle consisted of 12-hour culture, and the OD 600 The OD of the 12-hour culture solution used for the measurement was 600 = 0.01, and was subcultured (second time). Next, the culture solution after the second cycle was similarly newly cultured for subculture (third time). In the second and third times, the OD 600 was measured.

[0041] D. Separation, purification, and recovery of membrane vesicles After each cycle of rupture lysis, membrane vesicles were isolated, purified, and collected. The pelleted cells were used for the next cycle of culture. The rest of the procedure was the same as that described in Example 1(1)(c).

[0042] D. Quantification of membrane vesicles Quantification of membrane vesicles was performed in the same manner as described in Example 1(1)E, except that FM1-43 (Invitrogen) was used as the membrane lipid staining agent.

[0043] (2) Results The results are shown in Figure 2. It was confirmed that even when subculture was performed using a medium in which burst lysis was induced, membrane vesicles could be produced by burst lysis in the second and third cycles.

[0044] Example 3: MgSO 4 Effect of on membrane vesicle production In Example 3, the effect of adding MgSO4 to the medium on the production of membrane vesicles by burst lysis was examined.

[0045] (1) Method A bacterial body The bacterial cells described in Example 1(1)A were used.

[0046] B. Bacterial culture and induction of burst lysis The procedure was the same as that described in Example 1(1)(i), except that MgSO4 was added to the LB liquid medium to a concentration of 8 mM and calcein was not added.

[0047] C. Separation, purification, and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)(c).

[0048] D. Quantification of membrane vesicles Membrane vesicles were quantified using FM4-64 (Invitrogen) as a membrane lipid staining agent, in the same manner as described in Example 1(1)E.

[0049] (2) Results The results are shown in Figure 3. It was confirmed that when MgSO4 was added to the medium, the amount of membrane vesicles produced per cell was significantly increased.

[0050] Example 4: Effect of shaking conditions of bacterial culture on membrane vesicle production In Example 4, the effect of shaking conditions during culture on the production of membrane vesicles was examined.

[0051] (1) Method A bacterial body The bacterial cells described in Example 1(1)A were used.

[0052] B. Bacterial culture and induction of burst lysis The procedure was the same as that described in Example 1(1)(i), except that the shaking conditions during bacterial culture were 0 rpm, 100 rpm, and 190 rpm, and calcein was not added.

[0053] C. Separation, purification, and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)(c).

[0054] D. Quantification of membrane vesicles Quantification of membrane vesicles was performed in the same manner as described in Example 1(1)E, except that FM1-43 (Invitrogen) was used as the membrane lipid staining agent.

[0055] (2) Results The results are shown in Figure 4. It was confirmed that when the shaking condition was 190 rpm, the amount of membrane vesicles produced per cell was significantly increased.

[0056] Example 5: Effect of medium viscosity on the encapsulation of calcein into membrane vesicles In Example 5, the effect of medium viscosity on the encapsulation of calcein into membrane vesicles was examined.

[0057] (1) Method A bacterial body The bacterial cells described in Example 1(1)A were used.

[0058] B. Bacterial culture and induction of burst lysis The procedure was the same as that described in Example 1(1)B, except that Ficoll (Sigma) was added to the LB liquid medium to a concentration of 10%.

[0059] C. Separation, purification, and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)(c).

[0060] Quantification of membrane vesicles and calcein Membrane vesicles were quantified using FM4-64 as a membrane lipid staining agent, in the same manner as described in Example 1(1)(c). The fluorescence intensities of FM4-64 and calcein were measured using a fluorescence plate reader (Varioscan flash fluorometer, Thermo Scientific). The amount of calcein encapsulated per membrane vesicle was calculated as the fluorescence intensity of "calcein / FM4-64".

[0061] (2) Results The results are shown in Figure 5. It was confirmed that the amount of calcein encapsulated in membrane vesicles increased with increasing viscosity of the medium.

[0062] Example 6: Effect of medium pH on membrane vesicle production and inclusion of calcein in membrane vesicles In Example 6, the effect of medium pH on the production of membrane vesicles and the inclusion of calcein in the membrane vesicles was examined.

[0063] (1) Method A bacterial body The bacterial cells described in Example 1(1)A were used.

[0064] B. Bacterial culture and induction of burst lysis The same procedure as described in Example 1(1)(i) was carried out, except that the pH of the LB liquid medium was changed to pH 5, pH 7, or pH 9.

[0065] C. Separation, purification, and recovery of membrane vesicles The procedure was carried out in the same manner as described in Example 1(1)(c).

[0066] Quantification of membrane vesicles and calcein The procedure was the same as that described in Example 5(1)E.

[0067] (2) Results The results are shown in Figure 6. It was confirmed that the amount of calcein encapsulated in membrane vesicles decreased as the pH increased (Figure 6A), whereas the amount of membrane vesicles produced per cell increased as the pH increased (Figure 6B).

[0068] Example 7: Recovery of membrane vesicles using a magnet In Example 7, recovery of membrane vesicles using a magnet was examined.

[0069] (1) Method A bacterial body The bacterial cells described in Example 1(1)A were used.

[0070] B. Bacterial culture and induction of burst lysis The procedure was the same as described in Example 1(1)B, except that 1 mM iron oxide was added to the medium instead of calcein immediately before the addition of arabinose.

[0071] C. Recovery and quantification of membrane vesicles 900 μl of membrane vesicle solution (MV) and iron-loaded membrane vesicle solution (Fe-MV) were prepared so that the fluorescence intensity of FM1-43 was 3. Each solution was placed on a magnetic stand (Toyobo) and left to stand for 1 minute under static conditions, after which the supernatant was removed using an aspirator. Next, 900 μl of PBS was added and stirred using a vortex. The membrane vesicles in this solution were quantified by measuring the fluorescence intensity of FM1-43.

[0072] (2) Results The results are shown in Figure 7. It was confirmed that iron-encapsulating membrane vesicles could be collected using a porcelain stand.

[0073] Example 8: Encapsulation of bacterially produced substances into membrane vesicles In Example 8, the encapsulation of bacterially produced substances into membrane vesicles was examined.

[0074] (1) Method A bacterial body AHL synthesis gene-deficient strain, Chromobacterium violaceum ( Chromobacterium violaceum ) (strain: VIR24, Chromobacterium violaceum Bergonzini (ATCC12472) prepared according to the description in Microbes Environ. 2009;24(4):305-14.) was used as the bacterial cell.

[0075] B. Bacterial culture OD using LB medium 600 = 0.01, and cultured at 30°C for 12 hours at 100 rpm.

[0076] C. Induction of membrane vesicle production Mitomycin C (Nacalai tesque) was used to induce the production of membrane vesicles from Chromobacterium violaceum. The addition of mitomycin C reached OD 600 = 0.4 to 0.6, and added to give a concentration of 200 ng / ml.

[0077] Addition of signal substances The VIR24 strain cannot produce N-acyl-L-homoserine lactone (AHL) by itself, so purple pigment production is not induced (the culture medium does not turn purple when this strain is cultured). Therefore, we induced the production of the purple pigment (violacein) by the VIR24 strain by adding C10-HSL (Cayman Chemical), a type of AHL, to the strain. The concentration of C10-HSL added was 100 nM at the final concentration, added 1 hour after the start of culture.

[0078] Isolation of membrane vesicles and confirmation of the inclusion of bacterially produced substances in the membrane vesicles The culture solution was centrifuged (4°C, 30 minutes, 7000 x g) to pellet the cells. The supernatant was filtered through a 0.45 μm pore size filter to remove the cells, and then 7.0 ml of the sample was ultracentrifuged (4°C, 1 hour, 150,000 x g) to separate the membrane vesicles. The substance (violacein) produced by the cells was purple in color, so the presence or absence of its inclusion in the membrane vesicles was visually confirmed.

[0079] (2) Results The results are shown in Figure 8. The photographs show the pellets of membrane vesicles after ultracentrifugation. The photograph on the left shows the membrane vesicle pellets derived from the VIR24 strain with the addition of C10-HSL, and the photograph on the right shows the membrane vesicle pellets derived from the VIR24 strain without the addition of C10-HSL. The addition of a signal (C10-HSL) induced the production of violacein (purple) in the VIR24 strain, and it was confirmed that the produced violacein was encapsulated in the membrane vesicles.

Claims

1. A method for producing membrane vesicles in which a target substance is encapsulated, comprising: (A) a step of producing membrane vesicles by inducing bursting bacteriolysis in a gram-negative bacterium; and (X) a step of culturing the gram-negative bacterium before the induction of bursting bacteriolysis, wherein the gram-negative bacterium is cultured in a medium containing the target substance, the shaking conditions for the culture are 180 to 250 rpm, and the viscosity of the medium is 1 to 15 Pa s.

2. The method according to claim 1, wherein the burst lysis is induced in the presence of the target substance either outside or inside or both of the gram-negative bacteria.

3. The method according to claim 1 or 2, wherein the burst lysis is induced by a cell wall decomposing enzyme in a gram-negative bacterium.

4. 3. The method according to claim 1, wherein the burst lysis is induced by expression of a cell wall decomposing enzyme in a gram-negative bacterium and an enzyme that perforates the inner membrane of the gram-negative bacterium.

5. The method according to claim 3 or 4, wherein the Gram-negative bacterium has a genetic modification that induces expression of a cell wall-degrading enzyme.

6. The method according to claim 3 or 4, wherein the gram-negative bacterium has genetic modifications that induce expression of a cell wall-decomposing enzyme and an enzyme that perforates the inner membrane of the gram-negative bacterium.

7. The method according to claim 3 or 4, wherein the expression of the cell wall decomposing enzyme is induced by applying DNA damaging stress.

8. The method according to claim 3 or 4, wherein expression of the cell wall decomposing enzyme and the enzyme that perforates the inner membrane of a gram-negative bacterium is induced by applying DNA damaging stress.

9. The method according to claim 8, wherein the DNA-damaging stress is induced by mitomycin, a fluoroquinolone antibiotic, active oxygen and / or nitric oxide.

10. The method according to any one of claims 3 to 9, wherein the amount of membrane vesicles produced is adjusted by adjusting the expression level of the cell wall decomposing enzyme.

11. The method according to any one of claims 3 to 9, wherein the production amount of membrane vesicles is adjusted by adjusting the expression levels of a cell wall decomposing enzyme and an enzyme that perforates the inner membrane of a gram-negative bacterium.

12. The method according to any one of claims 3 to 11, wherein the cell wall decomposing enzyme is an endolysin.

13. The method according to any one of claims 3 to 12, wherein the enzyme that perforates the inner membrane of a gram-negative bacterium is a holin.

14. The method according to any one of claims 1 to 13, wherein the target substance is produced by a gram-negative bacterium.

15. The method according to any one of claims 1 to 14, further comprising a step (Y) of separating the produced membrane vesicles from the medium.

16. The method of claim 15, wherein the separating step uses magnetic force.

17. The method according to any one of claims 1 to 16, wherein the step of (X) culturing a gram-negative bacterium, the step of (A) producing membrane vesicles by bursting lysis, and the step of (Y) isolating the membrane vesicles are repeated one or more times.

18. The method according to any one of claims 1 to 17, wherein the medium contains a magnesium salt at a concentration of 1 to 30 mM.

19. The method according to any one of claims 1 to 18, wherein the pH of the medium is 4 to 10.

20. A method for encapsulating a target substance in membrane vesicles derived from gram-negative bacteria, comprising: a step of inducing bursting bacteriolysis in the gram-negative bacteria in the presence of the target substance; and a step (X) of culturing the gram-negative bacteria prior to the induction of bursting bacteriolysis, wherein the gram-negative bacteria are cultured in a medium containing the target substance, the shaking conditions for the culture are 180 to 250 rpm, and the viscosity of the medium is 1 to 15 Pa s.

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