A membraneless cell organelle and its construction method and application

Abstract

The present invention relates to the field of bioengineering technology, and specifically to the construction of a membraneless organelle and its application in regulating metabolic synthesis. By designing an octapeptide basic element, a recombinant disordered protein composed of repeated tandem peptide segments is constructed, which is introduced into an expression host to obtain a biomolecular condensate, i.e., a membraneless organelle. A membraneless organelle with good fluidity, stability, and temperature sensitivity is constructed and verified in Bacillus subtilis cells, proving that the constructed recombinant disordered protein has a high degree of programmability. The function of the membraneless organelle is tested using a visualization experiment. The constructed membraneless organelle can not only allow the extended penetration of small molecule compounds, but also recruit specific targeting proteins, and can be used to regulate the metabolism of target compounds.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a membraneless organelle and a construction method and application thereof. Background Art

[0002] How to achieve spatial level regulation of the intracellular metabolic network of microorganisms is a key prerequisite for building a new generation of high-performance cell factories. Studies have shown that recruiting and fixing pathway enzymes to form micro-reaction compartments can effectively shorten the spatial distance of biomolecules, increase the transmission rate of intermediate metabolites, and thus enhance the overall efficiency of the catalytic process. Biomolecular condensates are a new type of subcellular structure that plays an important role in regulating cell behavior and signal transduction. Biomolecular condensates are usually formed by liquid-liquid phase separation processes mediated by multivalent interactions of intrinsically disordered proteins or other biomolecules (nucleic acids, etc.). Compared with membrane-coated organelles or protein-coated microcompartments, biomolecular condensates can freely exchange target biomolecules with the external environment due to their inherent liquid properties. Therefore, biomolecular condensates can be used as membraneless organelles to achieve spatial dimensional regulation of biological processes.

[0003] As a GRAS (Generally Regard as Safe) Gram-positive host, Bacillus subtilis (B. subtilis) has been widely used for the biosynthesis of high value-added compounds such as L-asparaginase, hyaluronic acid and riboflavin. However, there are no reports on the construction and application of artificial membraneless organelles in B. subtilis. In addition, the use of natural intrinsically disordered proteins (IDPs) limits the programmability of biomolecular condensates and cannot achieve orthogonality. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to design a recombinant disordered protein that can form a biomolecular condensate and verify whether it can form a functional membraneless organelle in B. subtilis. The present invention provides a membraneless organelle based on a recombinant disordered protein and its application. First, an octapeptide basic element is designed from scratch and a recombinant disordered protein is obtained. Phase separation is induced in vitro to obtain a biomolecular condensate. The effects of temperature, NaCl concentration, recombinant disordered protein concentration and molecular crowding agent concentration on the formation and fluidity of the biomolecular condensate are systematically characterized. By changing the amino acid composition and sequence position of the octapeptide basic unit, a series of recombinant disordered proteins are designed and constructed to form different biomolecular condensates to verify the influence of amino acid composition and sequence on the fluidity of the condensate. The above-mentioned recombinant disordered protein is used to construct a membraneless organelle with good fluidity, stability and temperature sensitivity in Bacillus subtilis cells.

[0005] The first object of the present invention is to provide a recombinant disordered protein containing a repeated tandem peptide segment N8, wherein the peptide segment N8 consists of eight amino acids, including three glycines, one proline, one serine, one aspartic acid, one arginine, and one tyrosine or alanine.

[0006] Furthermore, the amino acid sequence of peptide segment N8 may be selected from any one of the following:

[0007] (1) RGYGSPDG (SEQ ID NO. 1);

[0008] (2) RGGDSPYG (SEQ ID NO. 2);

[0009] (3) RGSPYGDG (SEQ ID NO. 3);

[0010] (4) GYPSDGRG (SEQ ID NO. 4);

[0011] (5) DGRGSPYG (SEQ ID NO. 5);

[0012] (6) RGAGSPDG (SEQ ID NO. 6).

[0013] Furthermore, the sequence of the recombinant disordered protein is MSKGP-(N8) n -GY, n is any integer between 15 and 30.

[0014] Preferably, n is 20.

[0015] The second object of the present invention is to provide a membraneless organelle, construct an expression vector containing the above-mentioned recombinant disordered protein and introduce it into an expression host to express and form a membraneless organelle.

[0016] Furthermore, the recombinant disordered protein is expressed using an inducible promoter or a constitutive promoter.

[0017] Preferably, the recombinant disordered protein is composed of P grac Promoter expression.

[0018] Furthermore, IPTG was used to induce expression.

[0019] Furthermore, the IPTG concentration is 0.05-0.5 mM, the induction temperature is 37-42° C., and the induction time is 6-10 hours.

[0020] The third object of the present invention is to provide a host cell containing the above-mentioned recombinant disordered protein or the above-mentioned membrane-less organelle.

[0021] Furthermore, the host is Bacillus subtilis.

[0022] Preferably, the host is Bacillus subtilis 168.

[0023] The fourth object of the present invention is to provide a method for recruiting target proteins using the above-mentioned membraneless organelles.

[0024] Furthermore, membrane-less organelles are targeted to target proteins.

[0025] Furthermore, a polypeptide tag or a tag ligand is connected to the N-terminus or C-terminus of the target protein, and a tag ligand or a polypeptide tag is connected to the N-terminus or C-terminus of the membraneless organelle, and the polypeptide tag and the tag ligand are covalently bound.

[0026] Furthermore, the polypeptide tag is a RIDD short peptide or a CCDIA short peptide, and the tag ligand is a RIAD short peptide or a CCDIB short peptide.

[0027] Furthermore, the membraneless organelle is connected to the tag ligand via a linker peptide.

[0028] Furthermore, the sequence of the connecting peptide is as follows (GGGGS): n As shown, where n is an integer greater than or equal to 1.

[0029] The fifth object of the present invention is to provide the application of the above-mentioned membraneless organelles in regulating the metabolic synthesis of target compounds at the spatial level.

[0030] The above technical solution of the present invention has the following advantages compared with the prior art:

[0031] The present invention is based on the formation mechanism and principle of liquid-liquid phase separation, and for the first time, a membraneless organelle is constructed and applied in Bacillus subtilis. An octapeptide basic element is designed from scratch, and a recombinant disordered protein is obtained by repeating the peptide segment construction. The molecular simulation theory calculation and experimental verification are combined to verify that it can form a biomolecular condensate with liquid characteristics. The condensate can recover 83% of the fluorescence intensity within 210s after fluorescence bleaching. The amino acid sequence composition and order of the recombinant disordered protein are rationally designed to regulate the multivalent interaction strength, and then a series of recombinant disordered proteins that can form biomolecular condensates with different fluidities are obtained, realizing the programmability of biomolecular condensates. A membraneless organelle is constructed in B. subtilis cells, and experiments have proved that it has good fluidity, stability and temperature sensitivity. Visual experiments have further proved that the biomolecular condensates in B. subtilis cells allow the extended penetration of small molecule compounds and can recruit specific targeting proteins, realizing the regulation of the metabolic flux of target compounds in Bacillus. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0033] Figure 1 is the polymerization ability of FW1 calculated by molecular dynamics simulation in Example 1 of the present invention;

[0034] Figure 2 The in vitro formation of biomolecular aggregates in Example 2 of the present invention;

[0035] Figure 3 The FRAP experiment was used in Example 2 of the present invention to detect the recovery of the aggregates;

[0036] Figure 4 is the effect of protein and NaCl concentration on SDP1-GFP phase separation in Example 3 of the present invention;

[0037] Figure 5 is the phase separation behavior of the SDP1-GFP protein at different temperatures in Example 3 of the present invention;

[0038] Figure 6 The effect of different PEG addition concentrations on the properties of SDP1-GFP aggregates in Example 3 of the present invention;

[0039] Figure 7 The effect of changing the amino acid sequence position on the fluidity of the aggregate in Example 4 of the present invention;

[0040] Figure 8 The effect of changing the amino acid composition on the fluidity of the aggregate in Example 4 of the present invention;

[0041] Fig. 9 is the formation of SDP1-GFP aggregates in B. subtilis cells in Example 5 of the present invention;

[0042] Fig.10 is the position distribution of intracellular SDP1-GFP aggregates in Example 5 of the present invention;

[0043] Fig.11 The characteristics of the intracellular aggregates are verified by using urea in Example 5 of the present invention;

[0044] Fig.12 is the effect of the intracellular artificial aggregates on the host cells in Example 5 of the present invention;

[0045] Fig.13 is the effect of temperature and expression intensity on SDP1-GFP aggregate formation in Example 5 of the present invention;

[0046] Fig.14The co-localization analysis of the co-expressed target protein and the intracellular artificial aggregates in Example 6 of the present invention;

[0047] Fig.15 The co-localization analysis of the time-sequentially induced expression of the targeted protein and the intracellular artificial aggregates in Example 6 of the present invention;

[0048] Fig.16 In Example 6 of the present invention, the DV metabolic synthesis pathway is used to visualize the diffusion ability of small molecules in the aggregates, where "+" represents that the gene is expressed in the recombinant strain, "-" represents that the gene is not expressed in the strain, and "O" represents that the gene is recruited to the intracellular aggregates. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0050] Example 1: Design of basic units of recombinant disordered proteins

[0051] The octapeptide basic unit FW1 was designed, and its sequence was RGYGSPDG (shown in SEQ ID NO.1). The aggregation and slump of FW1 were calculated by molecular dynamics simulation to verify the feasibility of the design principle of FW1. The results are shown in Figure 1 It shows that the aggregation degree of FW1 is 0.95 and the slump is 0.21, which proves that it has high aggregation ability in theory.

[0052] Example 2: In vitro validation of biomolecular aggregates

[0053] (1) The FW1 unit was repeated 20 times to construct SDP1 (amino acid sequence: MSKGP-(RGYGSPDG) 20 -GY, as shown in SEQ ID NO.7). T7 Control GFP (with His tag) expression and P T7 The fragment controlling the fusion expression of SDP1 and GFP (containing His tag) (sequence as shown in SEQ ID NO.8) was connected to the pET28a plasmid to construct plasmids pET28a-GFP and pET28a-SDP1-GFP. The plasmids pET28a-GFP and pET28a-SDP1-GFP were transformed into Escherichia coli BL21 (DE3) and spread on LB solid medium supplemented with kanamycin, and the single colonies grown were Escherichia coli EC0 and EC1, respectively.

[0054] (2) Select the single colonies EC0 and EC1 grown on the plate in step (1) and inoculate them into 50 mL of TB medium. Cultivate at 220 rpm and 37°C until OD 600 After the pH value reached 0.6-0.8, 0.5 mM IPTG was added for induction, and the culture temperature was adjusted to 25°C for 20 h.

[0055] (3) The cultured cells were collected by centrifugation at 5000 × g for 10 min, washed twice with 20 mM Tris-HCl (pH 7.4), and resuspended in a lysis buffer (500 mM NaCl, 20 mM Tris-HCl, pH = 7.4). The cell suspension was then ultrasonically disrupted and centrifuged at 14000 × g for 30 min to collect the supernatant. Protein purification was performed using the FPLC protein purification system (GE Healthcare). Gradient elution was performed using an eluent (500 mM NaCl, 20 mM Tris-HCl, 500 mM imidazole, pH = 7.4) to collect the target protein peak; finally, desalting was performed in a dialysate (500 mM NaCl, 20 mM Tris-HCl, pH = 7.4, 5% (v / v) glycerol) to obtain purified proteins GFP and SDP1-GFP. The protein concentration was detected using a BCA kit.

[0056] (4) Use salt-free buffer (0 mM NaCl, 20 mM Tris, pH = 7.4) to dilute the purified proteins GFP and SDP1-GFP in step (3) to induce phase separation. According to the needs, different concentrations of PEG2000 are added to the buffer to simulate the intracellular environment.

[0057] (5) After 2 μL of protein solution was placed on a glass slide, the extracellular formation of biomolecule aggregates was immediately observed under a Leica DMi8 microscope equipped with a 100× oil immersion objective. The fusion protein containing GFP was observed using a FITC fluorescence filter. The observation results are as follows: Figure 2 As shown in Figure A, both SDP1-GFP and SDP1 can form spherical aggregates in vitro, while the GFP protein alone is diffuse. Figure 2 As shown in B, two separate SDP1-GFP aggregates can dissolve into a larger aggregate.

[0058] (6) A Leica TCS SP8 laser scanning confocal microscope was used to perform fluorescence photobleaching recovery experiments on SDP1-GFP aggregates. The protein droplet was excited with fluorescent protein at 488 nm; then, an area of ​​about 3 μm in the center of the biomolecule aggregate was circled for fluorescence photobleaching. After fluorescence photobleaching, fluorescence recovery was performed for 210 seconds, and a FRAP experiment was performed on the SDP1-GFP aggregate to further verify its liquid properties. The experimental results are shown in Figure 2. Figure 3 As shown, the fluorescence intensity of the bleached aggregates can recover 83% within 210 seconds. The above results prove that SDP1-GFP can form biomolecular aggregates with liquid properties in vitro.

[0059] Example 3: Systematic characterization of the properties of SDP1 biomolecular aggregates

[0060] (1) By adjusting the ratio of salt-free buffer, the concentration of NaCl and purified SDP1-GFP, the effects of SDP1-GFP protein concentration and NaCl concentration on aggregate formation were investigated. Figure 4 As shown, the higher the concentration of SDP1-GFP, the easier it is to form aggregates; the higher the concentration of NaCl, the more difficult it is to form aggregates. When the NaCl concentration is 50 mM, about 8 μM SDP1-GFP can form aggregates (pH = 7.4, 25°C).

[0061] (2) Further, by changing the temperature, the protein turbidity was detected by spectrophotometer at an absorbance of 600 nm. Figure 5 As shown, when the temperature is below 20°C, 50 μM SDP1-GFP protein will not phase separate; as the temperature continues to rise, SDP1-GFP protein phase separates and the solution gradually becomes turbid (pH=7.4, 150 mM NaCl). Simultaneous observation with a fluorescence microscope shows that the higher the ambient temperature, the more SDP1-GFP tends to form larger aggregates.

[0062] (3) In order to simulate the environment in which SDP1-GFP forms aggregates in cells, the molecular crowding agent PEG was added to the solution. Figure 6 As shown in the figure, after FRAP bleaching the aggregates under the same conditions, the higher the concentration of PEG added, the lower the fluidity of the aggregates formed by SDP1-GFP. When 5% PEG was added, the SDP1-GFP aggregates could recover 80% of the fluorescence intensity within 210s; while when the PEG addition concentration reached 13%, the aggregates could only recover 57% of the fluorescence intensity within 210s (150mM NaCl, pH=7.4, 25℃). In summary, temperature, molecular crowding agents and NaCl concentration can significantly affect the phase separation behavior and aggregate fluidity of SDP1-GFP.

[0063] Example 4: Effect of amino acid composition order on the formation and properties of biomolecular aggregates

[0064] (1) By changing the sequence position of the amino acid composition of FW1 while keeping it unchanged, four octapeptides, FW2 (RGGDSPYG, SEQ ID NO.2), FW3 (RGSPYGDG, SEQ ID NO.3), FW4 (GYPSDGRG, SEQ ID NO.4) and FW5 (DGRGSPYG, SEQ ID NO.5), were obtained. Then, the above four octapeptides were repeated 20 times to construct SDP2 (amino acid sequence: MSKGP-(RGGDSPYG) 20 -GY, SEQ ID NO.9), SDP3 (amino acid sequence: MSKGP-(RGSPYGDG) 20 -GY, SEQ ID NO.10), SDP4 (amino acid sequence: MSKGP-(GYPSDGRG) 20 -GY, SEQ ID NO.11), SDP5 (amino acid sequence: MSKGP-(DGRGSPYG) 20 -GY, SEQ ID NO.12), artificially synthesized with promoter P T7 The fragments of SDP2 and GFP (including His tag) fusion expression, SDP3 and GFP (including His tag) fusion expression, SDP4 and GFP (including His tag) fusion expression, and SDP5 and GFP (including His tag) fusion expression were controlled respectively, and connected to plasmid pET28a to construct plasmids pET28a-SDP2-GFP, pET28a-SDP3-GFP, pET28a-SDP4-GFP, and pET28a-SDP5-GFP. The above plasmids were transformed into Escherichia coli BL21 (DE3) respectively, and spread on LB solid culture medium supplemented with kanamycin, and the single colonies grown were Escherichia coli EC2, EC3, EC4, and EC5 respectively.

[0065] (2) Purified proteins SDP2-GFP, SDP3-GFP, SDP4-GFP, and SDP5-GFP were obtained according to the method in Example 2 and subjected to fluorescence photobleaching recovery experiments. The experimental results are shown in Figure 7As shown in the figure, when the amino acid composition is the same, the difference in its order position will change the fluidity of the aggregates formed. Compared with SDP1-GFP, 8μM SDP2-GFP and 6μM SDP3-GFP can form aggregates (150mM NaCl, pH=7.4, 25℃). However, the recovery of SDP2-GFP and SDP3-GFP aggregates is significantly lower than that of SDP1-GFP. After photobleaching, the SDP2-GFP aggregates can only recover 57% of the fluorescence intensity within 210s. The aggregates of SDP4-GFP and SDP5-GFP show better fluorescence recovery, and can recover more than 90% of the fluorescence intensity within 210s after photobleaching.

[0066] (3) The properties of the aggregates were further regulated by changing the amino acid composition. When Y was replaced by the hydrophobic amino acid alanine (A), the octapeptide FW6 (RGAGSPDG, SEQ ID NO.6) was obtained; when Y was replaced by threonine (T), the octapeptide FW7 (RGTGSPDG, SEQ ID NO.13) was obtained; and the aromatic amino acid Y was added to the C-terminus of FW1 to obtain the nonapeptide FW12 (RGYGSPDGY, SEQ ID NO.14). FW6, FW7 and FW12 were repeated 20 times to construct SDP6 (amino acid sequence: MSKGP-(RGAGSPDG) 20 -GY, SEQ ID NO.15), SDP7 (amino acid sequence: MSKGP-(RGTGSPDG) 20 -GY, SEQ ID NO.16) and SDP12 (amino acid sequence: MSKGP-(RGYGSPDGY) 20 -GY, SEQ ID NO.17). Artificially synthesized with promoter P T7 The fragments controlling the fusion expression of SDP6 and GFP (including His tag), the fusion expression of SDP7 and GFP (including His tag), and the fusion expression of SDP12 and GFP (including His tag) were connected to the pET28a plasmid to construct plasmids pET28a-SDP6-GFP, pET28a-SDP7-GFP, and pET28a-SDP12-GFP, and were transformed into Escherichia coli BL21 (DE3) respectively, and spread on LB solid culture medium supplemented with kanamycin. The single colonies grown were Escherichia coli EC6, EC7, and EC12, respectively. The purified proteins SDP6-GFP, SDP7-GFP, and SDP12-GFP were obtained according to the method in Example 2 and subjected to fluorescence photobleaching recovery experiments. The experimental results are shown in FIG. Figure 8As shown in Figure 2A, SDP6-GFP can also form aggregates. After photobleaching, SDP6-GFP can recover 80% of its fluorescence intensity within 210s, indicating that hydrophobic interaction can promote phase separation. Figure 8 As shown in -B, SDP7-GFP aggregates cannot recover fluorescence intensity after photobleaching. Figure 8 As shown in Figure 5-C, SDP12-GFP did not form aggregates with liquid properties, but instead formed irregular amorphous solid protein clusters.

[0067] Example 5: Construction and verification of membraneless organelles in Bacillus subtilis

[0068] (1) Artificially synthesized IPTG-inducible promoter P grac The fragment controlling the fusion expression of SDP1 and GFP (shown in SEQ ID NO.18) was connected to the vector pHT01 to construct the plasmid pHT01-P grac -SDP1-GFP, and transformed into the competent cells of B. subtilis 168, and then spread on LB solid medium plates supplemented with chloramphenicol, and cultured at 37°C for 12 h to obtain strain SC-1.

[0069] (2) The single colony SC-1 grown on the plate in step (1) was selected and inoculated into 2 mL of LB medium and cultured at 220 rpm and 37°C until OD 600 After the concentration of 0.5 mM IPTG was added to induce at 37°C for 2 h, 5 μL of bacterial solution was placed on a glass slide, and the formation of intracellular biomolecule aggregates was immediately observed under a Leica DMi8 microscope equipped with a 100× oil immersion objective. Fig. 9 As shown, SDP1-GFP and SDP1 can form spherical aggregates in vitro, and SDP1-GFP fusion protein can form aggregates in cells. When the induction time is prolonged and B. subtilis enters the sporulation stage, the spores will squeeze the SDP1-GFP aggregates spatially, causing the aggregates to change into arc shapes.

[0070] (3) The distribution of intracellular aggregates was calculated using ImageJ software. The calculation results are as follows: Fig.10 As shown, the aggregates in B. subtilis cells are mainly located in the cell center and at the two end poles.

[0071] (4) Take 2 mL of the strain induced to form aggregates, centrifuge and remove the supernatant, and wash repeatedly with PBS solution for 3 times; then, use PBS solution containing 0.5 M and 1 M urea to re-dissolve the bacteria, let it stand at 25°C for 20 min, and then observe and photograph it with a fluorescence microscope. Fig.11As shown. When the SDP1-GFP aggregates formed after IPTG induction for 2 hours were treated with 0.5M or 1M urea for 20 minutes, the intracellular fluorescent aggregates were decomposed and the aggregates could not be observed. When induced to 13 hours, the SDP1-GFP aggregates could still be destroyed and decomposed by 1M urea. Since low concentrations of urea can destroy multivalent interactions, the liquid aggregate structure is easily destroyed and disappears, while the structure of polymers such as inclusion bodies needs to be destroyed under high concentrations of urea. The above results prove that the intracellular SDP1-GFP aggregates are not inclusion bodies.

[0072] (5) Detect the OD of B. subtilis 168 and SC-1 strains at 0h, 2h, 4h, 6h, 8h and 10h after induction 600 The cell length was analyzed by ImageJ software. Fig.12 As shown in the figure, compared with B. subtilis 168, the growth of the recombinant strain SC-1 overexpressing SDP1-GFP was affected within 4 h after induction, and the growth rate decreased slightly, but the OD 600 It can still reach 5.19, which is basically consistent with B. subtilis 168. At the same time, the average length and width of the cells did not change significantly, with the cell length being 2.8 μm and the cell width being 0.7 μm, but the proportion of cells with a length of <2.5 μm that formed SDP1-GFP aggregates within the cells increased significantly.

[0073] (6) Inoculate strain SC-1 into 2 mL of LB medium and culture at 220 rpm and 37°C until OD 600 After the concentration of 0.5 mM IPTG was added at 30°C, 37°C and 42°C for 2 h, 5 μL of bacterial solution was placed on a glass slide, and the formation of intracellular biomolecule aggregates was immediately observed under a Leica DMi8 microscope equipped with a 100× oil-immersion objective. The formation of SDP1-GFP aggregates was statistically analyzed using ImageJ software. The statistical results are shown in the figure. Fig.13 As shown, the phase separation behavior of intracellular SDP1-GFP is also affected by the culture temperature. When the induction concentration of IPTG reaches 0.5mM and the culture temperature is 30°C, the formation rate of intracellular SDP1-GFP aggregates in strain SC-1 is only 2.26%. When the culture temperature is 37°C or 42°C, even if the induction concentration is reduced to 0.05mM, the formation rate of intracellular SDP1-GFP aggregates in strain SC-1 is still as high as 90.5% and 92.38%, respectively. The above results prove that compared with the expression intensity of SDP1-GFP, the formation of intracellular aggregates is more sensitive and dependent on temperature.

[0074] (7) Set the number of repetitions of the FW1 basic unit to 10 to build FW1 10 (Amino acid sequence: MSKGP-(RGYGSPDG) 10 -GY, as shown in SEQ ID NO.19), the number of repetitions of the FW1 basic unit was set to 15 to construct FW1 15 (Amino acid sequence: MSKGP-(RGYGSPDG) 15 -GY, as shown in SEQ ID NO.20), the number of repetitions of the FW1 basic unit was set to 30 to construct FW1 30 (Amino acid sequence: MSKGP-(RGYGSPDG) 30 -GY, as shown in SEQ ID NO.21). grac Control FW1 separately 10 Fusion expression with GFP, FW1 15 Fusion expression with GFP, FW1 30 The fragments fused with GFP were connected to the pHT01 vector to construct the plasmid pHT01-FW1 10 -GFP, pHT01-FW1 15 -GFP and pHT01-FW1 30 -GFP, respectively, were transformed into competent cells of B. subtilis 168, and then plated on LB solid medium plates supplemented with chloramphenicol and cultured at 37°C for 12 h to obtain strain SC-1 10 SC-1 15 and SC-1 30 .

[0075] (8) Select the single colony SC-1 grown on the plate in step (7) 10 SC-1 15 and SC-1 30 Inoculate into 2 mL of LB medium and culture at 220 rpm and 37°C until OD 600 After the concentration of 0.5 mM IPTG was added to induce at 30°C and 42°C for 2 h, 5 μL of bacterial solution was placed on a glass slide, and the formation of intracellular biomolecule aggregates was immediately observed under a Leica DMi8 microscope equipped with a 100× oil immersion objective. 10 No aggregates could be formed at 30℃ and 42℃; SC-1 15 No aggregates can be formed at 30°C, but aggregates can be formed at 42°C (formation rate 97.14%); SC-1 30Aggregates can be formed at both 30°C and 42°C (98.47% at 30°C and 96.91% at 42°C). The above results show that the temperature-dependent threshold range of SDP1 aggregates can be regulated by changing the molecular weight, thereby controlling the formation of intracellular aggregates. This result also proves that SDP1 is highly programmable.

[0076] Example 6: Functional testing of membraneless organelles in Bacillus subtilis

[0077] (1) Artificial synthesis of promoter P grac The fragment controlling the fusion expression of RIAD, SDP1 and GFP (shown in SEQ ID NO.22) was connected to the vector pHT01 to construct the plasmid pHT01-P grac -RIAD-SDP1-GFP, artificially synthesized under the xylose-inducible promoter P xylA The fragment controlling the fusion expression of mKate (red fluorescent protein) and RIDD (shown in SEQ ID NO.23) was connected to the vector p43NMK to construct the plasmid p43NMK-P xylA -mKate-RIDD. The two plasmids constructed above were simultaneously transferred into the competent cells of B. subtilis 168, and then plated on LB solid medium plates supplemented with chloramphenicol and kanamycin, and cultured at 37°C for 12 hours to construct strain SC2. Plasmid pHT01-P grac -SDP1-GFP (the construction process is shown in Example 5) and p43NMK-P xylA -mKate-RIDD was simultaneously transformed into the competent cells of B. subtilis 168, and then plated on LB solid medium plates supplemented with chloramphenicol and kanamycin, and cultured at 37°C for 12 h to construct strain SC3.

[0078] (2) Select the single colonies SC2 and SC3 grown on the plate in step (1) and inoculate them into 2 mL of LB medium. Cultivate at 220 rpm and 37°C until the OD 600 After the concentration of 0.6-0.8, 0.5mM IPTG and 30g / L xylose were added and induced at 37℃ for 2h. 5μL of bacterial solution was placed on a glass slide, and the formation of intracellular biomolecule aggregates and the distribution of red fluorescent protein mKate were immediately observed under a Leica DMi8 microscope equipped with a 100× oil immersion objective. The observation results are shown in Fig.14As shown, the red fluorescent protein mKate was selected as the targeting protein. When the SDP1-GFP condensate was not fused with the RIAD short peptide, the mKate-RIDD fluorescence was evenly distributed in the cytoplasm; when RIAD-SDP1-GFP was expressed, 84% of the mKate-RIDD was recruited into the condensate, indicating that biological macromolecules such as proteins can be recruited into the SDP1 condensate.

[0079] (3) According to step (2), when the expression is induced at the same time, mKate-RIDD can interact and bind with RIAD-SDP1-GFP before the formation of aggregates, which cannot directly prove that SDP1-GFP has the ability to recruit co-localized mKate after the aggregates are formed. In order to avoid this phenomenon, after the strain SC2 forms aggregates in the cell by IPTG induction, the culture medium is replaced with LB medium containing only 30g / L xylose. The results are shown in Fig.15 -A, when the medium contains only IPTG inducer, SDP1-GFP aggregates are formed in the strain SC2, and mKate-RIDD is not induced to express. When IPTG in the medium is replaced with 30g / L xylose, mKate-RIDD protein begins to express. At this time, 85% of mKate is still recruited and localized to SDP1-GFP aggregates. When the order of IPTG and xylose induction is changed, that is, the expression of mKate-RIDD is induced first, the results are as follows Fig.15 -B, when SDP1-GFP subsequently formed aggregates, 88% of mKate was still recruited. The above results prove that SDP1-GFP aggregates can effectively recruit target proteins and have high compatibility and universality.

[0080] (4) Artificial synthesis of promoter P 21 Gene fragments controlling the fusion expression of VioA and RIDD, VioB and RIDD, VioC and RIDD, and VioE and RIDD, P 21 -VioA RIDD -VioB RIDD -VioE RIDD The sequence is shown in SEQ ID NO.24, P 21 -VioA RIDD -VioB RIDD -VioE RIDD -VioC RIDD The sequence of the PCR product is shown in SEQ ID NO.25, and was connected to the pcrF11 vector in different combinations to construct plasmids pcrF11-55 (pcrF-P 21 -VioA RIDD -VioB RIDD -VioERIDD ) and plasmid pcrF11-56 (pcrF-P 21 -VioA RIDD -VioB RIDD -VioE RIDD -VioC RIDD ). grac -RIAD-SDP1-GFP (see step (1) for the construction process) was simultaneously transformed into the competent cells of B. subtilis168, and then plated on LB solid medium plates supplemented with chloramphenicol and kanamycin, and cultured at 37°C for 12 hours to construct strain SC4. pcrF11-56 was combined with plasmid pHT01-P grac -RIAD-SDP1-GFP was simultaneously transformed into the competent cells of B. subtilis168, and then plated on LB solid medium plates supplemented with chloramphenicol and kanamycin, and cultured at 37°C for 12 h to construct strain SC5.

[0081] (5) Inoculate strains SC4 and SC5 in step (4) into 2 mL of liquid LB medium, respectively, and culture at 37°C and 220 rpm for 8 h; secondly, inoculate the culture solution into a 250 mL baffled shake flask containing 20 mL of liquid LB medium at a 5% (v / v) inoculation rate, at 37°C and 220 rpm; after fermenting for 5 h, add IPTG at a final concentration of 0.5 mM to induce phase separation; finally, take 1.5 mL of the bacterial solution for visual detection after 24 h of fermentation. The fermentation color of the bacterial solution is as follows: Fig.16 As shown, when only VioABE is expressed intracellularly and the formation of aggregates is not induced, the cells appear light brown; when IPTG is added to induce the formation of SDP1 aggregates, VioABE is recruited into the aggregates, enhancing the metabolic synthesis efficiency of PDV, and the cell color changes to dark green. When VioABEC is expressed intracellularly and the formation of SDP1 aggregates is induced, the cell color also changes from pink to purple. The above results indicate that the small molecule L-tryptophan can diffuse into the intracellular aggregates and participate in the metabolic synthesis of DV. In addition, the significant change in cell color proves that artificial aggregates can regulate the metabolic synthesis of target compounds at the spatial level.

[0082] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant disordered protein, characterized in that: The recombinant disordered protein can form a biomolecular condensate, containing a repeated series of peptide segments N8, wherein the peptide segment N8 is composed of eight amino acids, including three glycines, one proline, one serine, one aspartic acid, one arginine, and one tyrosine or alanine; The amino acid sequence of the peptide segment N8 may be selected from any one of the following: (1) RGYGSPDG; (2) RGGDSPYG; (3) RGSPYGDG; (4) GYPSDGRG; (5) DGRGSPYG; (6) RGAGSPDG; The recombinant disordered protein contains the following sequence MSKGP-(N8) n -GY, n is any integer between 15 and 30.

2. A membraneless organelle, characterized in that: An expression vector containing the recombinant disordered protein according to claim 1 is constructed and introduced into an expression host to express and form the membraneless organelle.

3. The membraneless organelle according to claim 2, characterized in that: The recombinant disordered protein is expressed using an inducible promoter or a constitutive promoter.

4. The membraneless organelle according to claim 3, characterized in that: IPTG was used for inducible expression.

5. The membraneless organelle according to claim 4, characterized in that: The IPTG concentration is 0.05-0.5 mM, the induction temperature is 37-42°C, and the induction time is 6-10 hours.

6. A host cell containing the recombinant disordered protein according to claim 1 or the membrane-less organelle according to claim 2.

7. The host cell according to claim 6, characterized in that: The host is Bacillus subtilis.

8. A method for recruiting a target protein using the membraneless organelle described in claim 2.

9. The method according to claim 8, characterized in that: The membraneless organelle targets a protein of interest.

10. The method according to claim 9, characterized in that: A polypeptide tag or tag ligand is connected to the N-terminus or C-terminus of the target protein, and a tag ligand or polypeptide tag is connected to the N-terminus or C-terminus of the membraneless organelle, and the polypeptide tag and the tag ligand specifically bind.

11. The method according to claim 10, characterized in that: The polypeptide tag is a RIDD short peptide or a CCDIA short peptide, and the tag ligand is a RIAD short peptide or a CCDIB short peptide.

12. Use of the recombinant disordered protein according to claim 1, the membraneless organelle according to claim 2, the host cell according to claim 6 or the method according to claim 8 in regulating the metabolism of a target compound.

Images