Method for producing target molecule on basis of condensation-segregation technique, recombinant strain and use thereof

Abstract

Provided are a method for producing a target molecule on the basis of a condensation-segregation technique, a recombinant strain and the use thereof. By means of regulating the formation of biological condensates, high-concentration enrichment of target molecules at the cell end is achieved. By means of this step, highly-enriched target molecules are obtained and can be further accumulated by means of cell division. Compared with traditional methods, the method not only effectively improves the concentration of target molecules in a specific region within cells, but also realizes further accumulation of the enriched molecules by means of the mechanism of cell division.

Description

Method for producing target molecules based on coacervation-segregation technology, recombinant strain and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a method for producing target molecules based on coacervation-segregation technology, a recombinant strain and application thereof. BACKGROUND

[0002] In the prior art, biological macromolecules such as proteins, nucleic acids, polysaccharides and oils are important molecular components in organisms and play a key role in many biological processes and medical applications. However, the characteristics of biological macromolecules limit their application in production processes. Generally, these biological macromolecules have a large molecular weight, usually more than 3 kD. Due to their complex structure and properties, their extracellular secretion process is difficult, for example, nucleic acid substances are mainly limited in cells, polysaccharides and oil substances are often deposited or attached to the cell wall surface in cells, and proteins can be successfully secreted to the extracellular by the guidance of signal peptides. Intracellular accumulation of some biological macromolecules may cause toxicity, inhibit normal cell growth, and even affect the stability of biological processes in some cases. In addition, the biological macromolecules accumulated in cells are often affected by the intracellular degradation mechanism, resulting in unstable content. Therefore, the yield and efficiency of cell production of biological macromolecules are usually much lower than that of small molecule compounds. In order to improve the production efficiency of biological macromolecules, it is necessary to explore new ways to overcome the problems faced by the prior art and achieve higher yield and higher efficiency.

[0003] In rod-shaped bacteria, cell division usually occurs at the central position of the cell, forming two daughter cells of substantially equal size. This process is triggered by a structure called septum, which is composed of multiple interacting proteins such as FtsZ, FtsA, FtsK, FtsQ, ZipA, FtsL, FtsI, FtsW, etc. Among them, FtsZ can assemble into a division ring called Z-ring, which can generate a contraction force at the center of the cell. The cell division process is also finely regulated by the Min system, septum inhibition system, Z-ring regulation system, topological specificity factor, etc., including one or several of MinC, MinD, MinE, MinJ, DivIVA or proteins with similar functions. When some proteins related to cell division are deleted or their functions are impaired, Z rings may form at the center of the cell and at both ends of the cell, resulting in abnormal division of bacterial cells and the production of minicells containing no genomic DNA at the end of the parent cell. In fact, the pathways and mechanisms of minicell production are diverse.

[0004] Therefore, the present application proposes a method for effectively improving the production efficiency of target molecules based on the condensation-separation technology, so as to provide a beneficial solution for the technical innovation in the related field. SUMMARY

[0005] The present application aims to solve the technical problems of the prior art, and provides a method for producing target molecules based on the condensation-separation technology. First, the target molecules are formed into a condensed state in the cell to achieve high concentration enrichment of the target molecules at the end of the cell, and then the target molecules with high concentration enrichment are separated from the enriched end by using the cell division mechanism to produce small cells rich in target molecules, so that the target molecules are separated from the parent cell and can accumulate outside the parent cell, thereby achieving the purpose of improving the production efficiency of target molecules.

[0006] The present application also aims to solve the technical problem of providing a recombinant strain based on the condensation-separation technology.

[0007] The present application finally aims to solve the technical problem of providing the application of the above-mentioned recombinant strain based on the condensation-separation technology.

[0008] In order to solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0009] A method for producing target molecules based on the condensation-separation technology, first enriching the target molecules at the end of the cell, and then separating the target molecules from the enriched end by using the cell division mechanism.

[0010] In some embodiments of the present application, the target molecules are connected with a biological condensed state tag to obtain a target molecule gene fragment, and then the target molecule gene fragment is introduced into a small cell production chassis strain to separate the target molecules by producing small cells.

[0011] The target molecules are biological macromolecules or organic small molecules.

[0012] Preferably, the biological macromolecules include any one or combination of several of proteins, polypeptides, nucleic acids, polysaccharides, oils, biological particles, and intracellular polymers; and preferably, the organic small molecules include any one or combination of several of alcohols and aldehydes.

[0013] Specifically, the proteins include any one or combination of several of green fluorescent protein, xylose reductase, formate dehydrogenase, adenylyl cyclase, glucose dehydrogenase, sialidase, glutathione reductase, cellulase, xylanase, lysozyme, lipase, protease, saccharifying enzyme, amylase, laccase, beta-glucanase, L-aspartate enzyme, keratinase, glucose oxidase, pectinase, hyaluronidase, and collagen.

[0014] Specifically, the nucleic acid includes any one or combination of several of DNA, RNA or plasmid; wherein the DNA includes any one or combination of double-stranded DNA or single-stranded DNA, and the RNA includes any one or combination of several of double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), microRNA (miRNA), ribozyme, aptamer and combination thereof.

[0015] The nucleic acid described herein refers to a polymerization form of nucleotides of any length, ribonucleotides or deoxyribonucleotides, or structural analogs and derivatives thereof such as peptide nucleic acid.

[0016] Specifically, the polysaccharide includes any one or combination of several of cellulose, starch, dextran, oligosaccharide, polysaccharide, hyaluronic acid.

[0017] Specifically, the biological particle includes any one or combination of several of inclusion body, polyhydroxyalkanoate (PHA), poly-3-hydroxybutyrate (PHB), polylactic acid (PLA).

[0018] Specifically, the alcohol includes any one or combination of several of medium-chain (C6-C10) alcohol, ethanol, hexanol, octanol, 1,3-butanediol, 1,4-butanediol, etc.

[0019] Specifically, the aldehyde includes any one or combination of several of formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, etc.

[0020] In some embodiments of the present application, the biological macromolecule includes but is not limited to protein or nucleic acid; specifically, the protein includes green fluorescent protein, xylose reductase, formate dehydrogenase or adenylyl cyclase, and the nucleic acid is dsRNA.

[0021] In some embodiments of the present application, the organic small molecule includes but is not limited to alcohol, and specifically, the alcohol is 1,3-butanediol.

[0022] A recombinant strain based on the condensation-separation technology, specifically, a target molecule is connected with a biological condensed state tag to obtain a target molecule gene fragment, and the target molecule gene fragment is introduced into a production minicell chassis strain to construct.

[0023] The connection mode of connecting the target molecule with the biological condensed state tag includes any one of direct connection, connection through a flexible peptide, connection through a recruitment tag, and connection through gene fusion.

[0024] In some embodiments of the present application, the flexible peptide linker rich in G and S is used, such as (GGS)n, (GGGS)n, (GGGGS)n, (GGGSSS)n, or (RS)n, (GS)n, (EAAAK)n, wherein n = 1-6.

[0025] In some embodiments of the present application, the recombinant strain is constructed by connecting the target molecule gene with the biological condensed state tag, constructing a recombinant plasmid containing the target molecule gene fragment, and then introducing the recombinant plasmid into the chassis strain.

[0026] The recombinant plasmid containing the target molecule gene fragment is constructed by homologous recombination or enzyme digestion and connection into the corresponding expression vector (such as pETDuet-1 or pET28a plasmid).

[0027] The plasmid is any one of pET series plasmid, pCDF series plasmid, pBAD series plasmid, pT7 series plasmid, pTac series plasmid, pBFC series plasmid, pBV220 series plasmid, pJ23119 series plasmid, pBR322 series plasmid, pRSF series plasmid, pGEX series plasmid, pTrc99a, pCWJ, pRSETB series plasmid, pACY series plasmid.

[0028] In some embodiments of the present application, the plasmid includes but is not limited to any one of pETDuet-1, pET28a plasmid and pCWJ plasmid in pET series plasmid.

[0029] The biological condensed state tag includes any one or combination of several of liquid-liquid phase separation tag, end positioning tag, self-assembly tag.

[0030] Specifically, the liquid-liquid phase separation tag includes any one of I16, R32, V7, NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, ELP[KV7F-36], WT-20, I24, Y145, V5-N, V1-C, N1, HPB2-C, V2-C, tag2, tag1, FUS, TDP-43, LAF-1, ICP4, RGGRGG, CAG 40 The end positioning tag includes any one of EpsM, DivIVA or its truncated body, Div(60), PopZ, PodJ, SpmX, RodA, LcsA; and the self-assembly tag includes any one of Y15, L6KD, 18Awt.

[0031] Specifically, the liquid-liquid phase separation tag I16, R32, V7, NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, ELP[KV7F-36], WT-20, I24, Y145, V5-N, V1-C, N1, HPB2-C, V2-C, tag2, tag1, FUS, TDP-43, LAF-1, ICP4, RGGRGG, the corresponding amino acid sequences are shown in SEQ ID NO. 1-22, SEQ ID NO. 59 or variant amino acid sequences with similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more to SEQ ID NO. 1-22, SEQ ID NO. 59; the liquid-liquid phase separation tag CAG 40 , the corresponding nucleotide sequence is shown in SEQ ID NO. 67 or a variant nucleotide sequence with similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more to SEQ ID NO. 67.

[0032] Specifically, the end positioning tag EpsM, DivIVA, Div(60), the corresponding amino acid sequences are shown in SEQ ID NO. 47-49 or variant amino acid sequences with similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more to SEQ ID NO. 47-49; the DivIVA truncated body includes any one or combination of DivIVA 1-40 , DivIVA 1-60 , DivIVA 1-80 .

[0033] Specifically, the self-assembly tag Y15, L6KD, 18Awt, the corresponding amino acid sequences are shown in SEQ ID NO. 50-52 or variant amino acid sequences with similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more to SEQ ID NO. 50-52.

[0034] In order to realize efficient enrichment of proteins while reducing the length of molecular condensed state tags, a method based on short peptide tag interaction is developed to realize the recruitment of target proteins. Therefore, the target molecule gene can also be recruited to the biological condensed state tag by the recruitment tag, and the target molecule gene fragment is obtained.

[0035] Specifically, the recruitment tag includes any one group of RIAD and RIDD, P1 and P2, P3 and P4.

[0036] Specifically, the RIAD and RIDD have the amino acid sequences shown as SEQ ID NO. 53-54 or variant amino acid sequences with more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% similarity to SEQ ID NO. 53-54, respectively; the P1 and P2 have the amino acid sequences shown as SEQ ID NO. 55-56 or variant amino acid sequences with more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% similarity to SEQ ID NO. 55-56, respectively; the P3 and P4 have the amino acid sequences shown as SEQ ID NO. 57-58 or variant amino acid sequences with more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% similarity to SEQ ID NO. 57-58, respectively.

[0037] In some embodiments of the present application, the recruitment tag is RIAD and RIDD.

[0038] The production minicell chassis strain is a minicell-forming system chassis strain, which is constructed by modifying any one or a combination of several of the cell division minicell production related genes of an original strain of Escherichia coli.

[0039] Specifically, the cell division minicell production related genes include any one or a combination of several of the cell septum component genes, septum inhibition protein genes, Z-ring regulatory protein genes, cell division topological specificity factor genes, and fitting binding protein genes.

[0040] Specifically, the cell septum components include any one or a combination of several of FtsZ, FtsA, FtsK, FtsQ, ZipA, FtsL, FtsI, and FtsW; the septum inhibition proteins include any one or a combination of several of MinC or its truncated body, MinD, SulA, and SfiA; the Z-ring regulatory proteins include any one or a combination of several of ParA, ParB, and ParS; the cell division topological specificity factors include any one or a combination of several of MinE or its truncated body, DivIVA or its truncated body, and MinJ; and the fitting binding proteins include any one or a combination of several of HNS, HU, MukBEF, FisA, and Lsr2.

[0041] The MinC or its truncated body, MinE or its truncated body, and MinD also belong to the Min system genes.

[0042] In some embodiments of the present application, the Min system gene includes, but is not limited to, any one of MinC or its truncation, MinD, MinE or its truncation; the cell division gene includes, but is not limited to, FtsZ.

[0043] Specifically, the MinC truncation includes any one or combination of MinC 116-231 , MinC 122-231 , MinC 122-231 , wherein the residue R at the 172nd position of MinC 122-231 is replaced by A; the MinE truncation includes any one or combination of MinE 1-22 , MinE 1-33 , MinE 1-53 , MinE 1-62 , MinE 1-67 .

[0044] The E. coli includes any one of E. coli BL21, BL21(DE3), MG1655, MG1655(DE3), JM109, JM109(DE3), Rosetta, Rosetta(DE3), Lemo21, Lemo21(DE3).

[0045] In some embodiments of the present application, the E. coli includes, but is not limited to, E. coli BL21(DE3).

[0046] The chassis strain for producing minicells includes any one of the following chassis strains:

[0047] (A) knocking out the minC gene in the original strain;

[0048] (B) knocking out the minC, minD, minE genes in the original strain;

[0049] (C) knocking out the minC gene and expressing the ftsZ gene in the original strain;

[0050] (D) knocking out the promoter P BAD downstream structural genes rhaB, rhaA, rhaD and expressing the ftsZ gene in the original strain, to construct a chassis strain B-ΔBAD::ftsZ for producing minicells;

[0051] (E) knocking out the minC gene and the promoter P BAD downstream structural genes rhaB, rhaA, rhaD and expressing the ftsZ gene in the original strain;

[0052] (F) Overexpress any one or a combination of the genes MinC, MinC truncated, MinE, and MinE truncated in the original strain.

[0053] In some embodiments of the present invention, the chassis strain for producing small cells includes any one of the following chassis strains:

[0054] (a) Using Escherichia coli BL21(DE3) as the original strain, a chassis strain B-ΔC for producing small cells was constructed by knocking out the minC gene;

[0055] (b) Using Escherichia coli BL21(DE3) as the original strain, a chassis strain B-ΔCDE for producing small cells was constructed by knocking out the minC, minD and minE genes.

[0056] (c) Using Escherichia coli BL21(DE3) as the original strain, a chassis strain B-ΔC::ftsZ for producing small cells was constructed by knocking out the minC gene and expressing the ftsZ gene.

[0057] (d) Using Escherichia coli BL21(DE3) as the original strain, the promoter P was knocked out. BAD The downstream structural genes rhaB, rhaA, and rhaD simultaneously express the ftsZ gene to construct a chassis strain B-ΔBAD::ftsZ for producing small cells.

[0058] (e) Using Escherichia coli BL21(DE3) as the original strain, the minC gene and promoter P were knocked out. BAD The downstream structural genes rhaB, rhaA, and rhaD simultaneously express the ftsZ gene to construct a chassis strain B-ΔC-ΔBAD::ftsZ for producing small cells.

[0059] (f) Using Escherichia coli BL21(DE3) as the original strain, the corresponding chassis strain for producing small cells was constructed by overexpressing any one or a combination of MinC, MinC truncated, MinE, and MinE truncated genes.

[0060] Specifically, the gene knockout method described above was accomplished using CRISPR editing technology.

[0061] Specifically, the amino acid sequences of the minC gene, minD gene, minE gene, rhaB gene, rhaA gene, rhaD gene, and ftsZ gene are shown in SEQ ID NO. 60-66.

[0062] The method for constructing recombinant strains based on the above-mentioned agglomeration-dissection technology is also within the scope of protection of this invention.

[0063] In some embodiments of the present application, the construction method comprises the following steps:

[0064] (1) Intracellular protein enrichment: synthesizing a target molecule gene, connecting the target molecule gene with a biological condensation state tag to form a target molecule gene fragment;

[0065] (2) Introducing the target molecule gene fragment formed in step (1) into a linearized expression vector by enzyme digestion and connection for homologous recombination to obtain a recombinant plasmid;

[0066] (3) Construction of a minicell-forming chassis strain for producing small cells: taking E. coli as the original strain, constructing a chassis strain for producing small cells by modifying any one or a combination of several of Min system genes and cell division genes;

[0067] (4) Introducing the recombinant plasmid obtained in step (2) into the chassis strain for producing small cells constructed in step (3) to construct the recombinant strain.

[0068] Or,

[0069] (Ⅰ) Intracellular protein enrichment: synthesizing a target molecule gene, connecting the target molecule gene with a biological condensation state tag to form a target protein gene fragment;

[0070] (Ⅱ) Introducing any one of the fusion target molecule gene fragment obtained in step (Ⅰ) and the genes of MinC, MinC truncated body, MinE, MinE truncated body into a linearized expression vector by enzyme digestion and connection for homologous recombination to obtain a recombinant plasmid capable of making the host produce small cells;

[0071] (Ⅲ) Introducing the recombinant plasmid obtained in step (Ⅱ) into the host to construct the recombinant strain.

[0072] The above-mentioned recombinant strain based on the condensation-separation technology is also within the scope of protection of the present application in the application of culturing small cells and separating target molecules.

[0073] Among them, the target molecule is a biological macromolecule or an organic small molecule.

[0074] Preferably, the biological macromolecule includes any one or a combination of several of proteins, nucleic acids, polysaccharides, and biological particles; preferably, the organic small molecule includes any one or a combination of several of alcohols and aldehydes.

[0075] The culture medium contains any one or a combination of several of glucose, fructose, sucrose, galactose, and glycerol as carbon source, and the addition amount is 0-100 g / L; the nitrogen source contains any one of yeast powder, protein hydrolysate (such as peptone), and urea, and the addition amount is 0.5-100 g / L; the culture condition is that the temperature is 4-40 DEG C, preferably 15-30 DEG C.

[0076] The "biological condensed state label" has the characteristics of promoting the condensation of various biomolecules in cells and regulating various physiological activities in cells. By using the biological condensed state label, the condensate of proteins at the end of the cell can be formed. This molecular condensation strategy will provide a powerful solution to overcome the poor protein directional enrichment effect in the traditional biosynthesis system.

[0077] The "liquid-liquid phase separation label" refers to a molecular structure or sequence that can drive target molecules to aggregate in a liquid environment through intermolecular or intramolecular interaction forces to form liquid condensates or droplets, and separate the target molecules from the liquid mixture to condense into a liquid phase or even a solid phase. The design and application of such labels help to improve the production concentration of target substances and improve the separation efficiency. It should be noted that liquid-liquid phase separation relies on intermolecular or intramolecular multivalent forces, including covalent interaction, hydrogen bonding, electrostatic interaction, and hydrophobic interaction. Therefore, the length and position of the sequence of the liquid-liquid phase separation label, as well as the type of amino acid or base, can tolerate large changes while still having liquid-liquid phase separation ability. Therefore, the liquid-liquid phase separation label can be changed by sequence truncation or lengthening, amino acid substitution, base substitution, error-prone PCR random mutation, site-directed mutation, saturation mutation, computer-aided mutation, and other conventional technical methods in the art to generate new sequences. The sequence similarity is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, which can achieve similar effects as the original label sequence.

[0078] For example, in some embodiments of the application, Y145 can be truncated or mutated after optimization and mutation by artificial intelligence technology, wherein the remaining N-terminal sequence contains 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, 60 amino acids, 62 amino acids, 64 amino acids, 66 amino acids, 68 amino acids, 70 amino acids, 72 amino acids, 74 amino acids, 76 amino acids, 78 amino acids, 80 amino acids, 90 amino acids, 100 amino acids, 120 amino acids, 122 amino acids, and preferably any one of 66 amino acids, 72 amino acids, or 122 amino acids.

[0079] In addition, the Y145 partial amino acid mutation to more polar amino acids is more conducive to the performance of liquid-liquid phase function, such as mutation to serine (S), alanine (A), glycine (G), aspartic acid (N), threonine (T), glutamine (Q) or other amino acids such as isoleucine (I), phenylalanine (F).

[0080] In addition, the LAF-1 truncated body retains the N-terminal which can contain 50 amino acids, 100 amino acids, 150 amino acids, 168 amino acids, 200 amino acids, 250 amino acids, 336 amino acids, 400 amino acids, 500 amino acids, 550 amino acids, 600 amino acids, and preferably 168 amino acids, and the sequence is a sequence of a lower complexity RGG domain, rich in glycine (about 35%), arginine (about 14%), asparagine (about 14%), aspartic acid (about 10%), serine (about 7%) and tyrosine (about 7%).

[0081] The "end localization tag" described in the present application refers to a molecular structure or sequence that can localize or enrich target biomolecules to one end or both ends of a cell. They are usually located on the cell membrane or cell wall or can interact with other molecules on the cell membrane or cell wall at the bending site of the cell, thereby achieving localization at the end region of the cell. It should be noted that the localization of the cell end relies on the interaction between the functional region of the localization tag and the cell membrane structure or the cell wall structure or the intrinsic molecules therein, so such localization tags can tolerate a certain degree of sequence change in the functional region, and a larger degree of sequence change outside the functional region, and arbitrary truncation of the sequence length outside the functional region, and still have the ability of end localization. Therefore, the above-mentioned end localization tag can be changed by sequence truncation or lengthening, amino acid substitution, base substitution, error-prone PCR random mutation, site-directed mutation, saturation mutation, computer-aided mutation, etc. Conventional technical methods in the art to produce new sequences, sequence similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, can achieve similar effects to the original tag sequence. For example, DivIVA can be truncated to retain the N-terminal with membrane targeting function, containing at least 1-20 positions of the N-terminal sequence, which can also be 1-40 positions, 1-60 positions, 1-80 positions.

[0082] The "self-assembly tag" of the present application refers to a molecular tag or sequence that can assemble into a multi-molecular structure through intermolecular interactions. These tags are usually composed of some specific molecular fragments or functional groups that can interact with each other and orderly form stable three-dimensional structures or assemblies under appropriate conditions. The self-assembly tag is composed of 5-30 amino acids, or 10-100 nucleotides or deoxyribonucleotides, which can form structures such as α-helix, β-sheet, β-hairpin, group pairing, or the molecule has amphiphilic properties, while containing at least 1 hydrophobic amino acid and 1 hydrophilic amino acid, or containing at least 1 acidic amino acid and 1 basic amino acid. In some embodiments, the self-assembly tag includes Y15, L6KD, and 18Awt.

[0083] The "recruitment tag" of the present application refers to a molecular structure or sequence that can direct the binding of a protein, nucleic acid, polysaccharide or other target molecule containing a specific functional region or structure within a cell. The recruitment tag can also be any pair of sequences or structures that can bind to each other. By linking two recruitment tags that can bind to each other to any protein or molecule, the proximity and aggregation of these proteins or molecules can be achieved. The recruitment tag includes molecules that can form isopeptide bonds to bind to each other, such as SpyCatcher and SpyTag, molecules that can form non-covalent oligomer structures to bind to each other, such as GFP1-10 and GFP11, molecules that can bind to each other through disulfide bonds, molecules that can bind to each other through antigen-antibody interaction, and enzyme substrates and their binding domains, enzyme or protein binding substrates including protein, polysaccharide (such as starch, dextrin, cellulose, peptidoglycan, β-glucan, mannan), nucleic acid, lipid, PHA, etc. or insoluble particles. In some embodiments, the recruitment tag includes RIAD and RIDD, P1 and P2, P3 and P4. For different target molecules, changing the number or position of the recruitment tag linked to the target molecule can achieve different molecular aggregation effects. In the present application, two or more than two recruitment tags of the same or different can be connected in series at one end of the target molecule, or at both ends or internal regions of the target molecule.

[0084] The "droplet" of the present application, also known as "liquid condensate", aims to achieve a specific function or produce a specific effect (such as biomolecule enrichment function), and has a structure similar to organelles in natural cells. It is created by artificial means, and refers to a droplet formed by liquid-liquid phase separation to condense proteins, nucleic acids or other biomolecules in cells. Such microchambers can selectively concentrate certain molecules and exclude other molecules from material exchange with the surrounding environment.

[0085] The "protein enrichment" of the present application refers to the process of guiding or condensing the target protein from the cytoplasm to a specific region or location in the cell, which promotes the gradual transition of the target protein from the free state to the condensed state of the protein structure, thereby achieving the purposeful and directional enrichment in the cell. After protein enrichment, more protein synthesis may be stimulated, thereby increasing the accumulation of proteins. This process promotes the transition of the target molecule from the free state or single molecule state to the condensed state or multi-molecule state, thereby achieving directional aggregation and accumulation in the cell. "Enrichment" is a general term used to describe the process of condensing a substance or molecule from a dilute or mixed environment to a specific region or location. The condensed or condensed body of the enriched molecule can be liquid, solid, droplet, particle, macromolecular assembly structure, macromolecular complex, amorphous precipitate, endosome, crystal. In the present application, the concentration of the target molecule after enrichment is at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, or at least 20 times higher than that of the target molecule without enrichment.

[0086] The "protein accumulation" of the present application refers to the gradual increase in the number of target proteins in the cell through the promotion of protein synthesis and stability during the protein enrichment process, so that a high concentration of aggregated state is formed in a specific region or location. This accumulation may be achieved by forming condensed bodies after enrichment, which helps to achieve specific functions or effects.

[0087] The "minicell" of the present application is a nanoscale anucleate subcellular structure produced during abnormal cell division of Escherichia coli or Gram-positive bacteria, also known as small cells. Minicells contain all molecular components of the parent cell except DNA, including membranes, ribosomes, RNA, proteins, and plasmids, and their proportions are comparable to those of normal cells, so minicells cannot divide and grow, and their production does not interfere with the simultaneous division of normal cells. Compared with normal cells, minicells still maintain cell metabolic functions in the absence of a nucleus, showing superior stability and special tolerance. The production of minicells will increase the cytoplasmic space, which is the main place for the synthesis of cell factory materials in Escherichia coli, and plays a crucial role in the production and metabolism of cells.

[0088] The "Minicell-forming" of the present application is a minicell microcell platform. The present application edits the key genes of cell division of E. coli BL21(DE3) cells by CRISPR technology to regulate the cell division process and promote abnormal division of E. coli to produce minicells. Although minicells cannot grow and divide, they have functional metabolism and can be continuously produced by the parent cells. The present application uses minicells as a platform for encapsulating and enriching target molecules to increase the synthesis site of the target molecules and improve the accumulation of the target molecules. In some embodiments, the target molecules encapsulated by minicells are more active and stable than free target molecules.

[0089] The "condensation-separation" technology of the present application is based on the above-mentioned cell division, division and protein enrichment technology, which effectively integrates the two methods to further expand the synthesis space of the target molecules and improve the yield. Specifically, the present application "packs" the target molecules at the end of the cell through a biological condensate tag, and further divides and encapsulates them into minicells, thereby establishing a "condensation-separation" technology that significantly increases the enrichment amount of the target molecules. In some embodiments of the present application, the highly enriched target molecules are encapsulated and separated into minicells, and the concentration of the target molecules is increased by at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about 15 times, at least about 16 times, at least about 17 times, at least about 18 times, at least about 19 times, or at least about 20 times compared to the target molecules that have not been enriched and encapsulated. Advantages:

[0090] (1) Compared with the prior art, the present application creates a "condensation-separation" system based on cell division and molecular enrichment for the first time, which realizes the "packing" of biomolecules at the end of the cell and the division and encapsulation into minicells. This system effectively improves the accumulation of intracellular target molecules, reduces the difficulty of secretion of target molecules, and alleviates the problem of inhibition of normal bacterial growth and metabolism due to the accumulation of toxic molecules in the cell. This system not only improves the production efficiency of the target molecules, but even realizes the synchronization of the molecular condensation process and the cell division process. This strategy makes it possible to directionally enrich and divide and encapsulate target molecules into minicells, providing a novel approach for industrial fermentation to produce high-concentration compounds.

[0091] (2) The present application first provides a method for enhancing the enrichment of target molecules by molecular condensation strategy, thereby further promoting the production of target molecules. Secondly, the present application provides a method for producing target molecules by cell division, so that the target molecules are generated in microcells, thereby expanding the output of target molecules. Finally, the present application provides a method coupling the molecular condensation enrichment strategy and the cell division method, which provides a more efficient way for the production of target molecules. These innovative methods have significant application potential in solving the difficult problems in the production of target molecules. By controlling the formation of biological condensate state, the present application not only improves the enrichment degree of target molecules, but also further increases the accumulation amount by cell division. This technology provides beneficial technical support for the production of target molecules in the fields of biological technology and pharmaceutical manufacturing. At the same time, it provides an innovative solution to improve the production efficiency of target molecules, covering various production methods, and therefore has wide application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0092] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0093] FIG. 1 is a schematic diagram of the present application for constructing a strategy for improving the production efficiency of biological macromolecules.

[0094] FIG. 2 is a graph of the condensate formed after enrichment of EGFP based on liquid-liquid phase separation tag.

[0095] FIG. 3 is a graph of the optimization of liquid-liquid phase separation long peptide chain tag conditions. Wherein, A: protein fluorescence map of B-I16-EGFP strain under different inducer concentrations; B: protein fluorescence map of B-I16-EGFP strain under different induction times; C: protein fluorescence map of B-I16-EGFP placed at 4°C overnight after induction.

[0096] FIG. 4 is a graph of the optimization of liquid-liquid phase separation short peptide chain tag conditions. Wherein, A: the left two graphs are protein fluorescence maps of B-WT-20-EGFP strain at 10h and 22h after induction expression after adjusting the temperature to 15°C; the right one is a protein fluorescence map after changing the vector; B: mutation tag (Y1, Y2, Y4, Y24) forms EGFP condensate in E. coli cells.

[0097] FIG. 5 is a graph of the condensate formed after enrichment of EGFP based on different end positioning tags.

[0098] FIG. 6 is a fluorescence map of different end positioning tags (CLSM layer scanning map, each layer of picture is about 0.15mm apart).

[0099] Figure 7 is a graph of the formation of aggregates after enrichment of EGFP based on self-assembly of the tag. Wherein, A: schematic diagram of Y15-EGFP self-assembly; B: aggregate graph of Y15-EGFP complex in cells; C: SDS-PAGE electrophoresis graph of B-Y15-EGFP.

[0100] Figure 8 is a graph of the average gray value of the specific running program.

[0101] Figure 9 is a graph of the enrichment effect of proteins after fusion of various tags.

[0102] Figure 10 is a graph of the accumulation of proteins after fusion of various tags. Wherein, A: the average fluorescence intensity per unit biomass of EGFP protein after fusion of various tags is detected by a fluorescence analyzer; B: the average fluorescence intensity in 10,000 cells is detected and analyzed using a flow cytometer FITC-A channel.

[0103] Figure 11 is a schematic diagram of the recruitment tag construction. Wherein, A: V7-RIAD plasmid construction schematic diagram. RGGRGG-RIAD and Y145-RIAD are constructed by replacing the V7 tag position with RGGRGG and Y145 tag, respectively; B / C: similarly, RIAD-RGGRGG-RIAD and RIAD-Y145-RIAD, RGGRGG-RIAD-RIAD, and Y145-RIAD-RIAD are constructed.

[0104] Figure 12 is a fluorescence graph of EGFP protein recruitment based on RIAD-RIDD tag.

[0105] Figure 13 is a graph of the enrichment effect of EGFP protein based on different biological aggregation tag assembly forms. Wherein, A: under the conditions of TB medium, 22°C, 200 rpm, 12h, the cell concentration OD 600 ; B: average fluorescence intensity per unit biomass under different assembly modes; C: SDS-PAGE analysis of protein expression in different systems; D: protein enrichment effect characterization of different biological aggregation tag assembly forms.

[0106] Figure 14 is a schematic diagram of the construction strategy of the minicell-producing chassis strain in Escherichia coli.

[0107] Figure 15 is a physiological characterization of the minicell-forming system recombinant strain and the isolated minicells. Wherein, A: growth curve of the minicell-forming system recombinant strain; B: morphological diagram of minicells and parent cells produced by different minicell-forming recombinant strains; C: cell morphological diagram of B-EGFP and B-ΔC-EGFP after induction of EGFP expression at 22°C; D: size of minicells detected by a particle size analyzer; E: fluorescence intensity change graph of EGFP protein in minicells and bacterial cells.

[0108] Figure 16 is a diagram of the enrichment effect of EGFP protein in minicells produced by different ways. A: the average gray value intensity of EGFP protein in minicells; B: the left graph is the accumulation of EGFP protein induced by different recombinant strains in minicell-forming system, and the right graph is the accumulation of EGFP protein in minicells; C: the average fluorescence intensity per unit area in minicells.

[0109] Figure 17 is a diagram of cell division and encapsulation protein characterization. A: the average fluorescence intensity per unit biomass of cells detected by a fluorescence analyzer; B: the luminescence intensity of cells observed by a fluorescence inverted microscope under the same gain, the same exposure time and the same aperture conditions.

[0110] Figure 18 is a diagram of minicells produced by overexpression of fusion liquid-liquid phase separation tag MinE and MinC truncated body in E. coli.

[0111] Figure 19 is a diagram of specific end-division encapsulation of EGFP protein.

[0112] Figure 20 is a fluorescence diagram of EGFP protein cargo end-division encapsulation into minicells.

[0113] Figure 21 is a diagram of the average fluorescence intensity per unit biomass of minicells detected by a fluorescence analyzer.

[0114] Figure 22 is a diagram of xylose reductase and formate dehydrogenase applied in minicell-forming system. A: SDS-PAGE analysis of total protein expression of whole cell broken crude enzyme; B: the cell concentration of recombinant bacteria induced in LB medium, 0.2mM IPTG, 22℃, 200rpm for 24h; C: enzyme kinetics detected at 340nm by UV-visible spectrophotometer, and enzyme activity per unit biomass was calculated according to the formula.

[0115] Figure 23 is a diagram of minicell encapsulation of xylose reductase and formate dehydrogenase. A: the enzyme activity per unit biomass of teXR in different strains and systems under the conditions of coenzyme NADPH and NADH; B: the enzyme activity per unit biomass of FDH in different strains and systems.

[0116] Figure 24 is a diagram of SDS-PAGE analysis of adenylyl cyclase protein expression in minicell-forming system.

[0117] Figure 25 is a diagram of 1,3-butanediol applied in minicell-forming system. A: 1,3-butanediol production pathway; B: SDS-PAGE analysis of protein; C: 1,3-butanediol fermentation production in different chassis cells.

[0118] Figure 26 is the application of DivIVA and its truncated body in xylose reductase and formate dehydrogenase. Wherein, A: comparison of condensed enzyme activity and free teXR enzyme activity of DivIVA-teXR and Div(60)-teXR; B: SDS-PAGE analysis of proteins; C: enzyme activity after fusion of Div(60) tag to FDH.

[0119] Figure 27 is the packaging of xylose reductase into minicells based on Div(60) tag.

[0120] Figure 28 is the application of dsRNA in minicell-forming. Wherein, A: gel electrophoresis of dsRNA nucleic acid expressed by recombinant strains; B: dsRNA unit biomass concentration in recombinant strains.

[0121] Figure 29 is the packaging of dsRNA into minicells using the "condensation-separation" system. Wherein, A: CAG 40 and BcSAS1 gene three different fusion mode schematic diagram; B: gel electrophoresis of hairpin dsRNA successfully expressed in recombinant E. coli; C: dsRNA unit biomass yield in different strains; D: dsRNA yield per unit biomass after separation of encapsulated dsRNA condensed small cells. DETAILED DESCRIPTION

[0122] In the following examples, the experimental methods described are conventional methods unless otherwise specified; the reagents and materials described can be obtained from commercial sources unless otherwise specified.

[0123] The present application can be better understood according to the following examples, and Figure 1 is a schematic diagram of the strategy for improving the production efficiency of biological macromolecules. However, it is easy for those skilled in the art to understand that the content described in the examples is only used to illustrate the present application, and should not and will not limit the present application detailed in the claims.

[0124] In the following examples, the E. coli BL21(DE3) was purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd., the pETDuet-1 was purchased from Moli Plasmid Platform, the plasmids pET28a and pCWJ were preserved by the laboratory, and the recombinant gene fragments were all synthesized by Suzhou Jinyuzhi Technology and Biological Co., Ltd.

[0125] Example 1: Realize molecular condensation by liquid-liquid phase separation tag (LLPS) in cells.

[0126] The liquid-liquid phase separation (LLPS) tag of the embodiment includes I16, R32, V7, NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, ELP[KV7F-36], WT-20, I24, Y145, V5-N, V1-C, N1, HPB2-C, V2-C, tag2, tag1, FUS, TDP-43, LAF-1, ICP4, and the like, and the specific amino acid sequences of the tags are shown in SEQ ID NO. 1-22, and are shown in Table 1 in detail. Among them, I16, R32, and V7 are long-chain tags, and the rest are short-chain tags.

[0127] Table 1: Amino acid sequence table of liquid-liquid phase separation (LLPS) tag

[0128] First, the amino acid sequences of the LLPS tags are synthesized according to the codon optimization of Escherichia coli, and at the same time, the LLPS tags are connected with enhanced green fluorescent protein EGFP through a linker, i.e., a flexible peptide (GGS)4, to form LLPS tag fusion gene fragments (the synthesis process is completed by Suzhou Jinyizhi Technology Biotechnology Co., Ltd.). Then, each LLPS tag fusion gene fragment is inserted into the NcoI and SalI sites of the plasmid pETDuet-1 through enzyme digestion and ligation (the enzyme digestion and ligation systems are shown in Table 2 and Table 3, respectively), to obtain the corresponding tag fusion EGFP plasmid.

[0129] Among them, the construction method of the tag fusion EGFP plasmid is divided into two types: (1) tag-EGFP, i.e., the C-terminal of the tag is fused with the N-terminal of EGFP, and the tags used for C-terminal fusion include I16, R32, V7, NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, WT-20, Y145, and N1; (2) EGFP-tag, i.e., the N-terminal of the tag is fused with the C-terminal of EGFP, and the tags used for N-terminal fusion include ELP[KV7F-36], I24, V5-N, V1-C, HPB2-C, V2-C, tag1, and tag2.

[0130] Then, the obtained tag fusion EGFP plasmid is introduced into E. coli cells E. coli BL21 (DE3), so as to obtain different recombinant engineering strains capable of expressing LLPS tag fusion EGFP complex (all LLPS tag fusion gene fragments and the corresponding recombinant engineering strains are shown in Table 4).

[0131] Table 2 enzyme digestion system

[0132] Note: After mixing gently, centrifuge for 30 min at 37℃; the enzyme used in the application is from Takara.

[0133] Table 3 system for connecting plasmid vector and foreign fragment using T4 DNA ligase

[0134] Table 4 LLPS tag fusion gene fragments and corresponding recombinant engineering strains constructed

[0135] The single colonies of the recombinant engineering strains on the flat plate were picked into 50 mL centrifuge tubes containing 5 mL LB liquid medium (10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, containing 100 mg / L of ampicillin), and cultured overnight at 37℃ and 200 rpm. The next day, the activated bacterial liquid was inoculated into 250 mL conical flasks containing 100 mL of LB liquid medium at a 2% v / v inoculation amount for expansion culture, and when the OD 600 reached about 0.8, 0.2 mM IPTG inducer was added, and the protein expression was induced overnight at 22℃ and 200 rpm for 12 h. Figure 2 shows the fluorescence inverted microscope images of the liquid-liquid phase tag fusion proteins forming aggregates.

[0136] (1) Analysis and optimization of long-chain tag results

[0137] As can be seen from FIG. 2, under the condition of adding 0.2 mM IPTG inducer and culturing for 12 h at 22°C, the protein in the B-I16-EGFP strain did not undergo liquid-liquid phase separation to form droplets. Generally, the droplet formation of the LLPS tag can be controlled by adjusting the external environment, including the induction time and temperature. Therefore, the present application further optimizes the culture conditions of the I16 tag. First, the temperature for incubating the strain is adjusted from 22°C to 18°C, and the culture is induced at different inducer concentrations (0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM) for 12 h. However, it is found through fluorescence inverted microscope observation that as the inducer concentration increases, the fluorescent protein in B-ΔC-I16-EGFP does not condense to form condensates (as shown in FIG. 3A), indicating that lower temperature conditions still cannot promote the formation of condensates. At the same time, under the condition of 0.1 mM IPTG induction, prolonging the induction time also does not observe the condensation phenomenon (as shown in FIG. 3B). However, when the B-ΔC-I16-EGFP after induction is placed at 4°C and left overnight, fluorescent spots are observed at both ends of the cells under the fluorescence inverted microscope (as shown in FIG. 3C), indicating that lower temperature is conducive to promoting liquid-liquid phase separation. In addition, as can be seen from FIG. 2, the tags R32 and V7 can efficiently promote the liquid-liquid phase separation of EGFP protein at the ends of the cells to form droplets.

[0138] (2) Analysis and optimization of short-chain tag results

[0139] As can be seen from FIG. 2, the tags NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, N1, HPB2-C, V1-C, V2-C, V5-N, ELP[KV7F-36], I24, tag1 and tag2 do not form liquid-liquid phase separation in E. coli cells, and the tag Y145 can form droplets in E. coli cells.

[0140] WT-20 is an intrinsically disordered protein derived from the Drosophila melanogaster Rec-1 elastin, and studies have shown that it can form condensates in vitro and in vivo in E. coli cells, and can also form droplets after 10 h of low-temperature induction. However, under the condition of 22°C in the present application, the WT-20-EGFP fusion protein does not undergo liquid-liquid phase separation. Therefore, it is speculated that the high induction temperature leads to the failure of phase separation, and the culture temperature is adjusted to 15°C, but no condensates are observed (as shown in FIG. 4A). Therefore, it is speculated that the low expression level of the WT-20 tag inhibits the function of liquid-liquid phase separation. In order to improve the expression level of the WT-20 tag, the WT-20-EGFP fusion protein is replaced on the pET28a vector, and the results show that WT-20 successfully promotes the EGFP protein to form liquid-liquid phase separation condensates at one end of the cell (as shown in FIG. 4A).

[0141] In addition, the amino acid sequences of the Y145 tags are optimized and mutated, and a total of 24 mutant tags of Y1-Y24 of the Y145 tag are obtained (the amino acid sequences of the specific mutant tags are shown in Table 5), and the mutant tags of the amino acid sequences capable of liquid-liquid phase separation are successfully screened (Y1, Y2, Y4 and Y24), and the Y1, Y2, Y4, Y24 mutant tag fusion gene fragments and the corresponding recombinant engineering strains constructed are shown in Table 6. Figure 4B shows that these variants (Y1, Y2, Y4, Y24) form EGFP condensates in E. coli cells. It is found through analysis that the Y145 is still retained after being shortened from the original 122 AA to 66 AA and 72 AA, and the liquid-liquid phase separation function is still retained, wherein part of the amino acids of the original Y145 tag are mutated to serine (S), alanine (A), isoleucine (I), glycine (G), phenylalanine (F), aspartic acid (N), threonine (T), glutamine (Q), which is more conducive to the performance of liquid-liquid phase separation.

[0142] Table 5 Amino acid sequences of mutant tags of Y145 tags

[0143] Table 6 Y1, Y2, Y4, Y24 mutant tag fusion gene fragments and corresponding recombinant engineering strains constructed

[0144] In summary, compared with the free EGFP in the control group (B-EGFP-1), after screening, the tags I16, R32, WT-20, V7 and Y145 tags have obvious liquid-liquid phase separation behavior. The EGFP fused with the above tags can successfully realize the liquid-liquid phase separation function in E. coli cells, and the formation of LLPS can be promoted by adjusting the temperature, such as reducing the temperature from 22°C to 4°C, and by increasing the expression amount or optimizing the vector, such as replacing the pETDuet-1 vector with the pET28a vector. The effect is better after changing the conditions of I16 and WT-20 tags. However, too low temperature is not conducive to the growth of microbial cells, which will limit the application in industrial production, so the tag I16 will not be used in the subsequent research of the patent. Finally, R32, V7, Y145 and WT-20 four kinds of LLPS tags are screened out, which can perform liquid-liquid phase separation in BL21(DE3) cells and form stable liquid condensate structures. After fusing the LLPS tags, the EGFP protein can be promoted to form a condensed state at the end of the cell, which provides an effective strategy for the localization and enrichment of biological macromolecules at the end of the cell.

[0145] Example 2: Realization of protein site-specific condensation at the end of the cell by end-localization tag.

[0146] This embodiment investigates the effect of the end-localization tag DivIVA, Div(60), EpsM fused with EGFP gene to achieve enrichment of EGFP protein at the cell end. The host bacteria of the present application are the same as in Example 1, and the specific amino acid sequences of these tags are shown in Table 7.

[0147] Table 7 Amino acid sequence table of end-localization tags

[0148] First, the amino acid sequences of the end-localization tags are synthesized according to the codon optimization of E. coli, then the synthesized end-localization tags are fused with the EGFP gene sequence, and the specific connection mode is DivIVA-EGFP, Div(60)-EGFP, EGFP-Div(60), EGFP-EpsM, EpsM-EGFP. Among them, a flexible peptide Linker sequence (GGS)4 is inserted between the end-localization tag and the EGFP protein fusion, forming each end-localization tag fusion gene fragment (DivIVA-EGFP, EGFP-EpsM, EpsM-EGFP synthesized by Suzhou Jinyizhi Technology Biology Co., Ltd.). Then, the enzyme digestion and ligation method is used to insert it between the NcoI and SalI sites of the plasmid pETDuet-1, and finally the obtained end-localization tag recombinant plasmid is introduced into E. coli cells E. coli BL21(DE3), thereby obtaining different recombinant engineering strains that can express end-localization tag fusion EGFP complex.

[0149] The end positioning tag fusion gene fragments Div(60)-EGFP and EGFP-Div(60) are self-constructed. The specific construction process is as follows: (1) Div(60)-EGFP is obtained by PCR amplification of the Div(60) gene sequence fragment using DivIVA-EGFP plasmid as the template through primers Primer 1-F and Primer 1-R, and the remaining plasmid backbone is obtained by PCR amplification through primers Primer 2-F and Primer 2-R. Finally, the two fragments are connected into a circular plasmid by the one-step cloning kit of Beijing Quanshijin Biotechnology Co., Ltd., and are transformed into E. coli DH5α cloning host for expansion culture. Then, the plasmid is sent to Suzhou Jinyizhi Technology Biotechnology Co., Ltd. for sequencing verification. The verified plasmid is Div(60)-EGFP; (2) EGFP-Div(60) is obtained by PCR amplification of the Div(60) gene sequence using DivIVA-EGFP plasmid as the template through primers Primer 3-F and Primer 3-R. Then, the plasmid backbone containing the EGFP gene sequence is obtained by amplification of the EGFP-EpsM plasmid as the template through primers Primer 5-F and Primer 5-R, and the remaining plasmid backbone is obtained by amplification through primers Primer 4-F and Primer 4-R. Then, the fusion fragment EGFP-Div(60) and the remaining backbone are homologously recombined into a circular plasmid by PCR amplification of the fusion fragment EGFP-Div(60) and the EGFP gene sequence as the template through primers Primer 3-F and Primer 5-R. And the recombinant strain is transformed into E. coli DH5α cloning host for expansion culture. After verification, the extracted plasmid is EGFP-Div(60).

[0150] Div(60)-EGFP and EGFP-Div(60) are transformed into E. coli BL21(DE3) expression host to express the fusion protein, and the corresponding recombinant engineering strains are obtained. The primers used in the construction process are shown in Table 8.

[0151] Table 8 Primer sequences used in this example

[0152] All end positioning tag fusion gene fragments and the corresponding recombinant engineering strains are shown in Table 9. The culture conditions of the recombinant engineering strains are the same as those in Example 1.

[0153] Table 9 End positioning tag fusion gene fragments and corresponding recombinant engineering strains

[0154] Figure 5 shows that the end-localization tag fusion EGFP protein presents a condensed state at the cell end. Figure 6 shows the end-localization tag fluorescence map, the results show that (1) the formation of DivIVA-EGFP fluorescent protein condensates is found at both ends or in the middle of E. coli cells, and the CLSM layer scanning shows that the fluorescence of the middle layer cells of B-DivIVA-EGFP strain is wrapped around the cell membrane, which indicates that DivIVA is only anchored on the cell membrane and can keep the inside of the cell present a hollow state, which widens the space for the accumulation of target proteins. (2) Two different ways of fusing EGFP at the N- and C-termini of Div(60) were constructed, and different fusion modes have an important influence on the condensation and localization of EGFP protein. When EGFP is fused at the N-terminus of Div(60), the fusion protein does not condense at both ends of the cell, suggesting that the N-terminus of Div(60) is not suitable for fusion with EGFP protein, which may interfere with the membrane binding function, thereby causing the protein to fail to localize at the cell end. When EGFP is fused at the C-terminus of Div(60), Div(60) does not affect the cell morphology, and the Div(60)-EGFP fusion protein can localize at both ends of the cell. CLSM layer scanning found that Div(60)-EGFP condensates exist at both poles of the cell, further confirming that Div(60) also has a membrane binding function. In addition, two different ways of fusing EGFP at the N- and C-termini of EpsM were constructed, and when EGFP is fused at the N-terminus of EpsM, the protein condensate localizes at the cell end. However, when EGFP is fused at the C-terminus of EpsM, EGFP hardly emits light or EGFP protein in a few individual light-emitting cells is distributed freely in the whole cell, indicating that EpsM easily interferes with the function of the reporter protein EGFP.

[0155] In summary, compared with free EGFP, DivIVA, Div(60), and EpsM tag fusion EGFP proteins can form condensate structures at both ends of the cell. Therefore, the end-localization tags examined in this example include DivIVA, Div(60), and EpsM, all of which successfully exhibit end-localization function in the cell.

[0156] Example 3: Forming condensates of target proteins in the cell by self-assembly tags.

[0157] The self-assembly tag includes any one of Y15, L6KD, 18Awt, and a sequence with a homology of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%. In this example, the effect of the self-assembly tag Y15 fusion EGFP on the enrichment of EGFP protein at the cell end is investigated. The host bacteria of the present application are the same as in Example 1, and the specific sequences of the Y15, L6KD, and 18Awt tags are shown in Table 10.

[0158] Table 10 Amino acid sequence list of self-assembly tag

[0159] The amino acid sequence of Y15 tag was first synthesized according to the codon optimization of E. coli (synthesized by Suzhou Jinyuzhi Technology Biology Co., Ltd.), then the synthesized self-assembly tag was fused with EGFP, the self-assembly tag and EGFP fusion were connected by a flexible peptide linker sequence (GGS)4, forming a self-assembly tag fusion gene fragment Y15-EGFP (the above synthesis process was completed by Suzhou Jinyuzhi Technology Biology Co., Ltd.), then inserted into the Ncol and Sail sites of plasmid pETDuet-1 by enzyme digestion and ligation method, and then the obtained recombinant plasmid was introduced into E. coli cells E. coli BL21 (DE3), thereby obtaining a recombinant engineering strain capable of expressing self-assembly tag fusion EGFP complex. The self-assembly tag fusion gene fragment and the corresponding recombinant engineering strain are shown in Table 11. The culture conditions of the recombinant engineering strain are the same as those of Example 1.

[0160] Table 11 Self-assembly tag fusion gene fragment and corresponding recombinant engineering strain table

[0161] Figure 7A shows a schematic diagram of self-assembly peptides promoting the spontaneous assembly of macromolecules to form supramolecular aggregates. Figure 7B presents the aggregation state of Y15-EGFP complex in cells. The results show that after Y15 is fused with EGFP protein, it can promote the spontaneous assembly of proteins to form aggregates at both ends of the cell. To verify whether the Y15-EGFP aggregate presents a liquid state, SDS-PAGE analysis was performed on the supernatant and precipitate after ultrasonic disruption and centrifugation of B-Y15-EGFP strain cells. The research results are shown in Figure 7C, which shows that the target protein band can be observed in the precipitate, indicating that the Y15-EGFP complex is a solid aggregate.

[0162] Example 4: Evaluation of the enrichment effect of various end tags on proteins

[0163] (1) The average fluorescence intensity (represented by "average gray value") of the luminescence position in the cells was calculated and analyzed to evaluate the effect of the biological aggregation state tags (R32, V7, EpsM, DivIVA, Y145, Div(60), and Y15) screened in Examples 1-3 on the enrichment and accumulation of EGFP protein.

[0164] The recombinant engineering strains carrying different biological aggregation state tag fusion EGFP were inoculated into 100 mL LB medium, respectively, and cultured at 37°C, 200 rpm. When the OD600 of the E. coli fermentation broth reached 0.6-0.8, 1 mM IPTG was added to induce the expression of the fusion protein. After 4-6 hours of induction, the cells were collected by centrifugation at 6000 rpm for 5 minutes, and then resuspended in 1 mL of PBS buffer. The resuspended cells were subjected to ultrasonic disruption and centrifugation to obtain the supernatant and precipitate. 600When the OD600 reaches 0.6-0.8, 0.2 mM of isopropyl-beta-D-thiogalactoside (IPTG) is added to induce the expression of the relevant protein, and the culture is incubated at 22°C overnight. Subsequently, 200 μL of the overnight culture of E. coli fermentation broth is placed in a 96 light-proof well plate, and the average fluorescence intensity (represented by "average gray value") of the cells in the fermentation broth is detected by a fluorescence analyzer.

[0165] The average gray value is obtained by calculation, and the specific operation program is shown in FIG. 8. This program is used to calculate the average gray value (Average Gray Value) of the light-emitting cells in the fluorescence picture, and the program interface provided by the Open Source Computer Vision Library (OpenCV) is called by Python to achieve the expected function. In a gray image, the value 0 represents pure black, and the value 255 represents pure white. In the range of 0-255, the larger the value, the stronger the corresponding fluorescence intensity. In order to reduce the error in statistics, a gray threshold is set, and pixels below the threshold are not included in the statistics, at which time it is considered that the image at this position does not emit fluorescence at all. The running environment is: Python = = 3.7.6; OpenCV = = 4.5.5.

[0166] The results show that the R32, V7 and Y145 tags can form condensed states through liquid-liquid phase separation, and the fluorescence intensity of these condensed proteins is about 1.4 times, about 1.6 times and about 1.5 times that of the fluorescence intensity in the free state, respectively (as shown in FIG. 9). At the same time, the condensed proteins formed by the DivIVA, Div(60), EpsM end localization tags and self-assembling polypeptide Y15 screened in this embodiment are about 1.25 times, 1.5 times, 1.35 times and 1.33 times that of the fluorescence intensity in the free phase, respectively (as shown in FIG. 9). Therefore, these results show that different condensed tags can effectively realize the directional enrichment of protein molecules at the end of the cell, and promote the folding of protein structure from the free state to the condensed state.

[0167] (2) The content of EGFP protein enriched by different tags was investigated, and the average fluorescence intensity per unit biomass (OD 600 ) was analyzed to analyze the differences between various tags in the accumulation of EGFP protein content.

[0168] Specifically, for the LLPS tags, including R32, V7 and Y145, it was observed that they showed different effects on the accumulation of EGFP content. The results showed that the V7 and Y145 tags performed well, making the expression of EGFP protein higher, and the average fluorescence intensity per unit biomass was increased by about 1.75 and 1.9 times compared with the control strain B-EGFP-1, respectively. The R32 protein enrichment effect was second, which was increased by about 1.2 times compared with the control strain. For R32, the effect of increasing protein content was poor, which was speculated to be due to its larger protein molecular weight, which to some extent led to more consumption of cell resources during protein enrichment. Among the end-localization tags, the EpsM, DivIVA and Div(60) screened by the application showed different enrichment effects, and the accumulation effect of Div(60) protein was much higher than that of the other two, which was increased by about 4.3 times and about 12.5 times compared with DivIVA and EpsM, respectively. However, the effect of EpsM on protein accumulation was lower than that of the control strain B-EGFP-1, which indicated that EpsM might have an inhibitory effect on the expression of EGFP protein. The self-assembly tag Y15 also had a certain promoting effect on the expression ability of EGFP protein. FIG. 10A shows the difference in the accumulation ability of EGFP protein by different tags through the average fluorescence intensity per unit biomass (OD 600 ) of the control strain B-EGFP-1.

[0169] Subsequently, the required detection bacterial liquid sample cell concentration was diluted to about 1×10 6 cell / mL, and the flow cytometer control parameters were set to collect 10000 data points at the maximum particle collection rate of 2500 events / s, and the discard rate was maintained within 5%. The FITC-A channel was used to detect the EGFP protein mean fluorescence intensity (MFI). FIG. 10B shows the mean fluorescence intensity (MFI value) of EGFP protein at the single cell level, and the results show that the trend of the mean fluorescence intensity of EGFP protein is basically consistent with the average fluorescence intensity per unit biomass, which again confirms the molecular condensation ability of various tags.

[0170] In summary, some tags that can efficiently achieve the enrichment and accumulation of target proteins at the cell end are successfully screened from the LLPS tags, end localization tags and self-assembly tags, such as the LLPS tags R32, V7 and Y145, the end localization tags DivIVA, Div(60) and EpsM, and the self-assembly tag Y15. Among the three types of tags, except for EpsM, the rest of the tags can improve the yield of EGFP fluorescent protein. In particular, the V7, Y145 and Div(60) tags show certain advantages in the effect of enriching proteins and protein accumulation, which may be because these tags can promote the aggregation of proteins at the cell end, expand the cytoplasmic space, thereby freeing up more cell space for protein accumulation, while reducing the concentration of free proteins in the cell, which may ultimately stimulate the cell to produce proteins continuously. These tags provide an effective solution for protein enrichment in E. coli cells.

[0171] Example 5: Recruiting EGFP into the condensed state formed by the LLPS tag by recruiting tags in cells.

[0172] In order to realize efficient protein enrichment while reducing the length of the molecular condensed state tag, the target molecule gene can be recruited to the biological condensed state tag by recruiting tags to obtain a target molecule gene fragment. The recruiting tags include any one of RIAD and RIDD, P1 and P2, and P3 and P4. This embodiment investigates the effect of recruiting EGFP into the condensed state by fusing the short peptide tags (i.e. recruiting tags) RIAD and RIDD with the liquid-liquid phase separation tags (V7, RGGRGG or Y145) and EGFP protein, respectively. The amino acid sequences of all short peptide tags (recruiting tags) and the liquid-liquid phase separation tag RGGRGG are shown in Table 12. The host bacteria of the present application are the same as those in Example 1.

[0173] Table 12 Amino acid sequence table of short peptide tags (recruiting tags) and liquid-liquid phase separation tag RGGRGG

[0174] The RIDD short peptide is fused with EGFP through a flexible peptide Linker sequence (GGGGS)3 to form a recruiting tag fusion gene fragment EGFP-RIDD, and the RIAD short peptide is fused with the LLPS tag (V7, RGGRGG or Y145) through a flexible peptide Linker sequence (GGGGS)3, and three different connection modes of fusion gene fragments are set, such as LLPS-RIAD, RIAD-LLPS-RIAD or RIAD-RIAD-LLPS. Take the V7 tag in the LLPS tag as an example for description, and the construction modes of the remaining LLPS tags including RGGRGG and Y145 are consistent with those of the V7 tag.

[0175] Firstly, the EGFP-RIDD fusion gene fragment was inserted into the pETDuet-1 plasmid between the NcoI and SalI sites and between the NdeI and XhoI sites by enzyme ligation with the fusion gene fragments V7-RIAD, RIAD-V7-RIAD or RIAD-RIAD-V7, respectively; secondly, the T7 promoter upstream of the NdeI site was replaced with a weaker T7 promoter (sequence: ATATACGACTCACTATAGG) with an efficiency of only 83.7%; finally, the resulting recombinant plasmid was introduced into E. coli cells E. coli BL21 (DE3) to obtain a recombinant engineering strain that can express the recruitment tag fusion EGFP complex (Table 13). The culture conditions of the recombinant engineering strain were the same as in Example 1.

[0176] Table 13 Recombinant engineering strains expressing recruitment tag fusion EGFP complex

[0177] Figure 11 shows the V7 tag in the LLPS tag as an example, three different construction ways of the recruitment tag RIAD and the liquid-liquid phase separation tag LLPS, and the rest of the LLPS tags (RGGRGG, Y145) are constructed in the same way as the V7 tag. Figure 12 shows the formation of EGFP protein condensates under different recruitment conditions. The results show that after the liquid-liquid phase separation tag V7, RGGRGG or Y145 is fused with the recruitment tag RIAD-RIDD, EGFP protein can be effectively recruited into the condensed state, showing an eye-catching effect. Among them, the V7-RIAD fusion then recruits the EGFP-RIDD protein to have the best effect, and the three different construction ways of the V7 tag all show the best condensed state at the cell end. Overall, the short peptide tag RIAD and RIDD successfully realize the efficient recruitment of EGFP in the condensed state through the interaction between them.

[0178] Example 6: Comparison of EGFP protein enrichment effect by different assembly forms taking V7 tag as an example.

[0179] This example compares the effects of the assembly forms (B-V7-RIAD, B-RIAD-V7-RIAD, B-V7-RIAD-RIAD) of the V7 directly fused EGFP assembly in Example 1 and the V7 fusion short peptide tag recruitment EGFP protein into the condensed state in Example 5 on protein enrichment.

[0180] By analyzing the average fluorescence intensity of protein condensates in different assembly forms, it was found that the average fluorescence intensity of the unit biomass of V7-EGFP fusion protein was increased by about 1.5 times compared with the control strain (as shown in FIG. 13A). The results were consistent with the trend described in Example 4, which indicated that the V7 tag improved protein expression by forming condensates through liquid-liquid phase separation. In addition, B-V7-RIAD, B-RIAD-V7-RIAD and B-V7-RIAD-RIAD could express EGFP protein (as shown in FIG. 13A), but the average fluorescence intensity of the unit biomass of B-V7-RIAD, B-RIAD-V7-RIAD and B-V7-RIAD-RIAD was lower than that of the control strain by 18% to 43%. The more RIADs were connected in series on the V7 tag, the lower the fluorescence intensity of the EGFP protein (as shown in FIG. 13B). It was speculated that multiple RIADs in series would also affect the expression of EGFP.

[0181] To evaluate the protein expression at the single cell level under different assembly forms, flow cytometry was used to analyze the average fluorescence intensity (MFI value) at the single cell level. The results showed that the fluorescence intensity of EGFP protein in the condensate formed by V7 with one RIAD in series was basically consistent with that of the control strain (as shown in FIG. 13B). Therefore, compared with the assembly form with multiple RIADs in series, the V7 tag with one RIAD tag at the C-terminus had the least impact on protein expression, and it could successfully recruit proteins without affecting the expression of EGFP protein. In addition, by studying the SDS-PAGE gel electrophoresis diagram under different assembly forms, the size of the protein was analyzed. The protein gel diagram showed that the target protein band was correct, indicating that each assembly mode could successfully express the protein (as shown in FIG. 13C, red arrow). In FIG. 13D, EGFP protein condensates formed at one end in the cell could be observed, indicating that EGFP protein could be successfully recruited under various assembly modes, and various biological condensate tag assembly forms achieved the condensation of molecules at the end of the cell. In summary, a molecular recruitment method based on the interaction of short peptides RIAD-RIDD was established, which realized that molecular recruitment could replace gene fusion, successfully made EGFP protein enter the condensate formed by V7, and provided another innovative strategy for molecular condensation.

[0182] Example 7: Construction of a chassis strain for producing minicells by genetic modification.

[0183] Based on the development of new cell platform of physiological engineering, the gene inactivation of Min system and the regulation of cell division key gene ftsZ level in E. coli were carried out in the embodiment. E. coli BL21(DE3) was used as the target strain, and five different recombinant strains for producing minicells were constructed, and the construction process was defined as "minicell-forming" system. Fig. 14 is a schematic diagram of the construction of the minicell-producing chassis strain in the embodiment.

[0184] The specific molecular operation process is as follows:

[0185] 1. Construction of target strain E. coli BL21(DE3) containing pEcCas plasmid

[0186] The pEcCas plasmid (donated by Professor Yang Sheng of the Institute of Molecular Plant Science, Chinese Academy of Sciences) was heat-shocked and transformed into the competent cells of the target strain E. coli BL21(DE3), and after single colony picking and activation culture, the plasmid was extracted, PCR was performed using primers pEcCas-F and pEcCas-R, and the correct target strain containing the pEcCas plasmid was obtained through nucleic acid electrophoresis band verification.

[0187] 2. Construction of five kinds of recombinant strains for producing minicells

[0188] (1) Construction of the recombinant strain of minicells producing inactivated minC gene: The minC gene upstream homologous arm was amplified by PCR using primers ΔminC-1-F and ΔminC-1-R, and the minC gene downstream homologous arm was amplified by PCR using primers ΔminC-2-F and ΔminC-2-R, with the extracted E. coli BL21(DE3) genome as the template. Then, the Donor DNA fragment for knocking out the minC gene was constructed by PCR using the upstream and downstream homologous arms as the template and primers ΔminC-1-F and ΔminC-2-R. The linear recombinant plasmid pTarget F-1 was obtained by PCR amplification using the plasmid pTarget F (provided by Professor Yang Sheng of the Institute of Molecular Plant Science, Chinese Academy of Sciences) as the template and primers pTarget F-1-F and pTarget F-1-R. The linear recombinant plasmid pTarget F-1 was recovered after gel purification, and the original template was eliminated by Dpn I enzyme digestion. The correct pTarget F-1 plasmid containing the N20 sequence of the minC gene knockout was obtained by heat shock transformation into the cloning host E. coli Trans1-T1 (purchased from Beijing Full-Prime Biotechnology Co., Ltd.) and sequencing screening by Suzhou Jinweizhi Technology and Science Biotechnology Co., Ltd. The above-constructed Donor DNA and pTarget F-1 were simultaneously electroporated into the target strain containing the pEcCas plasmid at a concentration of 5:1 (100 ng / μL), and the electroporation instrument was set at an electric shock voltage of 2500 V and an electric shock time of 5-6 ms. After electric shock, LB liquid medium was added quickly, and the recovery was performed at 37℃ and 250 rpm for 2 h. After recovery, the culture was plated on LB solid medium containing streptomycin (40 mg / L) and kanamycin (50 mg / L), and incubated at 37℃ overnight. After activation, single colonies were selected and used for colony PCR amplification of the mutant fragment using primers ΔminC-Scr-F and ΔminC-Scr-R. The nucleic acid electrophoresis band was preliminarily verified, and the nucleic acid band size correct PCR product was further confirmed by sequencing by Suzhou Jinweizhi Technology and Science Biotechnology Co., Ltd., and the mutant recombinant strain B-ΔC was obtained.(2) Construction of a minicell-producing recombinant strain in which minCDE genes (i.e., minC, minD, and minE genes) are inactivated: Using the extracted E. coli BL21(DE3) genome as a template, primers ΔminCDE-1-F, ΔminCDE-1-R, ΔminCDE-2-F, and ΔminCDE-2-R were used to obtain Donor DNA in which minCDE was inactivated by the method of (1), and pTarget F-1 and the Donor DNA were simultaneously electroporated into a target strain containing a pEcCas plasmid, and primers ΔminCDE-Scr-F and ΔminCDE-Scr-R were used to obtain a mutant recombinant strain B-ΔCDE by the same screening method.

[0189] (3) Construction of a minicell-producing recombinant strain in which minC genes are inactivated and an FtsZ expression cassette is integrated: Using the extracted E. coli BL21(DE3) genome as a template, primers ΔC::ftsZ-1-F, ΔC::ftsZ-1-R, ΔC::ftsZ-4-F, and ΔC::ftsZ-4-R were used to obtain the upper and lower homologous arms of the minC gene by PCR amplification. Using the pBAD33 plasmid as a template, primers ΔC::ftsZ-2-F and ΔC::ftsZ-2-R were used to obtain an inducible T7 promoter sequence including a lacI protein by PCR amplification. Using the extracted E. coli BL21(DE3) genome as a template, primers ΔC::ftsZ-3-F and ΔC::ftsZ-3-R were used to obtain an FtsZ gene by PCR amplification. Using the T7 promoter sequence and the FtsZ gene sequence as templates, primers ΔC::ftsZ-2-F and ΔC::ftsZ-3-R were used to obtain an FtsZ protein expression cassette by PCR amplification. Using the upper and lower homologous arms and the FtsZ protein expression cassette as templates, primers ΔC::ftsZ-1-F and ΔC::ftsZ-4-R were used to obtain Donor DNA in which the minC gene was inactivated and the FtsZ protein expression cassette was integrated. pTarget F-1 and the Donor DNA were simultaneously electroporated into a target strain containing a pEcCas plasmid, and primers ΔminC-Scr-F and ΔminC-Scr-R were used to obtain a mutant recombinant strain B-ΔC::ftsZ by the same screening method.

[0190] (4) Construction of the recombinant strain producing minicells with inactivated BAD gene and integrated ftsZ expression cassette: The upstream and downstream homologous arms of the inactivated BAD gene were obtained by PCR amplification using primers ΔBAD::ftsZ-1-F, ΔBAD::ftsZ-1-R, ΔBAD::ftsZ-3-F and ΔBAD::ftsZ-3-R with the extracted E. coli BL21(DE3) genome as the template. The ftsZ gene sequence was obtained by PCR amplification using primers ΔBAD::ftsZ-2-F and ΔBAD::ftsZ-2-R. The Donor DNA sequence of the inactivated BAD gene and integrated ftsZ gene was obtained by PCR amplification using primers ΔBAD::ftsZ-1-F and ΔBAD::ftsZ-3-R with the upstream and downstream homologous arms and the ftsZ gene sequence as the template. The recombinant plasmid pTarget F-2 was obtained by PCR amplification using primers pTarget F-2-F and pTarget F-2-R with plasmid pTarget F as the template, and then recovered by gel purification. After eliminating the original template by Dpn I enzyme digestion, the recombinant plasmid pTarget F-2 was introduced into the cloning host E. coli Trans1-T1 (purchased from Beijing Joinnoble Biotech Co., Ltd.) by heat shock transformation, and then grown in a 37°C incubator for one night on LB solid medium containing streptomycin (40 mg / L). The next day, single colonies were picked for activation culture, and finally sent to Suzhou Joinnoble Biotech Co., Ltd. for sequencing screening to obtain the correct recombinant plasmid pTarget F-2 containing the N20 sequence with the knocked-out BAD gene. The successfully constructed Donor DNA and pTarget F-2 plasmid were simultaneously electroporated into the target strain E. coli BL21(DE3) containing pEcCas plasmid and the B-ΔC strain containing pEcCas plasmid, and then the mutant recombinant strains B-ΔBAD::ftsZ and B-ΔC-ΔBAD::ftsZ were obtained by colony PCR screening using primers ΔBAD::ftsZ-Scr-F and ΔBAD::ftsZ-Scr-R.

[0191] The original strain E. coli BL21(DE3) and the successfully mutated recombinant strains were prepared into electrotransformation competent cells, and then the pET28a-EGFP plasmid (Mingling plasmid platform) was electroporated into the corresponding recombinant strains to finally obtain six recombinant strains B-EGFP, B-ΔC-EGFP, B-ΔCDE-EGFP, B-ΔBAD::ftsZ-EGFP, B-ΔC-ΔBAD::ftsZ-EGFP and B-ΔC::ftsZ-EGFP.

[0192] The extraction of the E. coli BL21 (DE3) genome was performed according to the instructions of TaKaRa MiniBEST Bacterial Genomic DNA Extraction kit Ver. 3.0. The extraction of the plasmid was performed according to the instructions of AxyPrep Plasmid Miniprep Kit.

[0193] The primers used for the PCR to obtain the target gene fragments and the ligation fragments were designed using Snapgene software and synthesized by Beijing Genesee Biotech Co., Ltd. The reaction system for PCR amplification of the target gene fragments was 50 μL, ddH2O 9 μL, F primer 1.5 μL, R primer 1.5 μL, KOD enzyme 1 μL, 2 × KOD Buffer 25 μL, dNTPs 10 μL, and genomic template 2 μL. The PCR program was 96 °C pre-denaturation for 5-10 min, 98 °C denaturation for 30 sec, 60 °C annealing for 30 sec, 72 °C elongation at 1 kb / min, 35 cycles, and 72 °C sufficient elongation for 10 min. The product was stored in a refrigerator at 4 °C for standby.

[0194] The PCR product was recovered according to the instructions of TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0. The purified DNA fragments were connected by in vitro homologous recombination by PCR.

[0195] The Dpn I enzyme digestion system was 50 μL, Dpn I enzyme 1 μL, 10 × Buffer 5 μL, DNA fragments ≤1 μg, and ddH2O up to 50 μL. The digestion system was mixed gently, centrifuged momentarily, placed in a 37 °C water bath, and reacted for 2 h. After the DNA fragments were digested, the enzyme activity was inactivated at 90 °C for 10 min.

[0196] The amino acid sequences of the minC gene, minD gene, minE gene, rhaB gene, rhaA gene, rhaD gene, and ftsZ gene are shown in SEQ ID NOs. 60-66.

[0197] The primers used in this example are shown in Table 14.

[0198] Table 14 Sequences of the primers used in this example

[0199] The cell concentration OD 600To compare and evaluate their growth ability, the results of FIG. 15A show that the growth ability of the recombinant strains of the constructed "minicell-forming" system is consistent with the control strain BL21(DE3), indicating that the genetic modification does not affect the growth concentration. The production of minicells was detected by fluorescence inverted microscope, and FIG. 15B / C shows the production of minicells, and the results show that the above five recombinant strains constructed by genetic modification successfully realized the production of minicells from the parent E. coli, and the cell morphology is spherical. The size of minicells was detected by particle size analyzer, and FIG. 15D shows that the average particle size of minicells after purification is about 650 nm. In addition, in order to verify the stability of the protein in minicells, FIG. 15E evaluates the change of fluorescence intensity of EGFP protein in minicells and bacterial cells within 5 days, and the results show that the average fluorescence intensity of EGFP protein per unit biomass in minicells decreased by 3.797% on the 5th day, while the fluorescence intensity of EGFP in bacterial cells decreased by 56.439% under the same storage conditions. This shows that the stability of the protein in minicells is significantly better than that in bacterial cells.

[0200] In summary, the present embodiment demonstrates that the constructed minicell-forming chassis cells can produce spherical minicells, and this process does not adversely affect the production ability of normal cells. Minicells not only retain metabolic activity, but also have better stability in maintaining EGFP protein fluorescence.

[0201] Example 8: Production of minicells by different strains and their enrichment ability for EGFP protein.

[0202] The present example takes EGFP as a reporter protein to investigate the enrichment and accumulation of EGFP protein in minicells produced by different strains. By analyzing the average gray value of EGFP protein in minicells, it is found that the enrichment capacity of EGFP protein in minicells is increased by about 1.3 times compared with parent cells (as shown in FIG. 16A). Subsequently, the minicell production and protein production capacity of the five recombinant strains in Example 7 were compared. The induction conditions of the five recombinant strains are shown in Table 15. The results are shown in FIG. 16B and FIG. 16C. FIG. 16B shows the accumulation of EGFP protein induced by different recombinant strains of the minicell-forming system, and FIG. 16B shows the accumulation of EGFP protein in minicells. The results show that the total amount of protein expression in the minicell-forming system is increased by 1.1-1.3 times compared with the control strain, and the protein enrichment effect of the minicell population is increased by 1.5-2.8 times. The average fluorescence intensity per unit area in minicells was detected by flow cytometry. The results are shown in FIG. 16C, and the average fluorescence intensity per unit area in minicells is increased by 5-11 times compared with the control strain. Among them, the B-ΔC-EGFP recombinant strain has a slightly better ability to produce EGFP protein than other strains. The above results show that the minicells produced by the recombinant strains of the minicell-forming system constructed by the present application have a certain protein enrichment and accumulation capacity.

[0203] Table 15 Induction conditions of five recombinant strains

[0204] Example 9 Construction of "condensation-separation" system

[0205] In this example, the molecular condensation strategy and the method of producing minicells by cell division are combined to investigate the application effect of the biological condensate tag V7 fused reporter protein EGFP in minicell-forming, thereby constructing a "condensation-separation" system. The specific process is as follows:

[0206] The gene fragments EGFP and V7-EGFP were inserted into the Ncol and Sail sites of plasmid pETDuet-1 by enzyme digestion and ligation method, respectively, to obtain the corresponding fusion EGFP plasmid, and then the obtained fusion EGFP plasmid was introduced into the host E. coli BL21 (DE3) and B-ΔC, respectively, to finally obtain four different recombinant strains. The construction of the four recombinant strains is shown in Table 16.

[0207] Table 16 Construction of four recombinant strains in the present example

[0208] The four recombinant strains were cultured under induction conditions of 22℃, 200rpm, 24h, and the enrichment and aggregation state of EGFP protein in different systems were observed. The results are shown in Figure 17A. Compared with the control strain B-EGFP-1, the fusion of V7 tag with EGFP applied to the minicell-forming recombinant strain (B-ΔC-V7-EGFP) can promote the enrichment of intracellular EGFP protein to about 2.5 times. In addition, the minicell population (minicell-V7-EGFP) encapsulating V7-EGFP aggregation protein isolated from the strain can increase the EGFP protein enrichment capacity, i.e., the average fluorescence intensity per unit biomass, to about 15 times that of the control strain B-EGFP-1, and about 1.5 times that of the minicell population (minicell-EGFP-1) encapsulating free EGFP protein. Figure 17B shows the cell luminescence intensity observation results under fluorescence inverted microscope. The above results show that the protein molecular "condensation-separation" system is successfully constructed by combining the molecular condensation strategy with the minicell-forming system.

[0209] Example 10: Specific end split encapsulation of EGFP protein

[0210] To improve the loading rate of target proteins in minicells, another minicell-forming system production mode was constructed by regulating the expression of MinC or MinE protein in the Min system to achieve cutting at the specific cell end of E. coli. Taking the LLPS tag (V7) as an example, the LLPS tag (V7) was first inserted into the EcoR I and Hind III sites of the plasmid pCWJ by enzyme ligation (for specific ligation methods, see Table 2 and Table 3, except that the enzyme type was changed, and the corresponding linear fragment and vector were changed). Then it was introduced into E. coli BL21 (DE3). The MinC or its truncated body contains C-terminal sequence 116-231 (C-terminal sequence 116 amino acids), 122-231 (C-terminal sequence 110 amino acids), and 122-231 (the 172nd residue R is replaced by A, C-terminal sequence 110 amino acids). The MinE or its truncated body contains N-terminal sequence 1-22 (N-terminal sequence 22 amino acids), 1-33 (N-terminal sequence 33 amino acids), 1-53 (N-terminal sequence 53 amino acids), 1-62 (N-terminal sequence 62 amino acids), and 1-67 (N-terminal sequence 67 amino acids).

[0211] Figure 18 shows the overexpression of MinE 1-33 and MinC122-231 Fusion of truncations with LLPS tag (V7) and overexpression of MinE 1-33 and MinC 122-231 Fusion of truncations with LLPS tag (V7) can induce minicell production (indicated by circles in Fig. 18), and the observation of the rest of truncations is similar to Fig. 18. It is illustrated that overexpression of C-terminal of MinC and N-terminal of MinE in E. coli cells can both promote the establishment of another minicell-forming system.

[0212] MinC or its truncations, MinE or its truncations, and LLPS tag (V7) and EGFP are connected in the following ways, including MinC n -LLPS-EGFP or MinE n -LLPS-EGFP (n represents different number of amino acids).

[0213] In order to be able to encapsulate the above-mentioned minicells to the target protein, the reporter gene egfp of EGFP protein is further fused on the basis of the above-mentioned recombinant plasmid pCWJ (the fusion gene piece is synthesized by Suzhou Jinyuzhi Technology and Biology Co., Ltd.), that is, the liquid-liquid phase separation tag (V7), MinC 122-231 or MinE 1-33 truncations and the reporter gene egfp of EGFP protein are inserted into the EcoR I and Hind III sites of plasmid pCWJ by enzyme connection (for specific connection methods, please refer to Table 2 and Table 3, the difference is to change the enzyme type, and the corresponding linear fragment and vector), and then introduced into the host to construct different recombinant strains, so as to accurately regulate the minicell division site in the cell and achieve the segmentation at the end of the target protein aggregation. Fig. 19 shows a schematic diagram of parent cell specific end division encapsulating EGFP protein. Fig. 20 shows the fluorescence pictures of the recombinant strains and the corresponding minicell groups obtained by separation encapsulating EGFP protein in specific end division, involving red cell membrane stained by membrane-specific dye FM4-64 (2 μg / mL) for 1 h. The results show that overexpression of MinE 1-33 and MinC 122-231 protein and liquid-liquid phase separation tag (V7) fusion in E. coli achieves segmentation at the end of EGFP condensation and encapsulation in minicells, and the divided minicells are all loaded with cargos.

[0214] To determine the EGFP protein loading in minicells, Figure 21 shows the average fluorescence intensity of minicell unit biomass detected by a fluorescence analyzer, comparing the loading of EGFP protein under different minicell production methods. The results show that compared with the control minicell-V7-EGFP-2, overexpression of MinC or MinE and its truncated form in E. coli can effectively improve the accumulation of target protein EGFP, and the average fluorescence intensity per unit biomass is increased by 1.3 times and 2.8 times, respectively, compared with the control.

[0215] In summary, this embodiment successfully constructed a method for producing minicells by splitting at one end of the target protein condensate after fusing MinE 1-33 and MinC 122-231 protein with LLPS tag. At the same time, EGFP was used as a target protein to verify that this method can effectively split at the EGFP end and produce minicells encapsulating EGFP, further improving the production of EGFP protein in minicells. The synchronization of molecular condensation and cell division processes was initially achieved.

[0216] The construction of recombinant strains in this embodiment is shown in Table 17.

[0217] Table 17 Construction of recombinant strains in this embodiment

[0218] Example 11: Efficient production of xylose reductase and formate dehydrogenase in minicell-forming system

[0219] To study the practical application effect of minicell-forming system in enzyme production, this embodiment preliminarily investigated the production of xylose reductase (Xylose Reductase, teXR) and formate dehydrogenase (Formate Dehydrogenase, FDH) in the system.

[0220] First, the teXR and FDH genes were inserted between the EcoR I and Hind III sites of the plasmid pET28a by enzyme ligation (see Table 2 and Table 3 for the specific construction system). Then, the recombinant plasmids pET28a-teXR and pET28a-FDH were introduced into the minicell-forming chassis strain by chemical transformation method. Table 18 shows the construction of four recombinant strains in this embodiment.

[0221] The results are shown in Figure 22. SDS-PAGE analysis proved that teXR and FDH were successfully expressed (Figure 22A). The minicell-forming chassis cell concentration was slightly lower than the control strain after 24 h of culture in LB medium at low temperature 22℃ (Figure 22B). The whole cell lysate catalytic results showed that the enzyme activity of recombinant strain B-ΔC-teXR was about 1.8 times higher than that of the control strain B-teXR, and the enzyme activity of recombinant strain B-ΔC-FDH was also about 1.5 times higher than that of the control strain B-FDH (Figure 22C). Therefore, the minicell-forming system can promote the increase of enzyme activity in the production of enzyme macromolecular products, thereby establishing an efficient enzyme production system.

[0222] wherein the enzyme activity of teXR and FDH was detected at 340 nm using a UV-visible spectrophotometer. The catalytic system of teXR and FDH was reacted in 100 mM Tris-HCl buffer at pH 7.4, with 3 M xylose and 1 M sodium formate as substrates, and an appropriate amount of enzyme solution was added. One enzyme activity unit (U) was represented by the consumption or generation of NAD(P)H within 1 min. The calculation formula of teXR and FDH enzyme activity is as follows:

[0223] wherein ΔA: change in absorbance within 1 min; V: reaction liquid volume mL; 6220: molar absorption coefficient of NAD(P)H at 340 nm L·mol -1 ·cm -1 ; L: optical path distance 1 cm.

[0224] Table 18 Construction of four recombinant strains in this example

[0225] Example 12: Minicell encapsulation of xylose reductase and formate dehydrogenase

[0226] To verify the effect of small cell population on enzyme activity, this embodiment continues to investigate the enzyme activity encapsulated in small cells based on Example 11. The "small cell population" refers to the small cells obtained by separation of the minicell-forming system. Here, TB (peptone 12 g / L, yeast powder 24 g / L, disodium hydrogen phosphate 9.4 g / L, potassium dihydrogen phosphate 2.2 g / L, glycerol 4 mL / L) medium is selected. The results show that the teXR enzyme activity in the small cell population under the condition of NADPH cofactor is increased by about 3.6 times compared with the control strain, and the teXR enzyme activity of the minicell-forming system can be increased by about 2.8 times (as shown in Figure 23A), and it can be seen that the enzyme activity in the small cell is higher. Although the teXR enzyme activity under the condition of NADH cofactor decreases, the enzyme activity in the small cell is still the highest. In addition, for another enzyme FDH, the enzyme activity per unit biomass in the small cell is increased by about 4.3 times compared with the control strain, and the FDH enzyme activity of the minicell-forming system is increased by about 2 times (as shown in Figure 23B). Therefore, the small cells produced by cell division have advantages in enzyme production.

[0227] Example 13: Production of adenylyl cyclase in the minicell-forming system.

[0228] This embodiment investigates the application effect of adenylyl cyclase in the minicell-forming mixed system. To further evaluate the application effect of other types of enzymes in the minicell-forming system, this embodiment introduces the relatively less stable phosphatase adenylyl cyclase (AC) into the minicell-forming chassis strain for expression. As a key enzyme for synthesizing cyclic adenosine monophosphate (cAMP), adenylyl cyclase mainly functions to catalyze ATP to form cAMP, and is an important component of the cyclic adenosine monophosphate second messenger system. Table 19 shows the construction of two recombinant strains in this embodiment.

[0229] The experimental results show that the cAMP production of the recombinant strain B-ΔC-AC can reach 0.103 U / mL within 8 h by using ATP as the substrate and catalyzing by AC. Therefore, the minicell-forming system is suitable for the production of adenylyl cyclase and produces certain enzyme activity. The SDS-PAGE analysis of protein expression shows the successful expression of AC protein, and the protein molecular weight is about 42 kDa (as shown in Figure 24). In summary, the minicell-forming system also meets the production of adenylyl cyclase with poor stability, and has certain universality.

[0230] The catalytic reaction condition of adenylate cyclase (AC) is as follows: 30 mM ATP as substrate, 50 mM MgCl2and enzyme solution (total system 1 mL) are added in 100 mM Tris-HCl buffer with pH 8.0, reaction at 30°C for 15 min, then boiling in water for 5 min to terminate the reaction, centrifugal collection of supernatant through 0.22 μm water filter membrane, and HPLC detection of cAMP content. Enzyme activity is defined as follows: the amount of enzyme required for generating 1 μmol cAMP per minute at 30°C is defined as one enzyme activity unit (U). Agilent 1200 high performance liquid system is used in this example to analyze cAMP product content. The chromatographic column is Agilent reverse C18 [4.6 x 250 mm, 5 μm]. The mobile phase is prepared by V(methanol):V(pH 6.6 triethylamine phosphate solution) = 25:75, and the column oven temperature is controlled at 25°C. The UV detector wavelength is set at 254 nm, and the flow rate is finally stabilized at 0.8 mL / min. The standard sample concentration is 0.1 g / L.

[0231] Table 19 Construction of two recombinant strains in this example.

[0232] Example 14: Production of 1,3-butanediol in minicell-forming system

[0233] In this embodiment, E. coli BL21(DE3) was used as the chassis cell, and genetically engineered to produce 1,3-butanediol from glucose. In this embodiment, a metabolic pathway for the production of 1,3-butanediol was successfully constructed (Figure 25A), which included the key enzymes phaA (encoding acetyl-CoA acetyltransferase) from Ralstonia eutropha NBRC 102504, thil (encoding 3-ketoacyl-thiolase) from Clostridium beijerinckii, phaB (encoding NAD(P)H-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha NBRC 102504, bld (encoding aldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum ATCC 27012, and adh (encoding alcohol dehydrogenase) expressed endogenously, wherein phaA and thil belong to the same isozyme. Figure 25B shows that the four key enzymes were successfully expressed in E. coli, and the solubility of the enzymes was good. The plasmids involved in this pathway were synthesized by Suzhou Jinyizhi Technology Biotechnology Co., Ltd. The recombinant plasmids included pCDFDuet1-phaA-phaB-bld and pCDFDuet-thil-phaB-bld, wherein the fusion fragments were inserted between the Sal I and Not I sites of the vector pCDFDuet-1, and the RBS was used for connection; the bld fragment was inserted between the Nde I and Xho I sites. Table 20 shows the construction of the two recombinant strains in this embodiment.

[0234] The recombinant plasmids carrying the key enzymes were introduced into the E. coli BL21(DE3) chassis strain and the minicell-forming chassis strain B-ΔC, respectively, and the production capacity of 1,3-butanediol in the minicell-forming system was tested by fermentation. The results showed that the 1,3-butanediol production of the B-phaA-phaB-bld mutant strain was higher than that of the B-thil-phaB-bld mutant strain, and the difference was greater in the minicell-forming system (Figure 25C), which indicated that phaA and thil had different catalytic abilities although they belonged to the same isozyme. In addition, the 1,3-butanediol production in the minicell-forming system at the level of shake flask fermentation was increased by about 1.3 times compared with the control strain, and therefore the minicell-forming system had better advantages in the production of small molecule chemicals such as 1,3-butanediol, and showed higher performance in biochemical production. It can be seen that the production of minicells by cell division not only improves the production performance of the chassis, but also enhances the tolerance of the cells to toxic products.

[0235] In this embodiment, Agilent 1260 Infinity II high pressure liquid chromatography with differential refractive index detector (RID) was used to separate and quantitatively determine the 1,3-butanediol yield in the supernatant of the fermentation broth. The equipped chromatographic column was BIO-RAD Aminex HPX-87H anion exchange column, the mobile phase was 5 mM dilute sulfuric acid, the flow rate was 0.6 mL / min, and the column oven was set to 60°C. The sample to be tested was centrifuged at 13000 rpm for 5 min and the supernatant was collected. The supernatant was filtered through a 0.22 μm membrane to remove impurities and used for analysis. The standard concentration was 0.5 g / L.

[0236] Table 20 Construction of four recombinant strains in this embodiment

[0237] Example 15: Effect of molecular condensation strategy on enzyme activity.

[0238] In this embodiment, the effect of DivIVA and Div(60) tag fusion on enzyme activity was investigated. Div(60) is a truncated peptide of the N-terminal first 60 amino acids of DivIVA. Since it is less synthesized in cells and also relatively less consumes cell resources, this embodiment explores whether it is helpful for enzyme accumulation. After teXR is fused to DivIVA protein, although DivIVA-teXR condensate remains in cell precipitate, it still has enzyme activity (as shown in FIG. 26A / B). Under the condition of coenzyme NADPH, the condensate enzyme activity of DivIVA-teXR and Div(60)-teXR is about 1.1 times higher than that of free teXR (as shown in FIG. 26A). Therefore, after teXR is fused to DivIVA and Div(60), it can be localized at the end of the cell, and at the same time promote the increase of teXR enzyme activity. In addition, after FDH is fused to Div(60) tag, the enzyme activity of recombinant strain B-Div(60)-FDH is basically consistent with that of free FDH, indicating that the biological condensation tag does not produce negative effect on enzyme activity (FIG. 26C). It can be seen that DivIVA and its truncated body do not produce negative effect in the process of enzyme molecular condensation, and even have a certain promoting effect on the increase of enzyme activity. Among them, the construction of five recombinant strains in this embodiment is shown in Table 21.

[0239] Table 21 Construction of five recombinant strains in this embodiment

[0240] Example 16: Production of xylose reductase in the "condensation-separation" system.

[0241] This embodiment uses the combination of the end-positioning tag Div(60) and the minicell-forming system to produce xylose reductase as an example to investigate the actual production effect of the "aggregation-dissociation" system on the enzyme. The construction details of the four recombinant strains in this embodiment are shown in Table 22.

[0242] As shown in Figure 27, the enzyme activity of the recombinant strain B-ΔC-Div(60)-teXR was increased by about 2.6 times compared with the control strain B-teXR. This result supports the superiority of the method of generating small cells by cell division and encapsulating enzyme molecules into aggregates in the efficient production of enzymes, and confirms that the "aggregation-dissociation" system has successfully achieved efficient enzyme production.

[0243] Table 22 Construction details of the four recombinant strains in this example.

[0244] Example 17: Production of dsRNA in a “condensation-dissociation” system.

[0245] This embodiment investigates the production and application of dsRNA in a "condensation-dissociation" system. Currently, gray mold disease in plants caused by fungal pathogens mainly relies on chemical fungicides, but the frequent use of these chemical pesticides leads to ecotoxicological hazards of fungal pathogens, human health risks, and the development of drug resistance. This study selected to silence the BcSAS1 gene, a force-related gene of gray mold virus, which reduces hyphal development and spore formation. Normally, dsRNA produced in common Escherichia coli is easily degraded by intracellular RNase III; therefore, the HT115(DE3) RNase III-deficient strain is often used for large-scale dsRNA production. To address the RNase III enzyme degradation problem, a minicell-forming system was used. Furthermore, to better encapsulate dsRNA into small cells, this embodiment introduces the LLPS tag CAG. 40 The sequence of the tag is shown in SEQ ID NO.67 (SEQ ID NO.67: CAGCAGC ...

[0246] Firstly, the application effect of dsRNA in minicell-forming system was investigated. Figure 28A shows that the dsRNA nucleic acid electrophoretic bands expressed by H-L4440-BcSAS1, B-Δr-L4440-BcSAS1 and B-Δr-ΔC-L4440-BcSAS1 recombinant strains are all located at about 250 bp, indicating that the three RNase III-deficient chassis strains successfully transcribed the target gene BcSAS1. Then, the production of different chassis dsRNA was tested by 250 mL shake flask fermentation, and the mass concentration of extracted and purified dsRNA was detected by ultramicro spectrophotometer. The results are shown in Figure 28B. The unit OD 600 concentration of dsRNA in minicell-forming system (B-Δr-ΔC-L4440-BcSAS1) reached 8.25 μg / mL, which was 1.1-1.3 times higher than that of the control strains H-L4440-BcSAS1 and B-Δr-L4440-BcSAS1, indicating that the minicell-forming system could indeed effectively improve the accumulation of dsRNA. However, it should be noted that the unit OD 600 of free dsRNA packaged in minicells (minicell-BcSAS1) even reached 9.27 μg / mL, indicating that minicells further improved the production of dsRNA. This finding highlights the potential of the method of producing minicells based on cell division in dsRNA production, which can effectively increase the production of dsRNA and provide new methods for related applications.

[0247] Subsequently, in order to further improve the production of dsRNA, the "condensation-separation" system was adopted in this embodiment. Specifically, dsRNA was packaged at the end of the cell and encapsulated into minicells. Therefore, according to the LLPS tag CAG 40 and different fusion modes of BcSAS1 gene, three different fusion strategies were set to produce hairpin dsRNA (hpRNA) (as shown in Figure 29A) (the recombinant plasmid was synthesized by Suzhou Jinyuzhi Technology Biotechnology Co., Ltd. according to different sequences designed by dsRNA). In this embodiment, the hairpin dsRNA constructed by Suzhou Jinyuzhi Technology Biotechnology Co., Ltd. was successfully verified, and the nucleic acid band of about 250 bp was seen in the gel nucleic acid electrophoresis map, indicating that the hairpin dsRNA was successfully expressed in the recombinant Escherichia coli (as shown in Figure 29B). At the same time, in order to verify the effectiveness of the RNA extraction method, the strain containing the empty plasmid pET28a was subjected to dsRNA purification experiment, and the results showed that no target band was seen, indicating that the double-stranded nucleic acid purification method was feasible (as shown in Figure 29B). It was also found in this embodiment that when CAG 40The dsRNA production per OD of the minicell-forming system when fused in the middle of the BcSAS1 gene 600 The yield reached a maximum of about 25 μg / mL (as shown in FIG. 29C), which was about 1.4 times higher than the control strain B-Δr-BcSAS1-BcSAS1. However, when the small cells encapsulating the condensed state of dsRNA were separated, the dsRNA production per OD 600 was further improved, and the maximum could reach about 30 μg / mL, which was about 5 times higher than the non-fusion LLPS control group (as shown in FIG. 29D).

[0248] Therefore, based on the results observed in the experiment, it can be concluded that the nucleic acid dsRNA has a significant production and application effect in this innovative "condensation-separation" system. By fully exerting the advantages of molecular condensation strategy and cell division, the efficient production and accumulation of nucleic acid dsRNA are successfully achieved.

[0249] The E. coli HT115(DE3) strain and L4440 plasmid in this embodiment were purchased from the Mingling Plasmid Platform. The H-L4440 recombinant strain was obtained by introducing L4440 into the E. coli HT115(DE3) competent cells by heat shock transformation. The B-Δr and B-ΔC-Δr recombinant strains were obtained by knocking out the rnc gene based on the E. coli BL21(DE3) and B-ΔC strains, respectively, by CRISPR Cas9 technology. The specific molecular construction related operations are shown in Example 7, and the difference is the selection of the knocked-out gene. The construction of all recombinant strains in this embodiment is shown in Table 23.

[0250] Table 23 Construction of all recombinant strains in this embodiment

[0251] The present application provides a method for producing target molecules based on the condensation-separation technology, as well as the ideas and methods for the application of recombinant strains. There are many methods and ways to realize this technical solution, and the above description is only the preferred embodiment of the present application. It should be noted that for ordinary skilled persons in the technical field, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered as the protection scope of the present application. The components not explicitly described in this embodiment can be realized by existing technology.

Claims

1. A method for producing a target molecule based on a coacervation-fragmentation technique, characterized by, The target molecule is connected with the biological condensed state tag to obtain a target molecule gene fragment, and the target molecule gene fragment is introduced into a production minicell chassis strain to cut off the target molecule by the production minicell.

2. A recombinant bacterial strain based on coacervate-blebbing technology, characterized in that, The target molecule is connected with the biological condensed state tag to obtain a target molecule gene fragment, and the target molecule gene fragment is introduced into a production minicell chassis strain to cut off the target molecule by the production minicell.

3. The method of claim 1 or the recombinant bacterial strain of claim 2, wherein, The target molecule is a biological macromolecule or an organic small molecule; preferably, the biological macromolecule includes any one or combination of several of proteins, polypeptides, nucleic acids, polysaccharides, oils, biological particles, and intracellular polymers; preferably, the organic small molecule includes any one or combination of several of alcohols and aldehydes.

4. The method of claim 1 or the recombinant bacterial strain of claim 2, wherein, The biological condensed state tag includes any one or combination of several of a liquid-liquid phase separation tag, an end positioning tag, and a self-assembly tag.

5. The method or recombinant strain of claim 4, wherein, The liquid-liquid phase separation tag comprises any one of I16, R32, V7, NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, ELP[KV7F-36], WT-20, I24, Y145, V5-N, V1-C, N1, HPB2-C, V2-C, tag2, tag1, FUS, TDP-43, LAF-1, ICP4, RGGRGG, CAG 40 The end localization tag comprises any one of EpsM, DivIVA or its truncation, Div(60), PopZ, PodJ, SpmX, RodA, LcsA; the self-assembly tag comprises any one of Y15, L6KD, 18Awt.

6. The method or recombinant strain of claim 5, wherein, The liquid-liquid phase separation tag I16, R32, V7, NICD, mfp-3S, mfp-3S-pep-25aa, mfp-3S-pep-50aa, ELP[KV7F-36], WT-20, I24, Y145, V5-N, V1-C, N1, HPB2-C, V2-C, tag2, tag1, FUS, TDP-43, LAF-1, ICP4, RGGRGG, the corresponding amino acid sequence is shown in SEQ ID NO. 1-22, SEQ ID NO. 59 or the variant amino acid sequence with similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more to SEQ ID NO. 1-22, SEQ ID NO. 59; the liquid-liquid phase separation tag CAG 40 The corresponding nucleotide sequence is shown in SEQ ID NO. 67 or a variant nucleotide sequence with similarity of 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more to SEQ ID NO.

67.

7. The method or recombinant strain of claim 5, wherein, The end positioning label EpsM, DivIVA, Div(60), the corresponding amino acid sequence is shown as SEQ ID NO. 47-49 or a variant amino acid sequence with SEQ ID NO. 47-49 with more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% similarity; the DivIVA truncated body includes any one or a combination of several of DivIVA 1-40 , DivIVA 1-60 , DivIVA 1-80 .

8. The method or recombinant strain of claim 5, wherein, The self-assembly tag Y15, L6KD, 18Awt, the corresponding amino acid sequence is shown in SEQ ID NO. 50-52 or a variant amino acid sequence with a similarity of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% to SEQ ID NO. 50-52.

9. The method of claim 1 or the recombinant bacterial strain of claim 2, wherein, The target molecule is recruited to the biological condensed state tag by a recruitment tag to obtain a target molecule gene fragment.

10. The method or recombinant strain of claim 7, wherein, The recruitment tag includes any one group of RIAD and RIDD, P1 and P2, and P3 and P4.

11. The method or recombinant strain of claim 10, wherein, The RIAD and RIDD, the corresponding amino acid sequence is shown in SEQ ID NO. 53-54 or a variant amino acid sequence with a similarity of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% to SEQ ID NO. 53-54; the P1 and P2, the corresponding amino acid sequence is shown in SEQ ID NO. 55-56 or a variant amino acid sequence with a similarity of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% to SEQ ID NO. 55-56; the P3 and P4, the corresponding amino acid sequence is shown in SEQ ID NO. 57-58 or a variant amino acid sequence with a similarity of more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% to SEQ ID NO. 57-58.

12. The method of claim 1 or the recombinant bacterial strain of claim 2, wherein, The production minicell chassis strain is an Escherichia coli original strain, and is constructed by genetic modification of any one or combination of several of cell division production minicell related genes.

13. The method or recombinant strain of claim 12, wherein, The cell division production minicell related genes include any one or combination of several of a cell septum component gene, a septum inhibitor protein gene, a Z-ring regulatory protein gene, a cell division topological specificity factor gene, and a fitting binding protein gene.

14. The method or recombinant strain of claim 13, wherein, The cell septum component comprises any one or a combination of several of FtsZ, FtsA, FtsK, FtsQ, ZipA, FtsL, FtsI, and FtsW; the septum inhibitor protein comprises any one or a combination of several of MinC or its truncated form, MinD, SulA, and SfiA; the Z-ring regulatory protein comprises any one or a combination of several of ParA, ParB, and ParS; the cell division topological specificity factor comprises any one or a combination of several of MinE or its truncated form, DivIVA or its truncated form, and MinJ; and the fitting binding protein comprises any one or a combination of several of HNS, HU, MukBEF, FisA, and Lsr2.

15. The method or recombinant strain of claim 14, wherein, The MinC truncates include any one or a combination of MinC 116-231 , MinC 122-231 , MinC 122-231 , wherein the residue R at position 172 of MinC 122-231 is replaced by A; and the MinE truncates include any one or a combination of MinE 1-22 , MinE 1-33 , MinE 1-53 , MinE 1-62 , MinE 1-67 .

16. The method or recombinant strain of claim 12, wherein, The E. coli comprises any one of E. coli BL21, BL21(DE3), MG1655, MG1655(DE3), JM109, JM109(DE3), Rosetta, Rosetta(DE3), Lemo21, and Lemo21(DE3).

17. The method or recombinant strain according to any one of claims 12 to 16, characterized in that, The small cell-producing chassis strain comprises any one of the following chassis strains: (A) knocking out the minC gene in the original strain; (B) knocking out the minC, minD, and minE genes in the original strain; (C) knocking out the minC gene and expressing the ftsZ gene in the original strain; (D) Knocking out the promoter P in the original strain BAD Simultaneous expression of downstream structural genes rhaB, rhaA, rhaD and ftsZ gene, to construct the chassis strain B-ΔBAD::ftsZ for producing minicells. (E) Knocking out minC gene, promoter P BAD Simultaneous expression of the downstream structural genes rhaB, rhaA, rhaD with the gene ftsZ; (F) overexpressing any one or a combination of several of MinC, MinC truncated form, MinE, and MinE truncated form in the original strain.

18. The method or recombinant strain of claim 17, wherein, The minC gene, the minD gene, the minE gene, the rhaB gene, the rhaA gene, the rhaD gene, and the ftsZ gene have the amino acid sequences shown in SEQ ID NOs. 60-66.

19. Use of the recombinant strain of claim 2 in the production of small cells and the cleavage of target molecules.

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