Aptamer-rbp-based assembly for extracellular secretion of target chemicals, system for extracellular secretion of target chemical substance containing same, and production method of target chemicals using same

Abstract

The present invention relates to an assembly for extracellular secretion of a target chemical substance, which comprises a conjugate including an aptamer fused to RNA of the target chemical substance that can be produced intracellularly, and a transporter including an RNA-binding protein fused to a carrier protein; a gene construct encoding each of the transporter and the conjugate constituting the assembly; a vector carrying the gene construct; a host cell into having the vector introduced thereinto; a method for producing the target chemical substance, the method comprising a step of culturing the host cell; an extracellular secretion system of the target chemical substance constructed by introducing the gene construct or the vector into a microorganism; and a method for producing the target chemical substance by activating the system. By using the assembly and the system according to the present invention, it is possible to increase the intracellular production as well as extracellular secretion and excretion of the target chemical substance, and the invention can be advantageously applied to the production of microorganism-based useful chemical substances.

Description

Aptamer-RBP-based assembly for extracellular secretion of target chemicals, system for extracellular secretion of target chemicals including the same, and method for producing target chemicals using the same

[0001] The present invention relates to an aptamer-RBP-based assembly for extracellular secretion of a target chemical substance, a system for extracellular secretion of a target chemical substance comprising the same, and a method for producing a target chemical substance using the same.

[0002] Microbial-based production of valuable industrial and medical materials offers a promising and sustainable alternative to petroleum-based production methods, and its efficiency has a significant impact on the economics of bioprocesses.

[0003] Maximizing the potency, yield, or productivity of chemicals is a major challenge in microbial cell factories. Genetic engineering can address this through strain design and engineering, including DNA assembly, synthesis, gene editing, and the development of synthetic circuits. Furthermore, developing strategies to produce chemicals that surpass the inherent performance of engineered strains offers another avenue for enhancing productivity.

[0004] Among the chemicals produced by microorganisms, hydrophobic chemicals such as violacein derivatives and carotenoids often integrate or accumulate within cell membranes, limiting productivity. Furthermore, exposure to toxic environments, as in the case of bioindole pigments and carotenoids, can further reduce productivity. However, even if most major metabolites are secreted, enhancing extracellular secretion can improve overall productivity.

[0005] In this regard, there is a need to develop strategies to increase productivity by excreting chemicals produced by microorganisms. Excretion simplifies separation and purification, reduces bioproduction costs and downstream processing complexity, and enhances the potency of target chemicals. Furthermore, excretion of toxic chemicals minimizes disruption of membrane homeostasis and reduces intracellular accumulation, thereby maintaining cell growth and increasing potency.

[0006] Various strategies have been explored in secretion engineering studies using microorganisms or cells. One notable approach is exporter engineering, which involves discovering, designing, and overexpressing an exporter to enhance the secretion of a desired product. Exporter engineering not only mitigates the toxicity of a target chemical but also enhances the host strain's ability to produce the chemical by reducing intracellular product concentration, thereby maintaining product synthesis. However, finding a specific exporter suitable for each product or metabolite can be challenging, especially for heterologous products within the host strain.

[0007] Tailored engineering of cell morphology to expand intracellular space represents another approach to improving chemical accumulation within cell membranes and membrane-enclosed structures.

[0008] Membrane engineering enhances cell resistance to toxic internal or external environments, enabling them to utilize toxic substrates or produce toxic chemicals. While these engineering-based strategies simplify cell separation by altering cell size and shape, they often require additional steps, such as mechanical cell disruption, after separation, complicating downstream processing and increasing the cost of bioproduction.

[0009] Regulating target gene expression is essential for balancing cell growth, endogenous gene manipulation, and product formation. Furthermore, these strategies have limitations, as maximum potency cannot exceed the maximum intracellular storage capacity and limited membrane capacity. Excessive accumulation of intracellular chemicals can impose a metabolic burden on the host cell, negatively impacting cell physiology and metabolism, ultimately inhibiting cell growth and production.

[0010] Another approach to excreting intracellular products outside the cell involves constructing inducible cell lysis systems, such as the holin-endolysin system and the cell autolysis system, based on genes such as the lambda phage SRRz and the synthetic ribosome binding site. Strict control of cell lysis is crucial for the effective implementation of these systems.

[0011] Recent studies have shown that outer membrane vesicles can be repurposed for genetic material transport, detoxification, and interbacterial interactions and information transfer, offering a potential way to secrete accumulated chemicals into the extracellular medium. However, this approach requires careful consideration of the timing of vesicle formation and the prevention of unintended vesicle aggregation.

[0012] Despite these advances in secretion engineering strategies, they often require significant additional processing and lack specificity, potentially complicating the design process and reducing productivity. Therefore, designing platform-based release systems specifically targeting desired chemicals can streamline the design process, reduce time and costs, and improve purification processes. These systems can be applied to strains producing a variety of chemicals, offering performance advantages over existing strategies.

[0013] As a prior art related to a method for increasing the extracellular excretion and production of chemical substances using microorganisms, Korean Patent No. 10-2168039 discloses a microbial variant with improved extracellular production of heme, a complex salt that is increasingly used as a health food or food supplement for the treatment of porphyria, and a method for producing heme using the same. Korean Patent No. 10-2404998 discloses a microorganism comprising a multidrug efflux pump variant and a method for producing violacein or deoxyviolacein using the same.

[0014] In addition, Chinese patent CN 106591343 B discloses a method for extracellular secretion expression of a heterologous protein mediated by sfGFP (superfolder green fluorescent protein) in E. coli, and reveals that this method allows rapid optimization of expression conditions without affecting the function of the target protein, improves the yield of the target protein, and is suitable for large-scale production.

[0015] However, in the above patent documents, there is no report on an assembly composed of a conjugate of an aptamer and RNA, a transporter of a carrier and an RBP (RNA Binding Protein) fused thereto, which can be assembled within a microorganism (cell) producing a target chemical substance to enhance the extracellular secretion / excretion of the target chemical substance, and an extracellular secretion system of the target chemical substance including the same.

[0016] The present inventors have found that an RBP fused to an extracellularly secretable carrier protein, with or without a signal peptide, can function as a scaffolding molecule by specifically binding to an RNA fused to an aptamer specific for a target chemical, thereby enabling flexible and programmable secretion of a specific target chemical from cells.

[0017] In addition, the inventors of the present invention introduced a transporter including a carrier protein and an RBP fused thereto, and a conjugate including an aptamer specific for a target substance and an RNA fused thereto into E. coli as a testbed microorganism, and confirmed that these specifically bind to form an assembly through RBP-RNA interaction, and that the productivity and extracellular excretion of target chemicals such as protoporphyrin IX (PPIX), coproporphyrin III (CPIII), violacein (Vio), deoxyviolacein (Dvio), and prodeoxyviolacein (PDV) are improved by the assembly thus formed, thereby completing the present invention.

[0018] The assembly and system according to the present invention can be easily reconfigured simply by changing the aptamer sequence appropriate for the target chemical, thereby demonstrating high utility. Therefore, the assembly and system according to the present invention can not only improve the efficiency of extracellular secretion of target substances, but can also be universally applied to the development of strains for the production of various useful substances.

[0019] An object of the present invention is to provide an assembly comprising a transporter and a conjugate, which can improve extracellular secretion or excretion of a target substance to be produced within a cell.

[0020] Another object of the present invention is to provide a genetic construct comprising polynucleotides each encoding a transporter and a binding agent constituting the assembly.

[0021] Another object of the present invention is to provide a vector comprising the above genetic construct.

[0022] Another object of the present invention is to provide a host cell into which the above vector has been introduced.

[0023] Another object of the present invention is to provide a method for producing a target chemical substance, which comprises a step of culturing the host cell.

[0024] Another object of the present invention is to provide a system for extracellular secretion of a target chemical substance, constructed by introducing the above genetic construct into a microorganism.

[0025] Another object of the present invention is to provide a method for producing a target chemical substance using an extracellular secretion system.

[0026] To achieve the above purpose, the present invention provides an assembly for extracellular secretion of a target chemical, characterized in that the carrier protein comprises a transporter fused to an RNA binding protein (RBP), a binder fused to an aptamer, and the transporter and binder are assembled through RBP-RNA interaction.

[0027] Additionally, the present invention provides a genetic construct comprising a polynucleotide sequence encoding the transporter.

[0028] The present invention also provides a genetic construct comprising a polynucleotide sequence encoding the above complex.

[0029] In addition, the present invention provides a vector comprising the genetic construct.

[0030] In addition, the present invention provides a host cell into which the above vector has been introduced.

[0031] Additionally, the present invention provides a method for producing a target chemical, comprising a step of culturing the host cell.

[0032] In addition, the present invention provides a system for extracellular secretion of a target chemical substance, constructed by introducing the above genetic construct into a microorganism.

[0033] Additionally, the present invention provides a method for producing a target chemical using the above system.

[0034] An assembly comprising a transporter and a complex according to the present invention can stably form an assembly within a cell through RBP-RNA interaction, thereby easily and conveniently constructing a system for extracellular secretion / excretion of a target chemical substance.

[0035] The assembly and system according to the present invention can increase the intracellular productivity of a target chemical substance and improve the extracellular secretion efficiency, and are therefore useful for developing strains for producing various useful substances.

[0036] The assembly and system according to the present invention can select or design aptamers, RNAs, and RBPs according to host microorganisms and target chemicals, and thus have high value as a platform technology for producing target chemicals.

[0037] Figure 1 schematically depicts the structure of an assembly (abbreviated as 'BTSec assembly') for secretion of a target chemical substance, which comprises a binder (abbreviated as 'aptamer-RBP fusion binder', 'binding module') in which RNA is fused to an aptamer according to the present invention for secretion of a target chemical substance, and a transporter (transporter) in which a carrier protein is fused to an RNA binding protein (RBP) (abbreviated as 'carrier-RBP fusion transporter', 'transporter', or 'transport module') in which a carrier protein is fused to an RNA binding protein (RBP). The binding module and transport module constituting the BTSec assembly are combined through a specific interaction between RNA and RBP.

[0038] Figure 2 shows the results of examining the expression and secretion levels of four types of transporters according to the type of RBP using sfGFP as a carrier. (a) Schematic diagram of the transporters constituting the assembly. (b) SDS-PAGE experiment results on the degree of transporter release according to the type of RBP. (c) SDS-PAGE experiment results on the degree of transporter expression and secretion over time after IPTG induction. (d) Bar graph of the level of transporter secretion.

[0039] Figure 3 shows the predicted structures of sfGFP and sfGFP-binding RBPs modeled using the Protein 3D modeling program trRosseta and PyMOL. (a) sfGFP structure. (b) sfGFP:N22 structure. (c) sfGFP:Com structure. (d) sfGFP:MCP structure. (e) sfGFP:PCP structure.

[0040] Figure 4 is a diagram showing the structure and amino acid sequence of sfGFP-RBP analyzed using the Protein 3D modeling trRosseta program.

[0041] Figure 5 shows the results of an SDS-PAGE experiment showing whether rbGFP, a modified sfGFP-RBP, is released extracellularly.

[0042] Figure 6 shows the results of a protocatechuic acid (PCA) release experiment in an E. coli strain introduced with the AmiA, OmpA, and PelB signal peptides. (a) A bar graph showing the amount of protocatechuic acid produced when transporters and combinations using the AmiA, OmpA, and PelB signal peptides as carriers were introduced into the E. coli basic strain. (b) A bar graph showing the amount of protocatechuic acid produced according to the type of transporter.

[0043] Figure 7 schematically illustrates the complex constituting the assembly, and the predicted structures of each component of the complex that binds PPIX. (a) Components of the complex. (b) Predicted images of the PPIX-aptamer in Mfold and RNA Composer. (c) Predicted structures of the boxB RNA motif in Mfold and RNA Composer. (d) Predicted structures of the complex (binding module) that captures PPIX in Mfold and RNA Composer.

[0044] Figure 8 shows the intracellular and extracellular sfGFP:N22 expression levels when the BTSec assembly was applied to a PPIX-producing strain. (a) SDS-PAGE experiment results showing expression levels after L-arabinose induction. (b) Graph showing intracellular, extracellular, and total sfGFP:N22 expression levels.

[0045] Figure 9 illustrates an example of the application of the BTSec assembly to PPIX. (a) The biosynthetic pathway of PPIX. (b) A bar graph comparing total and extracellular PPIX production in a PPIX-producing strain and a strain into which the BTSec assembly was introduced. (c) A bar graph illustrating changes in total and extracellular PPIX production over time after induction. (d) A graph illustrating analysis of total and extracellular PPIX production during the culture period after induction.

[0046] Figure 10 shows the change in extracellular CPIII production when the BTSec assembly was applied to the PPIX-producing strain.

[0047] Figure 11 shows intracellular and extracellular sfGFP:N22 expression under various constitutive promoters. (a) SDS-PAGE results for sfGFP:N22 expressed under five promoters (PR(con), TacΔO, J23100, LacUV5, and J23114) 48 h after cell transfection. (b) Bar graph showing changes in relative secretion levels of the transporter under five promoters (PR(con), TacΔO, J23100, LacUV5, and J23114).

[0048] Figure 12 illustrates an example of the application of the BTSec assembly to CPIII. (a) Biosynthetic pathway of CPIII. (b) Bar graph comparing total and extracellular CPIII production in a CPIII-producing strain and a strain into which the BTSec assembly has been introduced.

[0049] Figure 13 illustrates examples of the application of BTSec assemblies to Vio, Dvio, and PDV. (a) Biosynthetic pathways of Vio, Dvio, and PDV. (b) Construction of an unknown RNA:aptamer library using overlap-PCR. (c) Construction of plasmids for chemical expression and screening of strains introducing BTSec assemblies and aptamer variants. (d) Bar graph of total and extracellular Vio production levels from the aptamer library. (e) Bar graph comparing total Vio production and extracellular production titers. (f) Bar graph of total and extracellular DVio production levels from the aptamer library. (g) Bar graph comparing total Dvio production and extracellular production titers. (h) Bar graph of total and extracellular PDV production levels from the aptamer library. (i) Bar graph comparing total PDV production and extracellular production titers.

[0050] Figure 14 shows the optimization of IPTG concentrations for sfGFP:N22 expression under the T7 promoter during application of BTSec assemblies to Vio, Dvio, and DPV producing strains. (a) Bar graph showing intracellular and extracellular sfGFP:N22 expression and extracellular sfGFP:N22 secretion levels at 24 h post-induction. (b) SDS-PAGE showing intracellular and extracellular sfGFP:N22 expression and extracellular sfGFP:N22 secretion levels at 36 h post-induction. (c) SDS-PAGE showing intracellular and extracellular sfGFP:N22 expression and extracellular sfGFP:N22 secretion levels at 48 h post-induction.

[0051] Figure 15 shows the relative gene expression levels of aptamers in the BL21(DE3)* strain. (a) A diagram showing aptamer expression in a strain cultured for 24 hours. (b) A diagram showing aptamer expression in a strain cultured for 48 hours. WT: wild-type strain; B: strain expressing PPIX aptamer conjugate; T: strain expressing sfGFP_N22 transporter; BT: strain co-expressing conjugate and transporter.

[0052] Hereinafter, the present invention will be described in detail.

[0053] As used herein, the term “fused” is used interchangeably with “binded,” “conjugated,” “jointed,” “linked,” “associated,” or similar terms, unless otherwise specified.

[0054] As used herein, the term “exocytosis” is used interchangeably with “exocytosis,” “exocytosis release,” or “exocytosis transport,” unless otherwise specified.

[0055] In one embodiment, the present invention relates to an assembly for extracellular secretion of a target chemical, comprising a carrier and a transporter including an RNA-binding protein (RBP) and a conjugate including an RNA and an aptamer, wherein the transporter and the conjugate are assembled through RBP-RNA interaction within a cell.

[0056] Unless otherwise specified, the term “transporter” is used interchangeably herein with the term “transport module” and the term “combination” is used interchangeably herein with the term “combination module”.

[0057] In the transporter, the carrier and RBP can have a structure in which they are directly bound or connected through a spacer.

[0058] RBP is directly bound to the N-terminus or C-terminus of the carrier or bound via a spacer, and one or more types of RBP corresponding to RNA constituting the complex may be bound singly or continuously.

[0059] In a specific embodiment, the transporter may have a structure of carrier-(RBP)n or carrier-spacer-(RBP)n or a structure in which the positions of the carrier and RBP are swapped, i.e., a structure of (RBP)n-carrier or (RBP)n-spacer-carrier, where n is an integer from 1 to 5. When n is 2 or more, the RBPs may be the same or different from each other.

[0060] The spacer is positioned between the RNA and the aptamer and may be 5 to 45 nucleotides in length, specifically 10 to 40 nucleotides in length, and more specifically 20 to 30 nucleotides in length, but is not limited thereto.

[0061] Carriers (or carrier proteins) serve to transport assemblies containing transporters or transporters and complexes out of the cell.

[0062] In the present invention, the carrier can be distinguished by the presence or absence of a signal peptide, and can be a non-signal peptide-based or signal peptide-based protein.

[0063] A non-signal peptide-based carrier refers to a carrier protein that can move out of the cell and be excreted without a signal peptide.

[0064] The non-signal peptide-based carrier may be, but is not limited to, superfolder green fluorescent protein (sfGFP) or a derivative thereof.

[0065] sfGFP may be, but is not limited to, the amino acid sequence represented by SEQ ID NO: 1 or an sfGFP having at least 90% sequence identity thereto.

[0066] sfGFP (superfolder green fluorescent protein) is a fluorescent protein designed to improve upon the properties of the existing GFP (green fluorescent protein) molecule. It has a unique beta-barrel structure. With this structural feature, sfGFP exhibits auto-secretion properties in Escherichia coli.

[0067] Various derivatives of sfGFP are known, including, for example, vsfGFP-9, oxGFP, moxGFP, vsGFP, usGFP, muGFP, vsfGFP-0, rsFolder, rsFolder2, pH-tdGFP, spGFP1-10, spGFP11, sfYFP, sfCFP, sfBFP, esGFP, sfpHluorin, mChartreuse, etc.

[0068] Sequence information, production methods, and acquisition methods of sfGFP and derivatives can be obtained through known databases, such as NCBI and FPbase.

[0069] In one embodiment, a protein (rbGFP) was designed in which the helix structure of sfGFP was replaced with the λN22 RBP structure.

[0070] rbGFP may be, but is not limited to, an rbGFP having the amino acid sequence represented by SEQ ID NO: 2 or having at least 90% sequence identity thereto.

[0071] In the case of transporters adopting rbGFP as a carrier, the extracellular excretion rate was found to increase.

[0072] Therefore, any protein that has a structure identical or similar to the beta barrel of sfGFP and has auto-crine activity can be used as a carrier in the transporter of the present invention.

[0073] In the present invention, the carrier may be a signal peptide-based carrier.

[0074] A signal peptide-based carrier protein refers to a carrier protein that contains a signal peptide.

[0075] In the present invention, the signal peptide may be a signal peptide used to export a protein in a microorganism such as E. coli, and is not limited thereto, but may be any one signal peptide selected from among phoA, PelB, DsbA, NSP4, NSP2, FhuD, MalE, OmpA, PhoE, OmpC, Lpp, LamB, OmpT, eltB, GH1, sta1, Xyn5, AnsB, MglB, TorT, sfmC, bla, npr, and AmiA, and more specifically, may be any one signal peptide selected from among PelB, OmpA, and AmiA. Relevant information including specific sequences for signal peptides can be obtained from the signal peptide DB site (http: / www.signalpeptide.de / ), Uniprot, etc.

[0076] In the present invention, the conjugate may have a structure in which RNA and aptamer are directly bound or connected through a spacer.

[0077] In the complex, RNA may be in the form of one or more types of RNA corresponding to RBPs constituting the transporter, either singly or sequentially bound.

[0078] In a specific embodiment, the complex may have a structure of aptamer-(RNA)p, aptamer-spacer-(RNA)p, or spacer-aptamer-spacer-(RNA)p, or a structure in which the positions of the aptamer and RNA are switched, i.e., a structure of (RNA)p-aptamer, (RNA)p-spacer-aptamer, or (RNA)p-spacer-aptamer-spacer, wherein the spacers may be the same or different, and p is an integer from 1 to 5. When p is 2 or more, the RNAs may be the same or different. In terms of maintaining a stable structure with RBP and maintaining a desirable three-dimensional shape for extracellular secretion, it may be preferable that p is 2 or 3.

[0079] In the conjugate, the spacer can be linked to the 5' end or the 3' end of the aptamer sequence.

[0080] In a specific embodiment, the conjugate may be a structure comprising a spacer between the RNA and the aptamer. In another specific embodiment, the conjugate may be a structure wherein a spacer, identical or different, is additionally linked to the end of the aptamer to which the spacer is not linked.

[0081] The spacer is not particularly limited in length and base sequence (continuity) as long as it can play a role in resolving steric hindrance and facilitating binding of RNA and RBP and binding of aptamer and target substance.

[0082] The phrase "resolves steric hindrance" in this specification means that the introduction of a spacer into the conjugate does not substantially affect the inherent structural, conformational, or functional stability of the RNA or aptamer. Known RNA structure prediction programs, such as Mfold and RNA composer, can be used to predict the suitability of the introduced spacer.

[0083] For example, the spacer may be from 5 to 45 nucleotides in length, specifically from 10 to 40 nucleotides in length, and more specifically from 20 to 30 nucleotides in length, but is not limited thereto.

[0084] In the present invention, RBP refers to a protein that binds to RNA of a complex, and specifically refers to a protein that specifically binds to one RNA.

[0085] The RBP may be composed of about 22 to about 133 amino acids and may have a size of about 2.7 to about 14.2 kDa, preferably composed of 22 to 130 amino acids and may have a size of 2.7 to 14 kDa, more preferably composed of 22 to 62 amino acids and may have a size of 2.7 to 7.4 kDa, and more preferably composed of 22 amino acids and may have a size of 2.7 kDa, but is not limited thereto.

[0086] RBP can be designed according to the desired RNA sequence, shape, or structure, or can be adopted from known ones.

[0087] Known RBPs include, but are not limited to, λN22 (abbreviated as N22), MCP (Escherichia MS2 coat protein), PCP (PP7 coat protein), and Com protein.

[0088] λN22 is a protein composed of 22 amino acids, MCP is a protein composed of 130 amino acids, PCP is a protein composed of 128 amino acids, and Com is a protein composed of 62 amino acids. Information on their specific sequences, structures, production methods, and availability can be obtained from publicly available databases such as NCBI and Uniprot.

[0089] In the present invention, RNA is RNA that can specifically bind to a corresponding RBP, and may be, for example, a single-stranded RNA having a hairpin structure. Examples of RNA include, but are not limited to, any one selected from MS2 RNA, box B RNA (also used interchangeably with box B RNA motif and abbreviated as box B), Com RNA, and PCP RNA.

[0090] MS2 RNA binds specifically to MCP, Box B RNA binds specifically to λN22, Com RNA binds specifically to Com protein, and PCP RNA binds specifically to PCP. Information on the sequence, structure, production method, and availability of these known RNAs can be obtained from known databases such as NCBI.

[0091] In addition to the above RBP and RNA, QCP (Qbeta coat protein) and its specific RNA, QCP RNA, Bacteriophage GA coat protein and its specific RNA, GA RNA, R17 coat protein and its specific RNA, R17 RNA, can be introduced into the assembly according to the present invention.

[0092] In the present invention, the "aptamer" is a "programmable" aptamer in that it can be appropriately designed and selected depending on the microorganism and the target chemical. Specifically, if an aptamer exhibiting appropriate binding activity to the target chemical is not known, the aptamer can be custom-made using a known aptamer library construction method; and if an aptamer already known to exhibit appropriate binding activity to the target substance exists, such an aptamer can be used without special modification or with appropriate modification.

[0093] The term "aptamer" in the present invention refers to a single-stranded oligonucleotide nucleic acid molecule that has binding activity toward a specific target substance. Aptamers may be, but are not limited to, 20 to 60 nucleotides in size, and may have various three-dimensional structures depending on the sequence and exhibit high affinity for a specific substance. The aptamer of the present invention may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be linear or cyclic.

[0094] Known aptamers include, but are not limited to, PPIX (protoporphyrin IX), CPIII (coproporphyrin III), organic dyes, D-tryptophan, L-valine, theophylline, cyanocobalamin, L-citrulline, flavin mononucleotide, flavin adenine dinucleotide, kanamycin A, kanamycin B, kanamycin, neomycin, tobramycin, libidomycin, nicotinamide adenine dinucleotide, riboflavin, biotin, L-arginine, dopamine, 7-methyl-guanosine, CCdApPuro, chloramphenicol, biomycin, sulforhodamine, streptomycin, L-isoleucine, 8-oxodG, xanthine, guanine, malachite green, phosphatidylcholine, cyclic adenosine monophosphate, adenosine Examples include aptamers specific for any one of triphosphate, L-tyrosine, S-adenosyl homocysteine, moenomycin A, sialyl Lewis X, tetracycline, adenine, morpholine-based GTP analogs, 4,4-methylenedianiline, isoleucine, L-histidine, codeine, mesomesoprotoporphyrin IX, thyroxine, 10-carboxy-2,7-di-t-butyl-trans-12c,12d-dimethyl-12c,12d-dihydrobenzo[e]pyrene, dimethylindole red, cyanine, aniline-substituted sulforhodamine analogs, atrazine, sphingosylphosphorylcholine, black hole quenchers, 4-dimethylaminobenzylidene imidazolinone, glutathione, and heteroaryldihydropyrimidines (M. McKeague et al,. Challenges and opportunities for small molecule aptamer development. Journal of nucleic acids. (2012) 1-20. reference).

[0095] An aptamer specific for PPIX or CPIII may be, but is not limited to, an aptamer comprising the base sequence of SEQ ID NO: 17, a base sequence having at least 90% sequence identity, or a sequence in which thymidine is substituted with uracil in the base sequence of SEQ ID NO: 17.

[0096] If a suitable aptamer for the target chemical is not known, a suitable aptamer can be obtained, for example, through random screening or SELEX (Systematic Evolution of Ligands of Exponential Enrichment).

[0097] In the case of random screening, for example, in the case of aptamers for Vio, Dvio, or PDV, an aptamer is designed using primers containing random sequences of SEQ ID NO: 24 and SEQ ID NO: 25, and then the aptamer is incorporated into a complex through overlapping-PCR, introduced into cells, and then cultured to select cells that increase the amount of the target substance released, thereby obtaining an aptamer specific for the target chemical.

[0098] For SELEX, suitable aptamers can be designed to bind to specific target molecules using known in vivo or in vitro selection techniques.

[0099] SELEX can be used to select aptamers with specific RNA sequences that have high affinity and specificity for a target chemical from a diverse RNA library (see Gopinath SC. Methods developed for SELEX. Anal Bioanal Chem. 2007 Jan;387(1):171-82).

[0100] In certain embodiments of the present invention, the aptamer may be an RNA aptamer specific for any one target chemical selected from Vio (violacein), Dvio (deoxyviolacein), and PDV (prodeoxyviolacein).

[0101] Aptamers specific for Vio may include, but are not limited to, aptamers comprising the base sequence of SEQ ID NOs: 26 to 31, a base sequence having at least 90% sequence identity, or a base sequence in which thymidine is substituted with uracil in the base sequence of SEQ ID NOs: 26 to 31.

[0102] As an aptamer specific to Dvio, an aptamer comprising a base sequence of SEQ ID NOs: 32 to 37, a base sequence having at least 90% sequence identity, or a base sequence in which thymidine is substituted with uracil in the base sequence of SEQ ID NOs: 32 to 37 can be used, but is not limited thereto.

[0103] Aptamers specific for PDV may include, but are not limited to, aptamers comprising a base sequence of SEQ ID NOs: 38 to 43, a base sequence having at least 90% sequence identity, or a base sequence in which thymidine is substituted with uracil in the base sequence of SEQ ID NOs: 38 to 43.

[0104] According to the present invention, an aptamer having a specific random sequence for Vio, Dvio, and PDV was integrated into a conjugate through overlapping PCR to form a conjugate library, through which a specific aptamer could be selected.

[0105] In another embodiment, the present invention provides a genetic construct for expression of a transporter constituting the assembly, wherein a polynucleotide encoding a carrier protein is operably linked to a polynucleotide encoding an RBP; and

[0106] As a genetic construct for expression of a combination constituting the above assembly, a polynucleotide encoding an aptamer may be a genetic construct operably linked to a polynucleotide encoding RNA.

[0107] The above genetic construct can be inserted into a carrier, e.g., a vector or plasmid, for expression in a cell or microorganism.

[0108] A vector is a DNA molecule that can introduce a desired DNA fragment into a host cell and propagate it. It is also called a cloning vehicle. A vector can be a plasmid, a phage particle, or simply a potential genomic insert. Since plasmids are currently the most commonly used form of vector, the terms "vector" and "plasmid" are sometimes used interchangeably herein.

[0109] The carrier used in the present invention may be any one of pET28a, pCop, pTac15K, p15A, pBBR1, pCloDF13, pT7, and pETM6.

[0110] A vector containing a gene construct for expression of the aptamer may additionally include a spacer that acts as a linker between the RNA-encoding sequence and the aptamer-encoding sequence.

[0111] The spacer may be 5 to 45 nucleotides in length, specifically 10 to 40 nucleotides in length, and more specifically 20 to 30 nucleotides in length.

[0112] In another embodiment, the present invention relates to a host cell introduced with a vector comprising nucleic acid molecules encoding a transporter and a binding agent, respectively, constituting an assembly.

[0113] Host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells.

[0114] In a particular embodiment of the present invention, the host cell into which the vector according to the present invention has been introduced may be a bacterial cell, for example, an Escherichia coli cell. More specifically, the E. coli cell may be a cell of the DH5α or BL21 (DE3) Star strain.

[0115] In another embodiment, the present invention relates to an extracellular secretion system of a target substance constructed by introducing into a microorganism a genetic construct encoding a transporter and a binder, each of which constitutes an assembly according to the present invention.

[0116] Microorganisms include, for example, Escherichia coli, lactic acid bacteria (e.g., Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus), Lactobacillus, Streptomyces, Gluconobacter, Acetobacter, Bacilus such as Bacilus thuringiensis, Chlamydia, Saccharomyces, Caproiciproducens, Clostridium, Mycobacterium, Enterococcus, Brucella, Propionibacterium, Aspergillum, These may include, but are not limited to, microorganisms used for industrial purposes, such as Listeria, Penicillium, and Sinorhizobium.

[0117] In a particular embodiment of the present invention, the microorganism into which the genetic construct according to the present invention has been introduced may be Escherichia coli. More specifically, the E. coli may be a DH5α or BL21 (DE3) Star strain.

[0118] In another embodiment, the present invention relates to a method for producing a target chemical, characterized in that it comprises a step of activating an extracellular secretion system of the target chemical.

[0119] As used herein, the phrase “activating the extracellular secretion system” means simply culturing a cell or microorganism into which a genetic construct or a carrier (vector or plasmid) containing the genetic construct has been introduced, or additionally culturing the cell or microorganism under various stimuli such as mechanical or electrical stimulation, to enhance the production level of the desired chemical substance.

[0120] As used herein, the phrase “produces a target chemical substance” means, for example, that the extracellular secretion system according to the present invention, i.e., a microorganism, is cultured in a suitable medium so that the system produces, secretes, or excretes the target chemical substance in an amount such that the target chemical substance can be recovered from the medium.

[0121] Examples of chemicals that can be produced by a system incorporating an assembly according to the present invention include nucleic acids, vitamins, antibiotics, growth factors, and physiologically active substances, as long as an excretion system for various amino acids, biosynthetic intermediates, or substrates exists. Furthermore, a system incorporating an assembly according to the present invention can produce chemicals that are currently unproducible by designing an appropriate aptamer, as long as a system exists for the intracellular uptake of byproducts of the target chemical or substrates of the biosynthetic system of the target chemical.

[0122] The above production method of the present invention may additionally include a step of separating and purifying the target substance.

[0123] In a particular embodiment of the present invention, the target chemical may be selected from the group consisting of PPIX, CPIII, Vio, Dvio, and PDV.

[0124] According to a specific embodiment of the present invention, the present inventors designed a BTSec assembly to promote the extracellular secretion of a specific chemical substance in E. coli. The assembly comprises a transporter and a conjugate (see Fig. 1). The transporter comprises a carrier and an RBP (see Fig. 2a). When sfGFP was used as a carrier, the excretion of transporters conjugated with four types of RBPs was compared. As a result, sfGFP:N22 was excreted at the highest level, followed by sfGFP:PCP, sfGFP:Com, and sfGFP:MCP in that order (see Fig. 2). Structural analysis of sfGFP:N22 confirmed that the beta-barrel structure of sfGFP was maintained (see Fig. 3). Based on these results, to confirm whether the beta-barrel structure of sfGFP is important for its role as a carrier, rbGFP was designed by substituting amino acids in the helix domain of sfGFP, and the extracellular excretion of rbGFP was found to increase. Accordingly, it was confirmed that a protein with a structure similar to that of sfGFP can be used as a carrier (Table 1; see Figs. 4 and 5). Subsequently, in order to confirm whether a signal peptide that has been widely used in the past can also be used as a carrier, it was introduced into E. coli, and as a result, it was confirmed that the target chemical substance was excreted extracellularly in the strain into which the signal peptide was introduced, confirming that it can be used as a carrier (Table 3; see Fig. 6). In addition, in order to determine whether the BTSec assembly can increase the production of the target substance, the assembly was introduced into a PPIX-producing strain, and the structure of the conjugate in this strain was analyzed, confirming that the structure of each component was maintained (see Fig. 7). In addition, it was confirmed that the expression level of the transporter in the PPIX-producing strain into which the assembly was introduced was higher than in the strain into which only the transporter was introduced (see Fig. 8), and the expression level of the transporter increased when both the transporter and the conjugate were introduced compared to when only the transporter was introduced.By introducing an assembly containing a transporter and a conjugate into a PPIX-producing strain and activating the BTSec assembly, the extracellular excretion and production were increased compared to the PPIX-producing strain, and it was confirmed that the excretion increased without reaching saturation over time (see Fig. 9). In addition, CPIII, which has a similar structure to PPIX, was also confirmed to have increased excretion and production when an assembly containing the same aptamer was introduced (see Figs. 10, 11, and 12). In order to investigate whether the assembly of the present invention can be applied even when the sequence of the aptamer is unknown, aptamer libraries were constructed for Vio, Dvio, and PDV, and specific aptamers for each were selected (see Fig. 13). Vio-, Dvio-, and PDV-producing strains were prepared, and it was confirmed that the excretion of the transporter increased in the strains (see Fig. 14). The conjugate containing the selected aptamer and the assembly containing the transporter were introduced into a host strain (E. coli) and the changes in the extracellular excretion and production of the target substance were measured. As a result, it was confirmed that both the extracellular excretion and production increased (see Fig. 13). As a result of checking whether the aptamer within the conjugate inserted into the cell was expressed, it was confirmed that the gene expression level of the aptamer increased in a strain that simultaneously expressed the conjugate and the transporter (see Fig. 15).

[0125] <Example 1> Materials and methods

[0126] <Example 1-1> Materials and strains

[0127] Vio was purchased from Sigma-Aldrich. CPIII and DVio were purchased from Santa Cruz Biotech (SCBT). Escherichia coli DH5α was used for molecular cloning and plasmid propagation. The cells were cultured in Luria-Bertani (LB) medium at 37°C. E. coli BL21 (DE3) Star was used for the production of Vio, DVio, PDV, PPIX, and CPIII derivatives.

[0128] <Example 1-2> Culture conditions

[0129] PPIX and CPIII producing strains were inoculated into 30 mL test tubes containing 5 mL of LB broth (10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract) with appropriate antibiotics and cultured overnight at 37°C. For flask culture, 500 μL aliquots of CPIII and PPIX starter cultures were transferred to 250 mL baffled flasks containing 50 mL of LB medium and cultured at 37°C for 48 h. Antibiotics (50 mg / L kanamycin) were added when necessary.

[0130] The strains producing Vio, Dvio, and PDV were inoculated into 30 mL test tubes containing 5 mL of LB broth (10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract) with appropriate antibiotics and cultured overnight at 37°C. For large-scale experiments, 50 μl of the seed culture was transferred to a 30 mL test tube containing 5 mL of LB medium, 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added, and the culture was incubated at 37°C for 48 h. For flask culture, an aliquot of the seed culture was transferred to a 250 mL baffled flask containing 50 mL of LB medium, and the OD 600 The culture was cultured to an IP value of 0.5 to 0.6, 0.1 mM IPTG was added, and then cultured at 30°C for 48 h. If necessary, antibiotics (ampicillin 100 mg / L, kanamycin 50 mg / L, chloramphenicol 25 mg / L, streptomycin 50 mg / L) were added.

[0131] When the transporter is in the inducible assembly, the culture conditions are similar to those above, but the inducer concentration and addition time are different. For IPTG and arabinose induction of the expressed transporter, an aliquot of the seed culture was transferred to the main culture medium, and then the OD 600 When nm reached 0.5 to 0.6, 0.1 mM IPTG and 0.1% arabinose were added, respectively.

[0132] <Example 1-3> Analysis method

[0133] The secretion efficiency of the transporter candidates was measured using SDS-PAGE analysis. Cells were cultured overnight in LB broth in a shaking incubator at 37°C and 200 rpm. The saturated culture was transferred to a 250-mL baffled flask containing 50 mL of LB medium. OD was collected every 12 h for up to 48 h. 600 Culture samples with a value of 5 were centrifuged at 6,000 rpm for 3 minutes. The pellets and supernatants were boiled in loading buffer for 10 minutes, separated by SDS-PAGE (12% acrylamide gel), and stained with Coomassie blue. Bands were quantified using ImageJ software, and band intensities in the Coomassie blue-stained gel were normalized to each control protein band in the gel.

[0134] For microplate reader analysis, the transformed strain library was cultured in 5 mL of LB medium at 30°C for 48 h. Each sample was centrifuged at 16,000 × g for 1 min, and the culture was separated into a pellet and supernatant. The culture was diluted 1 / 5 with DMSO and bleached in an ultrasonic bath (WUC.D06H, DAIHAN Scientific, Korea) at 60°C for 10 min. Total production was measured.

[0135] PPIX and CPIII were dissolved in HCl. Vio and Dvio were dissolved in DMSO and EtOH, respectively. The maximum absorption wavelength was determined by full-field scanning from 230 to 750 nm using a microplate reader (Synergy H1 Plate Reader, BioTek). The standard curves for each substance were measured at 409 nm for PPIX and CPIII, and at 575 nm for Vio and Dvio.

[0136] For HPLC analysis, the concentrations of PPIX, CPIII, Vio, Dvio, and PDV were measured using HPLC (1260 Infinity II, Agilent) equipped with a ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 5 μm, Agilent).

[0137] For PPIX analysis, the culture supernatant was separated from the medium by centrifugation at 16,000 g for 3 minutes, and an equal volume of 0.2 N HCl dissolved in methanol was added and mixed rigorously for 3 minutes. To quantify the total production of PPIX, 100 μl of cell culture medium was added to 900 μl of 0.1 N HCl dissolved in methanol (10-fold dilution) and bleached in an ultrasonic bath at 40°C for 60 minutes. All samples were filtered through a 0.2 μm PVDF syringe filter. For CPIII analysis, the culture supernatant was separated from the medium by centrifugation at 4,000 g for 10 minutes, and the supernatant was mixed with an equal volume of 1 N HCl and mixed for 3 minutes. To quantify the total production of CPIII, the pellet was resuspended in 1 mL of 1 N HCl and the cells were disrupted using an ultrasonic bath at 40°C for 60 minutes.

[0138] The mobile phase consisted of solvent A [0.1% trifluoroacetic acid] and solvent B [0.1% TFA in ACN] with the following gradient: 0–1 min, 90% solvent A and 10% solvent B; 1–3 min, linear gradient of 90% to 75% solvent A; 3–8 min, linear gradient of 75% to 55% solvent A; 8–10 min, linear gradient of 45% to 55% solvent B; 10–12 min, linear gradient of 55% to 100% solvent B; 12–15 min, linear gradient of 0% to 100% solvent A; 15–16 min, linear gradient of 100% to 90% solvent A. The mobile phase was delivered at 1 mL / min. Product concentration was measured using a diode array detector at 400 nm.

[0139] For extracellular Vio, Dvio, and PDV, a 0.5 mL aliquot of cultured cells was centrifuged at 16,000 g for 1 min, and the supernatant was mixed with a 1:1 equal volume of ethyl acetate. Vio, Dvio, and PDV were thoroughly mixed for 3 min to ensure sufficient dissolution in the organic solvent, and only the organic layer was filtered. To quantify the total production of Vio, Dvio, and PDV, 100 μl of cell culture medium was added to 400 μl of DMSO (5-fold dilution) and bleached in an ultrasonic bath at 60°C for 10 min. All samples were filtered through a 0.20 μm nylon syringe filter for HPLC analysis. The mobile phase consisted of solvent A [0.1% (v / v) formic acid in distilled water] and solvent B [acetonitrile] with the following gradient: 0–5 min, 95% solvent A; 5–8 min, linear gradient 55% to 45% solvent A; 8–10 min, linear gradient 95% solvent B; 10–13 min, 95% solvent A. The mobile phase was delivered at a rate of 1.2 mL / min, and the column temperature was maintained at 30 °C. Product concentration was measured at 570 nm using a diode array detector (G7117C, Agilent).

[0140] <Example 2> Design and construction of a synthetic BTSec assembly

[0141] <Example 2-1> Design of a synthetic BTSec assembly

[0142] A synthetic BTSec assembly was designed to promote the extracellular secretion of specific chemicals in Escherichia coli. This assembly consists of two functional modules: a transport module and a binding module. Components of each module were engineered to produce a fused protein (see Figure 1).

[0143] <Example 2-2> BTSec assembly containing sfGFP as a carrier

[0144] We constructed a transporter for chemical secretion in Escherichia coli cells. To promote extracellular secretion of the transporter, sfGFP, having the amino acid sequence represented by SEQ ID NO: 1, was utilized as a carrier (Fig. 2a; see Table 1).

[0145] Existing protein secretion systems typically rely on signal peptides or tags, but their effectiveness varies depending on the target protein's coding sequence. To enable more universal use, we selected sfGFP, a non-peptide, auto-secretory protein characterized by a beta-barrel structure and negative net charge.

[0146] Well-characterized viral RBPs were utilized as RBPs constituting the transporter, including λN22 plus (N22) with sequence number 3, MS2 coat protein (MCP) with sequence number 5, Com protein with sequence number 7, and PP7 coat protein (PCP) with sequence number 9. Since each protein can specifically recognize and bind to the viral RNAs corresponding to boxB with sequence number 4, MS2 with sequence number 6, com with sequence number 8, and PP7 with sequence number 10, these RNAs were used as RNAs of the binding module (see Table 2). MCP and PCP were expressed as ΔFG mutants lacking residues between the F and G β-strands to prevent capsid formation in E. coli. In addition, affinity-enhanced forms of MCP (ΔFG / V29I) and λN22 plus were used.

[0147] Each RBP was fused to sfGFP, and the extracellular secretion levels of these fusion proteins expressed under the control of the T7 promoter were quantified by measuring band intensities on SDS-PAGE (see Fig. 2b).

[0148] As a result, among the tested combinations, sfGFP:N22 and sfGFP:PCP showed secretion rates of approximately 120 to 150% compared to sfGFP alone, whereas sfGFP:Com and sfGFP:MCP showed decreased secretion rates (see Fig. 2b). When the secretion level of sfGFP:N22, the combination with the highest relative secretion rate compared to sfGFP alone, was examined over time, secretion began after 24 hours and continuously increased after 36 and 48 hours, showing the highest secretion rate. sfGFP:PCP also showed a high secretion rate over time (see Figs. 2c and 2d).

[0149] Analysis of the transporter structure revealed that sfGFP:N22 maintained the original shape of the β-barrel structure of sfGFP due to its small size and helical structure, and was confirmed to be suitable as a transporter for recruiting the binding module in subsequent experiments (see Figs. 3a, 3b). In addition, sfGFP:Com and sfGFP:PCP also appeared to partially maintain the original structure of sfGFP, whereas sfGFP:MCP did not maintain the original structure of sfGFP (see Figs. 3c, 3d, 3e).

[0150] <Example 2-3> Design and emission confirmation experiment of rbGFP

[0151] Based on the fact that proteins with a beta-barrel structure and a negative net charge are effective for excretion, the inventors analyzed the structure of a fused sfGFP and RBP using the trRosseta program. Since the analyzed RBPs, PCP and λN22, have a helical structure, we hypothesized that replacing the helix structure in sfGFP with the RBP structure would not affect the charge even if further fused with RBP.

[0152] To maintain the surface charge of sfGFP, the helix domain was modified through site-directed mutagenesis. Reanalysis of its structure confirmed that rbGFP contains the λN22 RBP (Table 1; see Fig. 4). After cloning rbGFP, which has the amino acid sequence represented by SEQ ID NO: 2, into a vector, the extent of its extracellular release was examined.

[0153] As a result, it was confirmed that the emission rate increased (see Fig. 5).

[0154] Amino acid sequence and base sequence of the helix domain of sfGFP and rbGFPsfGFPrbGFPamino acid sequenceMSKGEELFTGVVPILVELDGDVNGHKFSVREGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRPDHMKRHDFFKSAMPEGTVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO. 1)MGHHHHHHSGMSKGEELFTGVVPILVELDGDVNGHKFSVREGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYMNARTRRRRERRAEKQAQWKAANMPEGTVQERTISFKDDGTYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGS (SEQ ID NO: 2)

[0155] *The underlined part indicates the helix domain.

[0156] Base sequences of RBP and RNA used in the examplesRBPRNA base sequence (5'-3')Source and information acquisition Lambda N22 plus protein (λN22 plus)boxBλN22 plus: Atgaatgcacgcacacgccgccgcgaacgtcgcgcagagaaacaggctcaatggaaagcagcaaattaa (SEQ ID NO: 3)Designed Arginine-Rich RNA-Binding Peptides with Picomolar Affinity DOI: 10.1021 / ja026610bBoxB RNA:Gggccctgaagaagggccc (SEQ ID NO: 4)Bacteriophage MS2 coat protein (MCP)MS2 RNAMCP: Atggggcccgcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaacttcgctaacgggatcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaa tacaccatcaaagtcgaggtgcctaaaggcgcctggcgttcgtacttaaatatggaactaaccattccaattttcgccacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctacTGA (SEQ ID NO: 5) Zalatan et al., 2015 Cell.Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA ScaffoldsMS2 RNA:gcgcACATGAGGATCACCCATGTgc (서열번호 6)Com 단백질 (Com)Com RNACom: Atgaaatcaattcgctgtaaaaactgcaacaaactgttatttaaggcggattcctttgatcacattgaaatcaggtgtccgcgttgcaaacgtcacatcataatgctgaatgcctgcgagcatcccacggagaaacattgtgggaaaagagaaaaaatcacgcattctgacgaaaccgtgcgttattga (서열번호 7)Zalatan et al., 2015 Cell. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA ScaffoldsCom RNA:CTGAATGCCTGCGAGCATC (서열번호 8)박테리오파지PP7외피 단백질 (PCP)PP7 RNAPCP: Atgtccaaaaccatcgttctttcggtcggcgaggctactcgcactctgactgagatccagtccaccgcagaccgtcagatcttcgaagagaaggtcgggcctctggtgggtcggctgcgcctcacggcttcgctccgtcaaaacggagccaagaccgcgtatcgagtcaacctaaaactggatcaggcggacgtcgttgattgctccaccagcgtctgcggcgagcttccgaaagtgcgctacactcaggtatggtcgcacgacgtgacaatcgttgcgaatagcaccgaggcctcgcgcaaatcgttgtacgatttgaccaagtccctcgtcgcgacctcgcaggtcgaagatcttgtcgtcaaccttgtgccgctgggccgttaa(서열번호 9)Zalatan et al., 2015 Cell.Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA ScaffoldsPP7 RNA:aacaTAAGGAGTTTATATGGAAACCCTTAtg (서열번호 10)박테리오파지 Qbeta 외피 단백질 (QCP)QCP RNAQCP: Atggctaaattacaagctatcactttaagtggtattgggaagaacggtgacgttactctgaacctcaacccgcgtggggtaaatcccaccaacggtgttgccgcgctttcagaagcgggtgcagttcctgcattggagaagcgtgttacaatttctgtatcacagccttctcgcaatcgtaagaactacaaagttcaggtaaagatccagaacccaacctcttgcactgcaagcggtacttgtgacccttcagttactcgttcggcttatgctgacgtgacgttctcgttcacgcagtacagcactgatgaggaacgtgcactcgtacgaacagagcttaaagccctgttggcggatccaatgcttatcgatgctatcgataacttgaatccggcgtactga (서열번호 11)Francis Lim, Marc Spingola, David S.Peabody,The RNA-binding Site of Bacteriophage Qβ Coat ProteinQCP RNA:TAAGGATGAAATGCATGTCTAAGACAGCAT (서열번호 12)박테리오파지GA 외피 단백질GA RNAGA: Atggcaactttacgcagtttcgtactcgtcgataatggcggtacggggaatgttactgtcgttcctgttagcaatgccaacggcgtcgctgagtggctttctaataactcgcgcagtcaggcttatcgcgtgactgccagttatcgtgcgtcaggcgcggacaagcgcaaatatgccattaaacttgaagtaccgaaaatcgttacccaagttgtaaatggtgttgagctgcctggttccgcatggaaggcttatgcctctatcgacctgaccatccctatctttgctgcaaccgacgacgtgactgttatttccaagtcgctcgccggcctgttcaaagttgggaaccctatcgctgaagctatctcttcacagagtggcttctacgcgtaa(서열번호 13)Tars, K et al. "The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses." Journal of molecular biology vol. 271,5 (1997): 759-73. doi:10.1006 / jmbi.1997.1214GA RNA:AAACATAAGGAAAACCTATGTT (SEQ ID NO: 14) Bacteriophage R17 coat protein R17 RNAR17: Atggcttctaactttactcagtttgttctcgtcgacaatggcggaactggcgacgtgactgtcgctccaagcaacttcgctaacggggtcgctgaatg gatcagctctaactcgcgctcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccattaaagtcgaggtgc ctaaggtggcaactcagactgttggtggtgtagagcttcctgtagccgcatggcgttcgtacttaaatatggaattaactattccaattttcgctacgaactccgattgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctactaa (SEQ ID NO: 15) Zalatan et al., 2015 Cell. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA ScaffoldsR17 RNA:AACATAAGGAGTTTATATGGAAACCCTTATG (SEQ ID NO: 16).

[0157] *In the RNA base sequence, the base T (thymidine) represents U (uracil).

[0158] <Example 2-4> Experiment to confirm transporter release including signal peptide

[0159] The signal peptides of phoA, PelB, DsbA, NSP4, NSP2, FhuD, MalE, OmpA, PhoE, OmpC, Lpp, LamB, OmpT, eltB, GH1, sta1, Xyn5, AnsB, MglB, TorT, sfmC, bla, npr, and AmiA, which are widely used signal peptides, were initially selected from the Signal Peptide DB site (http: / www.signalpeptide.de / ) and Uniprot (see Table 3).

[0160] Secondarily, using the Signal P server and ExPASy server, we analyzed the water solubility, cleavage site, physicochemical characteristics, post-translational modification, and the pathway through which it is likely to be released, and selected the AmiA signal peptide, OmpA signal peptide, and PelB signal peptide, which are signal peptides involved in the Sec pathway and Tat pathway.

[0161] Experiments were conducted to determine whether the selected signal peptide could function as a carrier. A transporter was constructed by fusing the selected signal peptide with an RBP. Protocatechuic acid (PCA) was secreted in a protocatechuic acid (PCA)-producing strain (C), a strain introduced with the transporter into the producing strain (T), and a strain introduced with the transporter and a conjugate (BT) (see Figures 6a and 6b).

[0162] As a result, it was confirmed that the strain introducing only the transporter emitted PCA, but the PCA emission amount was lower than that of the baseline strain (see Fig. 6a). However, the strain introducing both the transporter and the binder showed an emission amount similar to or higher (400-600 mg / L) than the baseline strain (see Fig. 6a).

[0163] In addition, compared to sfGFP, which was used as a conventional transporter, the strain introducing only the transporter using the signal peptide as a carrier showed lower emissions than the strain introducing only the transporter using sfGFP as a carrier. It was confirmed that the E. coli strain introducing the conjugate also showed emissions similar to or higher (400-700 mg / L) than the strain introducing sfGFP as a carrier.

[0164] Therefore, when a signal peptide was used as a carrier, the successful release of PCA confirmed that the signal peptide could also serve as a carrier (see Figures 6a and 6b).

[0165] Types of tested signal peptides Number Source of signal peptide Uniprot code Protein name 1 phoAP00634 Alkaline phosphatase 2 PelBP14005 Plasma membrane pectate lyase 3 DsbAP0AEG4 Thiol: disulfide exchange protein 4 NSP4Q98VL4 Nonstructural glycoprotein 4 5 NSP2P89070 NSP2 protein 6 FhuDP07822 Iron (3+)-hydroxamate-binding protein 7 MalEP0AEX9 Maltose-binding outer plasma membrane protein 8 OmpAP0A910 Outer membrane protein A 9 StilP22542 Heat-stable enterotoxin II 10 OmpFP02931 Outer membrane protein F 11 PhoEP02932 Outer membrane pore protein E 12 OmpCP06996 Outer membrane protein C 13 LppP69776 Major outer membrane Lipoprotein 14LamBP02943Maltoporin 15OmpTP09169Proteinase 716eltBA7Y1X5LTB 17GH1B1A4G6Growth hormone 1 isoform 118sta1P01559Hematologically stable enterotoxin ST-IA / ST-P 19XynSQ59256Endo-1,4-beta-xylanase 20AnsBP00805L-Asparaginase 221MglBP0AEE5D-Galactose-binding outer plasma membrane protein 22TorTP38683Plasma membrane protein TorT 23TorBP0A855Protein TolB 24sfmCP77249Fimbrial Chaperone 25BlaP62593Beta-lactamase TEM 26NprP06832Bacillolysin 27AmiAP36548N-acetylmuramoyl-L-alanine amidase amiA

[0166] *Bold: Selected signal peptides

[0167] <Example 3> Evaluation of BTSec assembly using PPIX aptamer and PPIX-producing E. coli strain

[0168] To investigate the effect of the BTSec assembly on chemical production, PPIX was selected as the first target chemical, and application of this assembly was examined to determine whether emissions and additional production increased.

[0169] <Example 3-1> Preparation of PPIX-producing E. coli strain

[0170] First, hemA was added to plasmid pTac15K_MCS (p15A ori, CmR). fbr A PPIX-producing strain was constructed by sequentially introducing hemL, hemE, and hemF and expressing them in E. coli (see Fig. 9a). hemA derived from Salmonella typhimurium fbr The gene was heterologously co-expressed with the hemL, hemE and hemF genes after undergoing modifications to add two lysine residues at the second and third positions of the N terminus for protein stabilization.

[0171] As a result, the PPIX-producing strain produced 12.6 mg / L of PPIX, and 21.7% (2.37 mg / L) of the produced amount was excreted outside the cell (see Fig. 9b).

[0172] <Example 3-2> Structural analysis of a binding module containing PPIX aptamer

[0173] The binding module of the BTSec assembly was designed by linking an RNA specific for RBP and an aptamer for the target chemical PPIX (see Figure 7a). The transport module was composed of sfGFP:N22.

[0174] To apply the BTSec assembly to PPIX-producing strains, a binding module was designed by linking boxB RNA, spacer, and aptamer (Table 4; see Figs. 7b, 7c).

[0175] A spinach scaffold domain was used as a spacer to resolve steric hindrance (Ouellet J. RNA Fluorescence with Light-Up Aptamers. Front Chem. 2016 Jun 28; 4:29.; Abatemarco, J., Sarhan, MF, Wagner, JM et al. RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes. Nat Commun 8, 332 (2017)). The PPIX-specific aptamer was used with the base sequence of SEQ ID NO: 17 (5'-GCATTGTCTGCGTGTGGAGGCAGGAGGCAAGATAAGAGGTGATGCGGTTG-3') (Niles JC, Marletta MA. Utilizing RNA aptamers to probe a physiologically important heme-regulated cellular network. ACS Chem Biol. 2006, 1(8):515-24. (See also).

[0176] The spacer between the PPIX aptamer and the boxB RNA creates a gap between the aptamer and the RNA, which relieves steric hindrance and facilitates binding to the RBP and target chemical, respectively. The spacer attached to the 3' end of the aptamer interacts with the spacer located between the aptamer and the RNA, providing structural stability. This structural stability allows the spinach scaffold domain to bind to a fluorescent agent such as DFHBI, resulting in fluorescence, enabling imaging of the aptamer (see Table 4).

[0177] Afterwards, the structure of the binding module was analyzed using Mfold and RNA Composer, which are nucleic acid structure prediction programs.

[0178] As a result, it was confirmed that the predicted structure when boxB RNA, spacer, and aptamer were fused in the program was consistent with the structure of each independently existing component (see Figures 7b, 7c, and 7d).

[0179] PPIX aptamer and aptamer-containing conjugate sequence Base sequence (5'-3') Sequence number PPIX aptamer GCATTGTCTGCGTGTGGAGGCAGGAGGCAAGATAAGAGGTGATGCGGTTG17*Aptamer-containing conjugate gggccctgaagaagggcccGGATGTAACTGAATGAAATGGTGAAGGACGGGTCCGCATTGTCTGCGTGTGGAGGCAGGAGGCAAGATAAGAGGTGATGCGGTTGTGTTGAGTAGAGTGTGAGCTccgtaactagTTACATC18

[0180] *In the conjugate base sequence including the aptamer, the bold part is the boxB RNA of SEQ ID NO: 4, the underlined part is the aptamer sequence of SEQ ID NO: 17, and the remaining parts correspond to the spacer sequence. With respect to the RNA sequence, T (thymidine) in this sequence represents U (uracil).

[0181] <Example 3-3> Confirmation of sfGFP expression level in PPIX production strain introduced with BTSec

[0182] To apply the BTSec assembly to a PPIX-producing strain, a binding module with an aptamer sequence specific for the PPIX was utilized, and the transport module was expressed under an arabinose-inducible system. After culturing the strain into which the assembly was introduced in an induction-dependent manner, sampling was performed at 12, 24, and 36 h, and the expression level of sfGFP:N22 was examined using SDS-PAGE (see Fig. 8a).

[0183] As a result, it was confirmed that the expression level of sfGFP:N22 varied depending on the induction period (see Fig. 8b). Specifically, compared to the PPIX-producing strain, the strain expressing only the transporter and the strain expressing the transporter and the complex showed an approximately 1.2 to 2-fold increase in expression level (see Fig. 8b).

[0184] In addition, when comparing strains expressing only the transporter with strains expressing both the transporter and the conjugate, the total sfGFP:N22 expression level was found to be higher in the strain expressing both the transporter and the conjugate than in the strain expressing only the transporter. There was no significant difference in the expression level of extracellular sfGFP:N22, and the fact that the strain induced 36 hours after introduction had a higher expression level than the strain induced 24 hours after introduction can be seen as the release of sfGFP:N22 accumulated within the cell. On the other hand, the strain expressing both the transporter and the conjugate in intracellular expression was confirmed to have a higher expression level than the PPIX-producing strain, which was 1.5 to 2 times higher (see Fig. 8b). In addition, the strain induced 24 hours after introduction with the entire assembly showed the highest expression level in both intracellular and total expression levels, and the difference in expression level with the strain expressing only the transporter was also confirmed to be the largest (see Fig. 8b).

[0185] <Example 3-4> Confirmation of extracellular excretion and production in PPIX-producing strains introduced with BTSec

[0186] The strain into which the BTSec assembly was introduced was cultured in a time-dependent manner, and then sampled at 48 hours to investigate the extracellular excretion and PPIX production.

[0187] As a result, it was confirmed that the production titer and emission amount varied depending on the time of transporter induction (see Fig. 9b). It was confirmed that the strain expressing only the transporter showed an increase in emission amount when induced for 4 and 12 hours. However, although the emission amount relatively decreased after 24 and 36 hours of induction, it was confirmed that it showed a higher emission amount compared to the PPIX-producing strain. In addition, it was confirmed that the strain expressing both the transporter and the conjugate showed a tendency for the total titer to increase. The strain that showed the maximum production was a cell induced for 12 hours for transporter expression after introduction, and it was confirmed that the extracellular titer increased by 72% (4.7 mg / L) and the total titer increased by 30% (16.4 mg / L) compared to the PPIX-producing strain (see Fig. 9c).

[0188] Additionally, the production titer of PPIX was additionally confirmed at various sampling times after 12 hours of induction (see Figures 9c, 9d).

[0189] As a result, it was confirmed that the strain expressing the transporter alone showed a slight increase in total titer (15% increase, 20.42 mg / L) and extracellular titer (8% increase, 3.34 mg / L) compared to the PPIX-producing strain. In contrast, the strain expressing the transporter and the conjugate showed a dramatic increase in total titer and extracellular titer, with a 50% increase (26.24 mg / L) and a 44% increase (4.4 mg / L), respectively (see Fig. 9c). It was confirmed that the total titer was saturated at 12 and 24 h of induction sampling in all strains. Meanwhile, the extracellular titer was saturated at 24 h of sampling in the PPIX-producing strain and the transporter-expressing strain, whereas it was confirmed that it increased as the sampling time increased in the strain co-expressing the transporter and the conjugate. In the strain co-expressing the transporter and the conjugate, the aptamer binds to the target and is released. This process took approximately 24 hours, and an increase in chemical release was observed from 24 to 36 hours, reaching maximum release at 48 hours (see Fig. 9d).

[0190] These results suggest that BTSec assemblies can further enhance the total potency of cells producing low chemical secretion rates by enhancing secretion efficiency.

[0191] <Example 4> Application of BTSec assembly for CPIII production

[0192] When the transport module and binding module were introduced during the cultivation of the PPIX-producing strain of Example 3, the CPIII level was found to increase simultaneously (see Fig. 10). Since the PPIX aptamer binds to a compound with a four-pyrrole structure, it was assumed that CPIII, which shares this structure with PPIX, also exhibited enhanced excretion through the BTSec assembly for PPIX excretion. The present inventors further experimented to determine whether CPIII production could be enhanced through the BTSec assembly in the CPIII-producing strain.

[0193] <Example 4-1> Analysis of emission levels of transport modules according to promoter type

[0194] Transport modules were constructed using five promoters (PR(con), TacΔO, J23100, LacUV5, and J23114), and their expression levels were investigated. Information on the sequences and sources of the promoters is provided in the table below (see Table 5).

[0195] Sequence and source according to promoter type Promoter type sequence (5'-3)' source Note P R(con)taacaccgtgcgtgttgacAattttacctctggcggtTataatggttgc (서열번호 19)Kincade JM, deHaseth PL. Bacteriophage lambda promoters pL and pR: sequence determinants of in vivo activity and of sensitivity to the DNA gyrase inhibitor, coumermycin. Gene. 1991, 97(1):7-12.TacΔOTTGACAATTAATCATCGGCTCGTATAATG (서열번호 20)Registry of standard biological parts(https: / parts.igem.org / Part:BBa_K864400)Tac promoter 에서 operator가 제거된 버전J23100TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC (서열번호 21)Registry of standard biological parts(https: / parts.igem.org / Promoters / Catalog / Ecoli / Constitutive)LacUV5TTTACACTTTATGCTTCCGGCTCGTATAATG (서열번호 22)Registry of standard biological parts(https: / parts.igem.org / Part:BBa_K4941071)J23114TTTATGGCTAGCTCAGTCCTAGGTACAatgctagc (서열번호 23)Registry of standard biological parts(https: / parts.igem.org / Promoters / Catalog / Ecoli / Constitutive)

[0196] As a result, it was confirmed that sfGFP:N22 expressed under the control of PR(con) and TacΔO promoters showed higher emission levels than sfGFP:N22 expressed under other promoters at 48 hours. In addition, it was confirmed that almost no emission was observed under J23114 (see Figs. 11a and 11b).

[0197] <Example 4-2> Production of CPIII-producing E. coli strain

[0198] First, hemA was added to plasmid pTac15K_MCS (p15A ori, CmR). fbr , hemL, and hemE were sequentially introduced. Afterwards, a CPIII production strain was constructed through manipulation to express the plasmid in E. coli (see Fig. 12a).

[0199] As a result, it was confirmed that the strain produced 29.39 mg / L of CPIII, and 90% (26.49 mg / L) was secreted externally (see Fig. 12b).

[0200] <Example 4-3> Confirmation of extracellular excretion and production in CPIII-producing strains introduced with BTSec assembly

[0201] A binding module consisting of an aptamer, spacer, and RNA identical to the binding module used for PPIX production was constructed. The spacer and RNA play the same roles as those in the binding module used for PPIX production, and the aptamer specifically binds to CPIII in place of PPIX. The transport module was constructed with sfGFP:N22. When the assembly was introduced, the extracellular excretion and production amount in the CPIII-producing strain were confirmed.

[0202] As a result, it was confirmed that the use of the transport module expressed under the TacΔO promoter increased CPIII emission by 54% (40.79 mg / L). The introduction of the coupling module further increased emission by 28%, resulting in an overall increase of 82% (48.1 mg / L) compared to the CPIII-producing strain (26.49 mg / L) (see Fig. 12b).

[0203] Additionally, the increased emission contributed to the increase in total titer, with an 81% increase in CPIII emission (53.16 mg / L) compared to the CPIII-producing strain (29.39 mg / L) (see Fig. 12b).

[0204] These results demonstrate the potential for improving productivity and emissions of target chemicals in systems incorporating the assembly of the present invention.

[0205] <Example 5> Application of BTSec assembly for chemical production using arbitrarily designed aptamers

[0206] In the BTSec assembly according to the present invention, a previously unknown selected aptamer was adopted through a library containing random sequences of aptamer regions to test whether it could be used to produce a desired chemical substance (see FIG. 13b).

[0207] <Example 5-1> Production of Vio-producing E. coli strain, Dvio-producing E. coli strain, and PDV-producing E. coli strain

[0208] Strains producing Vio, Dvio, and PDV, whose aptamer sequences are unknown, were developed. To overexpress five genes (vioA, vioB, vioC, vioD, and vioE) derived from Chromobacterium violaceum in E. coli, pETM6-E12-vioABCDE (Plasmid #66537) purchased from Addgene was introduced into E. coli to construct a Vio-producing strain (see Fig. 13a). To overexpress four genes (vioA, vioB, vioE, and vioC) in E. coli, the four genes obtained from pETM6-E12-vioABCDE were sequentially introduced into plasmid CloDF13-MCS and expressed in E. coli to construct a Dvio-producing strain (see Fig. 13a). In order to overexpress three genes, vioA, vioB, and vioE, in E. coli, three genes obtained from pETM6-E12-vioABCDE were sequentially introduced into plasmid pET28a and expressed in E. coli to construct a PDV production strain (see Fig. 13a).

[0209] As a result, the Vio-producing strain produced 43.62 mg / L of Vio, of which 4.86 mg / L was detected extracellularly, indicating an observed secretion rate of approximately 11% (see Fig. 13e). Similarly, the Dvio-producing strain produced 77 mg / L of Dvio, of which 25.4 mg / L was detected extracellularly, indicating a secretion rate of approximately 33% (see Fig. 13g). In the case of the PDV-producing strain, HPLC data analysis showed that 208.35 areas (mAUs) of PDV were observed, and 44.57 areas (mAUs) were observed extracellularly, indicating a secretion rate of approximately 21% (see Fig. 13i).

[0210] <Example 5-2> Aptamer screening and BTSec assembly design specific for Vio, Dvio, and PDV

[0211] The sequence and structure of RNA aptamers that specifically bind to Vio, Dvio, and PDV are not yet known. An aptamer with a random sequence was designed using a forward primer having the nucleotide sequence represented by SEQ ID NO: 24 and a reverse primer having the nucleotide sequence represented by SEQ ID NO: 25. The aptamers were then integrated into a binding module through overlapping PCR to form a library of binders, and aptamers specific to Vio, Dvio, and PDV were selected (Tables 6, 7, and 8; see also Fig. 13c).

[0212] In addition, in the aptamer library specific for Vio, the primer binding sequence was used as a spacer to resolve steric hindrance (see Nutiu R, Li Y. In vitro selection of structure-switching signaling aptamers. Angew Chem Int Ed Engl. 2005 Feb 4;44(7):1061-1065.). The primer binding sequence is the sequence used in the amplification step during the SELEX process to select aptamers, and the sequence itself and its length do not significantly affect the function of the conjugate. Therefore, any spacer constituting the conjugate can be used without any special restrictions as long as it can alleviate the steric hindrance of the aptamer and thus help in stable binding between the conjugate and the transporter.

[0213] Primers for random aptamer designPrimer sequence (5'-3')UseAptamerForward primerAAGACTGAGCTCCATATGCTGGATCCTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTGGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTT (SEQ ID NO: 24)Random aptamer designAptamerReverse primerAACTGACTCGAGACGATGCGGGTGCCAAGCTTANNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGCTTAAGCTTGCGGAG (SEQ ID NO: 25)Random aptamer design

[0214] *N is a base randomly selected from A, T, C, and G.

[0215] Base sequence of aptamer specific for Vio, Dvio, and PDV Vio aptamer base sequence (5'-3') Sequence number V1 AAGCTCTCGTGTAATCGTTTCGTACAGTATACGAGGACCGCTGCTGTTTGTCCCA 26 V2 AAGCTTCTGTAGCAAGCAGCCAGCCCGGTGCAATACGTAACACAGAAGCTACGAAC 27 V3 AAGCTCAGAATGGTGAACCCCCCGAGAAAGACGCAAGCCTGTACGGCGGCATTGC 28 V4 AAGCTATACAATAACCGCTGAGTACGAAGTGCTGTCGCGCCGACAATAGGAGCGA 29 V5 AAGCTCATCCAGTCGACGCCCACTCCATCGGCCACGCTCAGCAAAAATGACCTCC 30 V6 AAGCTTCCCCTTAAGTGGCCCCCAAACCAAAACGATAGCAACAAACTTGCACCGAC 31 Dvio AptamerD1AAGCTTCTAGTCTGGTCAATACCCCCGATGAGCGAAGTGCTTCGTAACCCCGAGGC32D2AAGCTTAAATGTTAATCCAACGTTAACACCAAGGAGACACACGAGCGCAGACGACC33D3CAAGAGCCACATGACACACCACTACGCACAAGAGAACAAACC AGGAGCAC34D4ATACAATAACCGCTGAGTACGAAGTGCTGTCGCGCCGACAATAGGAGCGA35D5CTCGTGTAATCGTTTCGTACAGTATACGAGGACCGCTGCTGTTTGTCCCA36D6AACCAACGCGATGTGATCCAAAATGCCCCCACTAGAGTTTTCA37PDV압타머P1AAGCTTATGTCTAGCACTGCCGCGACCAGCCACCCCGACCTGACGAGCGCAGAATA38P2AGTCCACACGAATCTTCCCAGAACAGTCTGGAACGAAACAAGACCGGGAC39P3AAGCTTAGGCGCAACAGCGGCGGACACGGGGGCTAGCCGTCACAAATCACAACCCTA40P4AAGCTTAGGATTACACTACGACGCGAAACTGCTGGACGAAAAAGCAATTGCACAAT41P5AAGCTTAACCCACAGTCATAGGGCCCGGTATCATCCCCCTGAACGTAGTCCTCATA42P6CTCGTGTAATCGTTTCGTACAGTATACGAGGACCGCTGCTGTTTGTCCCA43

[0216] Base sequence of a conjugate containing an aptamer specific for Vio, Dvio, and PDV Base sequence of a conjugate containing an aptamer specific for Vio (5'-3') SEQ ID NO: V'1GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCTCGTGTAATCGTTTCGTACAGTATACGAGGACCGCTGCTGTTTGTCCCATAAGCTTGGCACCCGCATCGTCTCGAG44V'2GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCTGTAGC AAGCAGCCAGCCCGGTGCAATACGTAACACAGAAGCTACGAACTAAGCTTGGCACCCGCATCGTCTCGAG45V'3GGGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCAGAATGGTGAACCCCCCGAGAAAGACGCAAGCCTGTACGGCGGCATTGCTAAGCTTGGCACCCCGCATCGTCTCG AG46V'4GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTATACAATAACCGCTGAGTACGAAGTGCTGTCGCGCCGACAATAGGAGCGATAAGCTTGCACCCGCATCGTCTCGAG47V'5GGGCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCATCCAGTCGACGC CCACTCCATCGGCCACGCTCAGCAAAAATGACCTCCTAAGCTTGGCACCCGCATCGTCTCGAG48V'6GGGCCTGAAGAAGGCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCCCCTTAAGTGGCCCCCAAACCAAAACGATAGCAACAAACTTGCACCGACTAAGCTTGGCACCCGCATCGTCTCGAG49Dvio Contains specific aptamers결합체D'1GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCTAGTCTGGTCAATACCCCCGATGAGCGAAGTGCTTCGTAACCCCGAGGCTAAGCTTGGCACCCGCATCGTCTCGAG50D'2GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTAAATGTTAATCCAACGTTAACACCAAGGAGACACACGAGCGCAGACGACCTAAGCTTGGCACCCGCATCGTCTCGAG51D'3GGGCCCTTGAAGAAGGGCCCGGCCTGCCACGCTCGCAAGCTTAAGCTTCAAGAGCCACATGACACACCACTACGCACAAGAGAACAAACCAGGAGCACTAAAGCTTGGCACCCGCATCGTCTCGAG52D'4GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTATACAATAACCGCTGAGTACGAAGTGCTGTCGCGCCGACAATAGGAGCGATAAGCTTGGCACCCGCATCGTCTCGA53D'5GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCTCGTGTAATCGTTTCGTACAGTATACGAGGACCGCTGCTGTTTGTCCCATAAGCTTGGCACCCGCATCGTCTCGAG54D'6GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTAACCAACGCGATGTGATCCAAAATGCCCCCACTAGAGTTTTCATAAGCTTGGCACCCGCATCGTCTCGAG55PDV에 특이적인 압타머 포함ConjugateP'1GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTATGTCTAGCACTGCCGCGACCAGCCACCCCGACCTGACGAGCGCAGAATATAAGCTTGCACCCGCATCGTCTCGAG56P'2GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTAGTCCACACGAA TCTTCCCAGAACAGTCTGGAACGAAACAAGACCGGGACTAAGCTTGGCACCCGCATCGTCTCGAG57P'3GGGCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTAGGCGCAACAGCGGCGGACACGGGGGCTAGCCGTCACAAATCACAACCCTATAAGCTTGGCACCCGCATCGTCTCGAG 58P'4GGGCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTAGGATTACACTACGACGCGAAACTGCTTGGACGAAAAAGCAATTGCACAATTAAGCTTGGCACCCGCATCGTCTCGAG59P'5GGGCCCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTAACCCACAGTCAT AGGGCCCGGTATCATCCCCCTGAACGTAGTCCTCATATAAGCTGGCACCCGCATCGTCTCGAG60P'6GGGCCTGAAGAAGGGCCCGGCCTGCCACGCTCCGCAAGCTTAAGCTTCTCGTGTAATCGTTTCGTACAGTATACGAGGACCGCTGCTGTTTGTCCCATAAGCTTGGCACCCGCATCGTCTCGAG61

[0217] * With respect to the base sequences listed in the table above, the bold text represents boxB RNA of SEQ ID NO: 4, the underlined portion represents the aptamer sequence of SEQ ID NOs: 26 to 43, and the remaining portions represent spacer sequences. With respect to the RNA sequence, T (thymidine) in this sequence represents U (uracil).

[0218] The transporter was designed to be expressed under an IPTG-inducible promoter. Since genes involved in Vio, Dvio, and PDV production are expressed under the T7 promoter under IPTG induction, the transporter was designed to be expressed without a separate inducer. Before applying the assembly to the producer strain, the relationship between inducer concentration and secretion rate was further investigated. To measure the excretion levels of the sfGFP:N22 transporter at various concentrations of IPTG, cells were cultured and sampled at different times (24, 36, and 48 h), and the excretion of sfGFP:N22 was measured by analyzing the SDS-PAGE band intensity (see Figure 14).

[0219] As a result, it was confirmed that the excretion significantly increased 24 hours after treatment with 0.5 mM IPTG compared to treatments with other concentrations (see Fig. 14a). It was confirmed that samples treated with all concentrations of IPTG efficiently excreted sfGFP:N22 after 36 and 48 hours of induction (see Figs. 14b, 14c).

[0220] To ensure a sufficiently diverse strain library, initial screening was performed on a large scale using a microplate reader (see Figure 13c). Colonies were identified to exhibit a variety of extracellular excretion and total production of these chemicals (see Figures 13d, 13f, and 13h).

[0221] Aptamers were selected based on initial screening results and were confirmed to have a ≥10% increase in extracellular abundance compared to the production strain, while remaining unchanged or exhibiting a ≥10% increase in total abundance. The top seven aptamers were selected for flask-level testing, and their potency and release levels were determined by HPLC.

[0222] As a result, in the initial screening of the Vio-producing cell library, the total Vio titer was confirmed to be increased 1.99-fold compared to the Vio-producing strain. The extracellular titer detected in the medium was confirmed to be increased 2.50-fold compared to the Vio-producing strain (see Fig. 13d).

[0223] In flask culture, when only the transport module was introduced, the total titer of Vio increased 1.6-fold (67.64 mg / L) and the extracellular titer increased 2-fold (9.9 mg / L) compared to the Vio-producing strain. When the assembly was introduced, the highest total titer increased 2.1-fold compared to the Vio-producing strain (92.21 mg / L), and the highest extracellular titer increased 4.8-fold compared to the Vio-producing strain (23 mg / L) (see Fig. 13e).

[0224] For the Dvio cell library, the total Dvio titer was confirmed to increase 1.6-fold compared to the Dvio-producing strain. The extracellular titer detected in the medium was found to increase 2.06-fold compared to the Dvio-producing strain (see Fig. 13f). Similarly, when only the transport module was introduced in flask culture, the total Dvio titer increased 1.07-fold (82.39 mg / L) compared to the Dvio-producing strain, whereas the extracellular titer increased 1.39-fold (35.36 mg / L). When the assembly was introduced, the highest total titer showed a 1.2-fold increase compared to the Dvio-producing strain (91.72 mg / L), and the highest extracellular titer showed a 1.74-fold increase compared to the Dvio-producing strain (44.14 g / L) (see Fig. 13g).

[0225] For the PDV cell library, the total PDV titer was confirmed to increase 4.77-fold compared to the production strain. The extracellular titer detected in the medium was found to increase 12.22-fold compared to the PDV production strain (Fig. 13h). Similarly, when only the transport module was introduced in flask culture, the total PDV titer increased 3.5-fold (723.7 mAUs) compared to the PDV production strain (208.25 mAUs), while the extracellular titer increased 4.45-fold (198.41 mAUs).

[0226] When the assembly was introduced, the highest total titer was confirmed to be increased 5.27-fold (1097.58 mAUs) compared to the PDV producing strain, and the highest extracellular titer was confirmed to be increased 8.9-fold (397.08 mAUs) compared to the PDV producing strain (see Fig. 13i).

[0227] Therefore, the BTSec assembly and system disclosed in the present invention demonstrate potential as a platform technology capable of increasing the production of chemicals accumulated in cells while increasing their excretion, and also demonstrates the possibility of expanding production capacity through the application of aptamers targeting as-yet-unknown chemicals.

[0228] <Example 6> Confirmation of aptamer expression

[0229] To confirm whether the aptamer within the conjugate inserted into the cell was expressed, quantitative real-time PCR was performed to confirm the expression level. Five strains were used: BL21 (DE3)* strain, BL21 (DE3)* strain expressing only the transporter, BL21 (DE3)* strain expressing only the conjugate, BL21 (DE3)* strain expressing both the transporter and the conjugate, and BL21 (DE3)* strain expressing both the transporter and the conjugate but lacking the boxB module that mediates the conjugation. Each strain was cultured in LB medium, sampled at 24 and 48 hours, and total RNA was extracted, reverse transcribed into cDNA, and RT-PCR analysis was performed. Each condition was repeated three times, and the expression level of the intracellular RNA aptamer was quantitatively measured through the RT-PCR results. Transporter expression was induced by treating with 0.1% arabinose at 9 hours of incubation.

[0230] Specifically, total RNA (Total RNA) was extracted using the AccuPrep Bacterial RNA Extraction Kit (Bioneer, Cat# K-3140). Before the experiment, β-mercaptoethanol was added to the RB buffer to a final concentration of 1%, and lysozyme was added to the TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final concentration of 20 mg / mL. RS buffer equivalent to 0.5 times the total volume was added (Bacteria cell culture: RS buffer = 2:1), mixed well, and reacted at room temperature for 5 minutes. After centrifugation at 7,500 rpm for 10 minutes, the supernatant was removed, and a solution containing 100 μL of TE buffer (containing lysozyme) and 20 μL of proteinase K was added to the pellet. After complete resuspension by pipetting, the sample was reacted at room temperature for 10 minutes, sequentially treated with lysis buffer and binding buffer, and RNA was purified using a silica column. RNase was removed by DNase (Thermo Fisher Scientific, Cat# EN0521), and the final RNA was dissolved in RNase-free water, and the concentration and purity were measured using NanoDrop™ (Thermo Fisher Scientific). cDNA synthesis from total RNA was performed using PrimeScript™ RT Reagent Kit (Takara, Cat# RR037A). The total reaction volume was 20 μL per sample, and the reaction system below was prepared based on a 15-minute reaction. A common mixture for four samples was prepared in a 1.5 mL tube as follows.The mixture was mixed with 5× PrimerScript Buffer, PrimeScript RT Enzyme Mix I, Oligo dT Primer, and Random 6-mers at a ratio of 4:1:1:1. 7 μL of the mixture was dispensed per sample, and 3 μL of RNA was aliquoted and cDNA was synthesized using a Bio-Rad PCR system. After synthesis, it was quantified using NanoDrop™ (Thermo Fisher Scientific) and diluted with DEPC to a final RNA concentration of 400 ng / μL. qRT-PCR was performed using GoTaq® qPCR Master Mix (Promega, Cat# A6001). Each reagent, forward primer, reverse primer, and Master Mix, were mixed in a 1.5 mL tube at a ratio of 1:1:5 (see Table 9). PCR conditions were set at 90-95°C for enzyme activation and denaturation steps, approximately 60°C for the annealing step, and a total cycle number of 40. Relative quantification was analyzed using the ΔΔCt method using a housekeeping gene such as frr as an internal control. PCR efficiency was indirectly assessed from the slope.

[0231] The wild-type strain (WT) was set as the reference value with a relative expression value of 1.00 ± 0.04. The "B" treatment group was a strain expressing the PPIX conjugate and showed a relative expression increase of approximately 925.7 ± 127.2-fold compared to the wild-type strain, and the "T" treatment group was a strain that solely expressed the sfGFP_N22 transporter and showed a relatively low expression level of approximately 2.2 ± 0.23-fold. The "BT" treatment group was a strain that simultaneously expressed the conjugate and transporter and showed the highest gene expression increase of approximately 1480.2 ± 406.6-fold compared to the wild-type strain (see Figure 15). It was confirmed that the simultaneous expression of the conjugate and transporter induces the gene expression of the PPIX aptamer more effectively than the single expression condition.

[0232] Primer information used in qRT-PCR Primer name Sequence number Base sequence (5'->3') Purpose ppix_sybr green (Fw) 62 ACT CAA CAC AAC CGC ATC ACPPIX-specific aptamer gene expression confirmation ppix_sybr green (R) 63 TGA AAT GGT GAA GGA CGG GTfrr (Fw) 64 GAT CTT GGC CTG AAC CCG AAHousekeeping gene (frr) expression confirmation frr (R) 65 CTG CTT CAC CAC GAA CGA TTT

Claims

An assembly for extracellular secretion of a target chemical substance comprising a transporter in which a carrier is fused to an RNA binding protein (RBP), and a conjugate in which an RNA corresponding to the RBP is fused to an aptamer, An assembly for extracellular secretion of a target chemical, characterized in that the transporter and the complex are assembled through RBP-RNA interaction within the cell. An assembly for extracellular secretion of a target chemical substance, characterized in that the carrier in claim 1 is a non-signal peptide-based carrier protein. An assembly for extracellular secretion of a target chemical substance, characterized in that the non-signal peptide-based carrier protein in the second paragraph is sfGFP or a derivative thereof. An assembly for extracellular secretion of a target chemical, characterized in that in claim 3, the sfGFP derivative is rbGFP, vsfGFP-9, oxGFP, moxGFP, vsGFP, usGFP, muGFP, vsfGFP-0, rsFolder, rsFolder2, pH-tdGFP, spGFP1-10, spGFP11, sfYFP, sfCFP, sfBFP, esGFP, sfpHluorin or mChartreuse. An assembly for extracellular secretion of a target chemical substance, characterized in that the carrier in claim 1 is a signal peptide-based carrier protein. An assembly for extracellular secretion of a target chemical substance, characterized in that the signal peptide-based carrier protein in claim 5 is any one selected from the group consisting of PelB, OmpA, and AmiA. An assembly for extracellular secretion of a target chemical substance, characterized in that the RBP is composed of 22 to 133 amino acids and has a size of 2.7 to 14.2 kDa in the first paragraph. An assembly for extracellular secretion of a target chemical substance, characterized in that in claim 7, the RBP is any one selected from the group consisting of MCP, λN22, Com, PP7 coat protein, QCP, GA coat protein, and R17 coat protein. An assembly for extracellular secretion of a target chemical, characterized in that in claim 1, the complex further comprises a spacer of 5 to 45 nucleotides in length between the RNA and the aptamer. An assembly for extracellular secretion of a target chemical substance, characterized in that in claim 9, the conjugate has a spacer additionally linked to the aptamer end to which the spacer is not linked. An assembly for extracellular secretion of a target chemical substance, characterized in that in the first paragraph, the RNA specifically binds to the corresponding RBP and is a single-stranded RNA having a hairpin structure. An extracellular excretion system according to claim 11, characterized in that the RNA is selected from the group consisting of MS2 RNA, boxB RNA, Com RNA, Pcp RNA, QCP RNA, GA RNA, and R17 RNA. An assembly for extracellular secretion of a target chemical substance, characterized in that in the first paragraph, the complex comprises two or more types of RNAs connected in series. An assembly for extracellular secretion of a target chemical substance, characterized in that in the first paragraph, the transporter comprises at least two or more types of RBPs connected in series. An assembly for extracellular secretion of a target chemical substance, characterized in that, in the first paragraph, if an aptamer for the target chemical substance is known, the aptamer is selected from known aptamers. In claim 15, the target chemical substance is PPIX (protoporphyrin), CPIII (coproporphyrin), organic dye, D-tryptophan, L-valine, theophylline, cyanocobalamin, L-citrulline, flavin mononucleotide, flavin adenine dinucleotide, kanamycin A, kanamycin B, kanamycin, neomycin, tobramycin, libidomycin, nicotinamide adenine dinucleotide, riboflavin, biotin, L-arginine, dopamine, 7-methyl-guanosine, CCdApPuro, chloramphenicol, biomycin, sulforhodamine, streptomycin, L-isoleucine, 8-oxodG, xanthine, guanine, malachite green, phosphatidylcholine, cyclic adenosine monophosphate, adenosine triphosphate, L-tyrosine, An assembly for extracellular secretion of a target chemical, characterized in that the assembly is any one selected from the group consisting of S-adenosyl homocysteine, moenomycin A, sialyl Lewis X, tetracycline, adenine, a morpholine-based GTP analog, 4,4-methylenedianiline, isoleucine, L-histidine, codeine, mesomesoprotoporphyrin IX, thyroxine, 10-carboxy-2,7-di-t-butyl-trans-12c,12d-dimethyl-12c,12d-dihydrobenzo[e]pyrene, dimethylindole red, cyanine, an aniline-substituted sulforhodamine analog, atrazine, sphingosylphosphorylcholine, a black hole quencher, 4-dimethylaminobenzylidene imidazolinone, glutathione, and a heteroaryldihydropyrimidine. An assembly for extracellular secretion of a target chemical, characterized in that in the first paragraph, when an aptamer for the target chemical is not known, the aptamer is obtained through designing and screening an aptamer library for aptamers corresponding to the target chemical. An assembly for extracellular secretion of a target chemical, characterized in that the target chemical is selected from the group consisting of Vio (violacein), Dvio (deoxyviolacein), and PDV (prodeoxyviolacein), in claim 17. A genetic construct for the expression of a transporter constituting an assembly according to any one of claims 1 to 18, A genetic construct characterized in that a polynucleotide encoding a carrier protein is operably linked to a polynucleotide encoding an RNA binding protein. A genetic construct for the expression of a combination constituting an assembly according to any one of claims 1 to 18, A genetic construct characterized in that a polynucleotide encoding an aptamer is operably linked to a polynucleotide encoding RNA. A vector for transporter expression comprising a gene construct according to Article 19. A vector for expression of a conjugate, comprising a gene construct according to Article 20. A vector characterized in that it comprises a polynucleotide encoding RNA, a polynucleotide encoding an aptamer, and a spacer of 5 to 45 nucleotides in length, in claim 22. A host cell into which the vector of claim 21 and the vector of claim 22 have been introduced. In claim 24, the host cell is characterized in that it is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. A method for producing a target chemical, comprising the step of culturing the host cell of claim 25. An extracellular secretion system for a target chemical substance, constructed by introducing a genetic construct encoding an assembly according to any one of claims 1 to 18 into a microorganism. An extracellular secretion system for a target chemical substance, characterized in that in claim 27, the microorganism is Escherichia coli, lactic acid bacteria, Lactobacillus, Streptomyces, Gluconobacter, Acetobacter, Bacillus, Chlamydia, Saccharomyces, Capoteirobacter, Clostridium, Mycobacterium, Enterococcus, Brucella, Propionibacterium, Aspergillus, Listeria, Penicillium, or Sinorhizobium. A method for producing a target chemical, characterized in that it comprises a step of activating the extracellular secretion system of the target chemical according to Article 28. A method for producing a target chemical, characterized in that it additionally includes a step of separating and purifying the target chemical in Article 29. A method for producing a target chemical, characterized in that in claim 27, the target chemical is any one selected from the group consisting of PPIX, CPIII, Vio, Dvio, and PDV.

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