Engineered bacterial secretion system capable of efficiently delivering protein and application of engineered bacterial secretion system

Abstract

The invention belongs to the technical field of microbial genetic engineering and protein delivery, and particularly relates to an engineered bacterial secretion system for efficiently delivering protein and application of the engineered bacterial secretion system. Specifically, on the basis of analyzing a delivery mechanism of bacteroides fragilis GS086 type double-effect protein T6SS, engineering modification is performed on a toxic structural domain of the effect protein, and a bacterial secretion system capable of efficiently and simultaneously delivering a plurality of proteins is constructed. According to the system, a modified strain is a natural human intestinal symbiotic bacterium, the problems of high immunogenicity, poor stability and the like are effectively avoided, and protein can be delivered in situ in the intestinal tract to realize precise regulation and control of an intestinal flora structure and secretion of specific target protein, so that the system has a good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and protein delivery technology, specifically relating to an engineered, highly efficient bacterial secretion system for protein delivery and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The human gut microbiota is a complex microbial community closely related to host health. Bacteroides, as the most abundant Gram-negative bacteria in the gut, achieves long-term stable colonization through various interbacterial interaction mechanisms. Among them, the Type Six Secretion System (T6SS) is an important protein secretion machine in Bacteroides. It can not only inject effector proteins into sensitive strains through direct interbacterial contact to exert a killing effect, but also secrete effector proteins into the extracellular supernatant.

[0004] Bacteroides fragilis is a key component of the human gut symbiotic flora, primarily found in the distal colon. Bioinformatics analysis of the Bacteroides fragilis genome published by NCBI revealed that over 86% of Bacteroides fragilis encodes T6SS, indicating that T6SS is the main protein secretion pathway in Bacteroides fragilis, providing a natural advantage for constructing protein delivery systems based on this bacterium.

[0005] Traditional protein delivery systems have many limitations: viral vector delivery systems pose a safety risk due to high immunogenicity; chemical delivery methods such as liposome encapsulation have poor stability, are easily degraded by lysosomes, and have poor targeting; and delivery methods mediated by biomolecules such as cell-penetrating peptides and antibody conjugates have poor tissue specificity, may enter normal cells non-selectively or trigger a host immune response, leading to the clearance of the delivered protein.

[0006] In recent years, researchers have begun developing biological protein delivery systems. For example, Feng Zhang's team at MIT modified bacterial extracellular injection systems (eCISs) to transform bacterial effector proteins into Cas9, base editors, and bacterial toxins; in 2021, Tao Dong's team at the University of Calgary engineered the protein delivered by bacterial T6SS into Cre recombinase. However, these modifications to bacterial secretion systems still suffer from problems such as poor targeting, low delivery efficiency, and the ability to deliver only one effector protein at a time, limiting their application scope. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the inventors, through long-term technical and practical exploration, have provided an engineered, highly efficient bacterial secretion system for protein delivery and its applications. Specifically, based on the analysis of the delivery mechanism of the dual-effect protein T6SS from Bacteroides fragilis GS086, this invention engineered the toxic domain of the effector protein to construct a bacterial secretion system capable of efficiently delivering multiple proteins simultaneously. The modified strain in this system is a natural human intestinal symbiotic bacterium, effectively avoiding problems such as high immunogenicity and poor stability. It can deliver proteins in situ within the intestine to achieve precise regulation of the intestinal flora structure and the secretion of specific target proteins. Based on the above research results, this invention has been completed.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an engineered, highly efficient bacterial secretion system for protein delivery, the bacterial secretion system being based on the type 6 secretion system (T6SS) of Bacteroides fragilis, the T6SS comprising a core protein VgrG, accessory proteins, and multiple effector proteins; each effector protein having a conserved delivery domain and a replaceable toxicity domain, wherein the target protein to be secreted is fused with the conserved delivery domain for expression, thereby replacing the toxicity domain.

[0010] The core protein VgrG has the following characteristics:

[0011] a1) The amino acid sequence as shown in SEQ ID NO.1;

[0012] a2) Proteins with the same function as shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues.

[0013] The accessory protein has:

[0014] b1) The amino acid sequence as shown in SEQ ID NO.2;

[0015] b2) Proteins with the same function as shown in SEQ ID NO.2, by substitution and / or deletion and / or addition of one or more amino acid residues.

[0016] The plurality of effector proteins can be two, namely a first effector protein and a second effector protein, wherein the first effector protein has a delivery domain having:

[0017] c1) The amino acid sequence as shown in SEQ ID NO.3;

[0018] c2) Proteins with the same function as shown in SEQ ID NO.3, which have undergone substitution and / or deletion and / or addition of one or more amino acid residues.

[0019] The amino acid sequence of the toxic domain of the first effector protein is shown in SEQ ID NO.4.

[0020] The second effector protein has a delivery system that includes:

[0021] d1) The amino acid sequence as shown in SEQ ID NO.5;

[0022] d2) Proteins with the same function as shown in SEQ ID NO.5, which have undergone substitution and / or deletion and / or addition of one or more amino acid residues.

[0023] The amino acid sequence of the toxic domain of the second effector protein is shown in SEQ ID NO.6.

[0024] In this invention, the target protein can be an enzyme, antibody, toxin, or signaling molecule, and no specific limitation is made herein. In one specific embodiment of this invention, there are at least two target proteins, that is, a first target protein can replace the toxic domain of the first effector protein, and a second target protein can replace the toxic domain of the second effector protein.

[0025] In this invention, the replacement of the toxic domain of the effector protein can be achieved through gene knockout or knock-in of Bacteroides fragilis, which is a conventional operation in the art. In one specific embodiment of this invention, the gene encoding the target protein can be knocked out or knocked in without leaving a trace using a homologous double crossover method. Furthermore, the knockout or knock-in can be performed using pSIE series non-replicating vectors, which are not specifically limited here.

[0026] A second aspect of the invention provides a nucleic acid molecule encoding the aforementioned engineered, highly efficient protein delivery bacterial secretion system.

[0027] A third aspect of the present invention provides an engineered bacterium comprising the above-described engineered efficient protein delivery bacterial secretion system and / or nucleic acid molecules encoding the above-described engineered efficient protein delivery bacterial secretion system, thereby secreting and expressing a target protein.

[0028] Furthermore, the bacteria can be any one of the following genera: Bacteroides, Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0029] Furthermore, the bacteria are Bacteroides fragilis, Escherichia coli, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0030] Furthermore, in order to precisely regulate the secretory expression of the target protein, in one specific embodiment of the present invention, the engineered Bacteroides fragilis also includes an inducible expression system, which regulates the expression of the target protein by the bacterial secretion system by adjusting the concentration of the inducer.

[0031] In one specific embodiment of the present invention, the Bacteroides fragilis protein-inducible expression system integrates the rhamnosylprotein-inducible expression system and the target gene into a specific site (such as the attN site) on the genome of Bacteroides fragilis via a site-specific integrase on a pNBU2 series non-replicating vector. The chimeric expression and secretion are regulated by adjusting the concentration of the inducer rhamnosyl. Further, the final concentration of rhamnosyl used is 0-10 mM.

[0032] In another specific embodiment of the present invention, the *Bacteroides fragilis* can be *Bacteroides fragilis* GS086, which can be obtained from the Broad Institute-OpenBiome Microbiome Library (BIOML), sample ID: aa_0143_0055_e12. NCBI Biosample number: SAMN11943472.

[0033] A fourth aspect of the present invention provides the application of the above-described bacterial secretion system, nucleic acid molecules, and engineered Bacteroides fragilis in protein delivery.

[0034] The protein may be an enzyme, antibody, toxin, or signaling molecule, and no specific limitation is made here.

[0035] Furthermore, the applications also include in-situ delivery of proteins in the gut to regulate gut microbiota structure, or secretion of specific proteins in the gut.

[0036] In this context, the application can specifically refer to the use of the aforementioned bacterial secretion system, nucleic acid molecules, and engineered Bacteroides fragilis in the preparation of protein delivery products. The products can be pharmaceuticals or general laboratory reagents for non-pharmaceutical purposes.

[0037] Furthermore, the protein may be two or more enzyme systems with cascade activation effects, or bactericidal toxins with synergistic effects. No specific limitations are imposed here.

[0038] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) High-efficiency delivery: The modified secretion system can secrete the target protein of the fusion with levels comparable to or higher than those of the wild-type strain, and the secretion level can be further increased after the addition of an inducer.

[0041] (2) Simultaneous delivery of multiple proteins: This system can deliver two different target proteins simultaneously (fused with the conserved delivery domains of E1 and E2 respectively), breaking through the limitation of traditional bacterial secretion systems that can only deliver one protein at a time.

[0042] (3) Maintaining protein activity: Through co-culture experiments, it was verified that the target protein (such as bacteriocins) expressed by fusion can maintain normal physiological activity. For example, bacteriocins 1 expressed by fusion have a strong bactericidal effect when used alone, and the bactericidal effect is significantly enhanced when used in combination with bacteriocins 2.

[0043] (4) High biosafety: The modified strain is Bacteroides fragilis GS086, which is a natural symbiotic bacterium in the human gut. This avoids the high immunogenicity of viral vectors and the safety risks of chemical delivery systems, and reduces the risk of host immune response.

[0044] (5) Good stability: Based on the bacterial secretion mechanism, this system is more stable in the in vivo environment and is not easily degraded compared with chemical delivery systems such as liposomes.

[0045] (6) Wide range of applications: It can be used to deliver target proteins in situ in the intestine, achieve precise regulation of the intestinal flora structure (such as targeted killing of harmful bacteria), or secrete specific target proteins in the intestine (such as enzymes, antibodies, etc.), and has important application prospects in the fields of intestinal disease treatment, probiotic development, and biopharmaceutical production. Attached Figure Description

[0046] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0047] Figure 1 The gene cluster encoding the T6SS protein delivery system in this embodiment of the invention;

[0048] Figure 2 These are the genotypes of the three modified mutant strains used in this embodiment of the invention;

[0049] Figure 3 The secretion level of the mutant strain was detected in this embodiment of the invention;

[0050] Figure 4 The co-culture method was used in this embodiment of the invention to detect the physiological activity of bacteriocins expressed in fusion.

[0051] Figure 5The pSIE and pNBU2 series plasmids used in the embodiments of this invention. Detailed Implementation

[0052] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0053] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0054] Specifically, the core of this invention lies in the engineering modification of T6SS of Bacteroides fragilis GS086, and the specific technical solution is as follows:

[0055] 1. Composition of the bacterial secretion system

[0056] This bacterial secretion system is based on the T6SS of Bacteroides fragilis GS086, and its core components include:

[0057] VgrG protein, accessory protein, effector proteins E1 and E2.

[0058] 2. Modification of effector proteins

[0059] Using genetic engineering techniques, the replaceable toxic domains (black portions) of E1 and E2 are replaced with the target protein to be secreted, enabling the target protein to be fused with the conserved delivery domains (red portions) of E1 and E2 for expression. The modification process utilizes pSIE series non-replicating vectors, achieving seamless knockout or knock-in of the target gene based on homologous double crossover. The specific steps are as follows:

[0060] Gene knockout plasmid construction: Using the genome of Bacteroides fragilis GS086 strain as a template, DNA fragments of 1 kbp each upstream and downstream of the target gene (i.e., the E1 or E2 virulence domain) were obtained by PCR and gel recovery, which were used as the upstream homologous arm (HRF) and the downstream homologous arm (HRR), respectively. The HRF and HRR were seamlessly cloned into the pSIE gene knockout vector using the Gibson assembly method to construct the gene knockout plasmid.

[0061] Conjugation transfer and screening: The constructed gene knockout plasmid was transformed into E. coli S17-1λ-pir competent cells using the heat shock method to prepare donor strains; Bacteroides fragilis GS086 was activated and streaked on fresh BHI plates to prepare recipient strains; the gene knockout plasmid was transferred into the recipient strain by conjugation transfer at a donor-recipient ratio of 10:1; the mixed colonies were plated onto BHI forward selection plates containing chloramphenicol to screen for the first homologous recombination positive recombinants; after amplification of the positive recombinants, they were plated onto BHI reverse selection plates containing tetracycline to screen for the second homologous recombination positive mutant strains; the correctness of the mutant strains was verified by colony PCR and first-generation sequencing.

[0062] 3. Integration of inducible expression systems

[0063] To achieve precise regulation of the expression and secretion of the target protein, a chimera fused with the target protein and a rhamnose-induced expression system can be integrated into the attN site of the Bacteroides fragilis GS086 genome using the pNBU2 series of non-replicating vectors. The specific steps are as follows:

[0064] Construction of inducible expression plasmid: The chimeric gene fragment of the target protein was obtained by PCR and gel extraction. The fragment was placed after the rhamnose inducible expression element of the pNBU2 vector using the Gibson assembly method to construct the inducible expression plasmid.

[0065] Plasmid integration and validation: The inducible expression plasmid was transformed into E. coli S17-1λ-pir competent cells to prepare the donor strain; conjugation transfer was performed with Bacteroides fragilis GS086 recipient strain; mixed colonies were plated on BHI plates containing chloramphenicol, and site-specific integration-positive recombinants were screened; successful integration of the rhamnose-induced expression system and the target gene was verified by colony PCR and first-generation sequencing. The expression and secretion of the target protein were regulated by adjusting the final concentration of rhamnose in the culture medium (0-10 mM).

[0066] 4. The working mechanism of the endocrine system

[0067] When the modified Bacteroides fragilis GS086 is cultured under suitable conditions, the E1-target protein chimera and the E2-target protein chimera are expressed intracellularly. With the synergistic effect of VgrG protein, accessory proteins and other T6SS structural proteins, the chimera is secreted into the bacterial extracellular space via T6SS in a T6SS-dependent manner, thereby achieving the delivery of the target protein.

[0068] In summary, this invention successfully constructed an engineered, highly efficient bacterial secretion system for protein delivery. By modifying the T6SS domain of *Bacteroides fragilis* GS086, this system fuses the target protein with the conserved delivery domains of E1 and E2, enabling the efficient delivery of multiple target proteins while maintaining their normal physiological activity. This system offers advantages such as high efficiency, stability, safety, and the ability to deliver multiple proteins simultaneously. It can be used for gut microbiota regulation and in situ protein delivery within the gut, providing crucial technical support for research and applications in related fields.

[0069] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.

[0070] Example

[0071] 1. Experimental Methods

[0072] 1.1 Amino acid and nucleic acid sequences of the core components of this protein delivery system

[0073] 1.1.1 Amino acid sequence of VgrG

[0074] MASTNLDAVSVEIKVAGKVCDYVTMELFQSVSTHHRFKIKVNYRPDKPSVWAIGPDVIFKQLGEKVSIIMTHHESGEKTEFHGLISDIHVEGFDGNQGFVILEGGSPTILLDRDPAMDCYVEQNLNTIVSDILDKSGVKMNVTNNPKHTDIIPYVARYKETSYGFLSRLLRSYGEWFYYNGETLQIGNPEIETESRAGYDVDLTGVSINATIRSLNHSTYEFDPVNDKFYYDYSGTPKGATLGSRSAEKCSEPIFPTEAKLPSMRPAYSAMDLEHYGDAGFHRNYSQLSQIKASSRYCGIRLGELVVTRVPESFPGVKITDLGRYRITEITHTVDGQGRYSNTFCGVPGGTPVMPWGDAVMPVAYPEMARVVSNEDPKNQGRVKVQFMWQEVDGGESYWMRVQSPDAGKSDQVAKNRGFVFIPEPGDLVMVGFEQGNPDRPYVTGSLFYKANSQGAATDNTVKSIRTRSGHTLEFNDDEGGDWGITIKDRNGCMFHFDTKGKNIEITAPETMTLNAQNININAGEQLNTSSGKETVMQIGTDFQQDVGGNAEIAIGESLTESIAKDSTNSIAGNLSVTVDENLMYDAQDMTLTAQGGMKLLANAKIGLKSSEGVDIAQ(SEQ ID NO.1)

[0075] Nucleic acid sequence of VgrG

[0076]

[0077] 1.1.2 Amino acid sequence of Effector 1

[0078]

[0079] HFRGYKPFITKITFDKTFKGKIQ (Red indicates the conservative delivery domain, SEQ ID NO.3; black indicates the alternative toxicity domain, SEQ ID NO.4)

[0080] Nucleic acid sequence of Effector 1

[0081] (Red indicates nucleic acid sequences encoding delivery domains, and black indicates nucleic acid sequences encoding alternative toxic domains)

[0082] 1.1.3 Amino acid sequence of Effector 2

[0083] (Red indicates the conservative delivery domain, i.e., SEQ ID NO.5; black indicates the alternative toxic domain, i.e., SEQ ID NO.6)

[0084] Nucleic acid sequence of Effector 2

[0085]

[0086] (Red indicates nucleic acid sequences encoding delivery domains, and black indicates nucleic acid sequences encoding alternative toxic domains)

[0087] 1.1.4 Amino acid sequence of accessory proteins

[0088] MYKFRNLKGQYSINVSTTFPGSQASLYSAIIEVIASYQNVDDYFIFELRSRQEVKLNGNPPSQMVDLFMLRLSNTYYPMKLKVSTSGKIMEVINFTDIKERWEAECAKIVEEIPCIAYEQYIELSKSNM DTENVFLQALRKDSFIQFYFKEYLDDIDIVCYNFPRRGESTFYDLAIDSDGSFHKDIKTFHVKEYNSKRYSGKLICEYSEENDIFSLIAEFYYNTLDGQCKKKVSISVENRSVQKANKLKSFLFD(SEQ ID NO.2) Nucleic acid sequence of auxiliary protein

[0089] ATGTATAAATTTCGCAACCTAAAAGGACAATACAGTATAAATGTGTCCACGACTTTTCCCGGCTCGCAAGCAAGTCTGTACAGTGCAATTATAGAAGTAATTGCATCTTATCAGAATGTAGATGATTATTTCATTTTTGAATTGCGTTCCCGGCAGGAGGTAAAGTTGAATGGTAATCCACCCTCTCAAATGGTGGATCTATTTATGCTTCGTCTTTCTAATACGTATTATCCGATGAAGTTGAAAGTTTCTACGTCGGGAAAGATTATGGAAGTTATTAATTTTACTGATATAAAGGAAAGATGGGAAGCGGAATGTGCGAAAATCGTGGAGGAAATACCTTGTATAGCCTATGAGCAATATATTGAATTGTCAAAGTCAAATATGGATACTGAAAATGTTTTTTTACAGGCTTTGCGCAAAGACTCCTTTATCCAGTTCTATTTTAAGGAATACTTGGATGATATAGATATTGTTTGTTATAATTTTCCACGACGTGGTGAAAGTACTTTTTATGATTTGGCTATTGATAGCGATGGCTCATTTCATAAGGATATCAAGACATTTCACGTCAAAGAGTATAACAGTAAACGATATTCGGGTAAATTAATCTGTGAGTATTCCGAAGAAAATGATATATTTTCTTTGATTGCGGAATTTTATTATAATACTTTGGATGGACAGTGTAAAAAGAAAGTCAGTATTTCAGTAGAAAACCGTTCGGTTCAAAAAGCCAATAAATTAAAGAGTTTTTTGTTTGACTAA

[0090] 1.1.5 Amino acid sequence of bacteriocin 1

[0091] GFKSARPVYTTEELQAYKKQYENNSFDESVHALFSNAKDFEQPEPETDGEQQRDVVKDDYPVMEADKTRVDRTNYDLRTKLVRKRGKAIYSKEDKPSLKSLTPTSTLSDLERLKTTIKPGFNEDDALFEIFQNMASVFSITNAALRNNIIKMIHKFRSNSGGVYENPALTSAVEQHPSTERYCKELEEYMRNQLSKHKGDVGMLEDKQVYWKIKGSTKQTKDHRNTIRAGKNFSLTPQYDGGVKSEEKRKNLFDGVGIALGDIWATEISILDYQLHNDGSYKLTYQVTLLDHFGLNKEDLKWYFSLHSDIGNGFVSWFLLQHFHGYKPFITRIIFKRSFRGKIEKA

[0092] Nucleic acid sequence of bacteriocin 1

[0093]

[0094] 1.1.6 Amino acid sequence of bacteriocin 2

[0095] KEKKGKDCGGKYCITRSNYKEKNAGKLIQEINIRLAGFGGNIPTEEFTDRTEACIKQFQRDYMGVTATGKICGSLLKAIDEFCKNASYDFSFEQTKCPCTNGKNEAFGSYTLCSGYGNGKRNEHPGMHRSLLFILKTLLFYLKKTKS KYTLDCIYSAYRCTTDNNDSKRHTVRTTTNHMGDALDLHFKKDGIRTSSVDDMEAIRKDFFCKYMGAPEGKKNNTYYFGWETNKIGLEPKKFRDGGSGATSWVHVDVREFTNYKADDFYIKEKNLLAPKTLIQIAKELKLIDMCNCLK

[0096] bacteriocin 2 nucleic acid sequence

[0097] AAGGAAAAGAAAGGAAAAGACTGTGGTGGTAAGTATTGTATAACGAGAAGCAATTACAAAGAAAAGAATGCGGGTAAGTTGATACAAGAAATTAACATCCGTCTTGCTGGGTTCGGTGGAAATATACCAACTGAAGAATTCACAGATAGAACAGAAGCATGCATAAAACAATTCCAGAGAGATTATATGGGAGTTACTGCAACAGGGAAAATTTGTGGAAGTTTGCTTAAGGCCATTGATGAGTTTTGTAAAAATGCAAGTTATGATTTTAGCTTTGAGCAAACGAAATGTCCCTGTACAAATGGAAAGAATGAAGCATTTGGTAGTTATACCTTATGCAGTGGTTATGGTAATGGAAAAAGAAATGAACATCCGGGAATGCATAGATCGTTGCTGTTTATTTTAAAAACGTTGCTATTCTATTTAAAAAAAACAAAATCGAAATATACTCTTGATTGTATTTATTCAGCTTATCGATGCACTACAGATAATAATGATTCTAAAAGACATACAGTGAGAACAACTACCAATCACATGGGTGATGCATTGGATTTACACTTTAAGAAAGATGGTATTCGCACTTCGTCGGTAGATGATATGGAAGCAATAAGAAAGGATTTTTTCTGTAAATATATGGGGGCACCTGAAGGTAAGAAAAATAATACTTATTATTTTGGCTGGGAAACAAACAAAATAGGATTAGAACCCAAGAAATTTAGAGATGGTGGCTCGGGAGCAACATCATGGGTTCATGTTGATGTACGAGAGTTTACTAATTATAAGGCTGATGATTTTTACATTAAAGAGAAAAATCTACTTGCTCCAAAAACATTAATTCAAATAGCAAAAGAATTAAAATTAATAGATATGTGTAATTGTTTAAAATAA

[0098] 1.2 Growth conditions of Bacteroides fragilis GS086[[ID=~4]]

[0099] Bacteroides fragilis strain GS086 was cultured at 37°C in an anaerobic workstation with an anaerobic atmosphere consisting of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide. Liquid culture medium was prepared by adding 1 g / L L-cysteine ​​and 5 mg / L heme to Brain Heart Infusion Broth (BHI) medium. Solid culture medium was prepared by adding 15% m / v agarose to the liquid culture medium.

[0100] The protein delivery system coding strain described below is available from the Broad Institute-OpenBiome Microbiome Library (BIOML), sample ID: aa_0143_0055_e12. NCBI Biosample number: SAMN11943472.

[0101] 1.3 Modification of the Bacteroides fragilis GS086 T6SS protein delivery system

[0102] Bacteroides fragilis GS086 T6SS encodes two effector proteins, Effector 1 (E1) and Effector 2 (E2). E1 and E2 possess conserved delivery domains and variable toxicity domains, respectively. By replacing the toxicity domains of E1 and E2 with the target proteins, multiple target proteins can be delivered simultaneously via T6SS. The replacement of the toxicity domains in the Bacteroides fragilis GS086 E1 and E2 effector proteins is achieved through gene knockout or knock-in. Gene knockout or knock-in uses pSIE series non-replicating vectors. The pSIE series non-replicating vector plasmid structure and gene knockout procedure are described in [link to documentation]. Figure 5 .

[0103] 1.3.1 Constructing gene knockout plasmids using the Gibson assembly method

[0104] Gene knockout in Bacteroides fragilis is achieved based on the bacteria's endogenous homologous recombination ability. Using the pSIE series of non-replicating vectors, the target gene is knocked out or knocked in without leaving a trace through the homologous double exchange method.

[0105] The construction of the target gene knockout vector is as follows: First, using the genome of Bacteroides fragilis GS086 strain as a template, 1Kbp DNA fragments upstream and downstream of the target gene were obtained by PCR and gel extraction, denoted as the upstream homologous arm HRF and the downstream homologous arm HRR, respectively. Then, the HRF and HRR were seamlessly cloned into the pSIE gene knockout vector using the Gibson assembly method, completing the construction of the gene knockout plasmid. The plasmid construction method based on Gibson assembly is very mature and can be completed using mainstream commercial kits.

[0106] 1.3.2 Bacteroides fragilis GS086 gene knockout procedure

[0107] The gene knockout procedure for Bacteroides fragilis GS086 is as follows: Figure 5 Specifically, the constructed gene knockout plasmid was transformed into E. coli S17-1λ-pir competent cells using a heat shock method, and then plated onto LB agar plates containing Amp to prepare E. coli S17-1λ-pir donor strains carrying the positive plasmid. The Bacteroides to be genetically manipulated were activated and streaked onto freshly prepared BHI plates to prepare Bacteroides fragilis GS086 recipient strains. The following day, donor and recipient strains were mixed at a ratio of 10:1 to transfer the gene knockout plasmid from the donor strain E. coli S17-1λ-pir to the recipient strain Bacteroides fragilis GS086 via conjugation transfer.

[0108] By plating conjugated mixed colonies onto BHI positive selection plates containing chloramphenicol, positive Bacteroides fragilis GS086 recombinants that had successfully undergone their first homologous recombination were screened.

[0109] The positive Bacteroides fragilis GS086 recombinant was expanded and plated on BHI reverse selection plates containing dehydrotetracycline to screen for the positive Bacteroides fragilis GS086 mutant strains that successfully underwent a second homologous recombination.

[0110] Single colonies were selected for colony PCR identification, and the PCR products were sequenced in the first generation to identify the target gene knockout and screen out the positive Bacteroides fragilis GS086 mutant strain that successfully underwent a second homologous recombination.

[0111] 1.4 Assessment of secretion by the Bacteroides fragilis GS086 T6SS protein delivery system

[0112] After the target protein is fused with a conserved delivery domain, it is secreted into the bacterial extracellular space in a T6SS-dependent manner. Therefore, Western immunoblotting can be used to detect whether the intracellular fusion protein is normally expressed, whether it is secreted into the extracellular space, and to assess the amount of secretion.

[0113] 1) Preparation of seed culture: Streak wild-type Bacteroides fragilis GS086 or a mutant expressing the target protein onto a fresh BHI agar plate. Pick a single colony and incubate overnight in 1 mL of liquid BHI medium.

[0114] 2) Culture of secretory strains: The overnight seed culture obtained in step 1 was inoculated into 10 mL of fresh BHI medium at a ratio of 1:100 and cultured anaerobically for about 4.5 h until OD was reached. 600 It reaches 0.6-0.8.

[0115] 3) Preparation of whole-cell lysate and secretory supernatant samples: Centrifuge 10 mL of the collected culture at 9000 g, 4 °C for 10 min to separate the culture supernatant and bacterial pellet. Filter the culture supernatant through a 0.22 μm filter membrane and then concentrate it to approximately 200 μL (50× concentration) using an ultrafiltration tube (molecular weight cutoff 10 kDa). Boil the concentrated supernatant / resuspended bacterial culture in 200 μL of 2×SDS loading buffer (100 mM Tris 6.8, 200 mM DTT, 4% w / v SDS, 0.1% w / v bromothymol blue, 20% glycerol) to prepare the supernatant (SUP) sample.

[0116] 4) Western immunoblot detection of extracellular secretion: 5 μL of the supernatant was subjected to SDS-PAGE protein gel electrophoresis. The gel contents were transferred to a PVDF membrane using a wet transfer method, blocked with 5% skim milk powder-TBST, and incubated overnight at 4°C with primary antibody. The next day, after washing off the primary antibody, the membrane was incubated with a secondary antibody conjugated with horseradish peroxidase at room temperature for 1 hour. Western immunoblot substrate was then added, and after sufficient reaction, imaging was performed using a chemiluminescence imaging system.

[0117] 1.5 Assessment of the physiological activity of secreted proteins

[0118] Depending on the experimental objective, the target protein, after being fused with a conserved delivery domain, is secreted extracellularly into bacteria in a T6SS-dependent manner. Therefore, the methods for detecting the physiological activity of the target protein vary depending on the protein. For example, if the target protein is an enzyme, its presence in the secretory supernatant can be determined by enzyme activity assays. In this embodiment, the delivered target proteins are two novel bacteriocins with synergistic effects. Bacteriocin 1 alone has strong bactericidal activity, while bacteriocin 2 alone has almost no bactericidal activity. When used together, they achieve a synergistic bactericidal effect ("1+1>2"). Therefore, after fusing the bacteriocin with the delivery domain, the bactericidal phenotype is detected to determine whether the fused bacteriocin is correctly folded, secreted, and possesses normal physiological activity.

[0119] Detection of physiological activity of bacteriocins expressed in fusion using a co-culture method

[0120] 1) Using antibiotic resistance gene markers on recipient strains: In co-culture experiments, recipient strains need to be marked with antibiotic resistance genes, and antibiotic selection is used to isolate the recipient strains from the donor-recipient co-culture mixture. In this example, based on site-specific integrase, the chloramphenicol resistance gene is integrated into the recipient strain genome, making it resistant to chloramphenicol. Specifically, the pNBU2-Cmr series plasmids are transformed into recipient strain cells via conjugation transfer, and then the plasmids are linearized and inserted into a specific site (attN site) on the recipient strain genome using site-specific integrase. The recipient strains marked with the resistance gene are then re-stripened on BHI agar plates supplemented with chloramphenicol (final concentration 15 μg / mL) to confirm successful integration of the resistance gene.

[0121] 2) Preparation of seed culture: Streaking donor strains (toxin-secreting strains) and recipient strains (toxin-sensitive strains) onto fresh BHI agar plates. Single clones from each strain were picked and cultured overnight in 1 mL of liquid BHI medium.

[0122] 3) Preparation of donor and recipient strains for bacterial co-culture experiment: The seed cultures of the donor and recipient strains were inoculated into 10 mL of BHI liquid medium at a ratio of 1:100 and anaerobically grown at 37°C for about 4 h until the OD600 was approximately 0.4.

[0123] 4) Bacterial co-culture: Donor and recipient bacterial cultures were mixed at a 5:1 volume ratio to prepare a mixed culture, with a total volume of 12 mL. The mixed culture was centrifuged at 9000 g, 4°C for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in 200 μL of PBS. 20 μL of the resuspended culture was spotted onto a BHI agar plate. After anaerobic growth at 37°C for approximately 24 h, the bacterial cells on the plate were scraped off and resuspended in 1 mL of PBS buffer; this was recorded as the stock solution.

[0124] 5) Agar spot assay for bactericidal activity of bacteriocins: Dilute the resuspended bacterial solution obtained in the previous step 10-fold, and spot 2.5 μL of each dilution onto BHI agar plates supplemented with chloramphenicol (final concentration 15 μg / mL). Incubate anaerobically at 37°C for approximately 20 hours to assess the bactericidal activity of the bacteriocins.

[0125] 1.6 Upregulating the secretion of target protein using an inducible expression system

[0126] The basal expression or secretion levels of bacteria are relatively fixed, making precise control impossible in experiments requiring accurate regulation of expression and secretion. Therefore, by placing a chimera expressing the target protein under the control of an inducible expression system, the expression and secretion of the target protein can be regulated by adjusting the concentration of the inducer.

[0127] The Bacteroides fragilis protein inducible expression system integrates the rhamnosylprotein inducible expression system and the target gene into a specific site (attN site) on the Bacteroides fragilis genome via site-specific integrases on the pNBU2 series of non-replicating vectors. The chimeric expression and secretion are regulated by adjusting the concentration of the inducer rhamnosyl. The final concentration of rhamnosyl used is 0-10 mM.

[0128] The structure and integration process of pNBU2 series non-replicating vector plasmids are shown in [link to documentation]. Figure 5 .

[0129] 1.6.1 Construction of gene-inducible expression plasmids using the Gibson assembly method

[0130] Using the pNBU2 series of non-replicating vectors, the rhamnoglycoprotein inducible expression system and the target gene were integrated into the Bacteroides fragilis genome via site-specific integrase, thereby achieving the induction of the target gene expression.

[0131] The pNBU2 series of non-replicating vectors were constructed as follows: the target gene fragment to be induced for expression was obtained by PCR and gel extraction. Then, the target gene fragment was placed after a rhamnose inducible expression element using the Gibson assembly method, thus completing the construction of the gene-inducible expression plasmid. The plasmid construction method based on Gibson assembly is very mature and can be completed using mainstream commercial kits.

[0132] 1.6.2 Induced expression of target genes in Bacteroides fragilis

[0133] The induction expression protocol for the target gene in Bacteroides fragilis is as follows: Figure 5 Specifically, the constructed pNBU2 series of non-replicating vectors were transformed into E. coli S17-1λ-pir competent cells using a heat shock method, and then plated onto LB agar plates containing Amp to prepare E. coli S17-1λ-pir donor strains carrying positive plasmids. The Bacteroides to be genetically manipulated were activated and streaked onto freshly prepared BHI plates to prepare Bacteroides fragilis GS086 recipient strains. The following day, donor and recipient strains were mixed at a ratio of 10:1 to transform the pNBU2 series of non-replicating vectors from the donor strain E. coli S17-1λ-pir to the recipient strain Bacteroides fragilis GS086 via conjugation transfer.

[0134] By plating conjugated mixed colonies onto BHI positive selection plates containing chloramphenicol, positive Bacteroides fragilis GS086 recombinants that successfully underwent site-specific integration were screened. At this point, the Bacteroides fragilis rhamnose gene-inducible expression system had been integrated into the Bacteroides fragilis genome along with the pNBU2 series of non-replicating vectors.

[0135] Single colonies were selected for colony PCR identification, and the PCR products were subjected to first-generation sequencing to determine the rhamnose gene inducible expression system and the successful integration of the target gene.

[0136] Positive recombinants verified by sequencing were cultured in BHI medium containing rhamnose inducer, and the bacteria were harvested. The expression and secretion levels of the target gene were assessed as described in 1.4 and 1.5.

[0137] 2. Experimental Results

[0138] 2.1 T6SS protein delivery system gene cluster

[0139] The core proteins of the T6SS delivery system have been specified in Experimental Method 1.1, such as... Figure 1 As shown, the delivery domains (highlighted in red) of the key core proteins involved in delivery, including VgrG, accessory proteins, and effector proteins, all exhibit over 98% conservation, with highly conserved amino acid sequences. In contrast, the toxic domains of the effector proteins (highlighted in black, which can be replaced by the target protein) are variable and show no conservation among subspecies. Site-specific substitution of the toxic domain can achieve target protein delivery.

[0140] 2.2 Modification of the T6SS protein delivery system

[0141] 1. Two effector proteins were targeted for knockout using pSIE series plasmids, retaining only the conserved delivery domains. Specific gene clusters include... Figure 2 As shown.

[0142] 2. The toxic domains of two effector proteins were replaced with novel bacteriocins using pSIE series plasmids, with specific gene clusters as follows: Figure 2 As shown.

[0143] 3. Through pNBU2-P RhaR A series of plasmids regulate the expression and secretion of novel bacteriocins, with specific gene clusters such as... Figure 2 As shown.

[0144] 2.3 T6SS protein delivery system secretion detection

[0145] Wild-type strains were detected by Western blotting (wild-type strain genotype, such as...). Figure 1 (as shown) and each mutant strain (mutant genotype as shown) Figure 2 The extracellular secretion levels (as shown) are for each wild-type strain and mutant strain, as shown in the figure. Figure 3As shown, mutants retaining only the delivery domain (lanes 3 and 7), those fused with a single bacteriocin (lanes 4 and 5), and those fused with two bacteriocins (lanes 6 and 8) all showed detectable levels of modified protein in the supernatant that were comparable to or exceeded those of the wild-type strain. The secretion of the modified protein in the mutants was further upregulated upon the addition of an inducer (lanes 7 and 8).

[0146] 2.4 Detection of physiological activity of bacteriocins expressed in fusion using a co-culture method

[0147] GS086 encodes the homologous immune proteins I1 / I2 ( Figure 1 Therefore, it can resist the killing effect of E1 / E2. GS077, expressing two novel bacteriocin 1 / 2 proteins at a baseline, can effectively kill GS086. Modifying the E1 / E2 toxicity domains of GS086 and fusing them with bacteriocin 1 / 2 proteins from GS077 could theoretically give the GS086 mutant the ability to kill the wild-type GS086. The physiological activity of the fused bacteriocins was detected using a co-culture method, and the results are as follows: Figure 4 As shown, the GS086 mutant strain expressing bacteriocin 1 / 2 (Strain 6) or bacteriocin 1 alone (Strain 4) effectively killed the GS086 wild-type strain, while the mutant strain expressing bacteriocin 2 alone (Strain 5) showed no significant killing ability. After knocking out the essential accessory protein Adaptor, the T6SS delivery system no longer secreted effector proteins and lost its bactericidal activity.

[0148] The above results, based on secretion and physiological activity assays, indicate that after replacing the toxic domain with the target protein, the fusion protein can be normally secreted by the T6SS protein delivery system and maintain normal physiological activity.

[0149] 2.5 Potential Applications of Engineering Upgrading of GS086 Type T6SS Systems

[0150] Given that the GS086-type T6SS system can simultaneously fuse, express, and secrete two proteins, the engineered T6SS, after rational modification, can deliver two types of enzyme systems with cascade activation effects / multiple bactericidal toxins with synergistic effects. Therefore, it has potential applications in in vivo ELISA detection / targeted adjustment of gut microbiota structure.

[0151] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An engineered, highly efficient bacterial secretion system for protein delivery, characterized in that, The bacterial secretion system is based on the type 6 secretion system T6SS of Bacteroides fragilis. T6SS includes a core protein VgrG, accessory proteins, and multiple effector proteins. The effector proteins all have conserved delivery domains and replaceable toxic domains. In use, the target protein to be secreted is fused with the conserved delivery domain for expression, thereby replacing the toxic domain.

2. The bacterial secretion system as described in claim 1, characterized in that, The core protein VgrG has the following characteristics: a1) The amino acid sequence as shown in SEQ ID NO.1; a2) Proteins with the same function as shown in SEQ ID NO.1, which have one or more amino acid residues substituted and / or deleted and / or added; The accessory protein has: b1) The amino acid sequence as shown in SEQ ID NO.2; b2) Proteins with the same function as shown in SEQ ID NO.2, by substitution and / or deletion and / or addition of one or more amino acid residues.

3. The bacterial secretion system as described in claim 1, characterized in that, The plurality of effector proteins comprises two, namely a first effector protein and a second effector protein, wherein the delivery domain of the first effector protein has: c1) The amino acid sequence as shown in SEQ ID NO.3; c2) Proteins with the same function as shown in SEQ ID NO.3, by substitution and / or deletion and / or addition of one or more amino acid residues; The amino acid sequence of the toxic domain of the first effector protein is shown in SEQ ID NO.4; The second effector protein has a delivery system that includes: d1) The amino acid sequence as shown in SEQ ID NO.5; d2) Proteins with the same function as shown in SEQ ID NO.5, by substitution and / or deletion and / or addition of one or more amino acid residues; The amino acid sequence of the toxic domain of the second effector protein is shown in SEQ ID NO.

6.

4. The bacterial secretion system as described in claim 1, characterized in that, The target protein is an enzyme, antibody, toxin, or signaling molecule; further, there are at least two target proteins, namely, a first target protein replaces the toxic domain of the first effector protein, and a second target protein replaces the toxic domain of the second effector protein.

5. A nucleic acid molecule encoding the engineered, highly efficient protein delivery bacterial secretion system according to any one of claims 1-4.

6. An engineered bacterium, characterized in that, The bacteria comprise the engineered, highly efficient protein-delivering bacterial secretion system of any one of claims 1-4 and / or nucleic acid molecules encoding the engineered, highly efficient protein-delivering bacterial secretion system, thereby secreting and expressing the target protein.

7. The bacteria as described in claim 6, characterized in that, The engineered bacteria also include an inducible expression system, which regulates the expression of the target protein by adjusting the concentration of the inducer.

8. The bacteria as described in claim 7, characterized in that, The inducible expression system is a rhamnoglycoprotein inducible expression system.

9. The use of the bacterial secretion system according to any one of claims 1-4, the nucleic acid molecule according to claim 2, and the engineered bacteria according to any one of claims 6-8 in protein delivery.

10. The application as described in claim 9, characterized in that, The protein is an enzyme, antibody, toxin, or signaling molecule; Furthermore, the application specifically refers to the use of the bacterial secretion system, nucleic acid molecules, and engineered bacteria in the preparation of protein delivery products; the product is a pharmaceutical or a general laboratory reagent for non-pharmaceutical purposes.

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