CN117660505A

CN117660505A - Target product recovery method based on synthetic organelle, DNA construct and expression system

Info

Publication number: CN117660505A
Application number: CN202311503445.7A
Authority: CN
Inventors: 郭昊天; 陈炯霖
Original assignee: Kunshan Xunzhu Biotechnology Co ltd; Red Panda Co ltd
Current assignee: Kunshan Xunzhu Biotechnology Co ltd; Red Panda Co ltd
Priority date: 2023-11-13
Filing date: 2023-11-13
Publication date: 2024-03-08

Abstract

The invention relates to a target product recovery method based on a synthetic organelle, a DNA construct and an expression system. Specifically, the method comprises the following steps: constructing a synthetic organelle in a host; collecting a target product using the synthetic organelle; recovering the synthetic organelle containing the target product; releasing the target product from the synthetic organelle containing the target product, thereby obtaining the target product. The invention provides a general method for recovering and/or purifying a product, which has the advantages of simple steps, low cost, easy large-scale amplification and high-flux parallel treatment.

Description

Target product recovery method based on synthetic organelle, DNA construct and expression system

Technical Field

The invention relates to a target product recovery method based on a synthetic organelle, a DNA construct and an expression system. More particularly, the invention relates to a method for constructing a synthetic organelle through a host, controlling the process of collecting and releasing a target product by the synthetic organelle, and realizing separation, recovery and purification of the target product.

Background

At present, complex process flows are required for separating, recovering and purifying various target products. The process flows often need to be customized to arrange and combine various chemical unit operations (unit operations) such as extraction, chromatography, electrophoresis, centrifugation, membrane treatment and the like according to the physical, chemical and biological properties of the target product. Although these process flows can efficiently and specifically recover and purify the products, the process flows generally have the defects of complicated steps, long time, expensive required equipment consumables and the like, and the process flows lack of versatility, so that the separation, recovery and purification of different products are difficult to process in parallel in high throughput.

For target products such as recombinant proteins that can be engineered, purification can be performed by attachment of an affinity tag using affinity chromatography. Although such a process flow has a certain versatility, it involves removal of the affinity tag and secondary purification, and thus has a problem of complicated steps and high cost. The affinity tags for different target proteins are also not exactly identical and may require different column packing. Because of the different migration rates of different target proteins in the mobile phase, there are also significant differences in the flow rates used during the chromatography process and in the time taken for each step. Therefore, the recombinant protein affinity chromatography purification method based on the affinity tag is also difficult to operate in parallel.

Part of target products with special properties can be recycled by a recovery method with simple steps and low cost. For example, if the target product has high solubility in a specific solvent, the target product can be enriched by the solvent and then separated and recovered. If the target product is a natural organelle in a cell, the organelle may be obtained by disrupting the cell and then performing gradient density centrifugation. Further, if the target product is a molecule in a natural organelle, such as a mitochondrial genome, the target product may be recovered by first isolating the organelle and then disrupting the organelle. However, these methods are difficult to develop and difficult to reuse in recovery and purification of other products.

CN112575016a discloses a "construction of membraneless organelles in prokaryotes and application thereof", which utilizes recombinant spider silk proteins and meromelanoid elastin to successfully construct membraneless compartments in escherichia coli, and fuses the spider silk proteins or meromelanoid elastin with different cargo proteins through DNA recombination technology to realize intracellular co-localization of target functional proteins, thereby constructing membraneless compartments with bioactivity. The method directly expresses fusion proteins, and although successful to a certain extent, can form an enriched phase of cargo proteins in cells, the structure is disintegrated after the host cells are broken, so that the structure lacks the spatial independence of organelles, and cannot be separated and recovered. In addition, the design of direct expression of fusion proteins also results in low activity of the cargo protein being fused and limited specific expression of the assembly protein.

WO2023015190A1 discloses a method for controlling cellular processes in mammalian cells by synthesizing organelles, which uses arginine/glycine Rich (RGG) protein domains capable of liquid-liquid phase separation (LLPS) fused to high affinity helical tags (high-affinity helix tags), enriching endogenous proteins also fused to the helical tags, thereby controlling the behavior of the cells by sequestering or releasing the endogenous proteins. The method preferably ensures correct folding and activity of the target protein by fusing tags to the RGG protein and the target protein, respectively, which form organelles, but since the goal of the method is to control the behavior of mammalian cells, recruitment and release of the target protein by the synthetic organelles must occur within the mammalian cells, and thus the method cannot allow experimental procedures for isolation and recovery of the synthetic organelles from the host cells.

Thus, there is a need for a technique that can isolate, recover and utilize synthetic organelles.

Disclosure of Invention

In order to solve the above problems, the present invention provides a target product recovery method based on a synthetic organelle, which realizes separation, recovery and purification of a target product by constructing the synthetic organelle in a host and collecting and releasing the target product using the synthetic organelle.

Specifically, if a synthetic organelle can be constructed in a host for product recovery, if the target product can enter the synthetic organelle, or the target product itself is produced in the synthetic organelle, further, the target product can be collected using the synthetic organelle, and then recovered and/or purified by recovering the synthetic organelle, and finally, placing it in an eluent to release the target product. Thus, the present invention has been made.

The synthetic organelles useful in the present invention may be either artificially synthesized membrane-structured organelles or artificially synthesized membraneless organelles. Preferably, the host is a cellular system and the synthetic organelle is a membraneless organelle. Membraneless organelles are aggregates (biomolecular condensates) formed from biological macromolecules by phase separation (phase separation) with a distinct spatial separation. Synthetic biomacromolecules useful in the present invention for constructing membraneless organelles, including nucleotide sequences that are repeated 10-1000 times in succession, such as triplets CAG, CCUG, GGGAA, GGGGCC, etc.; amino acid sequences such as repeats of resinin-like peptides (RLP) that are repeated 3-500 times in succession; intrinsic non-structural regions, such as FUS protein, parB protein; multimerization of a multiplex biological macromolecule, such as PTB protein and UCUCU sequence repetition, an artificially designed binary protein two-dimensional structure, an artificially designed binary RNA two-dimensional structure.

The target product which can be used in the invention can be any molecule or structure which is intended to be prepared, and can be a sample obtained in the external environment or a product synthesized by artificially modifying a host. Can be produced in the same host cell as the synthetic organelle or in a different host. Preferably, the biosynthetic pathway of at least one component of the target product is encoded by heterologous DNA, comprising: heterologous biosynthetic small molecules such as deoxyviolacein (deoxyviolacein); recombinantly expressed polypeptides, proteins, RNAs, and DNAs, such as green fluorescent protein, red fluorescent protein, T4 DNA ligase, 16S rRNA; and multicomponent complexes, such as 30S ribosomal subunits, complexes of CRISPR-Cas with RNA.

In one aspect, the present invention provides a method for recovering a target product based on a synthetic organelle, comprising the steps of:

a. in the host, a synthetic organelle is constructed,

b. collecting a target product using the synthetic organelle;

c. recovering the synthetic organelle containing the target product;

d. releasing the target product from the synthetic organelle containing the target product, thereby obtaining the target product.

In one embodiment, the synthetic organelle refers to an artificially designed compartmentalized structure, preferably the synthetic organelle refers to a membraneless organelle, is a biomolecular aggregate (biomolecular condensates) formed by polypeptides and/or proteins and/or DNA and/or RNA and/or nucleic acid protein complexes in a host, more preferably the synthetic organelle is constructed from a biomacromolecule component selected from the group consisting of: nucleotide sequences repeated 10 to 1000 times in succession, such as triplets CAG, CCUG, GGGAA, GGGGCC, etc.); amino acid sequences such as repeats of resinin-like peptides (RLP) that are repeated 3-500 times in succession; intrinsic non-structural regions, such as FUS protein, parB protein; multimerization of a multiplex biological macromolecule, such as PTB protein and UCUCU sequence repetition, an artificially designed binary protein two-dimensional structure, an artificially designed binary RNA two-dimensional structure.

In one embodiment, the target product is one or more substances having a diameter in the range of 0.1-500 nm, preferably the target product is biosynthesized, more preferably the biosynthetic pathway of at least one component of the target product is encoded by heterologous DNA, comprising: heterologous biosynthetic small molecules such as deoxyviolacein (deoxyviolacein); recombinantly expressed polypeptides, proteins, RNAs, and DNAs, such as green fluorescent protein, red fluorescent protein, T4 DNA ligase, 16S rRNA; and multicomponent complexes, such as 30S ribosomal subunits, CRISPR-Cas9/gRNA complexes.

In one embodiment, step a comprises constructing a DNA construct (DNA construct) that, upon introduction into a host, directs the formation of a synthetic organelle. The host includes cell-free expression systems and cellular hosts, preferably prokaryotic hosts, more preferably E.coli.

In one embodiment, step b comprises collecting the target product using the synthetic organelle, which may be non-specific and/or specific binding, and which may originate from outside and/or be generated inside the synthetic organelle. This process can be achieved by nonspecific diffusion and adsorption; the method can also be realized by specific intermolecular interaction, and only needs to configure a receptor domain in a constructed synthetic organelle and connect a corresponding ligand on a target product, so that the synthetic organelle has a receptor, and the target product is connected with the ligand, thereby realizing that the synthetic organelle specifically binds to the target product and avoiding other impurity molecules from entering the synthetic organelle. The target product may be produced in the synthetic organelle, e.g., in one embodiment, deoxyviolacein is biosynthesized in a synthetic organelle; synthetic organelles can also be collected from the extracellular environment, for example, in one embodiment, by fusing the MS2 capsid protein at the N-terminus of the recombinant green fluorescent protein and the MS2 hairpin RNA at the 3' -terminus of the CAG repeat, and the resulting organelles formed from the latter by liquid-liquid phase separation (liquid-liquid phase separation) can specifically recruit GFP expressed in the cytoplasmic matrix. Preferably, the receptor/ligand pairs comprise: a complementarily paired nucleic acid molecule; capsid proteins of RNA phages such as MS2, PP7, qbeta, etc., and hairpin of translational operon RNA; protein N and B box (box B) RNA hairpin of DNA phage of lambda, 21, P22, etc.; CRISPR RNA hairpin and Cas6 proteins of bacteria such as pseudomonas aeruginosa (Pseudomonas aeruginosa); splitting two segments of the protein, such as split green fluorescent protein, alpha fragment and omega fragment of beta-galactosidase, split T7 RNA polymerase; an inducible dimerized or multimerized monomer, such as the light-controlled regulated multimeric green fluorescent protein Dronpa. Preferably, if the rate constant (rate constant) of the target product flowing into the synthetic organelle is higher than that of the target product flowing out in the host, the target product is enriched in the synthetic organelle, thereby achieving higher target product yield. This property can be achieved by the physicochemical properties of the synthetic organelles and the target product, such as the fact that various byproducts produced in the biosynthesis of small molecule deoxyviolacein and its biosynthetic pathway are poorly soluble in the aqueous phase and therefore can be enriched in intracellular synthesized RNA aggregates; may also be achieved with high affinity receptors and ligands.

In one embodiment, said step c comprises recovering from said host a synthetic organelle comprising said target product, preferably step c comprises isolation, recovery, purification of said synthetic organelle, preferably comprising the steps of:

c1. collecting and lysing the host;

c2. separating the precipitate containing the synthetic organelles, preferably by centrifugation and/or filtration, suction filtration;

c3. optionally, the precipitate is washed to remove impurities, thereby obtaining a purer synthetic organelle.

In one embodiment, said step d releases said target product from said synthetic organelle containing said target product. Preferably, step d comprises the separation, recovery, purification of the target product, preferably comprising the steps of:

d1. the synthetic organelle is placed in an eluent. Due to the permeability of the synthetic organelle to the target product, the target product spontaneously diffuses toward the eluent until equilibrium.

Preferably, if the rate of the target product flowing out of the synthetic organelle is higher than the rate of the target product flowing in the eluent, the synthetic organelle can be induced to release the target product in an accelerated manner, so that higher target product yield is realized. This property can be achieved by using a stronger affinity eluent, e.g., for deoxyviolacet, methanol, which is a higher affinity than the synthetic organelle RNA aggregates; it is also possible to alter the affinity of the synthetic organelles, for example by using pairs of receptors and ligands sensitive to temperature, optical signals, pH, etc. More preferably, the attachment of the target product to the ligand may be cut off such that the synthetic organelle loses specific binding to the target product, thereby allowing the synthetic organelle to release efficiently the ligand-tag-free target product, e.g., using nickel ion, factor Xa, enterokinase (Enterokinase), dithiothreitol (DTT), thrombin (Thrombin) or TEV protease, catalyzing cleavage of the corresponding fusion hinge (fusion linker) between protein and protein, inducing cleavage of the 3' end of PA14 RNA hairpin using imidazole dCsy4, catalyzing self cleavage using ribozymes or intein, etc.

d2. Separating the precipitate containing the synthetic organelle to obtain an eluent containing the target product, preferably by centrifugation and/or filtration and suction filtration;

d3. optionally, impurities in the eluent are removed to obtain a target substance with higher purity, preferably, by-products in the eluent are removed by separation and purification means, for example, broken recombinant proteins are removed by molecular sieves, and metal ion pollution is removed by dialysis.

In another aspect, the invention provides a DNA construct comprising the nucleotide sequence 1 as defined in the above method, directing the host to construct the synthetic organelle, and the nucleotide sequence 2 directing, regulating the production of the target product, preferably the DNA construct consists of the nucleotide sequence 1, 2 and a plasmid vector, such as pACYC184, pACYC177, pET28a (+), pET28b (+), pET-5a (+), pET43.1a, pET-37b (+), pCDFDuet-1, pCOLADuet-1, pRSFDuet-1, pETDuet-1, pUC57, pUC19, pBAD, pBluescript II SK (+), pTrcHisC, pTrcHis A, pTrcHis2C, pBV221, pQE-70, pCold III, pRSET-CFP, pR-BFP, pGFPuv, pKD, pKD4, pTYB1, pinPoint Xa-2, pTN 1, pRB 3, pSB4, pSB 5.

In yet another aspect, the present invention provides an expression system comprising the DNA construct described above, preferably the expression system refers to a system for simultaneously constructing the synthetic organelle and producing the target product in a prokaryotic cell, more preferably the expression system is an e.

The beneficial effects are that:

the recovery and purification of the target product of biosynthesis requires complex process flows, typically an array combination of various unit operations such as extraction, chromatography, electrophoresis, centrifugation, membrane processing, etc. The process flows have the defects of complicated steps, long time, expensive consumable materials of required equipment and the like. In the case of purification of recombinant proteins, chromatographic methods are generally used, but they have the following disadvantages: 1. the chromatographic column and the packing are expensive; 2. the chromatographic column has the service life limitation, and after each chromatographic packing is used, the chromatographic packing is cleaned before the next chromatographic packing is used; 3. different chromatographic methods (including ion exchange chromatography, gel chromatography, size exclusion chromatography, hydrophobic chromatography, affinity chromatography, etc.) are required according to the molecules with different properties. These disadvantages limit the versatility of chromatography and make it difficult to scale-up different process lines.

The method has universality and can be used for recovering and purifying small molecules or ionic compounds, biological macromolecules and the like; the operation is easy, the flow is simple, and the large-scale amplification is easy; low cost, generally does not require expensive equipment and/or consumables; highly standardized, allows for the use of substantially identical process flows for different products and thus allows for high throughput parallel processing.

Drawings

FIG. 1 shows gel electrophoresis identification images when the target product was purified to sfGFP using the rCAG-MS2/tdMCP system. Wherein, M, pre-dye protein Marker (gold, catalyst M00624); s1, inducing a host whole cell sample for 12h at 37 ℃, wherein a distinct band of about 51kDa is sfGFP fusion-connected with tdMCP-SNAC-tag; e1, cutting SNAC-tag by nickel ions, and recovering the obtained product from the organelle after 24 hours, wherein a distinct band of about 28kDa is the target product sfGFP; e2, cutting for 48 hours to obtain a product; e3, after 72 hours of cleavage, the product was obtained.

FIG. 2 shows the results of a relative quantitative analysis identified by green fluorescence activity when the target product was purified to sfGFP using the rCAG-MS2/tdMCP system. The left bar graph shows the purification efficiency of each operation. Wherein, the step c is a purification step of recovering the target product by the cell organelle after the whole cells are lysed; induced elution refers to the step d of adding an inducer to the organelle to elute the target molecule. The right panel shows the activity ratio of the target product to total protein in the different samples. Error bars (error bar) were calculated from standard deviations of three parallel replicates.

FIG. 3 shows gel electrophoresis identification images of the purification of target products to T4 DNA ligase protein using rCAG-MS2/tdMCP system. Wherein, 1-Lane C0 is whole cells which are not expressed in an induction way; 2-Lane C1, inducing whole cells at 37 ℃ for 5 hours; 3-Lane S1, inducing the supernatant of the first cell lysate for 5 hours at 37 ℃;4-Lane P1, inducing the precipitation of the first cell lysate for 5 hours at 37 ℃; inducing 5-Lane S2 at 37 ℃ for 5 hours, and lysing the obtained supernatant for the second time; inducing the secondary cell lysis of the cells for 5h at 37 ℃ to obtain a precipitate; 7-Lane W1, flushing the supernatant of the precipitated buffer solution; 8-Lane W2, rinsing the supernatant of the buffer solution after precipitation for the second time; 9-Lane F1: the supernatant of the product obtained after cutting; 10-Lane C3: residual sediment after cutting; lane M1 Pre-stained protein Marker (gold, catalog M00624).

FIG. 4 shows the results of a relative quantitative analysis identified by red fluorescence activity when the target product was purified to mKate2 using the RLP-GFP11/GFP (1-10) system. The left histogram shows the purification efficiency of each step, wherein the lysis separation refers to the purification step of precipitating organelle enriched molecules after whole cells are lysed; induced elution refers to the step d of adding an inducer to the organelle to elute the target molecule. The right panel shows the activity ratio of the target product to total protein in the different samples. Error bars (error bar) were calculated from standard deviations of three parallel replicates.

FIG. 5 shows agarose gel electrophoresis identification images of purification of 30S ribosomal subunits containing heterologous expression 16S rRNA with endogenous ribosomal proteins using a system of synthetic organelles constructed with ProteinB and ProteinA-dCsy4, PA14 CRISPR RNA hairpin as ligand. Wherein, the supernatant after lysis is RNA dispersion-like band released after whole cell lysis, and the signal is extremely weak; pre-elution pellet, which is a pellet of the cleaved organelles, has fewer complexes of free protein a-dCsy4 containing RNA with the target product; the eluted supernatant was the target product released after cleavage of PA14 CRISPR RNA by addition of imidazole-induced dCasy 4. The right arrow indicates the molecular size before and after cleavage.

FIG. 6 shows a thin layer chromatography identification image of nonspecifically recovered target product deoxyviolacein using synthetic organelles constructed with rCAG-Box B or rCAG-MS 2. Constructing a synthetic organelle in the left sample by using rCAG-Box B, and synthesizing a target product outside the organelle; in the right sample, rCAG-MS2 was used to construct a synthetic organelle in which the target product was biosynthesized. The right arrow indicates the eluted target product, as well as byproduct impurities.

Detailed Description

The present invention is described in more detail below.

1. Synthetic organelles

Cells generally have a complex spatial organization, one of which is called compartmentalization (compartmentalization), i.e., a spatial organization formed within cells, for performing specific functions and biochemical reactions. Such compartmentalized structures, also often referred to as organelles (organelles), are found in cells, like organs, in individuals.

Organelles, either individually enclosed in their own lipid bilayer, are known as membrane-bound organelles (membrane-organelles), such as organelles of mitochondria, chloroplasts, etc.; or spatially independent functional units, without a lipid bilayer surrounding, known as membraneless organelles (membraneless organelle), also a compartment observable under optical microscopy, found and revealed in 2009 that membraneless organelles are formed by phase separation (phase separation) of biological macromolecules within cells (1.C.P.Brangwynne et al., germline P granules are liquid droplets that localize by controlled dissolution/condensation.science 324,1729-1732 (2009)).

Synthetic organelles (synthetic organelle), which are an innovative bioengineering concept, are also known as "organelle-like structures" (artificial organelle), "artificial synthetic organelles" (artificial organelles). Synthetic organelles are intended to design and construct tiny functional units similar to natural organelles for compartmentalization in cells (or in vitro environments), for controlling biosynthesis, metabolic pathways or other biological processes. Construction of synthetic organelles involves introducing exogenous DNA constructs, including exogenous coding genes, non-coding DNA, regulatory elements, and the like, into host cells for directing cell-free expression systems or host cells to synthesize specific polypeptides, proteins, DNA, RNA, enzymes, or other biomolecules. One part of the molecules plays a role in constructing the structure of the synthetic organelle, and the other part of the molecules performs the preset functions of the synthetic organelle, such as biocatalysis, receptor recognition, protein sorting and the like. The construction of the synthetic organelles can be formed by self-assembly of heteromultimers of proteins, DNA, RNA, or by engineering the membrane structure of the cell. After the phase separation mechanism from membraneless organelles has been revealed, the construction of synthetic organelles can also be formed by aggregation of biological macromolecules capable of undergoing phase separation.

The present invention provides methods for recovering a target product using a synthetic organelle. Synthetic organelles may be essentially formed by the construction of polypeptides and/or proteins and/or DNA and/or RNA and/or nucleic acid protein complexes, e.g., repeated triplex ribonucleic acid (CAG) n spontaneously aggregates to form RNA aggregates/droplets (RNA aggregates/droples) by liquid-liquid phase separation (liquid-liquid phase separation) after intracellular transcription. The synthetic organelles may also contain other substances depending on the nature of the target product, for example, the P9 and P12 proteins expressing the phi 6 phage may recruit lipid molecules to form a membrane structure.

The biomacromolecule component for constructing the synthetic membraneless organelle is selected from the group consisting of: nucleotide sequences such as triplets CAG, CCUG, GGGAA, GGGGCC and the like, which are repeated 10 to 1000 times in succession; amino acid sequences such as repeats of resinin-like peptides (RLP) that are repeated 3-500 times in succession; intrinsic non-structural regions, such as FUS protein, parB protein; multimerization of a multiplex biological macromolecule, such as PTB protein and UCUCU sequence repetition, an artificially designed binary protein two-dimensional structure, an artificially designed binary RNA two-dimensional structure.

Of course, the synthetic organelles of the invention may also be constructed from polypeptides and/or proteins and/or DNA and/or RNA and/or nucleic acid protein complexes of other sequences, for example:

·RNA：

Repeating a triple ribonucleic acid, for example, (CAG) n, (GGU) n, (AUG) n, (CGG) n, (GUU) n, (CGU) n, (AUU) n, (ACG) n, (AAU) n, (AUC) n, (CCG) n, (AGC) n, (AGU) n, (ACU) n, wherein n is 10 to 1000;

repeating tetraribonucleic acids, for example (CCUG) n, where n is 10 to 1000;

repeating five-membered ribonucleic acids, e.g. (GGGAA) n, where n is 10-1000;

repeating hexahydric ribonucleic acids, for example GGGGCC (rGGGGCC), i.e. (GGGGCC) n, (GGAGCC) n, (GGGGAC) n, (aggaccc) n, where n is 10 to 1000;

first-element synthetic RNA scaffold, for example (underlined and bolded "XX-XX" represent sites where functional nucleic acid aptamers or ribozymes can be inserted): GGGTAGGCGCCTAGCCTAATGTACATTAAGTTATTTTTCCGGATGAATAGAATATATTCTAATAACGCAGGXX--XXCCTCGAATACGAGCTGGGXX--XXCCCAGGAAGTGTTCGCACTTCTCTCGTATTCGATTGCGACTAGT；

Two-component synthetic RNA scaffolds, for example (underlined and bolded "XX-XX" represent sites where functional nucleic acid aptamers or ribozymes can be inserted): d2-1GGGTCAGGAATCCTCCTGATAGCTATTTGGACAATTACGTACGTAGTTGATGACAACTACATGAAAATAAGGGXX- -XXCCCTCTTAGA and d2-2GGGTAGTTGTTATGGATTCCTGATTTATGGG XX--XXCCACTAGT；

·DNA：

A ssDNA sequence identical to the RNA sequence described above;

ssDNA capable of forming a nano-polymer, for example a nano-polymer of the following four ssdnas (underlined "XX-XX" represents sites where functional nucleic acid aptamers or ribozymes can be inserted): oligo-1TGCGCAATCC CXX-- XXCTTGAGCACGCCAACATCACCGTATTT，oligo-2GCGGCTAGCAGAGCATTCGGGAXX-- XXGACTATGGCTGTATTT，oligo-3GGCGTGCTCACXX--XXTCGGATTGCGCATGCTAGCCGCTATTT，oligo-4CGGTGATGTTCAGCCATAGTAGXX--XXGCCGAATGCTCTATTT, etc.;

protein:

self-assembled protein nanoparticles and nanostructures, for example: carboxylester (carboxomes) shell proteins; metabolome (metanolosomes) capsid proteins; bacterial micro-compartment (bacterial microcompartment, BMC) capsid proteins BMC-H, BMC-T, and BMC-P; yellow sea sessile Zaocysis (Haliangium ochraceum) bacterial micro-compartment capsid protein T1 (HO BMC Shell T1); thermotoga maritima (Thermotoga maritima) encapsulation protein encTm; mixing phage qβ virus-like particles; ferritin (Ferritin); artificially synthesized protein nanocages (nanocages), such as antibody Fc nanocages; artificially synthesized protein self-assembled fibers such as CC-Di-B-PduA protein; artificially synthesizing a protein self-assembled two-dimensional structure; artificially synthesized proteins self-assembled three-dimensional structures such as ATC-HL3 protein;

o intrinsic nonstructural proteins (intrinsically disordered protein, IDP), for example: resinoid-like peptides (RLP); elastin-like peptides (ELPs); RNA helicase LAF-1RGG domain repeats; RNA helicase DDX4; a candida (Caulobacter crescentus) nuclease E (rnase); IDP homologous protein sequence (GRGDSPYS) n, and mutant (GRGDSPYSGRGDSPYSGRGDSPYSGRGDSPVS) n, (GRGDSPYSGRGDSPVS) n, wherein n is 3-500; parB protein; FUS protein; ibpA protein; popZ protein; podJ protein; a conserved nucleoprotein fibrin FIB-1;

O protein amyloid pellet, for example: AEAEAKAKAEAEAKAK, LELELKLKLELELKLK the number of the individual pieces of the plastic,

b-amyloid peptide Ab42 of human origin (F19D);

other proteins that can produce aggregates, such as: coli (e.coli) beta-galactosidase; maltose binding protein mutant MalE31; clostridium cellulose (Clostridium cellulovorans) cellulose binding domain protein (CBD); clostridium thermocellum (Clostridium thermocellum) endoglucanase D (EGD); fusion proteins of the following proteins and fluorescent proteins derived from Brucella (Brucella) and PdhS-mCherry, fumC-YFP, divK-YFP; fusion proteins of the following proteins with fluorescent proteins from E.coli (E.coli), clpX-sfGFP, clpP-sfGFP, clpP-mCherry, clpX-mCherry, clpX-mCherry2, clpX-Venus, lacZ-Dronpa, tetR-Dronpa, P22C2-Dronpa, HKCI-Dronpa; tandem repeat SH3 (polySH 3) and tandem repeat PRM (polyPRM); tandem repeat SIM (polySIM) and tandem repeat SUMO (polySUMO); pentameric nucleoprotein Npm1, etc.;

nucleic acid protein multimerization complex:

long non-coding RNA (lncRNA) and RNA binding protein, wherein the nucleic acid scaffold is for example: mammalian paranuclear plaques in paraplaques assemble transcript 1isoform 2 (mammalian nuclear paraspeckle assembly transcript 1 isosporm 2); intergenic spacer lncRNA in amyloids; human satellite III (SatIII) lncRNA in nuclear stressors; drosophila heat shock RNA (Hsr) omega; schizosaccharomyces meiRNA; RNA binding proteins such as: PTB protein; FUS protein; histone BRD4

The omicron polypeptide sequence and polynucleotide, e.g., polypeptide RRASLRRASL and polyuridylic acid (polyuridylic acid, polyU) having a molecular weight of 600-1,000 kDa; synthetic polypeptides RP3, SR8, polyU, and the like.

The synthetic organelle may be constructed from biomolecules which are completely synthesized by the host guided by the exogenous DNA construct, or may be constructed from a part of exogenous molecules combined with endogenous molecules of the host, or may be constructed from modification of the original endogenous organelle or cytoskeletal structure in the host, for example, in a method for achieving codon expansion using synthetic organelles disclosed in EP 19157257.7, the synthetic organelle is formed by exogenously expressed Pyles:: FUS:: KIF16B1-400 fusion protein, MCP: EWSR1:: KIF16B1-400 fusion protein and endogenous microtubule cytoskeletal structure.

It should be noted that although the synthetic organelles according to the present invention may be constructed in various ways and there is no particular requirement as to the source of their constituent components, the synthetic organelles should be capable of being present as a spatially independent structure and thus of being isolated and recovered. For example, in one embodiment, the host is an E.coli cell and the synthetic organelle is constructed from 46 repeats of the triple ribonucleic acid CAG, which is located in the pellet and impurities such as cytoplasmic matrix are located in the supernatant after cell disruption and centrifugation. In contrast, if a specific spatial distribution is formed in the host by the biomacromolecule, but the biomacromolecule fails to aggregate into an independent spatial structure, the biomacromolecule cannot be separated from the cytoplasmic matrix, for example, the membraneless compartment disclosed in CN112575016A, construction and application of membraneless organelles in prokaryotes, is a soluble protein-enriched phase formed by recombinant spider silk proteins or meromorphic elastin, and after host cell disruption, a fusion protein composed of the spider silk proteins or meromorphic elastin and the target functional protein is mainly present in the supernatant after centrifugation, which means that the membraneless compartment is not independent of the structure in which the cytoplasmic matrix exists, and therefore, although also referred to as a "membraneless organelle", the membraneless compartment cannot be separated and recovered, and does not belong to the category of the synthetic organelles described in the present invention.

2. Collection of target product by synthetic organelle

Organelles generally have selective permeability and properties of sorting biomolecules, so that the composition of biomolecules within a compartment of the organelle and their physicochemical properties are significantly different from those of components outside the organelle (e.g., the cytoplasmic matrix), thereby enabling the organelle to perform specialized functions. The invention allows the synthetic organelles to collect the target product by tailoring the properties of the synthetic organelles and/or the target product.

In the invention, the collection of the target product in the synthetic organelle can be realized by nonspecific diffusion and adsorption, and preferably, the collection can also be realized by specific biological macromolecule interaction, namely, a receptor domain is configured in the constructed synthetic organelle, a corresponding ligand is connected to the target product, the ligand specifically targets the receptor, and the synthetic organelle can specifically bind to the target product through the action. Both the receptor and the ligand may be selected as desired, for example, in one embodiment the ligand recruited is a tandem dimer MS2 capsid Protein (tdMCP), the receptor is an MS2 hairpin RNA aptamer (MS 2 hairpin RNA aptamer), and in another embodiment the ligand is Protein N (Protein N), the receptor is a box b hairpin RNA aptamer. Other ligand/receptor pairs may also be used as desired, for example:

Any sequence complementary pair of nucleic acid molecules;

RNA binding proteins and corresponding RNAs, for example:

the capsid protein of RNA phage and the RNA hairpin of translation operon, and the RNA phage comprises MS2, PP7, qbeta and the like;

protein N and B box (box B) RNA hairpin of DNA phage, DNA phage includes lambda, 21, P22, etc.;

CRISPR Cas protein and CRISPR RNA hairpin, e.g. Csy4 (Cas 6 f) from pseudomonas aeruginosa (Pseudomonas aeruginosa) PA14 strain and PA14 RNA hairpin;

other binding pairs, such as PUF-8 and 5 '-UGUANUA-3'; FBF-2 and 5 '-UGURRNNAUA-3'; protein a and protein a binding aptamer; archaebacteria RNA binding protein L7Ae and RNA C/D box, etc.;

DNA binding proteins and corresponding DNA, for example: tetR and tetO operons; cI repressor proteins and cI operons; gal4 and the corresponding DNA; zif268 DNA binding domain and GATGCTGCA sequence; gli-1 DNA binding domain and GACCACCCAAGACGA sequence; lexA DNA binding domain and CTGTATATATATACAG sequence; a transcription activator-like (TAL) domain and corresponding DNA;

homodimers or multimers, for example: a reversible green fluorescent protein Dronpa;

split protein (split protein), for example: split fluorescent proteins, such as split green fluorescent proteins GFP1-10 and GFP11; cleavage of beta-galactosidase (split beta-galactose); cleavage of T7polymerase (split T7 polymerase); split esterases (split esterases); cleaving TEV protease (split TEV protease); cleaving dihydrofolate reductase (split dihydrofolate reductase); cleavage of beta-lactamase (split beta-lactamase); a disrupted firefly luciferase (split firefly luciferase); cleavage of thymidine kinase (split thymidine kinase); a split chorismate mutase (split chorismate mutase); cleaving CRISPR-Cas9; breaking horseradish peroxidase (split horseradish peroxidase), etc.;

Antigens and specific antibodies, for example: his tag and anti-His antibody; a flag tag and an anti-flag antibody; an HA tag and an anti-HA antibody; myc tag and anti-Myc antibody; GST protein and anti-GST antibody;

other protein-protein binding pairs, for example: a SYNZIP peptide pair; SZ17 and SZ18 paired; fos and Jun pair; arabidopsis thaliana (Arabiodopsis thaliana) photosensitive B (PhyB) and PIF3 protein or PIF6 protein;

of course, the receptor and ligand need not be pairs between two molecules, but complex multi-component complexes can be formed between multiple components, such as CRISPR-Cas9 proteins, crRNA, tracrRNA, quaternary complexes with crRNA-targeted DNA; ternary complexes of FKBP proteins, FRB proteins and rapamycin (rapamycin).

In a synthetic organelle, the receptor may be covalently linked to a component that constructs the synthetic organelle structure, e.g., in one embodiment, the receptor MS2 hairpin RNA aptamer and CAG repeat nucleotide that constructs the synthetic organelle structure constitute a fusion RNA molecule, e.g., in another embodiment, the receptor dCsy4 and protein a that constructs the synthetic organelle structure constitute a fusion protein molecule. Of course, receptors can also be incorporated into synthetic organelles by non-covalent intermolecular interactions. For example, CAG repeat nucleotides constructing a synthetic organelle structure are fused with an MS2 hairpin RNA aptamer, the MS2 hairpin RNA aptamer is non-covalently bound to tdMCP-Histag, the Histag is non-public and binds to fusion proteins of anti-His anti and PhyB, phyB serves as a receptor, and the synthetic organelle can specifically bind to a protein to which a ligand PIF3 or PIF6 is attached. The synthetic organelle may possess one to more receptors of different nature.

The linkage of the ligand to the target product may be covalent, e.g., in one embodiment, the ligand tdMCP and the target product green fluorescent protein comprise a fusion protein, e.g., in another embodiment, the ligand CRISPR RNA hairpin structure and the target product 16S rRNA comprise a fusion RNA molecule. Of course, the ligand may also be attached to the target product by non-covalent intermolecular interactions. For example, dronpa145N mutant target product can be specifically recruited by synthetic organelles with MS2 hairpin RNA and ligand tdMCP-Dronpa145K by intermolecular interaction to form heterodimers, which can be then isolated and recovered to the synthetic organelles, and the dissociation of Dronpa145N from tdMCP-Dronpa145K is induced by 500nm light irradiation to obtain Dronpa145N. The target product may also be linked to one or more ligands.

In the present invention, the target product may be obtained from outside the synthetic organelle and collected by the synthetic organelle during the collection in step b, for example, in one embodiment, the heterologous green fluorescent protein GFP is expressed in the cytoplasmic matrix; it can also be produced directly in the synthetic organelle, for example in one embodiment, the biosynthesized small molecule deoxyviolacein is biosynthesized directly in the synthetic organelle composed of RNA.

In the invention, in the collecting process of the step b, the rate constant of the target product flowing into the synthetic organelle is not necessarily higher than the rate constant of the target product flowing out of the synthetic organelle, and the target product is also partially distributed in the synthetic organelle, thereby realizing the collecting of the target product by the synthetic organelle. But preferably, if the inflow rate constant is higher than the outflow, the target product will be enriched in the synthetic organelle, thereby achieving a higher yield of the target product. The collection of the target product in the synthetic organelle may be achieved by physicochemical properties, e.g., in one embodiment, the biosynthetic small molecule deoxyviolacein is poorly soluble in the aqueous phase and in the organic phase, so that in the cell deoxyviolacein is enriched in the synthetic organelle composed of RNA; this can also be achieved by a high affinity receptor and ligand, for example, in one embodiment, the recruited ligand is a CRISPR RNA hairpin structure and the receptor is the corresponding CRISPR Cas protein Csy4. Step b may be performed in a specific buffer, but more preferably step b occurs in the host simultaneously with the production of the target product, to shorten the time required for the process flow and to reduce the complexity of the operation.

3. Release of target products by synthetic organelles

The invention can release the target product after the synthetic organelle is mixed with the eluent by designing the properties of the synthetic organelle and/or the target product and/or the eluent.

In the invention, in the eluent, the rate constant of the target product flowing out of the synthetic organelle is not necessarily higher than the rate constant of the target product flowing in, and the target product can be partially distributed in the eluent, so that the recovery of the target product is realized. But preferably, if the rate of outflow is higher than the inflow in the eluent, the synthetic organelle can be induced to accelerate the release of the target product, thereby achieving higher target product yields.

This property may be achieved by using a stronger affinity eluent, for example in one embodiment, the deoxyviolacet is more soluble in the eluent methanol than the synthetic organelle and can therefore be released from the synthetic organelle.

This property can also be achieved by altering the affinity of the synthetic organelle for the target product. If the synthetic organelle uses a receptor to specifically recruit a target product to which the ligand is attached, then altering the affinity between the receptor and the ligand accelerates the release of the target product, e.g., using a temperature, optical signal, pH, etc. sensitive receptor and ligand pair, thereby inducing the synthetic organelle to release the target product via the signal.

More preferably, the attachment of the target product to the ligand may be disrupted such that the synthetic organelle loses affinity for the target product, thereby inducing the synthetic organelle to release the ligand-tagged target product efficiently, e.g., in one embodiment, SNAC-tag attachment between the ligand protein and the target product protein is used, SNAC-tag cleavage is induced using nickel ions such that the synthetic organelle releases the target product protein; for example, in another embodiment, the target product 16S rRNA forms a fusion RNA molecule with a ligand that is a CRISPR repeat hairpin RNA, which is bound by the receptor dCsy4, and imidazole is used to induce cleavage of the 3' end of the CRISPR repeat hairpin RNA, such that the synthetic organelle releases the target product RNA. Disruption of the attachment of the target product to the ligand may also be accomplished in other ways, for example, by cleavage of the corresponding polypeptide sequence using factor Xa, enterokinase (Enterokinase), dithiothreitol (DTT), thrombin (Thrombin) or TEV protease, by catalytic self-cleavage using ribozymes or inteins, and the like.

4. Target product

The target product referred to in the present invention may be any molecule or structure intended to be produced. The target product need not be a biosynthetic molecule, for example, the target product may be a heavy metal ion in industrial wastewater or glucose absorbed from the environment by the host cell.

Preferably, the target product is a biosynthetic molecule. For example, biosynthesized small molecules, natural compounds derived from microorganisms such as violacein, deoxyviolacein, antibiotics, and nupharin, and natural compounds derived from animals and plants such as carotenoid, indigo, artemisinin, 4-methyl octanoic acid, 4-methyl nonanoic acid, 8-10 carbon branched unsaturated fatty acid, cochineal, and taxol; or recombinant polypeptides or proteins, such as superfolder GFP, sfGFP, red fluorescent protein mKate2, T4 DNA ligase, artificially designed polypeptides; or recombinant RNA, such as E.coli 16S rRNA; or DNA, e.g., recombinant plasmid, single-stranded DNA; or complexes of recombinant macromolecules with endogenous macromolecules, such as 30S ribosomal subunits formed by the assembly of recombinantly expressed 16S rRNA with endogenous ribosomal proteins; or other cellular structures, such as cytoskeleton, other organelles, other than the synthetic organelles.

Preferably, the biosynthesis pathway of at least one component in the target product is encoded by the heterologous DNA, so that the biosynthesis of the target product can be conveniently regulated and controlled, the target product is connected with a proper ligand, and the recovery efficiency of the target product is optimized.

The target product may be a mixture of substances, such as a 1:1 mixture of green fluorescent protein and red fluorescent protein.

The target product may be biosynthesized either in the synthetic organelle or in a matrix other than the synthetic organelle.

DNA construct

The present invention introduces an artificially designed DNA construct (DNA construct) into a host. A DNA construct is a polynucleotide comprising a plurality of coding genes, non-coding DNA, regulatory elements, and the like.

The DNA construct is capable of directing the host to construct a synthetic organelle. For example, in one embodiment, the synthetic organelle is constructed from an RNA molecule that is a CAG triplet nucleotide repeat, and the DNA construct comprises a nucleotide sequence that expresses the RNA molecule, including a CAG triplet nucleotide repeat, a transcription terminator, and a promoter that regulates induction of expression.

The DNA construct may also or alternatively comprise a nucleotide sequence that regulates the production of the product of interest by the host. For example, in one embodiment, the DNA construct comprises genes encoding 4 enzymes VioA, vioB, vioE, vioC that synthesize deoxyviolacein. For example, the DNA construct may comprise synthetic RNA that modulates host metabolic pathways to increase tryptophan production by the host.

The DNA constructs of the present invention may be one or more plasmids, cosmids, artificial chromosomes, single or multiple sites integrated into the host genome, and permutations and combinations of the above. Preferably, the DNA construct is a plasmid, and the nucleotide sequence for constructing the synthetic organelle and producing the target product from the directing host is constituted with a plasmid vector. The plasmid vector may be a commercial vector such as pACYC184, pACYC177, pET28a (+), pET28b (+), pET-5a (+), pET43.1a, pET-37b (+), pCDFDuet-1, pCOLADuet-1, pRSFDuet-1, pETDuet-1, pUC57, pUC19, pBAD, pBluescript II SK (+), pTrcHisC, pTrcHis A, pTrcHis2C, pBV221, pQE-70, pCold III, pRSET-CFP, pRSET-BFP, pGFPuv, pKD, pKD4, pTYB1, pinPoint Xa-2, pTWIN1 or pRSET C, or an altered construct of a commercial vector, or a non-vector such as pSB1C3, pSB3C5, pSB4K5, or other nucleotide sequences designed artificially to be able to replicate stably in host cells. For example, in one embodiment, the DNA construct is a two plasmid system in which one plasmid directs the host to synthesize a synthetic organelle constructed from CAG triplex nucleotide repeat RNA molecules, the vector is a variant of pACYC184, and the other plasmid directs the host to express the recombinant protein sfGFP linked to the ligand tdMCP, the vector is pET28a (+).

6. Expression system

Introducing the above DNA construct into a host for expression, directing the host to construct a synthetic organelle, and/or regulating the host to produce the desired product. The expression system may be a cell-free expression system and/or a cellular expression system. Wherein the cell expression system comprises prokaryotic cells and eukaryotic cells, eukaryotic cells such as yeast, fungal cells, animal cells or plant cells, or prokaryotic cells such as E.coli, B.subtilis, lactic acid bacteria, bifidobacteria, streptomyces. The prokaryotic expression system has the advantages of rapid cell proliferation, low culture cost, high yield and the like, and is preferable, and escherichia coli is more preferable. For example, in one embodiment, the DNA construct is a three plasmid, the expression system is an e.coli expression system, the three plasmids are co-expressed in e.coli, one directing the host to construct a synthetic organelle, and the other two directing the host to synthesize deoxyviolacein. Expression systems may also involve the use of multiple systems. For example, small molecule compounds having greater toxicity to cell growth, such as paclitaxel, are biosynthesized using a cell-free expression system, synthetic organelles capable of recruiting paclitaxel are constructed using an E.coli expression system, and then E.coli is lysed and mixed with the former cell-free expression system, and then the synthetic organelles are recovered, and finally paclitaxel is recovered from the synthetic organelles.

7. Target product recovery method

The invention provides a target product recovery method based on a synthetic organelle, which comprises the following steps:

a. in the host, a synthetic organelle is constructed,

b. collecting a target product using the synthetic organelle;

c. recovering the synthetic organelle;

d. releasing the target product from the synthetic organelle, thereby obtaining the target product.

More specifically, said step a is directed to introducing a DNA construct into a host, directing the host to construct a synthetic organelle; step b refers to the collection of the target product using the synthetic organelle; step c includes separation, recovery, purification of the synthetic organelles; step d includes releasing the target product from the synthetic organelle for separation, recovery, and purification of the target product.

Preferably, steps a and b are processes of simultaneously constructing synthetic organelles in a single host cell, biosynthesis of a target product, and specific enrichment of the target product by the synthetic organelles, step c is separation, recovery and purification of the synthetic organelles, and d is separation, recovery and purification of the target product, comprising:

c1. collecting and lysing the host;

c2. separating the pellet containing the synthetic organelles, preferably by centrifugation and/or filtration;

c3. Optionally, washing the precipitate to remove impurities, thereby obtaining purer synthetic organelles;

d1. placing the synthetic organelle in an eluent and inducing the synthetic organelle to release the target product, preferably in a cleavage reaction, cleavage of the binding site of the ligand and the target substance using a catalyst such as nickel ion, imidazole-induced dCsy4, factor Xa (factor Xa), enterokinase (Enterokinase), dithiothreitol (DTT), thrombin (Thrombin) or TEV protease;

d2. separating the precipitate containing the synthetic organelle to obtain an eluate containing the target product, preferably by centrifugation and/or filtration;

d3. optionally, impurities in the eluent are removed, and a target substance with higher purity is obtained.

Examples

EXAMPLE 1 purification of fluorescent proteins Using nucleic acid phase separation Synthesis of CAG repeats

System design

The molecules constituting the synthetic organelles in this example are46CAG repeated RNA fragments, which can be separated by interactions to produce liquid-liquid phase separations. In this example, a superfolder green fluorescent protein (superfolder green fluorescent protein, sfGFP) was enriched by tandem dimer MS2 capsid protein (tdMCP) as ligand and MS2 RNA hairpin as receptor. SNAC-tag, which is self-cleaving induced by divalent Nickel ions, was used as a protein fusion hinge between tdMCP and sfGFP (f use linker) and cleavage site (clear site). In this example, two plasmids pGFP and prCAG-MS2 were used together to express tdMCP-SNAC-sfGFP fusion protein and rCAG-MS2 RNA, respectively. The experiment was performed as follows.

Experimental procedure

pGFP and prCAG-MS2 were co-transformed into E.coli BL21 (DE 3) competent cells (full gold). The monoclonal was picked and cultured at 37℃and 220rpm with shaking for 16 hours. Inducer was added to the flask to 0.5mM isopropyl thioβ -galactoside (IPTG) (manufacturing), 150ng/ml anhydrotetracycline hydrochloride (aTc) (carbosulfan), and expression was induced and cultured overnight for about 12 hours. And centrifuging the bacterial liquid and removing the supernatant. Adding lysate (one-step method schizolysis kit, manufacturing) into the precipitate, blowing and mixing uniformly, reacting for 30min at room temperature, centrifuging, discarding the supernatant, and recovering the precipitate. And adding a cutting eluent containing nickel ions into the precipitate obtained in the last step, and cutting at room temperature on a rotary mixer. And centrifuging the sample subjected to the cleavage reaction, and recovering the supernatant, namely the solution containing the target product protein.

Thereafter, protein samples were detected using SDS PAGE electrophoresis (SurePAGE ^TM Gold sri) for assessing the size, yield and purity of the recovered protein.

And total protein/corresponding fluorescence were detected by taking whole cells/supernatant after first lysis, precipitation/elution, respectively. After the data were uniformly subtracted from the background, three indicators of characteristic activity (specific activity, SA), purification efficiency (purification factor, PF) and yield (yield) were calculated. Wherein the characteristic activity represents the ratio of the target product to the total protein in the sample of each step, and the purification efficiency is the ratio of the characteristic activity in the sample before and after each step and is used for describing the efficiency of each step. The yield was calculated as the ratio of the total amount of target product contained in each sample to the total amount without purification, and was used to evaluate the yield of final purification.

Results

The results are shown in fig. 1 and 2. The gel diagram shows that the sfGFP obtained by final elution has higher purity and no obvious impurity band. Semi-quantitative analysis shows that the product purification efficiency of the step of recovering the synthetic organelle is about 1.5 times, the product purification efficiency of the step of recovering the target product is about 2 times, and the characteristic activity of the product is obviously improved in both steps, so that the system for combining the synthetic organelle and the corresponding flow of the example can be used for product purification.

Sequence details

Wherein, sfGFP size: the 26kDa protein has the sequence shown in SEQ ID NO. 1:

MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHG(SEQ ID NO:1)。

the rCAG-MS2 RNA sequence is shown in the following SEQ ID NO: 2:

cagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagagacggagcucgucgacgcggccgcaagcuugguaccgagcucggauccuaagguaccuaauugccuagaaaacaugaggaucacccaugucugcaggucgacucuagaaaacaugaggaucacccaugucugcaguauucccggguucauuagauccuaagguaccuaauugccuagaaaacaugaggaucacccaugucugcaggucgacucuagaaaacaugaggaucacccaugucugcaguauucccggguucauuagauccuaagguaccuaauugccuagaaaacaugaggaucacccaugucugcaggucgacucuagaaaacaugaggaucacccaugucugcaguauucccggguucauuagauccuaagguaccuaauugccuagaaaacaugaggaucacccaugucugcaggucgacucuagaaaacaugaggaucacccaugucugcaguauucccggguucauuagauccuaagguaccuaauugccuagaaaacaugaggaucacccaugucugcaggucgacucuagaaaacaugaggaucacccaugucugcaguauucccggguucauuagauccuaagguaccuaauugccuagaaaacaugaggaucacccaugucugcaggucgacucuagaaaacaugaggaucacccaugu

the protein sequence of tdMCP is shown in the following SEQ ID NO: 3:

MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY(SEQ ID NO:3)

the SNAC-tag protein sequence is shown in the following SEQ ID NO. 4:

GSHHW(SEQ ID NO:4)

EXAMPLE 2 phase separation of synthetic organelle purified enzyme Using nucleic acid composed of CAG repeats

System design

The mechanism of cleavage of the released products was the same as in example 1 except that the target product was T4 DNA ligase (T4 DNA ligase) in this example, two plasmids pT4 and prCAG-MS2 were used together to express tdMCP-SNAC-T4 DNA ligase fusion protein and rCAG-MS2 RNA, respectively. The experiment was performed as follows.

Experimental procedure

Co-transformation of competent cells of E.coli BL21 (DE 3) (gold of full format) with pT4 and prCAG-MS 2. The monoclonal was picked and cultured at 37℃and 220rpm with shaking for 16 hours. The inducer was added to the flask to 0.5mM isopropyl thioβ -galactoside (IPTG) (manufacturing), 150ng/ml anhydrotetracycline hydrochloride (aTc) (carbosulfan), and the expression was induced and cultured for about 5 hours. And centrifuging the bacterial liquid and removing the supernatant. Adding lysate (one-step method schizolysis kit, production) into the precipitate, blowing and mixing uniformly, reacting for 30min at room temperature, namely, first cracking, centrifuging, and separating supernatant and precipitate. Adding the sediment obtained in the last step into the lysate, blowing and mixing uniformly, reacting for 30min at room temperature, namely, performing secondary pyrolysis, and then centrifuging, and separating the supernatant and the sediment. Adding the sediment obtained in the last step into a 10-fold diluted lysate, blowing and mixing uniformly, centrifuging, and separating the supernatant and the sediment, namely, flushing for the first time. Adding the obtained precipitate into the lysate diluted by 10 times again, blowing and mixing uniformly, centrifuging, and separating supernatant and precipitate, namely washing for the second time. And adding a cutting eluent containing nickel ions into the precipitate obtained in the last step, and cutting at room temperature on a rotary mixer. And centrifuging the sample subjected to the cleavage reaction, and recovering the supernatant, namely the solution containing the target product protein. Thereafter, protein samples were detected using SDS PAGE electrophoresis (SurePAGE ^TM Gold sri) for assessing the size, yield and purity of the recovered protein.

Results

The results are shown in FIG. 3. The gel diagram shows that the purity of the finally eluted T4 DNA ligase is higher, and no obvious impurity band exists. Multiple flushes can remove small amounts of impurities.

Sequence details

Size of T4 DNA ligase: 55.3kDa, its protein sequence is shown in SEQ ID NO. 5:

MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL(SEQ ID NO:5)

EXAMPLE 3 Synthesis of organelle purification proteins Using protein phase separation made up of RLP repeat sequences

System design

The molecules that make up the synthetic organelle in this example are polypeptide fragments of 20 repeats of resinin-like peptides (RLP), which can be separated by interactions to produce liquid-liquid phase separations. In this example, the target red fluorescent protein mKate2 was enriched by cleaving two portions of GFP1-10 and GFP11 of the green fluorescent protein (split GFP) as ligand and receptor, respectively. SNAC-tag ligation was used between GFP1-10 and mKate2. In this example, two plasmids, pET28a-RLP-sfGFP (1-10) and pCDF-sfGFP11-SNAC-mKate2, were used together to express the 20xRLP-7xGFP11 fusion protein, and the GFP (1-10) -SNAC-mKate2 fusion protein, respectively.

Experimental operation

E.coli BL21 (DE 3) competent cells (full gold) were transformed with the pET28a-RLP-sfGFP (1-10) and pCDF-sfGFP11-SNAC-mKate2 plasmids together. The monoclonal cells were picked up and cultured at 37℃and 220rpm for 16 hours. The inducer was added to the flask to 0.5mM IPTG (Industry), 150ng/ml aTc (carbofuran) and incubated overnight for about 12 hours. And centrifuging the bacterial liquid and removing the supernatant. Adding lysate (one-step method schizolysis kit, production) into the precipitate, blowing and mixing uniformly, and reacting for 30min at room temperature. Adding a nickel ion-containing cutting buffer into the precipitate obtained in the last step, and placing the mixture on a rotary mixer for eluting at room temperature. And centrifuging the sample subjected to the cleavage reaction, and recovering the supernatant, namely the solution containing the target product protein.

And then taking the whole cells, respectively, precipitating after the first lysis, and respectively detecting total protein and corresponding fluorescence from the supernatant after elution. After the background is uniformly subtracted from the data, three indexes of characteristic activity, purification efficiency and yield are calculated. Wherein the characteristic activity represents the ratio of the target product to the total protein in the sample of each step, and the purification efficiency is the ratio of the characteristic activity in the sample before and after each step and is used for describing the efficiency of each step. The yield was calculated as the ratio of the total amount of target product contained in each sample to the total amount without purification, and was used to evaluate the yield of final purification.

Analysis of results

The specific results are shown in FIG. 4. Semi-quantitative analysis shows that the purification efficiency of the mKate2 product reaches 1.76, and the characteristic activity of the product is obviously different from that of the product before purification, which proves that the system and the flow can be used for recovering and purifying the product when the protein is used as a receptor/ligand for synthesizing a cellular organelle. In particular, the characteristic activity of the product of this step of recovering the synthetic organelles was reduced, with a product purification efficiency of about 0.6 times, indicating that the ratio of mKate2 in the synthetic organelles is actually significantly smaller than its ratio in the total cells. Thus, the results demonstrate that step b can be used for recovery and purification of the target product in the present invention even if the affinity of the synthetic organelle is insufficient to achieve enrichment of the target product in the synthetic organelle relative to the extracellular cytoplasmic matrix.

Sequence details

Wherein the 7xGFP11 protein has the sequence shown in the following SEQ ID NO. 6:

RDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSGGRDHMVLHE YVNAAGITGGSGGRDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGS GGRDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGIT(SEQ ID NO:6)

the GFP1-10 protein sequence is shown in the following SEQ ID NO. 7:

MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK(SEQ ID NO:7)

the protein sequence of mKate2 is shown in the following SEQ ID NO. 8:

MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHR(SEQ ID NO:8)

the RLP size of 20 repeats was 17kDa, and the protein sequence was shown in SEQ ID NO 9 as follows:

SKGPGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGRGDSPYSGY(SEQ ID NO:9)

EXAMPLE 4 purification of RNA Using synthetic organelles constructed with an artificially designed two-dimensional binary protein structure

System design

In the example, the molecules constituting the synthetic organelle are two proteins A and B which are designed artificially, and the proteins A/B can self-assemble in cells to form a two-dimensional planar nanostructure. The receptor used in this example was a conditionally inactivated mutant dCsy4 of the CRISPR Cas protein Csy4 protein selected from pseudomonas aeruginosa (Pseudomonas aeruginosa) PA14 strain, the ligand was the corresponding CRISPR RNA repeated hairpin sequence (PA 14 RNA), and the target product was a 30S ribosomal subunit formed by the assembly of the recombinant expressed 16S rRNA selected from escherichia coli (e.coli) with endogenous ribosomal proteins. The PA14 RNA was directly linked to 16S rRNA to form a fusion RNA molecule. The dCasy 4 protein fused with the protein A can specifically recognize and bind to the PA14 RNA, and cut the 3' -end of the PA14 RNA under the induction of imidazole to release a target product. In this case, two plasmids were used to express ProteinB, proteinA-dCasy 4 fusion protein, and PA14-16S rRNA, respectively. The experiment was performed as follows.

Experimental procedure

The two plasmids were transformed together into E.coli BL21 (DE 3) competent cells (gold of the full format), respectively. The monoclonal is picked up, and the bacterial liquid is cultured for 16 hours at 37 ℃ and 220 rpm. The inducer was added to the flask to 0.5mM IPTG (Bio-), 150ng/ml aTc (carbofuran) and incubated overnight. And centrifuging the bacterial liquid and removing the supernatant. Adding lysate (one-step method schizolysis kit, and the like) into the precipitate, blowing and mixing uniformly, and reacting for 30min at room temperature. The precipitate obtained in the previous step is added with an eluent containing 300mM imidazole and placed on a rotary mixer for elution at room temperature. And centrifuging the sample subjected to the cleavage reaction to obtain an eluted supernatant, namely a solution containing the target product protein. The whole cells/the supernatant after the first lysis and precipitation/elution were taken separately for agarose electrophoresis gel detection.

Results

The specific results are shown in FIG. 5. In this example, the eluted product had a clear and bright band with no apparent bands. The results demonstrate that the design and experimental framework can be used for recovery and purification of RNA and its complexes. The precipitate recovered before elution had a clearly migrating band, but the signal was very weak, indicating that there was a small amount of complex bound by the target product free from the synthetic organelle and protein A-dCasy 4 fusion protein before cleavage reaction.

Sequence details

Wherein, the corresponding RNA sequence of the 16S rRNA template is shown in the following SEQ ID NO. 10:

aguuugaucauggcucagauugaacgcuggcggcaggccuaacacaugcaagucgaacgguaacaggaagcagcuugcugcuuugcugacgaguggcggacgggugaguaaugucugggaaacugccugauggagggggauaacuacuggaaacgguagcuaauaccgcauaacgucgcaagcacaaagagggggaccuuagggccucuugccaucggaugugcccagaugggauuagcuaguaggugggguaacggcucaccuaggcgacgaucccuagcuggucugagaggaugaccagcaacacuggaacugagacacgguccagacuccuacgggaggcagcaguggggaauauugcacaaugggcgcaagccugaugcagccaugcngcguguaugaagaaggccuucggguuguaaaguacuuucagcggggaggaagggaguaaaguuaauaccuuugcucauugacguuacccgcagaagaagcaccggcuaacuccgugccagcagccgcgguaauacggagggugcaagcguuaaucggaauuacugggcguaaagcgcacgcaggcgguuuguuaagucagaugugaaauccccgggcucaaccugggaacugcaucugauacuggcaagcuugagucucguagagggggguagaauuccagguguagcggugaaaugcguagagaucuggaggaauaccgguggcgaaggcggcccccuggacgaagacugacgcucaggugcgaaagcguggggagcaaacaggauuagauacccugguaguccacgccguaaacgaugucgacuuggagguugugcccuugaggcguggcuuccggannuaacgcguuaagucgaccgccuggggaguacggccgcaagguuaaaacucaaaugaauugacgggggccgcacaagcgguggagcaugugguuuaauucgaugcaacgcgaagaaccuuaccuggucuugacauccacggaaguuuucagagaugagaaugugccuucgggaaccgugagacaggugcugcauggcugucgucagcucguguugugaaauguuggguuaagucccgcaacgagcgcaacccuuauccuuuguugccagcgguccggccgggaacucaaaggagacugccagugauaaacuggaggaagguggggaugacgucaagucaucauggcccuuacgaccagggcuacacacgugcuacaauggcgcauacaaagagaagcgaccucgcgagagcaagcggaccucauaaagugcgucguaguccggauuggagucugcaacucgacuccaugaagucggaaucgcuaguaaucguggaucagaaugccacggugaauacguucccgggccuuguacacaccgcccgucacaccaugggaguggguugcaaaagaaguagguagcuuaacuucgggagggcg(SEQ ID NO:10)

the corresponding RNA sequence of PA14 is shown in the following SEQ ID NO. 11:

guucacugccguauaggcagcuaagaaa(SEQ ID NO:11)

the dCasy 4 corresponding protein sequence is shown in the following SEQ ID NO. 12:

MDHYLDIRLRPDPEFPPAQLMSVLFGKLAQALVAQGGDRIGVSFPDLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQAKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPLQVTAEEGGFTCYGLSKGGFVPWF(SEQ ID NO:12)

the corresponding protein sequence of the ProteinB is shown in the following SEQ ID NO. 13:

MGSLITLVELEWLEHQLIVQLSERLKGQIAKVGELLCECLKKGGKILICGNGGSAADAQHFAAELSGRYKKERKALAGIALTTDTSALSAIGNDYGFEFVFSRQVEALGNEKDVLIGISTSGKSPNVLEALKKAKELNMLCLGLSGKGGGMMNKLCDHNLVVPSDTARIQEMHILIIHTLCQIIDESFLEHHHHHH(SEQ ID NO:13)

the corresponding protein sequence of the protein A is shown in the following SEQ ID NO. 14:

MGHHHHHHGGLALVATGNDTTTKPDLYYLKNSEAINSLALLPPPPAVGSIAFLNDQAMYEQGRLLRNTERGKLAAEDANLSSGGVANAFSGAFGSPITEKDAPALHKLLTNMIEDAGDLATRSAKDHYMRIRPFAFYGVSTCNTTEQDKLSKNGSYPSGHTSIGWATALVLAEINPQRQNEILKRGYELGQSRVICGYHWQSDVDAARVVGSAVVATLHTNPEFQAQLIKAKIEFKQHQKEL(SEQ ID NO:14)

EXAMPLE 5 recovery of deoxyviolacein Using nucleic acid phase separation Synthesis of the CAG repeats

System design

The target product in this example is Deoxyviolacein (DV), which is obtained by sequentially reacting four enzymes VioA, vioB, vioE, vioC derived from Chromobacterium violaceum (Chromobacterium violaceum) to convert tryptophan (L-trptophan) into purple deoxyviolacein having antibacterial and anticancer effects, and has an absorption peak at about 570 nm. In this example, strain 1, using three plasmids, co-expressed in the host, wherein prCAG-MS2 was used to construct a synthetic organelle consisting of CAG repeats and MS2 RNA hairpin repeats, pvioambem and pVioCM were co-expressed, each with the VioA, vioB, vioE, vioC enzyme tdMCP linked at the C-terminus; strain 2, using three plasmids, was co-expressed in the host, wherein prCAG-Box B was used to construct a synthetic organelle consisting of CAG repeats and Box B RNA hairpin repeats, and pVioAMBMEM and pVioCM co-expressed the four tdMCP and enzyme fusion proteins described above. The sequences of the pVioAMBMEM and pVioCM plasmids are from the literature Guo, haotian, et al, "Spatial engineering of E.coli with addressable phase-isolated RNAs," Cell 185.20 (2022): 3823-3837. Due to the high specificity of the box b and MS2 systems, the enzyme VioA, vioB, vioE, vioC will be located inside and outside the synthetic organelles, respectively, among strain 1 and strain 2, such that the sites of DV production are located in the synthetic organelles and the cytoplasmic matrix, respectively. In this case, the collection of DV by the synthetic organelle is not subject to specific receptor/ligand binding, but is distributed inside and outside the synthetic organelle by the diffusion and non-specific lysis, adsorption of DV within the host cell.

Experimental procedure

The designated plasmid set was co-transformed into E.coli BL21AI competent cells to obtain strain 1 and strain 2. The monoclonal was picked up, shake-cultured at 37℃and 150rpm for 16 hours, then diluted 1:200 times into 5mL of culture medium, and cultured at 37℃and 150rpm for 4 hours to logarithmic phase, and aTc was added to a final concentration of 50ng/mL, followed by further culturing for 20 hours. And centrifuging the bacterial liquid and removing the supernatant to obtain a cell-containing precipitate. The cells were washed by resuspension and centrifugation with phosphate buffer (Phosphate Buffered Saline, PBS) and repeated 2 times. The cells were resuspended in PBS and added with lysis solution (Bugbuster, sigma-Aldrich), stirred and mixed well, reacted for 20min at room temperature with shaking at 900rpm, then centrifuged, the supernatant was discarded, the precipitate was recovered, methanol was added, shaking was performed for 30 seconds, and the supernatant was centrifuged to obtain the eluent containing the target product. 200 μl of the eluate was taken and analyzed by thin layer chromatography on a Silica gel thin layer chromatography plate (TLC Silica gel 60, merck, germany) using a 6:1 chloroform methanol solution.

Results

The results are shown in FIG. 6, and the color development of the chromatography under the visible light shows that the target product DV is obtained through methanol elution and recovery, and the target product DV and a small amount of other byproduct impurities are marked by arrows respectively, and the recovery rate of the target product is relatively similar between the two strains. Proved by the design and experimental framework, the method can be used for recovering small molecular compounds; and the design and experimental framework are proved to be insensitive to the synthesis position of the target product, and can be arranged in the cell or outside the cell.

Sequence details

Wherein the rCAG-Box B corresponding RNA sequence is shown in the following SEQ ID NO. 15:

cagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagagacggagctcgtcgacgcggccaagcttggtaccgagctcgggccctgaagaagggcccctcgactaagtccaactactaaactgggccctgaagaagggcccatatagggccctgaagaagggcccctcgactaagtccaactactaaactgggccctgaagaagggcccatatagggccctgaagaagggcccctcgactaagtccaactactaaactgggccctgaagaagggcccatatagatcaagcttggtaccgagctcgggccctgaagaagggcccctcgactaagtccactactaaactgggccctgaagaagggcccatatagggccctgaagaagggcccctcgactaagtccaactactaaactgggccctgaagaagggcccatatagggccctgaagaagggcccctcgactaagtccaactactaaactgggccctgaagaagggcccatatatcgagtaaggatct

the amino acid sequence corresponding to VioA is shown as the following SEQ ID NO. 16:

MKHSSDICIVGAGISGLTCASHLLDSPACRGLSLRIFDMQQEAGGRIRSKMLDGKASIELGAGRYSPQLHPHFQSAMQHYSQKSEVYPFTQLKFKSHVQQKLKRAMNELSPRLKEHGKESFLQFVSRYQGHDSAVGMIRSMGYDALFLPDISAEMAYDIVGKHPEIQSVTDNDANQWFAAETGFAGLIQGIKAKVKAAGARFSLGYRLLSVRTDGDGYLLQLAGDDGWKLEHRTRHLILAIPPSAMAGLNVDFPEAWSGARYGSLPLFKGFLTYGEPWWLDYKLDDQVLIVDNPLRKIYFKGDKYLFFYTDSEMANYWRGCVAEGEDGYLEQIRTHLASALGIVRERIPQPLAHVHKYWAHGVEFCRDSDIDHPSALSHRDSGIIACSDAYTEHCGWMEGGLLSAREASRLLLQRIAA

the corresponding amino acid sequence of VioB is shown as the following SEQ ID NO. 17:

MSILDFPRIHFRGWARVNAPTANRDPHGHIDMASNTVAMAGEPFDLARHPTEFHRHLRSLGPRFGLDGRADPEGPFSLAEGYNAAGNNHFSWESATVSHVQWDGGEADRGDGLVGARLALWGHYNDYLRTTFNRARWVDSDPTRRDAAQIYAGQFTISPAGAGPGTPWLFTADIDDSHGARWTRGGHIAERGGHFLDEEFGLARLFQFSVPKDHPHFLFHPGPFDSEAWRRLQLALEDDDVLGLTVQYALFNMSTPPQPNSPVFHDMVGVVGLWRRGELASYPAGRLLRPRQPGLGDLTLRVNGGRVALNLACAIPFSTRAAQPSAPDRLTPDLGAKLPLGDLLLRDEDGALLARVPQALYQDYWTNHGIVDLPLLREPRGSLTLSSELAEWREQDWVTQSDASNLYLEAPDRRHGRFFPESIALRSYFRGEARARPDIPHRIEGMGLVGVESRQDGDAAEWRLTGLRPGPARIVLDDGAEAIPLRVLPDDWALDDATVEEVDYAFLYRHVMAYYELVYPFMSDKVFSLADRCKCETYARLMWQMCDPQNRNKSYYMPSTRELSAPKARLFLKYLAHVEGQARLQAPPPAGPARIESKAQLAAELRKAVDLELSVMLQYLYAAYSIPNYAQGQQRVRDGAWTAEQLQLACGSGDRRRDGGIRAALLEIAHEEMIHYLVVNNLLMALGEPFYAGVPLMGEAARQAFGLDTEFALEPFSESTLARFVRLEWPHFIPAPGKSIADCYAAIRQAFLDLPDLFGGEAGKRGGEHHLFLNELTNRAHPGYQLEVFDRDSALFGIAFVTDQGEGGALDSPHYEHSHFQRLREMSARIMAQSAPFEPALPALRNPVLDESPGCQRVADGRARALMALYQGVYELMFAMMAQHFAVKPLGSLRRSRLMNAAIDLMTGLLRPLSCALMNLPSGIAGRTAGPPLPGPVDTRSYDDYALGCRMLARRCERLLEQASMLEPGWLPDAQMELLDFYRRQMLDLACGKLSREA

the amino acid sequence corresponding to VioE is shown as the following SEQ ID NO 18:

MENREPPLLPARWSSAYVSYWSPMLPDDQLTSGYCWFDYERDICRIDGLFNPWSERDTGYRLWMSEVGNAASGRTWKQKVAYGRERTALGEQLCERPLDDETGPFAELFLPRDVLRRLGARHIGRRVVLGREADGWRYQRPGKGPSTLYLDAASGTPLRMVTGDEASRASLRDFPNVSEAEIPDAVFAAKR

the amino acid sequence corresponding to VioC is shown as the following SEQ ID NO 19:

KRAIIVGGGLAGGLTAIYLAKRGYEVHVVEKRGDPLRDLSSYVDVVSSRAIGVSMTVRGIKSVLAAGIPRAELDACGEPIVAMAFSVGGQYRMRELKPLEDFRPLSLNRAAFQKLLNKYANLAGVRYYFEHKCLDVDLDGKSVLIQGKDGQPQRLQGDMIIGADGAHSAVRQAMQSGLRRFEFQQTFFRHGYKTLVLPDAQALGYRKDTLYFFGMDSGGLFAGRAATIPDGSVSIAVCLPYSGSPSLTTTDEPTMRAFFDRYFGGLPRDARDEMLRQFLAKPSNDLINVRSSTFHYKGNVLLLGDAAHATAPFLGQGMNMALEDARTFVELLDRHQGDQDKAFPEFTELRKVQADAMQDMARANYDVLSCSNPIFFMRARYTRYMHSKFPGLYPPDMAEKLYFTSEPYDRLQQIQRKQNVWYKIGRVN 。

Claims

1. a method for recovering a target product based on a synthetic organelle, comprising the steps of:

a. in the host, a synthetic organelle is constructed,

b. collecting a target product using the synthetic organelle;

c. recovering the synthetic organelle containing the target product;

2. The method according to claim 1, wherein the host comprises a cell-free expression system and a cellular host, preferably a prokaryotic host, more preferably e.

3. The method according to claim 1, wherein the synthetic organelle refers to an artificially designed compartmentalized structure, preferably the synthetic organelle is a membraneless organelle, is a biomolecular aggregate (biomolecular condensates) formed by the construction of polypeptides and/or proteins and/or DNA and/or RNA and/or nucleic acid protein complexes in a host, more preferably the synthetic organelle is formed by the construction of a biomolecular component selected from the group consisting of: nucleotide sequences such as triplets CAG, CCUG, GGGAA, GGGGCC and the like, which are repeated 10 to 1000 times in succession; amino acid sequences such as repeats of resinin-like peptides (RLP) that are repeated 3-500 times in succession; intrinsic non-structural regions, such as FUS protein, parB protein; multimerization of a multiplex biological macromolecule, such as PTB protein and UCUCU sequence repetition, an artificially designed binary protein two-dimensional structure, an artificially designed binary RNA two-dimensional structure.

4. The method according to claim 1, wherein the target product is one or more substances having a diameter in the range of 0.1-500 nm, preferably the target product is biosynthesized, more preferably the biosynthetic pathway of at least one component of the target product is encoded by heterologous DNA, comprising: heterologous biosynthetic small molecules such as deoxyviolacein (deoxyviolacein); recombinantly expressed polypeptides, proteins, RNAs, and DNAs, such as green fluorescent protein, red fluorescent protein, T4 DNA ligase, 16S rRNA; and multicomponent complexes, such as 30S ribosomal subunits, complexes of CRISPR-Cas with RNA.

5. The method according to any one of claims 1 to 4, wherein the collection in step b is in a non-specific and/or specific binding, the target product is derived from and/or generated within the synthetic organelle, preferably the synthetic organelle has a receptor, the target product has a ligand attached thereto such that the synthetic organelle is capable of specifically binding to the target product, preferably the receptor/ligand pair comprises: a complementarily paired nucleic acid molecule; capsid proteins of RNA phages such as MS2, PP7, qbeta, etc., and hairpin of translational operon RNA; protein N and B box (box B) RNA hairpin of DNA phage of lambda, 21, P22, etc.; CRISPR RNA hairpin and Cas6 proteins of bacteria such as pseudomonas aeruginosa (Pseudomonas aeruginosa); splitting two segments of the protein, such as split green fluorescent protein, alpha fragment and omega fragment of beta-galactosidase, split T7 RNA polymerase; an inducible dimerized or multimerized monomer, such as the light-controlled regulated multimeric green fluorescent protein Dronpa.

6. The method according to any one of claims 1 to 5, wherein step c comprises the isolation, recovery, purification of the synthetic organelles, preferably comprising the steps of:

c1. collecting and lysing the host;

7. The method according to any one of claims 1 to 5, wherein step d comprises the separation, recovery, purification of the target product, preferably comprising the steps of:

8. A DNA construct comprising nucleotide sequence 1 directing the host to construct a synthetic organelle as defined in the method of any one of claims 1 to 7 and/or nucleotide sequence 2 directing, regulating the production of a target product as defined in the method of any one of claims 1 to 7, preferably the DNA construct consists of said nucleotide sequence 1, 2 with a plasmid vector, such as pACYC184, pACYC177, pET28a (+), pET28b (+), pET-5a (+), pet43.1a, pET-37b (+), pcdfduret-1, pcoladat-1, prsduet-1, petduet-1, pUC57, pUC19, pBAD, pBluescript II SK (+), ptrcc, pthis 2C, pBV, pQE-70, pCold III, set-p, pr BFP, pGFPuv, pKD46, d4, pTYB1, ppn-2, p-5 b, pSB3, pSB 5C 4, pSB 5C.

9. An expression system comprising the DNA construct of claim 8, preferably the expression system refers to a system that constructs the synthetic organelle and produces the target product simultaneously in a prokaryotic cell, more preferably the expression system is an e.