WO2023093883A1 - 一种经遗传修饰的微生物及其应用 - Google Patents

一种经遗传修饰的微生物及其应用 Download PDF

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WO2023093883A1
WO2023093883A1 PCT/CN2022/134672 CN2022134672W WO2023093883A1 WO 2023093883 A1 WO2023093883 A1 WO 2023093883A1 CN 2022134672 W CN2022134672 W CN 2022134672W WO 2023093883 A1 WO2023093883 A1 WO 2023093883A1
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polypeptide
genetically modified
amuc
modified microorganism
microorganism
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French (fr)
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郭雨奇
杨国雪
向斌
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Commbio Therapeutics Co Ltd
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Commbio Therapeutics Co Ltd
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Priority to EP22897964.7A priority Critical patent/EP4438049A4/en
Priority to US18/713,062 priority patent/US20250017982A1/en
Priority to CA3238993A priority patent/CA3238993A1/en
Priority to JP2024531338A priority patent/JP2024544610A/ja
Priority to CN202280076857.4A priority patent/CN118541470A/zh
Publication of WO2023093883A1 publication Critical patent/WO2023093883A1/zh
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    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a genetically modified microorganism and its application.
  • Inflammation is the body's normal response to remove pathogenic or damaged cells from the body to aid in tissue repair. Disorders of the immune system often result in abnormal inflammation, and inflammatory diseases involving the immune system often underlie a large class of human diseases.
  • autoimmune disease is a condition in which there is an abnormal immune response to normal body parts. At least 80 autoimmune diseases affect 24 million people in the United States alone. Current treatments include nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressants. However, these treatments can only relieve symptoms, not cure them completely. Moreover, these treatments often cause serious side effects.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • immunosuppressants include immunosuppressants. However, these treatments can only relieve symptoms, not cure them completely. Moreover, these treatments often cause serious side effects.
  • the present disclosure provides a genetically modified microorganism comprising at least two exogenous genes encoding polypeptides selected from the group consisting of: a) Amuc_1100 polypeptide; b) IL-10 polypeptide; and c) IL- 22 polypeptides.
  • the genetically modified microorganism comprises exogenous genes encoding the following polypeptides, respectively: a) Amuc_1100 polypeptide and IL-10 polypeptide; b) IL-10 polypeptide and IL-22 polypeptide; or c) Amuc_1100 polypeptide and IL-22 polypeptide.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide, an IL-10 polypeptide, and an IL-22 polypeptide, respectively.
  • the present disclosure provides a combination of genetically modified microorganisms, wherein the combination of genetically modified microorganisms comprises at least two different genetically modified microorganisms, wherein each genetically modified microorganism expresses at least A different exogenous gene; wherein the exogenous genes respectively encode polypeptides selected from the group consisting of: a) Amuc_1100 polypeptide; b) IL-10 polypeptide; and c) IL-22 polypeptide.
  • the polypeptide encoded by the exogenous gene can be expressed by the microorganism and/or can be secreted outside the cell/microorganism after expression.
  • the microorganism is capable of expressing and/or secreting the polypeptide encoded by the exogenous gene in the intestinal tract of a human or an animal.
  • the secretion is via a native or non-native secretion system of the microorganism.
  • the Amuc_1100 polypeptide, IL-10 polypeptide and/or IL-22 polypeptide is a polypeptide with its own signal peptide removed.
  • the Amuc_1100 polypeptide, IL-10 polypeptide and/or IL-22 polypeptide that have removed their own signal peptide are also connected to the signal peptide respectively, such as Amuc_1100 polypeptide, IL-10 polypeptide and/or IL-22 polypeptide respectively It is connected with the first signal peptide, the second signal peptide or the third signal peptide, and the first signal peptide, the second signal peptide and the third signal peptide can secrete the polypeptide outside the microorganism.
  • the present disclosure provides a nucleotide sequence comprising at least one recombinant expression cassette comprising i) at least one or at least two encoding Amuc_1100 polypeptides, IL-10 polypeptides and/or IL-22 polypeptides respectively exogenous genes, and ii) one or more regulatory elements operably linked to said at least one or at least two exogenous genes.
  • the nucleotide sequence comprises exogenous genes encoding Amuc_1100 polypeptide and IL-10 polypeptide respectively, exogenous genes encoding IL-10 polypeptide and IL-22 polypeptide respectively, or encoding Amuc_1100 polypeptide and IL-10 polypeptide respectively The exogenous gene of -22 polypeptide.
  • the nucleotide sequence comprises exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide respectively.
  • the present disclosure provides a composition, wherein the composition comprises: (a) as an active ingredient a genetically modified microorganism described in the present disclosure; and (b) a physiologically or pharmacologically acceptable Carrier.
  • the composition is a pharmaceutical composition.
  • the composition is an oral formulation.
  • the present disclosure provides a use of the genetically modified microorganism or composition described in the present disclosure for the preparation of a medicament for treating or preventing an inflammatory disease or an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of inflammatory bowel disease, graft-versus-host disease (GvHD), systemic lupus erythematosus (SLE), arthritis, asthma, or combinations thereof.
  • GvHD graft-versus-host disease
  • SLE systemic lupus erythematosus
  • the present disclosure provides a use of the genetically modified microorganism or composition described in the present disclosure in the preparation of a medicament for improving the therapeutic effect of a medicament for treating an inflammatory disease or an autoimmune disease.
  • the present disclosure provides a method for preparing the genetically modified microorganism of the present disclosure, wherein the preparation method includes the step of: introducing the nucleotide sequence described in the present disclosure into the microorganism, so that the nucleoside Foreign genes in the acid sequence can be expressed in the microorganism.
  • the present disclosure provides a method of treating or preventing an inflammatory disease or an autoimmune disease in an individual in need thereof, comprising: administering to the individual an effective amount of the genetically modified microorganism described in the present disclosure , or a composition of the present disclosure.
  • the present disclosure provides a method of improving the effect of a drug treatment in an individual undergoing drug therapy, including a drug for the treatment of an inflammatory disease or an autoimmune disease, the method comprising: administering to the individual An effective amount of a genetically modified microorganism described in the present disclosure, or a composition described in the present disclosure.
  • the present disclosure provides a use of a genetically modified microorganism in the preparation of a medicament for treating an inflammatory disease or an autoimmune disease, wherein the inflammatory disease or an autoimmune disease is selected from the group consisting of: inflammation Infectious bowel disease (IBD), graft-versus-host disease (GvHD), systemic lupus erythematosus (SLE), arthritis, and asthma; wherein the genetically modified microorganisms comprise at least one, at least two, or at least three, respectively A foreign gene encoding a polypeptide selected from the group consisting of: a) Amuc_1100 polypeptide; b) IL-10 polypeptide; and c) IL-22 polypeptide.
  • IBD Infectious bowel disease
  • GvHD graft-versus-host disease
  • SLE systemic lupus erythematosus
  • arthritis and asthma
  • the genetically modified microorganisms comprise at least one, at least two, or at least three, respectively A foreign
  • Figure 1 shows a schematic diagram of the insertion of exogenous gene expression cassettes into the EcN genome.
  • Fig. 2 Comparison results of aerobic and anaerobic conditions on the production of IL-10 expressed by strain CBT4078.
  • Figure 3 shows the comparison results of the expression of Amuc_1100 (Y259A) with different copy numbers (Panel A), and the comparison results of improving the stability of Amuc_1100 by modifying the outer membrane of cells (Panel B).
  • column A and column B in figure A represent only one Amuc_1100 (Y259A) copy under the control of promoter BBA_J23101 bacterial strain CBT4101
  • C column represents only one Amuc_1100 (Y259A) copy under the control of promoter BBA_J23110 bacterial strain CBT4107
  • D represents the strain CBT4102 with two copies of Amuc_1100 (Y259A) under the control of promoter BBA_J23110
  • column E represents the strain CBT4103 with three copies of Amuc_1100 (Y259A) under the control of promoter BBA_J23101
  • column F represents one copy of Amuc_1100 (Y259A) under the control of promoter BBA_J
  • Figure 4A shows the EcN chromosomal map expressing the combination of IL-10, IL-22 and Amuc-1100;
  • Figure 4B shows the growth status of single expression, double-gene combination expression, and three-gene combination expression strains
  • Figure 4C demonstrates the biological activity of secreted IL-10
  • Figure 4D shows the biological activity of secreted IL-22
  • Figure 4E shows the biological activity of secreted Amuc_1100
  • Standard (40 ⁇ g/mL) in the figure represents Amuc_1100 standard substance, and its concentration is 40 ⁇ g/mL
  • EcN represents Amuc_1100 secreted by bacterial strain EcN
  • CBT4080 (28.8 ⁇ g/mL)
  • Amuc_1100 of bacterial strain CBT4080 which The concentration was 28.8 ⁇ g/mL.
  • Figure 5 shows the expression level of IL-10 in the medium supernatant after adding different concentrations of sodium salicylate and continuing to culture for 1-4 hours.
  • Figure 6A shows the comparison results of the colon length of the efficacy endpoint of different bacterial strains in the mouse enteritis model induced by T cell transplantation
  • Figure 6B shows the results of HE staining of colon histopathological sections using different strains in the T cell transplantation-induced enteritis model in mice.
  • Figure 7A shows the body weight changes of the DSS-induced mouse enteritis model
  • Figure 7B shows the DAI score of the mouse enteritis model induced by DSS
  • Figure 7C shows the colon length of the pharmacodynamic end point of the DSS-induced mouse enteritis model.
  • Figure 8 shows the survival rate of mice in each experimental group in the GvHD animal model.
  • Figure 9A shows the scores of renal tubule damage in mice of each experimental group in the SLE animal model
  • Figure 9B shows the concentration of anti-double-stranded DNA antibody IgG in the mouse serum of each experimental group in the SLE animal model
  • Figure 9C shows the concentration of albumin in the urine of mice of each experimental group in the SLE animal model
  • Fig. 9D shows the glomerular damage shown by the PAS staining results of the renal histopathological sections of mice in each experimental group in the SLE animal model.
  • Figure 10A shows the thickness of the mouse footpads of each experimental group in the CIA animal model.
  • Figure 10B shows the disease scores of mice in each experimental group in the CIA animal model.
  • Figure 11A shows the results of counting the total number of cells in the alveolar lavage fluid (BALF) of each experimental group of mice in the asthma animal model;
  • Figure 11B shows the eosinophil count results in the alveolar lavage fluid (BALF) of each experimental group of mice in the asthma animal model;
  • Figure 11C shows the macrophage count results in the alveolar lavage fluid (BALF) of each experimental group of mice in the asthma animal model;
  • Figure 11D shows the neutrophil count results in the alveolar lavage fluid (BALF) of each experimental group of mice in the asthma animal model;
  • Figure 11E shows the results of lymphocyte counts in the alveolar lavage fluid (BALF) of mice in each experimental group in the asthma animal model.
  • Figure 12 shows the nucleotide sequence (SEQ ID NO: 136) that the pCBT001 plasmid that expresses Cas9 protein has.
  • Figure 13 shows the nucleotide sequence (SEQ ID NO: 137) that pCBT003 plasmid has.
  • Figure 14 shows the nucleotide sequence (SEQ ID NO: 138) that pMUT2-kana plasmid has.
  • Figure 15 shows the nucleotide sequence (SEQ ID NO:215) that pCBT012 plasmid has.
  • Figure 16 shows the nucleotide sequence (SEQ ID NO:216) that pCBT013 plasmid has.
  • a and “the” are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article.
  • a protein means one protein or more than one protein.
  • amino acid refers to an organic compound containing amine ( -NH2 ) and carboxyl (-COOH) functional groups, as well as side chains characteristic of each amino acid.
  • Amino acid names are also referred to in this disclosure by standard one-letter or three-letter codes, a summary of which is shown in Table 1.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • a “conservative substitution” in relation to an amino acid sequence refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, between amino acid residues with hydrophobic side chains (such as Met, Ala, Val, Leu and Ile), amino acid residues with neutral hydrophilic side chains (such as Cys, Ser, Thr, Asn and Gln), between amino acid residues with acidic side chains (such as Asp, Glu), between amino acid residues with basic side chains (such as His, Lys, and Arg), or between amino acid residues with aromatic side chains (eg Trp, Tyr and Phe) with conservative substitutions. As is known in the art, conservative substitutions generally do not cause a significant change in the conformational structure of a protein, and thus preserve the biological activity of the protein.
  • conservative substitutions generally do not cause a significant change in the conformational structure of a protein, and thus preserve the biological activity of the protein.
  • mutant refers to a polypeptide or polynucleotide having at least 70% sequence identity to a parent sequence.
  • a mutant may differ from a parent sequence by one or more amino acid residues or by one or more nucleotides.
  • a mutant may have a substitution (including but not limited to conservative substitutions), addition, deletion, insertion or truncation, or any combination thereof, of one or more amino acid residues or one or more nucleotides of a parental sequence.
  • the mutant Amuc_1100 has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99% or more sequence identity.
  • fragment refers to a partial sequence of any length of a parent polypeptide or parent polynucleotide. A fragment may still retain at least part of the function of the parent sequence.
  • fusion/fused when applied to amino acid sequences such as peptides, polypeptides or proteins refers to the combination of two or more amino acid sequences into a single non-naturally occurring amino acid, for example by chemical bonding or recombinant means sequence. Fusion amino acid sequences can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotides into a microorganism.
  • derivative refers to a chemically modified polypeptide or polynucleotide in which one or more well-defined number of substituents have been covalently linked to one or more specific An amino acid residue or one or more specific nucleotides of a polynucleotide.
  • exemplary chemical modifications to a polypeptide can be, for example, alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation, or conjugation of one or more moieties of one or more amino acids.
  • Exemplary chemical modifications to polynucleotides may be (a) terminal modifications, such as 5' or 3' end modifications, (b) nucleobase (or “base”) modifications, including substitution or removal of bases , (c) sugar modification, comprising modification at the 2', 3' and/or 4' position, and (d) backbone modification, comprising modification or replacement of a phosphodiester bond.
  • homologous refers to at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, A nucleic acid sequence (or its complement) or an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • % sequence identity with respect to amino acid sequences (or nucleic acid sequences) is defined as the number of amino acids in a candidate sequence to that in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids) The percentage of amino acid (or nucleic acid) residues that are identical (or nucleic acid) residues. In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical to the reference sequence to which it is compared by the number of amino acid residues (or bases) in the candidate sequence or reference sequence The total number of residues (or bases), whichever is shorter.
  • amino acid residues may or may not be considered identical residues. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (found at the U.S. National Center for Biotechnology Information ; NCBI), see also Altschul S.F. et al., Journal of Molecular Biology, 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389- 3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins D.G.
  • BLASTN Altschul S.F. et al., Journal of Molecular Biology, 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389- 3402 (1997)
  • ClustalW2 available at the European Bioinformatics Institute website, see also Higgins D.G.
  • nucleotide sequence As used herein, the terms “nucleotide sequence”, “nucleic acid” or “polynucleotide” include oligonucleotides (ie short polynucleotides). It also refers to synthetic and/or non-naturally occurring nucleic acid molecules (eg, comprising nucleotide analogs or modified backbone residues or linkages). The term also refers to deoxyribonucleotide or ribonucleotide oligonucleotides in either single- or double-stranded form. The term encompasses nucleic acids containing analogs of natural nucleotides. The term also encompasses nucleic acid-like structures having synthetic backbones.
  • a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequences.
  • degenerate codon substitution can be achieved by generating sequences in which one or more selected (or all) codons are substituted at the third position by mixed bases and/or deoxyinosine residues (see Batzer et al.
  • the term “encoded/encoding” means capable of being transcribed into mRNA and/or translated into peptide or protein.
  • the term “coding sequence” or “gene” refers to a polynucleotide sequence that encodes a peptide or protein. These two terms may be used interchangeably in this disclosure.
  • the coding sequence is a complementary DNA (cDNA) sequence reverse transcribed from messenger RNA (mRNA).
  • mRNA messenger RNA
  • the coding sequence is mRNA.
  • operably linked refers to the juxtaposition of two or more biological sequences of interest, with or without a spacer or linker, such that they are in a relationship that allows them to function in their intended manner .
  • polypeptides When used in reference to polypeptides, it means that the polypeptide sequences are linked in a manner that allows the linked product to have the desired biological function.
  • antibody variable regions can be operably linked to constant regions to provide a stable product with antigen binding activity.
  • the term can also be used in reference to polynucleotides.
  • a polynucleotide encoding a polypeptide when operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it means that the polynucleotide sequence allows expression of the regulatory polypeptide from the polynucleotide way to connect.
  • a regulatory sequence e.g., a promoter, enhancer, silencer sequence, etc.
  • vector means a vehicle into which a genetic element is operably inserted to effect expression of said genetic element, thereby producing a protein, RNA or DNA encoded by said genetic element, or The genetic element is replicated.
  • Vectors can be used to transform, transduce, or transfect host cells (eg, microorganisms) such that the genetic elements they carry are expressed within the host cells. Different vectors may be suitable for different host cells.
  • vectors examples include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage; and animal virus.
  • Vectors can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes.
  • the vector may contain an origin of replication.
  • a vector may also contain materials that facilitate its entry into cells, including, but not limited to, virions, liposomes, or protein coatings.
  • a vector may be an expression vector or a cloning vector.
  • the application provides a genetically modified microorganism, which comprises at least one exogenous gene encoding selected from the group consisting of a gene encoding an Amuc_1100 polypeptide expressed, or a gene encoding an IL-10 polypeptide expressed, or an encoding gene expressing an IL-10 polypeptide A gene expressing IL-22 polypeptide, or any combination of the above.
  • the application provides a genetically modified microorganism comprising at least two exogenous genes respectively encoding a polypeptide selected from the following group: (i) Amuc_1100 polypeptide, (ii) IL-10 polypeptide, and (iii) ) IL-22 polypeptide.
  • the genetically modified microorganisms comprise: a) exogenous genes encoding Amuc-1100 polypeptides and IL-10 polypeptides respectively; b) exogenous genes encoding IL-10 polypeptides and IL-22 polypeptides respectively; Or c) exogenous genes encoding Amuc_1100 polypeptide and IL-22 polypeptide respectively.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide, an IL-10 polypeptide, and an IL-22 polypeptide, respectively.
  • the genetically modified microorganism comprises an exogenous gene encoding an Amuc-1100 polypeptide, an IL-10 polypeptide, or an IL-22 polypeptide.
  • a "foreign gene” is a gene introduced by genetic modification in the microorganism.
  • Exogenous genes may include heterologous genes that are not originally expressed in the microorganism, or may include endogenous genes that are originally expressed in the microorganism introduced through genetic modification (for example, for the purpose of increasing the expression level).
  • the polypeptide expressed by the exogenous gene in the microorganism is also referred to as exogenous polypeptide.
  • the exogenous polypeptide is selected from: Amuc_1100 polypeptide, IL-10 polypeptide, or IL-22 polypeptide.
  • the exogenous polypeptide is a heterologous polypeptide not originally expressed in the microorganism, for example, human IL-10 polypeptide, human IL-22 polypeptide, etc.
  • Amuc_1100 may also be a heterologous polypeptide when the microorganism used does not endogenously express Amuc_1100.
  • Microorganisms that can be used in the present invention include bacteria, archaea, fungi (eg yeast, filamentous fungi) and algae.
  • the microorganisms provided herein comprise probiotic microorganisms or non-pathogenic microorganisms.
  • non-pathogenic microorganisms refers to microorganisms that are not capable of causing disease or adverse reactions in a host.
  • the non-pathogenic microorganism does not contain lipopolysaccharide (LPS).
  • the non-pathogenic microorganisms are commensal bacteria.
  • the non-pathogenic microorganisms are attenuated pathogenic bacteria.
  • the microorganisms used in the invention are probiotic microorganisms.
  • a "probiotic microorganism” means, when administered in an effective amount, a beneficial effect on the health or well-being of an individual, including, for example, health benefits associated with improving the balance of the human or animal microbiota, and/or for use in Restoration of normal microbiota.
  • all probiotic microorganisms have a proven non-pathogenic profile.
  • probiotics help the gut microbiome maintain (or rediscover) its balance, integrity, and diversity.
  • the effects of probiotics can be strain-dependent.
  • probiotic microbial organisms include preparations of probiotic microbial cells (e.g., live microbial cells).
  • the probiotic microorganism is a probiotic bacterium or a probiotic yeast.
  • the probiotic bacteria are selected from the group consisting of Bacteroides, Bifidobacteria (e.g., Bifidobacterium bifidum), Clostridium, Escherichia, Lactobacilli (e.g., Lactobacillus acidophilus, Lactobacillus bulgaricus) Bacillus, Lactobacillus paracasei, Lactobacillus plantarum) and Lactococcus.
  • the probiotic bacterium belongs to the genus Escherichia.
  • the probiotic bacterium is of the species E. coli strain Nissle 1917 (EcN).
  • the probiotic yeast is selected from the group consisting of Saccharomyces cerevisiae, Candida utilis, Kluyveromyces lactis, and Saccharomyces kini.
  • the probiotic microorganism is E. coli strain Nissle 1917 (EcN).
  • Escherichia coli As a model strain for the study of microbial genetics, physiology and metabolism, Escherichia coli has become one of the important chassis bacteria or host bacteria due to the advantages of diverse genetic manipulation tools and clear genetic background.
  • the probiotic bacteria are bacteria normally present in the human gut.
  • the probiotic yeast is selected from the group consisting of Saccharomyces cerevisiae, Candida utilis, Kluyveromyces lactis, and Saccharomyces carlsbergensis).
  • the term “genetic modification” refers to the introduction of modification or modification in DNA and/or RNA or the introduction of exogenous DNA or RNA in cells (such as microorganisms) through artificial intervention.
  • the modification or modification can be introduced by way of recombinant nucleic acid expression vector, or introduced by way of mutation, or introduced by way of gene editing.
  • the term "genetically modified microorganism” refers to a microorganism into which an exogenous gene or exogenous expression cassette (including a vector containing the expression cassette) has been introduced.
  • Genetically modified microorganism is also used interchangeably with “genetically engineered microorganism,” “engineered microorganism,” “genetically modified microorganism,” or “genetically engineered microorganism.”
  • the exogenous expression cassette comprises a polynucleotide encoding the polypeptide or protein of interest and can allow its expression.
  • Introduction of expression cassettes into microorganisms can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation (Davis, L., Dibner, M., Battey, I., Methods in Basic Molecular Biology (Basic Methods in Molecular Biology) "(1986)), can also be realized by gene editing technology (for example, CRISPR technology).
  • Genetically modified microorganisms also encompass any progeny of the microorganisms provided herein or derivatives thereof. It is understood that all progeny may differ from the parent cell due to mutations that may occur during replication.
  • the present application also provides a nucleotide sequence comprising at least one recombinant expression cassette, which comprises i) at least one or at least two exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and/or IL-22 polypeptide respectively , and ii) operably linked to one or more regulatory elements of said at least one or at least two exogenous genes.
  • the present disclosure also provides a combination of genetically modified microorganisms comprising the disclosed microorganisms comprising at least one (eg, comprising at least one, at least two, or at least three) exogenous genes. Combinations of genetically modified microorganisms.
  • the combination of genetically modified microorganisms comprises two different genetically modified microorganisms, wherein each genetically modified microorganism expresses a different exogenous gene; wherein,
  • the polypeptides encoded by the exogenous gene are respectively: a) Amuc_1100 polypeptide and IL-10 polypeptide; b) IL-10 polypeptide and IL-22 polypeptide; or c) Amuc_1100 polypeptide and IL-22 polypeptide.
  • the combination of genetically modified microorganisms comprises three different genetically modified microorganisms, wherein each genetically modified microorganism expresses a different exogenous gene; or the The combination of genetically modified microorganisms comprises two different genetically modified microorganisms, wherein the two genetically modified microorganisms respectively express an exogenous gene and two other exogenous genes; wherein, the exogenous genes encode
  • the polypeptides are respectively: Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide.
  • Amuc_1100 is known as an outer membrane protein present in the bacterial species Akkermansia muciniphila ("A. muciniphila"). Along with two other members, Amuc_1099 and Amuc_1101, Amuc_1100 is located within a gene cluster involved in type IV pili-like formation (Ottman et al., PLoS One, 2017). An exemplary amino acid sequence of Amuc_1100 is published in GenBank: ACD04926.1. In this disclosure, the terms "Amuc_1100” and “AMUC_1100” are used interchangeably.
  • Amuc_1100 broadly encompasses Amuc_1100 polypeptides, as well as Amuc_1100 polynucleotides, eg, DNA or RNA sequences encoding Amuc_1100 polypeptides. As used herein, the term “Amuc_1100” further encompasses wild type Amuc_1100, as well as functional equivalents that are functionally equivalent to the wild type Amuc_1100 polypeptide.
  • wild type when used to describe a polypeptide or polynucleotide, it means that the sequence of said polypeptide or polynucleotide is identical to those found in nature.
  • a wild-type polypeptide or polynucleotide may be a native or naturally occurring polypeptide or polynucleotide sequence, and also broadly includes fragments thereof, even though the fragments themselves may not be found in nature.
  • polypeptide or polynucleotide when used to describe a polypeptide or polynucleotide, it encompasses all kinds of different forms of said polypeptide or polynucleotide, including but not limited to fragments of naturally occurring polypeptides or polynucleotides , mutants, fusions, derivatives, mimics, or any combination thereof.
  • wild-type Amuc_1100 means that the sequence of the Amuc_1100 polypeptide or polynucleotide is the same as or those sequences found in nature. Wild-type Amuc_1100 can be a native or naturally occurring Amuc_1100 sequence, and fragments thereof, even though the fragments themselves may not be found in nature. Wild-type Amuc_1100 may also comprise naturally occurring variants, such as mutants or isomers or different native sequences found in different bacterial strains. The wild-type full-length Amuc_1100 polypeptide has a length of 317 amino acid residues.
  • the exemplary amino acid sequence of the wild-type Amuc_1100 includes but is not limited to the amino acid sequence Amuc_1100 (1-317) shown in SEQ ID NO: 1, the 31-317th in the amino acid sequence shown in SEQ ID NO: 1 Amuc_1100 (31-317) shown and Amuc_1100 (81-317) shown in the 81-317th positions in the amino acid sequence shown in SEQ ID NO:1.
  • Amuc_1100 (1-317) amino acid sequence (SEQ ID NO: 1):
  • the Amuc_1100 polypeptide comprises a functional equivalent of a wild-type Amuc_1100 polypeptide.
  • a functional equivalent of wild-type Amuc_1100 refers to any Amuc-1100 variation that at least partially retains one or more biological functions of wild-type Amuc_1100 despite differences in amino acid sequence or polynucleotide sequence or chemical structure body.
  • wild-type Amuc_1100 include but are not limited to a) regulating and/or promoting the intestinal immune system function of mammals, b) maintaining, restoring and/or increasing the physical integrity of the intestinal mucosal barrier of mammals, c) activating TLR2 , d) increasing the immune response to cancer immunotherapy (e.g., an immune checkpoint modulator) in a mammal, and e) reducing, delaying and/or preventing resistance to one or more immune checkpoint modulators in a mammal sex.
  • cancer immunotherapy e.g., an immune checkpoint modulator
  • functional equivalents of Amuc_1100 retain substantial biological activity of the parent molecule.
  • functional equivalents of Amuc_1100 described herein still retain substantially similar functions to wild-type Amuc-1100, for example, functional equivalents of Amuc-1100 may retain at least a portion (e.g., at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) such as modulation of intestinal immunity and/or activation of TLR2 active.
  • a functional equivalent of an Amuc_1100 polypeptide may comprise a mutant, fragment, fusion, derivative, or any combination thereof of wild-type Amuc_1100.
  • the functional equivalent of Amuc_1100 may include a variant produced by artificially modifying wild-type Amuc_1100, such as an artificial polypeptide sequence obtained by recombinant methods or chemical synthesis.
  • Functional equivalents of Amuc_1100 may comprise non-naturally occurring amino acid residues without affecting activity.
  • Suitable unnatural amino acids include, for example, ⁇ -fluoroalanine, 1-methylhistidine, ⁇ -methyleneglutamic acid, ⁇ -methylleucine, 4,5-dehydrolysine, hydroxyl Proline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, etc.
  • Amuc_1100 variants can encompass all kinds of different forms of Amuc_1100, including but not limited to fragments, mutants, fusions, derivatives, mimics or any combination thereof of wild-type Amuc_1100.
  • the Amuc-1100 fragment has the amino acid sequence shown in SEQ ID NO:2.
  • the Amuc-1100 fragment has the amino acid sequence shown in SEQ ID NO:3.
  • the Amuc_1100 polypeptide comprises a sequence as shown in SEQ ID NO:5, or comprises at least 80% sequence identity with the sequence shown in SEQ ID NO:5 and still maintains the regulation of intestinal immunity and/or Or an amino acid sequence that activates the activity of toll-like receptor 2 (TLR2).
  • TLR2 toll-like receptor 2
  • the Amuc-1100 polypeptide comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % sequence identity and still maintain the amino acid sequence that modulates intestinal immunity and/or activates toll-like receptor 2 (TLR2) activity.
  • TLR2 toll-like receptor 2
  • the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5 is shown in SEQ ID NO:4.
  • the Amuc-1100 polypeptide has a Y259A or Y259S mutation at position 259, and the numbering at position 259 is based on the numbering of the sequence shown in SEQ ID NO:5.
  • the Amuc-1100 polypeptide having the Y259A mutation has the amino acid sequence shown in SEQ ID NO:7.
  • the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:7 is shown in SEQ ID NO:6.
  • the Amuc-1100 polypeptide having the Y259S mutation has the amino acid sequence shown in SEQ ID NO:9.
  • the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:9 is shown in SEQ ID NO:8.
  • the Amuc_1100 polypeptide further comprises a tag or amino acid extension at the N- or C-terminus.
  • the tag can be used for the purification of Amuc_1100 polypeptide.
  • Amino acid extensions can be used to increase stability or reduce clearance. Any suitable tag or extension can be used, such as His-tag (eg 6 ⁇ His tag), protein A, lacZ ( ⁇ -gal), maltose binding protein (MBP), calmodulin binding peptide (CBP), intein - Chitin binding domain (intein-CBD) tags, streptavidin/biotin based tags, tandem affinity purification (TAP) tags, epitope tags, reporter gene tags, etc.
  • the Amuc-1100 polypeptide further comprises an enzymatic digestion site that separates the tag from the rest of the Amuc-1100 polypeptide.
  • Enzymatic digestion sites are available for tag removal if desired. Examples of suitable enzymatic digestion sites include enterokinase recognition sites (eg, DDDDK), factor Xa recognition sites, genenase I recognition sites, furin recognition sites, and the like.
  • Amuc_1100 DNA sequence (SEQ ID NO: 4)
  • Amuc_1100(Y259A) DNA sequence (SEQ ID NO: 6)
  • Amuc_1100(Y259A) amino acid sequence (SEQ ID NO: 7)
  • Amuc_1100(Y259S) DNA sequence (SEQ ID NO: 8)
  • Amuc_1100 is a membrane protein
  • the signal peptide sequence at its N-terminal will promote the membrane localization of the Amuc_1100 polypeptide. Therefore, in order to achieve the purpose of this disclosure, that is, to realize the secretion of the Amuc_1100 polypeptide, the self-signal peptide of Amuc_1100 needs to be replaced by the Other signal peptides capable of secreting the Amuc_1100 polypeptide to the outside of the microorganism, such as the USP45 signal peptide.
  • the genetically modified microorganisms provided by the present disclosure can also express and secrete one or more cytokines.
  • the cytokines are IL-10 and IL-22.
  • the cytokine can also be IL-17A, IL-19, IL-23, IL-35, IL-37 or TGF-beta.
  • Interleukin-10 (IL-10)
  • IL-10 is an anti-inflammatory cytokine that maintains the balance of the immune response and is synthesized by many types of cells, including B cells, monocytes, dendritic cells, natural killer cells and T cells.
  • IL-10 is recognized by specific receptors expressed by hematopoietic cells and belongs to class II cytokines.
  • IL-10 transduces signals through two receptors IL-10R1 and IL-10R2 and the downstream JAK/STAT pathway, and finally activates the expression of anti-inflammatory response genes.
  • IL-10 can inhibit the activity of macrophages and dendritic cells, and indirectly inhibit the activation of T cells and the function of effectors.
  • IL-10 has a protective effect on inflammatory bowel disease and allergic reactions. Defects in IL-10 and/or its receptors are associated with IBD and intestinal sensitivity (Nielsen, 2014).
  • Interleukin-10 is a pleiotropic cytokine produced by several cell types such as macrophages, monocytes, Th2-type and regulatory T cells and B cells.
  • IL-10 is a cytokine with immunosuppressive and anti-inflammatory properties; it regulates the activity of many myeloid and lymphoid cells and directly inhibits the production of several inflammatory cytokines by T cells and NK (natural killer) cells.
  • IL-10 was originally described as a cytokine synthesis inhibitory factor (CSIF) produced by Th2 cells, which inhibit Th1 cells from producing pro-inflammatory cytokines, such as ⁇ -interferon (IFN- ⁇ ), interleukin-1 - ⁇ (IL-1 ⁇ ), Interleukin-1- ⁇ (IL-1 ⁇ ), Interleukin-2 (IL-2), and Tumor Necrosis Factor- ⁇ (TNF- ⁇ ).
  • cytokines such as ⁇ -interferon (IFN- ⁇ ), interleukin-1 - ⁇ (IL-1 ⁇ ), Interleukin-1- ⁇ (IL-1 ⁇ ), Interleukin-2 (IL-2), and Tumor Necrosis Factor- ⁇ (TNF- ⁇ ).
  • IFN- ⁇ ⁇ -interferon
  • IL-1 ⁇ interleukin-1 - ⁇
  • IL-1 ⁇ Interleukin-1- ⁇
  • IL-2 Interleukin-2
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • IL-10 has a strong inhibitory effect on the activation of Th1 cells and the production of pro-inflammatory cytokines led to the hypothesis that IL-10 is a potent immunosuppressant of cell-mediated immune responses.
  • Other authors have proposed the use of this cytokine to treat acute and chronic inflammation and to treat autoimmune diseases. For these reasons, this cytokine has been used in several autoimmune diseases such as psoriasis, rheumatoid arthritis and Crohn's disease.
  • autoimmune diseases such as psoriasis, rheumatoid arthritis and Crohn's disease.
  • infectious processes or cancer it has side effects because it prevents the induction of therapeutically beneficial Th1 responses. Examples of these processes include leprosy, tuberculosis, leishmaniasis and viral infections.
  • IL-10 has been documented to be abundantly expressed in chronic infection by hepatitis C virus.
  • This cytokine can be produced by Th2 cells as a result of stimulation with HCV antigens. It can also be produced by regulatory T cells (D4 and CD8) that suppress the formation of Th1 -type antiviral effector cells.
  • DC infected dendritic cells
  • monocytes exposed to HCV proteins produced greater amounts of IL-10 than non-infected cells, which favored Th2 responses while impeding viral elimination.
  • the genetically modified microorganisms provided by the present disclosure can express and secrete IL-10.
  • IL-10 broadly encompasses IL-10, as well as IL-10 polynucleotides, such as DNA or RNA sequences encoding IL-10.
  • IL-10 further encompasses wild-type IL-10, as well as functional equivalents that are functionally equivalent to wild-type IL-10.
  • functional equivalents of wild-type IL-10 refer to those that at least partially retain one or more biological functions of wild-type IL-10 despite differences in amino acid sequence or polynucleotide sequence or chemical structure. Any IL-10 variant.
  • the one or more biological functions of the wild-type IL-10 include, but are not limited to, the ability to bind IL-10R1/IL-10R2 and function through the receptor-JAK-STAT signaling pathway.
  • the IL-10 comprises the amino acid sequence shown in SEQ ID NO: 11, or comprises at least 80%, at least 85%, at least 90%, at least 95% of the sequence shown in SEQ ID NO: 11 Or an amino acid sequence that is at least 99% sequence identical and still maintains the activity of modulating immune cells (eg, macrophages, dendritic cells).
  • modulating immune cells eg, macrophages, dendritic cells.
  • the IL-10 comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % sequence identity and still maintain amino acid sequences that modulate the activity of immune cells (eg, macrophages, dendritic cells).
  • immune cells eg, macrophages, dendritic cells.
  • the IL-10 comprises a mutation of P2A, wherein the numbering is relative to SEQ ID NO: 13; that is, has the amino acid sequence shown in said SEQ ID NO: 13.
  • variants of IL-10 encompass all kinds of different forms of IL-10, including but not limited to fragments, mutants, fusions, derivatives, mimics or any combination thereof of IL-10.
  • a genetically modified microorganism provided by the present disclosure can comprise any suitable gene encoding IL-10 (eg, human IL-10).
  • the gene encoding IL-10 comprises the nucleotide sequence shown in SEQ ID NO:10, or comprises at least 80%, at least 85%, at least 90%, at least 90% of the sequence shown in SEQ ID NO:10, A nucleotide sequence that is at least 95% or at least 99% sequence identical and whose encoded protein still maintains the activity of modulating immune cells (eg, macrophages, dendritic cells).
  • the gene encoding IL-10 comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86% of the sequence shown in SEQ ID NO: 10 , at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and the encoded protein still maintains a nucleotide sequence that regulates the activity of immune cells (eg, macrophages, dendritic cells).
  • immune cells eg, macrophages, dendritic cells
  • the gene encoding IL-10 may include modifications and/or mutations, eg, to enhance stability under inducing conditions, increase expression of IL-10, and/or increase anti-inflammatory efficacy.
  • the gene encoding IL-10 comprises the nucleotide sequence shown in SEQ ID NO:12.
  • the genetically modified microorganisms provided herein are capable of producing IL-10 under inducing conditions (eg, under conditions induced by microenvironmental factors in inflamed tissues). In some embodiments, the genetically modified microorganism is capable of producing IL-10 under hypoxic or anaerobic conditions. In some embodiments, the genetically modified microorganism is capable of secreting at least 300 ng, at least 350 ng, at least 400 ng, at least 450 ng, at least 500 ng, at least 550 ng, at least 600 ng, or at least 650 ng of IL- 10.
  • Interleukin-22 (IL-22)
  • Interleukin-22 or "IL-22” is a T-cell-secreted glycoprotein that was named IL-10-related T-cell-derived inducible factor (IL-10-related T cell-derived inducible factor (IL-TIF), due to its unique function, has become one of the most intensively studied IL-10 family members.
  • IL-10-related T cell-derived inducible factor IL-10-related T cell-derived inducible factor
  • IL-22 is mainly secreted by adaptive immune cells (CD4 positive T cells, CD8 positive T cells, etc.) and innate immune cells (LTi cells, NK cells, etc.). Numerous transcription factors have been found, such as signal transducer and activator of transcription 3 (STAT3), retinoic acid-related orphan nuclear receptor ⁇ (ROR ⁇ ), aryl hydrocarbon receptor (AhR), etc., as well as cytokines such as IL-23, IL-6, IL-17, TNF- ⁇ , TNF- ⁇ , etc. can affect the expression of IL-22.
  • STAT3 signal transducer and activator of transcription 3
  • ROR ⁇ retinoic acid-related orphan nuclear receptor ⁇
  • AhR aryl hydrocarbon receptor
  • cytokines such as IL-23, IL-6, IL-17, TNF- ⁇ , TNF- ⁇ , etc. can affect the expression of IL-22.
  • IL-22 exerts biological functions by binding to IL-22RI receptor and IL-10R2 receptor. Unlike other immune cytokines that mainly act on hematopoietic cells, IL-22 mainly functions in non-blood-derived cells in tissues such as endothelial cells, stromal cells, fibroblasts, etc., and IL-22 is widely distributed in many tissues. In tissues such as lung, liver, kidney, thymus, breast, intestine, skin and synovium.
  • IL-22 activates the STAT signal transduction pathway to cause downstream proliferation and anti-apoptotic effects to participate in the maintenance and repair of the mucosal barrier, although IL-22 can enhance the repair of epithelial cells after injury and improve the value of epithelial cells through anti-apoptosis and promoting proliferation.
  • the continuous high expression of IL-22 can cause pathogenic effects such as tissue damage and chronic inflammation in certain diseases such as tumors and autoimmune diseases.
  • IL-22 can also induce the production of antimicrobial peptides so as to resist the invasion of microorganisms and parasites. It is precisely because of such extensive and complex protective and pathogenic functions of IL-22 that many studies in recent years have confirmed that IL-22 plays a unique role in infections, autoimmune diseases and tumors.
  • the genetically modified microorganisms provided by the present disclosure express and secrete IL-22.
  • the IL-22 includes a polypeptide having SEQ ID NO: 15 and functional equivalents thereof.
  • the term “IL-22” broadly encompasses IL-22, as well as IL-22 polynucleotides, such as DNA or RNA sequences encoding IL-22.
  • the term “IL-22” further encompasses wild-type IL-22, as well as functional equivalents that are functionally equivalent to wild-type IL-22.
  • a functional equivalent of wild-type IL-22 refers to one or more biological functions of wild-type IL-22 that are at least partially retained despite differences in amino acid sequence or polynucleotide sequence or chemical structure Any variant of IL-22.
  • the one or more biological functions of wild-type IL-22 include, but are not limited to, the ability to bind IL-22R1/IL-10R2, signal through Janus kinases (associated with IL-22R subunits), and STAT molecules. active.
  • the IL-22 comprises the sequence shown in SEQ ID NO: 15, or comprises at least 80% sequence identity with the sequence shown in SEQ ID NO: 15 and still maintains the binding IL-22 receptor And regulate the amino acid sequence of the activity of IL-22 receptor expressing cells.
  • the IL-22 comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, At least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % sequence identity and still maintain the amino acid sequence that binds IL-22 receptor and regulates the activity of IL-22 receptor expressing cells.
  • variants of IL-22 encompass all kinds of different forms of IL-22, including but not limited to fragments, mutants, fusions, derivatives, mimics or any combination thereof of IL-22.
  • Interleukin-17A IL-17A
  • Interleukin-17 or "IL-17A” is produced by activated T cells and mediates an inflammatory response in conjunction with IL-17R, which is ubiquitously expressed on all cell types. However, the exact mechanism of IL-17A signaling is still not fully elucidated. In chronic inflammation including RA, IL-17A can cause tissue damage by directly degrading the matrix, or indirectly increasing the number of activated inflammatory cells and inducing other pro-inflammatory cytokines including IL-1b and TNF-a into the inflammatory tissue .
  • IL-17A activates all three members of MAPKs (mitogen-activated protein kinases), namely extracellular signal-regulated kinases (ERK1 and ERK2) p44 and p42, Jun protein N-terminal kinase (JNK) and p38.
  • MAPKs mitogen-activated protein kinases
  • ERK1 and ERK2 extracellular signal-regulated kinases
  • JNK Jun protein N-terminal kinase
  • IL-17A can induce the activation of NF-kb in human fibroblasts, intestinal epithelial cells and cultured chondrocytes, which may be related to TNF receptor-associated factor (TRAF)-6.
  • TNF receptor-associated factor (TRAF)-6 TNF receptor-associated factor-6.
  • IL-17A induces tyrosine phosphorylation of certain members of the Janus kinase (JAN) and signal transducer and activator of transcription (STAT) pathways, including Tyk2, JAK1,2 and 3, STAT1, 2, 3 and 4, suggesting that the JAK/STAT pathway may be involved in regulating the biological effects of IL-17A.
  • JAN Janus kinase
  • STAT signal transducer and activator of transcription
  • IL-19 mainly transmits biological signals through the IL-20R1/R2 receptor complex to play a biological role.
  • IL19 is a member of the IL-10 family of cytokines, but the role of IL-19 is not yet clear, and whether IL-19 is a pro-inflammatory or anti-inflammatory cytokine is still controversial and uncertain.
  • IL-19 up-regulates the production of IL-6 and TNF- ⁇ in monocytes, showing that IL-19 has pro-inflammatory characteristics.
  • IL-20R1 Although no expression of IL-20R1 was detected in immune cell populations, there are many studies clearly showing the effect of IL-19 on these cells. Most studies have shown that IL-19 is part of the Th2 system. Studies have shown that IL-19 plays an anti-inflammatory effect in myocarditis. Endogenous IL-19 exhibits a protective effect in the inflammatory gut, and some scholars have established a DSS colitis model of IL-19 mice and shown that these mice are more susceptible to colitis than those mice with intact immunity. In active Kern's disease, defective IL-19 expression and lack of response to it favor the development of disease inflammation.
  • adenovirus-mediated IL-19 fusion gene transfer into rats can effectively reduce damage and activate MAPK; it was further found that some cellular inflammatory factors can induce the production of IL-19.
  • IL-19 can reduce the inflammatory response of vascular smooth muscle by reducing the stability of mRNA species encoding inflammatory proteins.
  • Some in vitro studies have shown that IL-19 can induce CD4+ T cells to produce Th2 cytokines, IL-19 in peripheral blood monocytes increases the production of IL-10, and T cells exposed to IL-19 for a long time will down-regulate IFN- ⁇ , upregulates IL-4 and IL-13.
  • IL-19 up-regulates the production of pro-inflammatory factors IL-6 and TNF- ⁇ in monocytes, and IL-19 and its two receptor chains IL-20R1/ IL-20R2 expression, further studies found that treatment that reduces IL-19 levels is effective.
  • Interleukin-23 (IL-23)
  • interleukin-23 or "IL-23” is a heterodimeric cytokine composed of IL-12p40 subunit and IL-23p19 subunit, wherein the p40 subunit is IL-23 and IL-23 12 shared.
  • a functional receptor for IL-23 has been identified and consists of IL-12R ⁇ 1 and IL-23R.
  • IL-23 plays a role in the immune response of type 1 polarized T cells. Although IL-12 can potently activate naive T cells, initial reports suggested that IL-23 could preferentially act on memory T cells to promote IFN- ⁇ secretion and proliferation, suggesting an important role for IL-23 in the control of bacterial infection .
  • IL-23 has been further described as a key cytokine controlling inflammation in peripheral tissues. Overexpression of p19 is associated with inflammation in multiple organs as well as epithelial tissues including skin. In addition, IL-23 is also involved in inflammation of the central nervous system and various autoimmune diseases.
  • IL-23 (IL-12p40 subunit) DNA sequence (SEQ ID NO: 221)
  • IL-23 (IL-12p40 subunit) amino acid sequence (SEQ ID NO: 222)
  • IL-23 (IL-23p19 subunit) DNA sequence (SEQ ID NO: 223)
  • IL-23 (IL-23p19 subunit) amino acid sequence (SEQ ID NO: 224)
  • Interleukin-35 (IL-35)
  • Interleukin-35 or "IL-35” is a heterologous protein consisting of Epstein-Barr virus-induced gene 3 (EBI3) protein and IL-12p35 (IL-12A) subunit.
  • EBI3 Epstein-Barr virus-induced gene 3
  • IL-12A IL-12p35
  • Studies have confirmed that IL-35 is mainly secreted by regulatory T cells (Treg), and is one of the main cytokines for Treg to negatively regulate immunity.
  • Treg regulatory T cells
  • autoimmune demyelinating inflammation chronic hepatitis, diabetes, tumors, etc.
  • participate in the immune regulation of inflammation and are closely related to the occurrence and development of the disease.
  • IL-35 (EBI3 protein) DNA sequence (SEQ ID NO: 225)
  • IL-35 (EBI3 protein) amino acid sequence (SEQ ID NO: 226)
  • IL-35 (IL-12p35 subunit) DNA sequence SEQ ID NO: 227)
  • IL-35 (IL-12p35 subunit) amino acid sequence SEQ ID NO: 228)
  • Interleukin-37 IL-37
  • Interleukin-37 or "IL-37” has five different subtypes (IL-37a-e), and current research shows that only IL-37b has biological functions and anti-inflammatory effects in various diseases .
  • IL-37 can not only be secreted into the extracellular space as a cytokine and bind to receptors to regulate cell activity, but also enter the nucleus as a transcriptional regulator.
  • IL-37 has high homology with L-18, and can bind IL-18-binding protein (IL-18BP) to have a certain inhibitory effect on IL-18 signaling pathway.
  • IL-18BP IL-18-binding protein
  • TGF-beta is a multifunctional cytokine that can be produced and secreted by many types of cells in the human body.
  • TGF- ⁇ is involved in the mechanism of many diseases, playing a dual role of inhibiting or promoting diseases, including wound healing, tissue fibrosis, atherosclerosis, cancer occurrence and metastasis, autoimmune diseases, diabetic complications, and Alzheimer's disease Nerve damage caused by various diseases, etc., are closely related to TGF- ⁇ .
  • TH1 helper T cells
  • TH1 cells If the function of TH1 cells is excessively strong, the secretion of cytokines that cause inflammatory reactions, such as interleukin-2 (Interleukin-2, IL-2) and interferon- ⁇ (Interferon- ⁇ , IFN- ⁇ ), etc., will enhance the cellular immune response , attack specific tissues or special cells of the human body, causing long-term damage to certain tissues or organs of the human body, especially autoimmune diseases and organ transplant rejection.
  • TH2 type 2 helper T cells
  • TH2 cells If the function of TH2 cells is too strong, the secretion of TH2 cell hormones will be too high, prompting B cells to produce a large amount of allergic antibody IgE, IgE will induce mast cells or basophils to release inflammatory substances, such as histamine, interleukin, cell Hormone, platelet activating factor, etc. act on cells or blood vessels, causing vasodilation and smooth muscle contraction, leading to allergic symptoms such as allergic asthma, allergic rhinitis, and atopic dermatitis. These two types of immunity are presented in a balance of balance in the human body.
  • inflammatory substances such as histamine, interleukin, cell Hormone, platelet activating factor, etc.
  • TH1 and TH2 balance each other, and are jointly regulated by regulatory cells (Treg) and immune regulatory factors (TGF- ⁇ ), allowing the body's immune defense
  • Treg regulatory cells
  • TGF- ⁇ immune regulatory factors
  • the balance of TH1 and TH2 in the system can achieve the effect of preventing autoimmune diseases and allergy-related diseases.
  • Common genetic allergic diseases such as: atopic dermatitis, asthma, allergic rhinitis, etc., all lack TGF- ⁇ immune regulatory factors in the body.
  • Studies have shown that long-term supplementation of TGF- ⁇ can increase the concentration in the body, improve physical fitness, reduce inflammation, slow down the allergic index, repair tissues, prolong the protection of breast milk for infants and young children, improve the body's tolerance to food, and maintain digestive tract functions.
  • TGF-beta DNA sequence (SEQ ID NO: 231)
  • TGF- ⁇ amino acid sequence (SEQ ID NO: 232)
  • expression cassette refers to a DNA sequence capable of directing the expression of a specific nucleotide sequence in a suitable microorganism, comprising a promoter operably linked to a nucleotide sequence of interest, said nucleotide sequence of interest
  • the nucleotide sequence is operably linked to a termination signal. It also generally contains sequences required for proper translation of the nucleotide sequence.
  • the coding region typically encodes a protein of interest, but may also encode a functional RNA of interest in the sense of an antisense orientation, such as antisense RNA or untranslated RNA.
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the term "recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., a "recombinant expression cassette"), and refers to a polynucleotide produced in a cell or other biological system using a recombinant polynucleotide or recombinant expression cassette. product, or refers to a polypeptide encoded by a recombinant polynucleotide.
  • a “recombinant expression cassette” encompasses nucleic acid molecules from different sources joined into an expression cassette or vector to express, for example, fusion proteins; or proteins produced by inducible or constitutive expression of polypeptides.
  • a recombinant expression cassette encompasses a recombinant polynucleotide operably linked to one or more regulatory elements.
  • the at least two exogenous genes are contained in (at least one) exogenous expression cassette.
  • the genetically modified microorganism described in the present application comprises at least one, at least two or all three exogenous expression cassettes selected from the group consisting of: the first exogenous expression cassette (in this disclosure can also be referred to as "Amuc_1100 expression cassette”, “exogenous expression cassette A” or “expression cassette A”, the above names are used interchangeably in the present disclosure and have the same meaning), comprising the nucleoside encoding Amuc_1100 polypeptide acid sequence; the second exogenous expression cassette (also referred to as “IL-10 expression cassette” in the present disclosure, “exogenous expression cassette B” or “expression cassette B”, and the above-mentioned names may be used in the present disclosure used interchangeably and have the same meaning), comprising a nucleotide sequence encoding IL-10; and a third exogenous expression cassette (which may also be referred to as "IL-22 expression cassette” in this disclosure, “exogenous Expression Cassette C” or “Expression Cassette C”, the above
  • the at least one exogenous expression cassette comprises one or more regulatory elements (or "expression regulatory elements", both of which have the same meaning herein), which are operably linked to the exogenous Gene.
  • the regulatory element comprises one or more elements selected from the group consisting of a promoter, a ribosome binding site (RBS), a cistron, a terminator, and any combination thereof.
  • promoter refers to a non-translated DNA sequence, usually upstream of a coding region, that contains a binding site for RNA polymerase and initiates transcription of the DNA.
  • the promoter region may also contain other elements that act as regulators of gene expression.
  • the promoter is suitable for initiating a polynucleotide encoding an Amuc_1100 polypeptide, IL-10 and/or IL-22 in a genetically modified microorganism.
  • the promoter is a constitutive promoter or an inducible promoter.
  • constitutive promoter refers to a promoter capable of promoting continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked.
  • Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23119, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23109, BBa_J23110, BBa_J23114, BBa_J23117, USP45_promoter, Amuc_1102_promoter, OmpA _promoter, BBa_J23100, BBa_J23104, BBa_J23105, BBa_I14018, BBa_J45992, BBa_J23118, BBa_J23116, BBa_J23115, BBa_J23113, BBa_J23112, BBa_J23111, BBa_J23
  • the nucleotide sequence of an exemplary constitutive promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 16-56 as shown in Table 3, and which has at least 80% (e.g., at least 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity homologous sequences.
  • the constitutive promoter comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-56.
  • the promoter is active in vitro, eg, under culture, expansion and/or manufacturing conditions.
  • the promoter is active in vivo, eg, under conditions present in an in vivo environment, eg, the intestinal tract and/or an inflammatory microenvironment.
  • the nucleotide sequences of exemplary constitutive promoters are shown in Table 3.
  • inducible promoter refers to a regulated promoter that can be activated in one or more cell types by external stimuli, such as chemicals, light, hormones, stress, or pathogens.
  • inducible promoters and variants are well known in the art and include, but are not limited to, PLteto1, galP1, PLlacO1, Pfnrs, Psal, Pvan.
  • the nucleotide sequence of an exemplary inducible promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 57-61 as shown in Table 4, and having at least Homologous sequences with 80% (eg, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • the promoter is an endogenous promoter or an exogenous promoter.
  • a foreign promoter refers to a promoter that is operably combined with a coding region, wherein the promoter is not the promoter naturally associated with the coding region in the genome of an organism.
  • a promoter naturally associated with or linked to a coding region in the genome is referred to as the endogenous promoter of the coding region.
  • the promoter is operably linked upstream or downstream of the transcription initiation site of the gene of interest.
  • the promoter is selected from: BBa_J23101, BBa_J23108, BBa_J23110, PfnrS, Psal, Pvan, BBa_J23119, BBa_J23102, or BBa_J23114.
  • RBS ribosome binding site
  • SD Shine-Dalgarno
  • CDS coding sequence
  • the nucleotide sequence of an exemplary RBS comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 62-65 as shown in Table 5, and having at least 80% (e.g., at least 85%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity homologous sequences.
  • the ribosome binding site is operably linked to 8-13 nucleotides upstream of the coding region of the gene of interest.
  • the term "cistronic” refers to a segment of a nucleic acid sequence that is transcribed and encodes a polypeptide. Cistrons and variants are well known in the art and include, but are not limited to, GFP, BCD2, luciferase, MBP.
  • the nucleotide sequence of an exemplary cistron comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 66-75 as shown in Table 6, and having at least 80% (e.g., at least 85%) , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity homologous sequences.
  • the cistron is operably linked to the N-terminus of the coding region of the gene of interest.
  • the term "terminator” refers to a nucleotide sequence that provides an RNA polymerase transcription termination signal, which prevents subsequent incorporation of nucleotides into the resulting polynucleotide chain, and thereby interrupts polymerase-mediated elongation.
  • the terminator used in the present disclosure is a T7 terminator.
  • the terminator comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76-79 as shown in Table 7, and which has at least 80% (e.g., at least 85%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity homologous sequences.
  • a terminator is operably linked to the 3' end of the encoding gene.
  • the terminator used in the present disclosure is the rrnB_T1_T7Te_terminator, and the rrnB_T1_T7Te_terminator includes the sequence obtained by concatenating the rrnB_T1_terminator and the T7Te_terminator, and the specific sequence is as follows: caaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctactagag tcacactggctcacc ttcgggtgggcctttctgcg (SEQ ID NO :242).
  • the polynucleotide sequence encoding Amuc_1100 polypeptide, IL-10 and/or IL-22 is operably linked to a signal peptide.
  • the term "signal peptide” or “signal sequence” refers to a protein that can be used to secrete a heterologous polypeptide into the periplasm or culture medium of a cultured bacterium or secrete Amuc_1100 polypeptide, IL-10 and/or IL22 into the periplasm. of peptides.
  • the signal of the heterologous polypeptide may be homologous to the bacterium, or it may be heterologous, comprising a signal native to the polypeptide produced in the bacterium.
  • the signal sequence is usually endogenous to the bacterial cell, but it need not be, so long as it is effective for its purpose.
  • Non-limiting examples of secreted peptides include USP45, OppA (ECOLIN_07295), OmpA, OmpF, cvaC, TorA, fdnG, dmsA, PelB, HlyA, Adhesin (ECOLIN_19880), DsbA (ECOLIN_21525), Gltl (ECOLIN_03430), GspD ( ECOLIN_16495), HdeB (ECOLIN_19410), MalE (ECOLIN_22540), PhoA (ECOLIN_02255), PpiA (ECOLIN_18620), TolB, tort, mglB and lamB.
  • the secreted peptide is USP45.
  • the signal peptides provided herein for Amuc-1100, IL-10 and/or IL22 secretion comprise USP45, OmpA, DsbA, pelB, Cel-CD, sat or Amuc-1100, IL-10 and/or IL22 Endogenous signal peptide (or called “self signal peptide", in this article, “endogenous signal peptide” and “self signal peptide” have the same meaning, both refer to the original or naturally occurring signal peptide sequence in the wild-type polypeptide sequence) .
  • the nucleotide sequence of the exemplary signal peptide comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 123-135 as shown in Table 9, and it has at least 80% (such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity of homologous sequences.
  • the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 80-122 as shown in Table 8, and which has at least 80% (e.g., at least 85%, 90%, Homologous sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity.
  • the signal peptide is operably linked to the N-terminus of the expressed target protein, in some embodiments, the self signal peptide sequence of the target protein (such as Amuc_1100, IL-10 and/or IL-22) is removed, Then at the N-terminus, it is connected with a selected signal peptide (the first signal peptide as described herein, the second signal peptide or the third signal peptide, such as USP45 signal peptide) (that is, the self signal peptide is replaced by the selected signal peptide) peptides) for protein secretion.
  • the self signal peptide sequence of the target protein such as Amuc_1100, IL-10 and/or IL-22
  • the expression cassette comprises a nucleotide sequence encoding Amuc_1100, IL-10 and/or IL-22 operably linked to a signal peptide and an autotransporter domain, wherein the signal peptide and the autotransporter domain Operably linked to the opposite end of Amuc_1100, IL-10 and/or IL-22, for example the signal peptide is operably linked to the N-terminus of Amuc_1100, IL-10 and/or IL-22, and the autotransporter domain can Operably linked to the C-terminus of Amuc_1100, IL-10 and/or IL-22.
  • the constructs can be used for type V autocrine-mediated secretion in which the N-terminal signal peptide is removed upon translocation of the precursor protein from the cytoplasm into the periplasmic compartment by a native secretion system such as the Sec system, and additionally , once the autosecretory translocates across the outer membrane, the C-terminal autotransporter domain can be removed by autocatalytic or protease-catalyzed cleavage of e.g. released into the extracellular environment.
  • a native secretion system such as the Sec system
  • signal peptides can be processed by export pathways and/or secretion systems present in genetically modified microorganisms (eg, genetically modified bacteria).
  • the signal peptide is usually at the N-terminus of the exported precursor protein and can direct the precursor protein to the export pathway in the plasma membrane of the bacterial cell.
  • the secretion system is capable of removing signal peptides from precursor proteins prior to secretion of mature proteins from engineered microorganisms such as bacteria.
  • secretion system is used interchangeably herein with “export system” and “export pathway” and refers to the ability to secrete or export expressed polypeptide products (such as Amuc_1100 polypeptides) from microorganisms (such as bacterial cytoplasm) ) native or non-native secretion mechanism.
  • polypeptide products such as Amuc_1100 polypeptides
  • microorganisms such as bacterial cytoplasm
  • OM outer membrane
  • IM inner membrane
  • OM is an extremely effective and selective permeability barrier.
  • OM is a lipid bilayer composed of phospholipids at the inner lobe and glycolipids at the outer lobe as well as lipoproteins and ⁇ -barrel proteins. OM is anchored to the underlying peptidoglycan by a lipoprotein called Lpp. The periplasm is densely packed with proteins and is more viscous than the cytoplasm.
  • the IM is a phospholipid bilayer and the main location houses membrane proteins that function in energy production, lipid biosynthesis, protein secretion and transport.
  • the Sec pathway processes higher molecular weight precursor proteins in the unfolded state, where a signal peptide targets the substrate protein to the membrane-bound Sec translocase.
  • the precursor target protein is delivered to the translocase and passed through the SecYEG pore by SecA, SecD and SecF.
  • the chaperone proteins SecB, GroEL-GroES, DnaK-DnaJ-GrpE assist in target protein trafficking.
  • the Tat pathway typically transports proteins in a fully folded or even oligomeric state and consists of components TatA, TatB and TatC in both Gram-negative and positive bacteria. Following membrane translocation, the signal peptide is removed by signal peptidase and the mature protein is secreted.
  • secretion systems include Sat secretion system, type I secretion system (T1SS), type II secretion system (T2SS), type III secretion system (T3SS ), type IV secretion system (T4SS), type V secretion system (T5SS), type VI secretion system (T6SS) and the resistance-nodulation-division (RND) family of multi-drug efflux pumps, Various single-membrane secretion systems.
  • T1SS type I secretion system
  • T2SS type II secretion system
  • T3SS type III secretion system
  • T4SS type IV secretion system
  • T5SS type V secretion system
  • T6SS type VI secretion system
  • RTD resistance-nodulation-division
  • the secretion system is a system native or non-native to the genetically modified microorganism.
  • “Native” for a microorganism means that the secretion system is normally present in the microorganism
  • non-native for a microorganism means that the secretion system is not normally present in the microorganism, such as additional secretion systems, such as from different species, strains or subspecies of bacteria or viruses.
  • the genetically modified microorganisms of the present disclosure further comprise one or more genetic modifications in the genome of the microorganism.
  • the one or more genome modifications comprise engineering and/or optimization of the secretion system such that at least one gene encoding an outer membrane protein is deleted, inactivated or suppressed.
  • the secretion system is engineered and/or optimized such that at least one outer membrane protein-encoding gene is deleted, inactivated, or suppressed in the genetically modified microorganism.
  • the outer membrane protein is selected from the group consisting of OmpC, OmpA, OmpF, OmpT, pldA, pagP, tolA, Pal, TolB, degS, mrcA, and lpp.
  • Inducible promoters can be used to replace the endogenous promoters of one or more genes of choice to minimize negative effects on cell viability.
  • “Deletion/deletion” or “inactivation” or “inhibition” of a gene or coding region means that the enzyme or protein encoded by said gene or coding region is not produced, or is produced in a microorganism in an inactive form, or at a rate lower than Amounts found in the wild-type form of the microorganism are produced in the microorganism under the same or similar growth conditions. This can be achieved, for example, by one or more of the following approaches: (1) homologous recombination, (2) RNA interference-based techniques, (3) ZFNs and TALENs, (4) CRISPR/Cas systems.
  • the secretion system is engineered such that at least one chaperonin-encoding gene is amplified, overexpressed, or activated.
  • Chaperones are involved in many important biological processes, such as protein folding and aggregation of oligomeric protein complexes, maintaining protein precursors in an unfolded state to facilitate protein transport across membranes, and enabling depolymerization and repair of denatured proteins. It is mainly to assist other peptides to maintain the normal conformation to form the correct oligomeric structure, so as to exert normal physiological functions.
  • Various chaperones are well known in the art.
  • the chaperone protein is selected from the group consisting of dsbA, dsbC, dnaK, dnaJ, grpE, groES, groEL, tig, fkpA, surA, skp, PpiD, and DegP.
  • the chaperone protein is selected from the group consisting of Ssa1p, Ssa2p, Ssa3p, and Ssa4p from the cytoplasmic SSA subfamily of 70kDa heat shock proteins (Hsp70), BiP, Kar2, Lhs1, Sil1, Sec63, protein Disulfide bond isomerase Pdi1p, in some embodiments, the chaperone consists of dsbA, dsbC, dnaK, dnaJ, grpE, groES, groEL, tig, fkpA, and surA.
  • “Overexpressed/overexpression” of a gene or coding region means that the enzyme or protein encoded by said gene or coding region is brought to a level higher than that found in the wild-type form of the microorganism under the same or similar growth conditions Produced in microorganisms.
  • the "overexpressed" gene or the “overproduced” protein may be native to the microorganism, or it may be transplanted into the microorganism from a different organism by means of genetic modification, in the latter case, the enzyme or protein and the encoding enzyme or The gene or coding region of the protein is said to be “foreign” or “heterologous”. Foreign or heterologous genes and proteins are overexpressed and overproduced because they are not present in the unengineered microorganism.
  • the secretion system mainly consists of nascent protein translocation, protein folding in the endoplasmic reticulum (ER), glycosylation, protein sorting and trafficking.
  • ER endoplasmic reticulum
  • SRPs signal recognition particles
  • Exemplary yeast secretion signal peptides for secretion of heterologous proteins include Saccharomyces cerevisiae ⁇ -Mating Factor ( ⁇ -Mating Factor, ⁇ -MF) pre-pro-peptide, from Kluyveromyces marxianus
  • ⁇ -Mating Factor ⁇ -MF
  • the exogenous expression cassette is integrated into the genome of the genetically modified microorganism.
  • the exogenous expression cassette is contained in a plasmid, and the plasmid is introduced into the microorganism and is suitable for expression in the microorganism.
  • the genetically modified microorganisms described herein are capable of stable growth, stable expression of non-natural genetic material, such as genes encoding Amuc_1100, IL-10 and/or IL-22, or expression of these polypeptides/proteins Exogenous expression cassette.
  • non-natural genetic material is introduced into the genome of the microorganism or replicated on an extrachromosomal plasmid, so that the non-natural genetic material is retained, transcribed and expressed.
  • a stable microorganism (such as a bacterium) can be a genetically modified microorganism that expresses and secretes Amuc_1100, IL-10, and/or IL-22, and carries a gene encoding Amuc_1100, IL-10, and/or IL-22.
  • the plasmid or chromosome of the exogenous expression cassette of -22 is stably maintained in the genetically modified microorganism, so that the exogenous expression cassette can be expressed in the genetically modified microorganism, and the genetically modified
  • the microorganisms are capable of surviving and/or growing in vitro and/or in vivo.
  • a stable microorganism is capable of surviving and growing in vitro (eg, in culture) or in vivo (eg, in the digestive tract).
  • copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.
  • the genetically modified microorganism comprises one or more gene sequences and one or more gene cassettes
  • the gene sequences may be present on one or more plasmids
  • the gene cassettes may be present on the microorganism chromosomes, and vice versa. Additionally, multiple copies (e.g., one, two, three, four, five, six, or more) of any gene, gene cassette, or regulatory region may be present in a microorganism in which the gene, gene cassette, or regulatory One or more copies of a region may be mutated or otherwise altered as described herein.
  • a genetically modified microorganism is engineered to contain multiple copies of the same gene, gene cassette or regulatory region to enhance copy number.
  • a genetically modified microorganism is engineered to comprise a variety of different components of a gene cassette that performs a variety of different functions.
  • a genetically modified microorganism is engineered to contain one or more copies of different genes, gene cassettes, or regulatory regions to produce expression of more than one therapeutic molecule and/or to perform more than one Functional genetically modified microorganisms.
  • copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material.
  • the two or more gene sequences are multiple copies of the same gene. In some embodiments, the two or more gene sequences are sequences encoding different genes. In some embodiments, the two or more gene sequences are sequences encoding multiple copies of one or more different genes.
  • the genetically modified microorganism comprises one or more gene cassettes of molecules useful in the treatment or prevention of inflammatory or autoimmune diseases.
  • a genetically modified microorganism may contain two or more gene cassettes for molecules useful in the treatment or prevention of inflammatory or autoimmune diseases. In some embodiments, the two or more gene cassettes are multiple copies of the same gene cassette. In some embodiments, the two or more gene cassettes are different gene cassettes for the production of the same or different molecules for treating or preventing an inflammatory or autoimmune disease. In some embodiments, the two or more gene cassettes are gene cassettes for multiple copies of the molecule.
  • any regulatory region, promoter, gene, and/or gene cassette may be present in the genetically modified microorganism, wherein one or more of the regulatory region, promoter, gene, and/or gene cassette The copy can be mutated or otherwise altered as described herein.
  • genetically modified microorganisms are engineered to contain multiple copies of the same regulatory region, promoter, gene, and/or gene cassette to enhance copy number or to contain gene cassettes that perform multiple different functions. Multiple different components or to contain one or more copies of different regulatory regions, promoters, genes, and/or gene cassettes to produce genetically modified genes that express more than one therapeutic molecule and/or perform more than one function of microorganisms.
  • the exogenous expression cassette is integrated into the genome of the genetically modified microorganism through the CRISPR-Cas genome editing system.
  • Any suitable microorganism provided herein can be engineered to allow integration of an exogenous expression cassette into the genome.
  • the genetically modified microorganism is E. coli strain Nissle 1917 (EcN), and the exogenous expression cassette is integrated into a site in the EcN genome.
  • suitable integration sites in the EcN genome are the integration sites agaI/rsmI, lacZ, kefB, malP/T, yicS/nepl, rhtB/C, maeB, malE/K, yieN, lldD, maeA, pflB or araB/C.
  • Amuc_1100, IL-10 and/or IL-22 used in the present invention are integrated in the EcN genomic locus.
  • the genomic site of the disclosed EcN is advantageous for insertion of the expression cassettes for Amuc_1100, IL-10 and/or IL-22 by at least one of the following features: (1) The one or more bacterial genes affected by the engineering of the site are not essential for EcN growth and do not alter the biochemical and physiological activity of the host bacterium, (2) the site can be easily edited, and (3 ) the Amuc_1100 gene cassette, IL-10 and/or IL-22 gene cassette in said locus can be transcribed.
  • the microorganisms of the present disclosure further comprise inactivation or deletion of at least one auxotrophy-associated gene.
  • auxotroph refers to the growth of a microorganism (eg, a strain of microorganism) that requires an external source of specific metabolites that cannot be synthesized due to an acquired genetic defect.
  • auxotrophy-associated gene refers to a gene required for the survival of a microorganism (eg, a microorganism such as a bacterium). Auxotrophy-associated genes may be required for the microbial organism to produce nutrients necessary for survival or growth, or may be required for the detection of signals in the environment that regulate the activity of transcription factors, the absence of which would cause cell death.
  • an auxotrophic modification is intended to cause a microbial organism to die in the absence of an exogenously added nutrient necessary for survival or growth because the microbial organism lacks one or more genes necessary to produce the essential nutrient.
  • any of the genetically modified bacteria described herein further comprise deletions or mutations of genes required for cell survival and/or growth.
  • auxotrophy-associated genes in bacteria are well known in the art.
  • Exemplary auxotrophy-related genes include, but are not limited to: thyA, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, uraA, dapF, flhD, metB , metC, proAB, yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS , argS, pgsA, yeflA
  • the essential gene thyA is deleted or replaced by another gene, making the genetically modified bacteria dependent on exogenous thymine for growth or survival.
  • the microorganism is auxotrophic for one or more substances selected from the group consisting of: uracil, thymine, diaminopimelic acid, leucine, histidine, tryptophan , lysine, methionine, adenine, and non-naturally occurring amino acids.
  • the non-naturally occurring amino acid is selected from the group consisting of 1-4,4'-biphenylalanine, p-acetyl-1-phenylalanine, p-iodo-1-phenylalanine amino acid and p-azido-l-phenylalanine.
  • the microorganism comprises an allosterically regulated transcription factor capable of detecting a signal in the environment that modulates the activity of the transcription factor, wherein the absence of the signal causes cell death.
  • the "signal transduction molecule-transcription factor" pair may comprise any one or more selected from the group consisting of: tryptophan-TrpR, IPTG-LacI, benzoate derivatives-XylS, ATc-TetR , galactose-GalR, estradiol-estrogen receptor hybrid protein, cellobiose-CelR and homoserine lactone-luxR.
  • the genetically modified bacteria have a deletion of one or more endogenous plasmids.
  • chassis bacterial hosts contain one or more endogenous plasmids that consume considerable resources for their transcription. Without being bound by any theory, it is believed that these endogenous plasmids are deleted to free resources that can be better utilized for heterologous gene expression.
  • EcN comprises two endogenous plasmids, pMUT1 and pMUT2, which can be removed from EcN by any suitable method.
  • One exemplary way is through CRISPR-Cas9-mediated DNA double-strand cleavage.
  • a guide RNA gRNA or single-stranded guide RNA, sgRNA
  • a nucleic acid sequence such as a plasmid
  • the induced DNA double-strand cleavage destroys pMUT1 or pMUT2.
  • the pMUT2 in the EcN can also be replaced with a recombinant pMUT2 plasmid expressing a specific resistance gene (such as a kanamycin resistance gene), for example, by screening the resistant recombinant EcN. Then introduce the gRNA or sgRNA that specifically binds to the resistance gene and the nucleic acid sequence (such as a plasmid) expressing the Cas9 protein, so that the DNA double-strand cleavage mediated by the Cas9 protein in the EcN destroys the replaced recombinant pMUT2 plasmid.
  • a specific resistance gene such as a kanamycin resistance gene
  • the inventors of the present application have surprisingly found that the genetically modified microorganisms (such as bacteria) described in the present disclosure can not only grow stably and maintain vitality, but also can simultaneously integrate at least two exogenous genes in their genome and remain stable.
  • the polypeptides/proteins encoded by these at least two foreign genes are efficiently expressed and secreted.
  • the microorganisms expressing and secreting a new combination of exogenous polypeptides (at least two combinations of Amuc_1100/IL-10/IL-22) exhibited anti-inflammatory effects on various inflammatory diseases in animal models through intestinal administration. Or the therapeutic effect of autoimmune diseases (such as, inflammatory bowel disease) is even an unexpected synergistic effect, so the engineered microorganisms of the present disclosure have broad clinical application prospects as living medicines.
  • the present invention also provides the preparation method of the genetically modified microorganism, wherein the preparation method comprises the step of: introducing into the microorganism a protein that can exogenously express Amuc-1100, IL-10 and/or IL-22 of the present invention; Nucleotide sequence, so that the foreign gene in the nucleotide sequence can be expressed in the microorganism, so as to obtain the genetically modified microorganism.
  • the exemplary optimized Amuc_1100 expression cassette of the present invention has the structure shown in any one of the following sequences SEQ ID NO: 139 to SEQ ID NO: 143.
  • the IL-10 expression cassette has the structure shown in any one of the following sequences SEQ ID NO: 144 to SEQ ID NO: 156 and 235.
  • the IL-22 expression cassette has the structure shown in any one of the following sequences SEQ ID NO: 157 to SEQ ID NO: 163.
  • the present application provides a variety of sequences of specific expression cassettes of Amuc_1100 containing different regulatory elements (such as different promoters, different signal peptides, different cistrons, etc.), specifically as follows.
  • the present application also provides a genetically modified microorganism (such as EcN) comprising any one of the Amuc_1100 specific expression cassette sequences.
  • An example of the sequence of the Amuc_1100 specific expression cassette provided by the application is as follows:
  • BBa_J23101_USP45_Amuc_1100(WT)_rrnB_T1 (mutant)_T7Te comprising the corresponding expression cassettes of strains CBT4068, CBT4069, CBT4067 and CBT4070 in the embodiments of the present disclosure, SEQ ID NO: 141)
  • PfnrS_USP45_Amuc_1100(WT)_rrnB_T1_T7Te (included in the corresponding expression cassettes of strains CBT4080, CBT4096, CBT4088, CBT4098 and CBT4111 of the embodiments of the present disclosure, SEQ ID NO: 143)
  • the present application provides sequences of various IL-10-specific expression cassettes containing different regulatory elements (eg, different promoters, different signal peptides, and different cistrons), as shown below.
  • the present application also provides a genetically modified microorganism (eg, EcN) comprising any one of the IL-10 specific expression cassette sequences.
  • EcN genetically modified microorganism
  • BBa_J23110_USP45_IL-10_rrnB_T1_T7Te (contained in the corresponding expression cassette of the disclosed embodiment bacterial strains CBT4005, CBT4071, CBT4072, CBT4073, CBT4074, CBT4112, CBT4020, CBT4075, CBT4076 and CBT4077, SEQ ID NO: 14 5)
  • BBa_J23108_USP45_IL-10_rrnB_T1_T7Te comprising the corresponding expression cassette of strain CBT4004 of the embodiment of the present disclosure, SEQ ID NO: 146)
  • BBa_J23101_YebF_IL-10_rrnB_T1_T7Te comprising the corresponding expression cassette of strain CBT4013 of the embodiment of the present disclosure, SEQ ID NO: 150
  • BBa_J23110_BCD2_USP45_IL-10_rrnB_T1_T7Te (comprising the corresponding expression cassettes of strains CBT4028, CBT4067, CBT4068, CBT4063 and CBT4062 in the embodiments of the present disclosure, SEQ ID NO: 152)
  • BBa_J23110_luciferase_USP45_IL-10_rrnB_T1_T7Te (comprising the corresponding expression cassette of strain CBT4030 of the embodiment of the present disclosure, SEQ ID NO: 154)
  • PfnrS_BCD2_USP45_IL-10_rrnB_T1_T7Te comprising the corresponding expression cassette of strain CBT4078 of the embodiment of the present disclosure, SEQ ID NO: 155)
  • PfnrS_USP45_IL-10_rrnB_T1_T7Te (included in the corresponding expression cassettes of strains CBT4084, CBT4088, CBT4110 and CBT4111 of the embodiments of the present disclosure, SEQ ID NO: 156)
  • the present application provides sequences of various IL-22-specific expression cassettes containing different regulatory elements (eg, different promoters), as shown below.
  • the present application also provides a genetically modified microorganism (eg, EcN) comprising any one of the IL-22 specific expression cassette sequences.
  • a genetically modified microorganism eg, EcN
  • An example of the sequence of the IL-22 specific expression cassette provided by the application is as follows:
  • BBa_J23108_USP45_IL-22_rrnB_T1_T7Te comprising the corresponding expression cassette of strain CBT4043 of the embodiment of the present disclosure, SEQ ID NO: 160
  • BBa_J23110_USP45_IL-22_rrnB_T1_T7Te comprising the corresponding expression cassettes of strains CBT4039, CBT4016, CBT4110 and CBT4111 in the embodiments of the present disclosure, SEQ ID NO: 161)
  • PfnrS_USP45_IL-22_rrnB_T1_T7Te comprising the corresponding expression cassettes of strains CBT4095 and CBT4098 in the embodiments of the present disclosure, SEQ ID NO: 163
  • Psal_USP45_IL-17A_rrnB_T1_T7Te (comprising the corresponding expression cassette of strain CBT4114 of the embodiment of the present disclosure, SEQ ID NO:236)
  • Psal_USP45_IL-19_rrnB_T1_T7Te (comprising the corresponding expression cassette of strain CBT4114 of the embodiment of the present disclosure, SEQ ID NO:237)
  • Psal_USP45_IL-12p40_linker1_IL-23p19_rrnB_T1_T7Te (comprising the corresponding expression cassette of strain CBT4114 of the embodiment of the present disclosure, SEQ ID NO:238)
  • Psal_USP45_IL-37_rrnB_T1_T7Te (comprising the corresponding expression cassette of strain CBT4114 of the embodiment of the present disclosure, SEQ ID NO:240)
  • Psal_sTRSV-HHRz_USP45_TGF- ⁇ _rrnB_T1_T7Te (comprising the corresponding expression cassette of strain CBT4114 of the embodiment of the present disclosure, SEQ ID NO:241)
  • the present application also provides various genetically modified EcN strains having the genotypes shown in Table 10 below. As shown in Table 10, each bacterial strain is named after the genotype name, wherein EcN represents Escherichia coli E.Coli Nissle 1918, ⁇ maeB represents the maeB site in the strain for insertion, and the underlined part represents the specific expression described in this application
  • Cassette names such as BBa_J23101_USP45_IL-10 represent that the expression cassette has a BBa_J23101 promoter operably linked to the coding sequence for the USP45 signal peptide and the coding sequence for the IL-10 polypeptide.
  • the nucleic acid sequence of the specific expression cassette is shown in the sequence numbers SEQ ID NO: 139 to SEQ ID NO: 163 of the present application.
  • Some strains also lack one or more outer membrane proteins, as reflected in their genotype designations, eg ⁇ LPP, which stands for LPP outer membrane protein deletion.
  • Some strains also overexpress one or more molecular chaperones, which are reflected in their genotype names, such as ⁇ yicS/nepI::BBa_J23114_dsbA_dsbC_dnaK_dnaJ_grpE, which represents an expression cassette for expressing molecular chaperone_dsbA_dsbC_dnaK_dnaJ_grpE inserted at the yicS/nepI site, which The expression cassette has an operably linked BBa_J23114 promoter sequence.
  • the invention provides a composition comprising: (a) a genetically modified microorganism comprising the invention; and (b) a physiologically acceptable carrier.
  • composition refers to a preparation of a genetically engineered bacterium of the invention with other components such as a physiologically suitable carrier.
  • physiologically acceptable carrier used interchangeably refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered genetically modified microorganism.
  • the bacteria may be present in the composition in an amount ranging from about 104 to about 1013 colony forming units (CFU).
  • CFU colony forming units
  • the effective amount of the microorganism can be about 10 5 CFU to about 10 13 CFU, preferably about 10 6 CFU to about 10 13 CFU, preferably about 10 7 CFU to about 10 12 CFU, more preferably about 10 8 CFU to an amount of about 10 12 CFU.
  • Microorganisms can be living cells or can be dead cells. Microbial effectiveness correlates with the presence of Amuc_1100, IL-10 and/or IL-22 provided herein.
  • a composition disclosed herein may be formulated for oral administration and may be a nutritional or nourishing composition, such as a food, food supplement, feed or feed supplement, such as a dairy product, such as a fermented dairy product , such as yogurt or yogurt drinks.
  • the composition may comprise a nutritionally acceptable carrier, which may be a suitable food base.
  • a nutritionally acceptable carrier which may be a suitable food base.
  • Those skilled in the art are aware of a variety of formulations that may encompass live or dead microorganisms and may be presented as food supplements (eg, pills, tablets, etc.) or functional foods (eg, beverages, fermented yoghurts, etc.).
  • the compositions disclosed herein can also be formulated as medicaments in capsules, pills, liquid solutions, eg, as encapsulated lyophilized bacteria, and the like.
  • the composition is a probiotic composition.
  • compositions disclosed herein can be formulated to be effective for a given individual in a single administration or in multiple administrations.
  • a single administration is substantially effective to reduce the monitored symptoms of the targeted disease condition in the mammalian subject administered the composition.
  • the composition is formulated such that a single oral dose contains at least or at least about 1 ⁇ 10 4 CFU of bacterial and/or fungal entities, and a single oral dose will typically contain about or at least 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 bacterial and/or fungal entities . If known, e.g. the concentration of cells of a given strain or the sum of all strains is e.g.
  • the composition comprises 1 ⁇ 10 8 -1 ⁇ 10 12 CFU of the genetically modified microorganism of the invention.
  • the composition contains at least about 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, by mass, 90% or greater than 90% of the microorganisms of the present disclosure.
  • the administered dose does not exceed 200, 300, 400, 500, 600, 700, 800, 900 mg or 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 grams of the microorganisms provided by the application.
  • Physiologically acceptable carriers for compositions disclosed herein can include, for example, physiologically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, Buffers, antioxidants, suspending/dispersing agents, sequestering/chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof .
  • the compositions described in the present disclosure are liquid formulations, solid formulations, semi-solid formulations.
  • the liquid formulation is selected from the group consisting of solution products or suspension products.
  • aqueous vehicles may include, for example, Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles may include Comprising, for example, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; the antimicrobial agent may be at a bacteriostatic or fungistatic concentration, and/or may be added to the composition in a multi-dose container, the Container contains phenol or cresol, amalgam, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride.
  • Isotonic agents can contain, for example, sodium chloride or dextrose; buffers, such as phosphate or citrate buffers; antioxidants, such as sodium bisulfate; suspending and dispersing agents, such as carboxymethylcellulose Sodium, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; chelating agents may include, for example, ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Suitable excipients may comprise, for example, water, saline, dextrose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may comprise, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins. reagents.
  • the compositions provided herein can be pharmaceutical compositions.
  • the compositions provided herein can be food supplements.
  • the compositions provided herein may comprise, in addition to the genetically modified microorganisms provided herein, a pharmaceutically, nutritionally/alimentarily or physiologically acceptable carrier.
  • the preferred form will depend on the intended mode of administration and (therapeutic) application.
  • the carrier can be any material suitable for delivering the genetically modified microorganisms provided herein to the gastrointestinal tract of a mammal, such as a human, preferably near or within the intestinal mucosal barrier, more preferably the colonic mucosal barrier, of a mammal. Contains a physiologically acceptable non-toxic substance.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of powders, powders, tablets, sugar-coated agents, capsules, granules, suspensions, solutions, syrups, drops, sublingual slices, or combinations thereof.
  • the composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder.
  • Oral formulations can contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the present invention provides a kit comprising the composition of the present invention.
  • the kit may further comprise one or more of various conventional pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as described for the It will be apparent to those skilled in the art. Instructions may also be included in the kit, either as an insert or as a label, indicating the amounts of the components to be administered, directions for administration and/or directions for mixing the components.
  • the present invention provides a method for treating or preventing an inflammatory disease or an autoimmune disease in an individual in need thereof, the method comprising: administering to the individual an effective amount of the genetically modified Microorganisms, or compositions of the present invention.
  • the genetically modified microorganism expresses at least one exogenous gene selected from the group consisting of exogenous genes encoding and expressing Amuc-1100 polypeptides, or exogenous genes encoding and expressing IL-10 polypeptides, or encoding An exogenous gene expressing IL-22 polypeptide, or an exogenous gene in any combination of the above.
  • the present invention also provides a method of improving the efficacy of drug therapy in an individual receiving drug therapy, including drugs for the treatment of inflammatory diseases or autoimmune diseases, the method comprising: administering to the individual An effective amount of a genetically modified microorganism described herein, or a composition described herein, is administered.
  • the genetically modified microorganism expresses at least one exogenous gene selected from the group consisting of exogenous genes encoding and expressing Amuc-1100 polypeptides, or exogenous genes encoding and expressing IL-10 polypeptides, or encoding An exogenous gene expressing IL-22 polypeptide, or an exogenous gene in any combination of the above.
  • the use of the genetically modified microorganism as described in the present invention or the composition of the present invention in the preparation of medicines for treating or preventing inflammatory diseases or autoimmune diseases is also provided.
  • the inflammatory disease or autoimmune disease is inflammatory bowel disease (IBD).
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-10 polypeptide, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding IL-10 polypeptides and IL-22 polypeptides, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-22 polypeptide, respectively.
  • the genetically modified microorganism comprises an exogenous gene encoding an IL-10 polypeptide.
  • the genetically modified microorganism comprises an exogenous gene encoding an Amuc_1100 polypeptide. In some embodiments, the genetically modified microorganism comprises an exogenous gene encoding an IL-22 polypeptide. In some embodiments, the genetically modified microorganism comprises exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide respectively.
  • the inflammatory disease or autoimmune disease is systemic lupus erythematosus (SLE).
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-10 polypeptide, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding IL-10 polypeptides and IL-22 polypeptides, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-22 polypeptide, respectively.
  • the genetically modified microorganism comprises an exogenous gene encoding an IL-10 polypeptide.
  • the genetically modified microorganism comprises an exogenous gene encoding an Amuc_1100 polypeptide. In some embodiments, the genetically modified microorganism comprises an exogenous gene encoding an IL-22 polypeptide. In some embodiments, the genetically modified microorganism comprises exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide respectively.
  • the inflammatory or autoimmune disease is arthritis.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-10 polypeptide, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding IL-10 polypeptides and IL-22 polypeptides, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-22 polypeptide, respectively.
  • the genetically modified microorganism comprises an exogenous gene encoding an IL-10 polypeptide.
  • the genetically modified microorganism comprises an exogenous gene encoding an Amuc_1100 polypeptide. In some embodiments, the genetically modified microorganism comprises an exogenous gene encoding an IL-22 polypeptide. In some embodiments, the genetically modified microorganism comprises exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide respectively.
  • the inflammatory disease or autoimmune disease is asthma.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-10 polypeptide, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding IL-10 polypeptides and IL-22 polypeptides, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-22 polypeptide, respectively.
  • the genetically modified microorganism comprises an exogenous gene encoding an IL-10 polypeptide.
  • the genetically modified microorganism comprises an exogenous gene encoding an Amuc_1100 polypeptide. In some embodiments, the genetically modified microorganism comprises an exogenous gene encoding an IL-22 polypeptide. In some embodiments, the genetically modified microorganism comprises exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide respectively.
  • the inflammatory disease or autoimmune disease is graft versus host disease (GvHD).
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-10 polypeptide, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding IL-10 polypeptides and IL-22 polypeptides, respectively.
  • the genetically modified microorganism comprises exogenous genes encoding an Amuc_1100 polypeptide and an IL-22 polypeptide, respectively.
  • the genetically modified microorganism comprises an exogenous gene encoding an IL-10 polypeptide.
  • the genetically modified microorganism comprises an exogenous gene encoding an Amuc_1100 polypeptide. In some embodiments, the genetically modified microorganism comprises an exogenous gene encoding an IL-22 polypeptide. In some embodiments, the genetically modified microorganism comprises exogenous genes encoding Amuc_1100 polypeptide, IL-10 polypeptide and IL-22 polypeptide respectively.
  • the subject has a condition/disease (eg, those described herein) or disorder of an inflammatory disease or an autoimmune disease, or has a predisposition to the disease/condition. Its purpose is to treat, cure, alleviate, relieve, change, correct, alleviate, improve or affect a disease/disorder, or the cause of the disease/disorder.
  • treatment includes preventing or ameliorating a disease, disorder or condition, slowing the rate of onset or progression of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the , development of symptoms associated with a disease, disorder or condition, reduction or termination of symptoms associated with a disease, disorder or condition, complete or partial regression of a disease, disorder or condition, cure of a disease, disorder or condition, or some combination thereof.
  • the term "effective amount” refers to an amount and/or dosage and/or dosage regimen of one or more agents necessary to produce a desired result, e.g., sufficient to moderate in an individual the disease condition associated with the individual being treated An amount of one or more symptoms associated, or an amount sufficient to lessen the severity or delay the progression of a disease condition in an individual (e.g., a therapeutically effective amount), sufficient to reduce the risk of or delay the onset of a disease condition in an individual, and/or An amount that reduces its ultimate severity (eg, a prophylactically effective amount).
  • Effective amounts will vary depending on the severity of the condition being treated, individual patient parameters including age, physical condition, height, sex and weight, duration of treatment, nature of the concurrent treatment, if any , and other factors within the knowledge and experience of the general practitioner or other medical practitioner. These factors are well known in the art and can be determined by no more than routine experimentation. It is generally preferred to use the maximum dose of the individual components or combination thereof which can be safely determined by sound medical judgment. However, as will be appreciated by those of ordinary skill in the art, patients may insist on lower or tolerable doses for medical, psychological, or virtually any other reasons.
  • the term "individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates such as mammals and non-mammals such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles.
  • the terms "patient” or “individual” are used interchangeably herein.
  • inflammatory disease or autoimmune disease includes, but is not limited to, inflammatory disease and autoimmune disease.
  • the inflammatory disease may include an autoimmune disease, such as inflammatory bowel disease.
  • autoimmune disease such as inflammatory bowel disease.
  • IBD inflammatory bowel disease
  • IBD is used interchangeably herein to refer to a class of disorders associated with inflammation of the digestive tract, characterized by significant localized inflammation in the gastrointestinal tract, often driven by T cells and activated macrophages and Impaired epithelial barrier function is characterized (Ghishan et al., 2014), including but not limited to, Crohn's disease, ulcerative colitis, Behcet's disease, lymphocytic colitis, collagenous colitis, Metastatic colitis and indeterminate colitis.
  • Autoimmune disease as used herein also includes graft-versus-host disease (GvHD), systemic lupus erythematosus, arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), asthma (such as allergic asthma or neutrophilic asthma).
  • GvHD graft-versus-host disease
  • arthritis such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis
  • asthma such as allergic asthma or neutrophilic asthma.
  • graft versus host refers to the development of an immune response of transplanted (donor) cells against microorganisms and tissues.
  • graft versus host disease refers to an abnormal state (both acute and chronic) caused by the effects of transplanted (graft) cells produced by GVH on microorganisms and tissues.
  • GvHD Acute graft-versus-host disease (GvHD) is a disorder caused by donor immune cells in patients transplanted with allogeneic bone marrow or blood cells.
  • GvHD The gut epithelium and liver are frequently affected tissues, and in severe cases, GvHD can cause blistering of the skin or excessive diarrhea and wasting. Inflammation in the liver caused by the donor's immune cells can lead to blockages that cause jaundice. Other tissues such as the lungs and thymus may also be affected.
  • the diagnosis of GvHD can usually be confirmed by looking microscopically at a small patch of skin, liver, stomach or intestine for specific inflammatory features.
  • SLE Systemic lupus erythematosus
  • AID autoimmune disease
  • Symptoms of SLE vary from person to person and can range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth sores, swollen lymph nodes, feeling tired, and a red rash, most often on the face. There are usually periods of flare-ups and periods of remission with fewer symptoms.
  • the cause of SLE is unknown. It is thought to be related to genetic and environmental factors.
  • Rheumatoid arthritis is a long-term autoimmune disease that primarily affects the joints. RA typically causes joints to be warm, swollen, and painful, and the pain and stiffness often worsen with rest. The wrists and hands are most commonly affected, usually the same joints on both sides of the body. RA can also affect other parts of the body, including the skin, eyes, lungs, heart, nerves, and blood. This can lead to low red blood cell counts, inflammation around the lungs and around the heart, and possibly fever and low energy. Typically, symptoms develop gradually over weeks to months. Although the cause of rheumatoid arthritis is unknown, it is believed to involve a combination of genetic and environmental factors.
  • the underlying pathogenesis involves the body's immune system attacking the joint, which causes inflammation and thickening of the joint capsule, which also affects the underlying bone and cartilage.
  • the diagnosis of RA is primarily based on the patient's signs and symptoms. In a specific diagnosis, X-rays and laboratory tests may also be combined to support the diagnosis or rule out other diseases with similar symptoms.
  • the autoimmune disease is selected from the group consisting of inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), graft-versus-host disease (GvHD), systemic lupus erythematosus, arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), asthma (such as allergic asthma or neutrophilic asthma), or a combination thereof.
  • the autoimmune disease comprises graft versus host disease (GvHD).
  • the autoimmune disease includes inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), systemic lupus erythematosus, asthma, multiple sclerosis and/or rheumatoid arthritis.
  • gut autoimmune diseases such as inflammatory bowel disease
  • non-gut autoimmune diseases such as multiple sclerosis
  • intestinal flora dysbiosis is closely related to the pathogenesis of autoimmune diseases.
  • changes in microbial composition can be found in the early stages of rheumatoid arthritis and inflammatory bowel disease.
  • gut microbiota dysbiosis in both Spanish and Chinese SLE patients.
  • the balance of gut bacteria is critical to the regulation and development of the immune system.
  • impaired intestinal barrier function under inflammatory conditions promotes the leakage of bacteria from the mucosal layer into blood vessels, thereby stimulating local and systemic autoimmune pathways.
  • the inventors of the present application constructed engineered microorganisms (such as engineered E. Combination, because the engineered bacteria can continuously express and secrete exogenous polypeptide factors in the intestine, they can continuously act on intestinal cells for a period of time, thereby enhancing the intestinal barrier and/or improving intestinal immunity, thereby improving the symptoms of immune diseases . If the lesion is located in the intestinal tract, the genetically modified microorganisms of the present disclosure are expected to be distributed near the lesion, and the polypeptide factors secreted by it can even directly act on the cells of the lesion, thereby exerting a therapeutic effect.
  • engineered microorganisms such as engineered E. Combination, because the engineered bacteria can continuously express and secrete exogenous polypeptide factors in the intestine, they can continuously act on intestinal cells for a period of time, thereby enhancing the intestinal barrier and/or improving intestinal immunity, thereby improving the symptoms of immune diseases .
  • the genetically modified microorganisms of the present disclosure are expected to be distributed near
  • the inventors first screened exogenous gene combinations that are suitable for microbial expression and secretion, and can exert therapeutic effects in local delivery in the intestinal tract. It should be noted that living drugs need to maintain the vitality of microorganisms so that they can grow normally and play a therapeutic role in the body, but the continuous expression of exogenous polypeptides by microorganisms, especially two or more exogenous polypeptides will inevitably bring certain effects on cell stability.
  • the exogenous polypeptides expressed by microorganisms should not have too much adverse effect on the activity of the microorganisms themselves, otherwise, the poor state of the microorganisms will affect their ability to express and secrete therapeutic exogenous polypeptides, and even the microorganisms may pass through the internal
  • the stress mechanism reduces or even shuts down the expression of foreign genes, which leads to the failure of engineered microorganisms.
  • the inventors did find that some exogenous factors were not suitable for expression and secretion by the engineered microorganism of the present disclosure, so they finally gave up (such as IL-19, IL-23, IL-37, etc.).
  • the signal peptides of these polypeptides (replacing their own signal peptides) and the expression control elements in the expression cassette (for example, select Appropriate promoters, cistrons, ribosome binding sites, etc.) and the structure of chassis bacteria (selecting to knock out specific membrane proteins to improve membrane permeability) have been fully optimized, so that engineered bacteria can efficiently stabilize
  • the expression of the above-mentioned brand-new exogenous gene combination has also been confirmed in multiple animal disease models. All or part of the engineered bacteria expressing the exogenous factor combination has produced unexpected curative effects on various inflammatory diseases or autoimmune diseases. It shows a synergistic effect and has broad clinical application prospects.
  • Example 1 Editing schemes for bacterial genomes
  • N20NGG 20bp sequence connecting the NGG PAM sequence on the two strands of the target integration site sequence and blast the EnN genome.
  • the 300-500bp sequences upstream and downstream of the sgRNA were selected as the left homology arm (LHA) and right homology arm (RHA).
  • the sgRNA sequence was added to the 5' end of the gRNA backbone reverse primer, amplified by PCR and digested with restriction endonucleases PstI and SpeI, and the digested PCR product fragment was combined with the same digested plasmid pCBT003 (SEQ ID NO: 137) were ligated to form pCBT003_sgRNA plasmid.
  • the foreign gene to be integrated into the EcN genome is synthesized by GeneScript on a cloning plasmid (eg pUC57).
  • the exogenous gene was amplified using the synthesized plasmid as a template, and the LHA and RHA of the selected integration site were amplified using the EcN genome as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected by overlapping PCR, and the resulting PCR product containing LHA-exogenous gene-RHA is used as a donor gene fragment.
  • a single colony of EcN/pCBT001 on the resistant LB agar plate was inoculated into 3 mL of LB liquid medium supplemented with 50 ⁇ g/mL spectinomycin and streptomycin, cultured overnight at 30° C. with shaking (220 rpm), and then 300 ⁇ L of this overnight culture was inoculated into 30 mL of LB liquid medium supplemented with 50 ⁇ g/mL of spectinomycin and streptomycin, and cultured at 30° C. with shaking (220 rpm). IPTG was added to the cultures at a concentration of 1 mM after 1 hour.
  • the cells were washed 3 times with 20 mL, 10 mL and 5 mL of 10% glycerol (4°C) and finally resuspended in 300 ⁇ L of 10% glycerol (4°C) and aliquoted in 100 ⁇ L per tube into a 1.5mL centrifuge tube.
  • Example 3 A protocol for removing redundant plasmids
  • the primers used for validation were designed upstream of LHA and downstream of RHA. Select a single colony grown on the transformation plate to perform colony PCR with verified primers, and select clones that have correctly integrated the exogenous gene into the genome by the size of the PCR product.
  • the selected correct clones were inoculated into 3 mL of LB liquid medium supplemented with 50 ⁇ g/mL spectinomycin, streptomycin and 10 mM arabinose, cultured overnight at 30°C with shaking (220 rpm), and then the culture was diluted 10 6 100 ⁇ L was spread on LB agar plates supplemented with 50 ⁇ g/mL spectinomycin and streptomycin, and incubated overnight at 30°C.
  • the pCBT003_sgRNA depleted clone was inoculated in LB liquid medium, cultured overnight at 42° C. with shaking (220 rpm), and then the culture was diluted 10 6 times and 100 ⁇ L was spread on an LB agar plate. Pick a single colony and spot on LB agar plate and LB agar plate supplemented with 50 ⁇ g/mL spectinomycin and streptomycin. Only the colonies growing on the LB agar plate were pCBT001-eliminated colonies.
  • the plasmid pCBT003_pMUT1_sgRNA expressing the sgRNA of pMUT1 was transformed into EcN/pCBT001.
  • the pMUT1 elimination verification primers are specifically homologous to pMUT1 but not to the EcN genome. PCR is performed with the pMUT1 verification primers, and the colonies that cannot obtain PCR products are pMUT1 elimination colonies.
  • the kanamycin resistance gene was connected to pMUT2 to obtain the plasmid pMUT2-kana, the nucleotide sequence of which is shown in SEQ ID NO:138.
  • the plasmid pMUT2-kana was transformed into EcN ⁇ pMUT1, and the transformed cells were spread on LB agar plates supplemented with 10 ⁇ g/mL kanamycin, and the grown colonies were EcN ⁇ pMUT1 containing plasmid pMUT2-kana.
  • EcN ⁇ pMUT1/pMUT2-kana was passaged several times in LB liquid medium supplemented with 10 ⁇ g/mL kanamycin, the plasmid was extracted, and the electrophoresis band was used to determine whether pMUT2 was completely replaced by pMUT2-kana. Then pCBT001 was transferred into EcN ⁇ pMUT1/pMUT2-kana in which pMUT2 was completely replaced, and then the plasmid pCBT003_Kana-sgRNA expressing kanamycin resistance gene sgRNA was transferred into EcN ⁇ pMUT1/pMUT2-kana/pCBT001. The obtained colonies were subjected to PCR with primers specific to the kanamycin resistance gene, and the colonies that could not obtain PCR products were pMUT2-kana eliminated colonies.
  • sequence of the sgRNA targeting pMUT1 is shown in SEQ ID NO:164; the sequence of the sgRNA targeting the kanamycin resistance gene is shown in SEQ ID NO:165
  • sgRNA sequence targeting pMUT1 SEQ ID NO:164:
  • the design method of the target membrane protein sgRNA that needs to be knocked out and the construction method of the sgRNA plasmid are the same as those described in 1.1 and 1.2 in Example 1.
  • the 300-500bp sequences upstream and downstream of the target membrane protein coding gene were selected as the left homology arm (LHA) and right homology arm (RHA).
  • LHA and RHA of the selected knockout sites were amplified using the genome of EcN as a template.
  • the PCR primers used to amplify these fragments have 15-20 bp of homologous sequences to each other, so they can be joined by overlapping PCR, and the obtained PCR products containing LHA-RHA are used as donor fragments.
  • the PCR product of the obtained donor fragment and the pCBT003_sgRNA plasmid expressing the knockout site sgRNA were transformed into competent cells containing pCBT001 at the same time, and the obtained single colony was amplified with the verification primers for the knockout site.
  • the size of the band screens the clones for which the target protein is successfully knocked out from the genome.
  • the membrane proteins knocked out in this example include tolQ, tolR, tolA, pal, lpp, mrcA and ompT.
  • the sequence of the sgRNA at the tolQ knockout site is shown in SEQ ID NO: 166, and the homology arm sequences on both sides of the tolQ knockout site are shown in SEQ ID NO: 167 and 168 respectively;
  • the sgRNA at the tolR knockout site The sequence of the tolA knockout site is shown in SEQ ID NO:169, and the homology arm sequences on both sides of the tolR knockout site are shown in SEQ ID NO:170 and 171 respectively;
  • the sequence of the sgRNA at the tolA knockout site is shown in SEQ ID NO:
  • As shown in 172, the homology arm sequences on both sides of the tolA knockout site are shown in SEQ ID NO: 173 and 174 respectively;
  • the sequence of the sgRNA at the pal knockout site is shown
  • sgRNA sequence targeting tolQ site SEQ ID NO:166:
  • LHA left homology arm sequence of the tolQ site
  • RHA right homology arm sequence of the tolQ site
  • sgRNA sequence targeting tolR site (SEQ ID NO:169):
  • LHA left homology arm sequence (SEQ ID NO: 170) of tolR site:
  • RHA right homology arm sequence (SEQ ID NO: 171) of tolR site:
  • sgRNA sequence targeting tolA site SEQ ID NO:172:
  • LHA left homology arm sequence (SEQ ID NO: 173) of tolA point:
  • RHA right homology arm sequence of the tolA site
  • the left homology arm (LHA) sequence of the pal site (SEQ ID NO: 176):
  • RHA right homology arm sequence of the pal site
  • the sgRNA sequence targeting the lpp site (SEQ ID NO: 178):
  • LHA left homology arm sequence (SEQ ID NO:179) of lpp site:
  • RHA right homology arm sequence (SEQ ID NO:180) of lpp site:
  • LHA left homology arm sequence of the mrcA site
  • RHA right homology arm sequence (SEQ ID NO: 183) of the mrcA site:
  • sgRNA sequence targeting ompT site SEQ ID NO:184:
  • LHA left homology arm sequence of the ompT site
  • RHA right homology arm sequence of the ompT site
  • Promoter BBa_J23114 and ribosome binding site (RBS) were amplified using synthetic plasmids as templates, groES, groEL, tig coding sequences were amplified using plasmid pG-TF2 as templates, fkpA, surA coding sequences and insertion site yieN LHA and RHA were amplified using the EcN genome as a template, the transcription terminator was amplified using the plasmid pCBT003 as a template, and the sgRNA expression sequence targeting the yieN site was amplified using the sgRNA expression plasmid pCBT003_yieN_sgRNA as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected by overlapping PCR to obtain donor gene fragment expression cassettes, molecular chaperones, which are respectively connected to LHA and RHA-yieN_sgRNA sequences on both sides
  • the elements on the expression cassette are arranged in the following order from 5' to 3': 5'-promoter-ribosome binding site (RBS)-groES-groEL-tig-fkpA-surA coding sequence-terminator.
  • the pCBT012 plasmid backbone is amplified using the plasmid pCBT010 as a template.
  • the PCR primers used to amplify the pCBT012 plasmid backbone have a 15-20bp homologous sequence to the molecular chaperone expression cassette, so the kit ClonExpress Ultra One Step Cloning Kit (Vazyme) can be used Ligated with the chaperone expression cassette to form plasmid pCBT012 (SEQ ID NO: 215).
  • Plasmid pCBT010 sequence (SEQ ID NO: 187):
  • the promoter BBa_J23114 and the ribosome binding site (RBS) were amplified using the synthetic plasmid as a template, the dnaK, dnaJ, and grpE coding sequences were amplified using the plasmid pKJE7 as a template, and the dsbA, dsbC coding sequences and the insertion site yicS/nepI were amplified.
  • LHA and RHA were amplified using the EcN genome as a template, the transcription terminator was amplified using the plasmid pCBT003 as a template, and the sgRNA expression sequence targeting the yicS/nepI site was amplified using the sgRNA expression plasmid pCBT003_yicS/nepI_sgRNA as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected by overlapping PCR to obtain the donor gene fragment expression cassettes that are respectively connected to the LHA and RHA-yicS/nepI_sgRNA sequences on both sides,
  • the elements on the molecular chaperone expression cassette are arranged in the following order from 5' to 3': 5'-promoter-ribosome binding site (RBS)-dsbA-dsbC-dnaK-dnaJ-grpE coding sequence-terminator.
  • the pCBT013 plasmid backbone is amplified using the plasmid pCBT010 as a template.
  • the PCR primers used to amplify the pCBT013 plasmid backbone have a 15-20bp homologous sequence to the molecular chaperone expression cassette, so the kit ClonExpress Ultra One Step Cloning Kit (Vazyme) can be used Ligated with the chaperone expression cassette to form plasmid pCBT013 (SEQ ID NO: 216).
  • the plasmids pCBT012 and pCBT013 expressing molecular chaperones were respectively transformed into competent cells containing pCBT001, and the obtained single colony was amplified with the verification primers of the insertion site, and the molecular chaperone expression cassette was successfully inserted into the genome by screening the size of the amplified band clone.
  • the sequence accession numbers of each molecular chaperone are shown in Table 12.
  • the sequence of the sgRNA targeting the yieN insertion site is shown in SEQ ID NO: 188, and the sequences of the homology arms on both sides of the yieN insertion site are shown in SEQ ID NO: 189 and 190, respectively.
  • the sequence of the sgRNA targeting the yicS/nepI insertion site is shown in SEQ ID NO:191, and the sequences of the homology arms on both sides of the yicS/nepI insertion site are shown in SEQ ID NO:192 and 193, respectively.
  • the sgRNA sequence targeting the yieN site (SEQ ID NO: 188):
  • the left homology arm (LHA) sequence of the yieN site (SEQ ID NO: 189):
  • RHA right homology arm sequence of the yieN site
  • sgRNA sequence targeting yicS/nepI site (SEQ ID NO:191):
  • LHA left homology arm sequence of the yicS/NepI site
  • RHA right homology arm sequence of the yicS/NepI site
  • Embodiment 6 the construction of bacterial strain
  • the IL-10 coding sequence, signal peptide, promoter, ribosome binding site (RBS) and cistron were synthesized by GeneScript on a cloning plasmid (such as pUC57).
  • the IL-10 coding sequence, signal peptide, promoter, ribosome binding site (RBS) and cistron were amplified using the synthetic plasmid as a template, and the LHA and RHA at the insertion site were amplified using the EcN genome as a template.
  • the transcription terminator was amplified using plasmid pCBT003 as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected by overlapping PCR to obtain the expression cassettes of the donor gene fragments that are respectively connected to the LHA and RHA sequences on both sides.
  • the elements are arranged in the following order from 5' to 3': 5'-Promoter-Ribosomal Binding Site (RBS)-Cistron-Signal Peptide-IL-10 Coding Sequence-Terminator (each donor gene constructed
  • RBS 5'-Promoter-Ribosomal Binding Site
  • Cistron-Signal Peptide-IL-10 Coding Sequence-Terminator each donor gene constructed
  • the sequence structure of the fragment expression cassette is shown in SEQ ID NO: 144-156, 235).
  • the PCR product of the donor gene fragment expression cassette with LHA and RHA sequences on both sides and the pCBT003_maeB_sgRNA plasmid expressing sgRNA were transformed into competent cells containing pCBT001 at the same time, and the obtained single colony was tested with the verification primer of the insertion site. Amplification, the clones with successful insertion of IL-10 into the genome were screened by the size of the amplified band. The production of IL-10 was detected by ELISA kit (Sino Biological, KIT10947A).
  • the IL-10 coding sequence in this embodiment is the sequence that encodes wild-type human IL-10 (SEQ ID NO: 11);
  • the inserted site is maeB of the EcN genome, and the sequence of the sgRNA targeting the maeB site is as follows As shown in SEQ ID NO: 194, the sequences of homology arms on both sides of the maeB site are shown in SEQ ID NO: 195 and 196, respectively.
  • LHA left homology arm sequence of the maeB site
  • RHA Right homology arm sequence of maeB site
  • strains expressing IL-10 obtained in this example and the information of the insertion elements and knockout elements therein are shown in Table 13 below.
  • sequences of related elements are shown in Table 2-Table 9.
  • Table 14 shows the production of IL-10 expressed in the medium supernatant and intracellular of the engineered bacteria using different signal peptides after culturing in LB for 24 hours.
  • the dsbA signal peptide was used in the strain CBT4007; the ompA signal peptide was used in the strain CBT4009; the pelB signal peptide was used in the strain CBT4011; the yebF signal peptide was used in the strain CBT4013, and the USP45 signal peptide was used in the strain CBT4003.
  • the concentration of IL-10 in engineered bacteria samples using OmpA, PelB, and YebF signal peptides was lower than the detection limit.
  • USP45 signal peptide was used, the production of IL-10 was significantly higher than that of DsbA signal peptide.
  • Table 14 The genotypes of engineered bacteria using different signal peptides and the production of IL-10
  • the production of IL-10 expressed in the medium supernatant and in the cells of the engineered bacteria using different promoters after culturing in LB for 24 hours is shown in Table 15.
  • the BBa_J23101 promoter is used in the strain CBT4003; the BBa_J23108 promoter is used in the strain CBT4004; the BBa_J23110 promoter is used in the strain CBT4005.
  • the results show that the production of IL-10 can be regulated by selecting a suitable promoter (such as BBa_J23101 promoter).
  • Table 15 The genotypes of engineered bacteria using different promoters and the production of IL-10
  • Table 16 shows the yield of IL-10 expressed in the medium supernatant after the engineering bacteria using different cistrons were cultured in LB for 24 hours.
  • the cistron T7g10 is used in the strain CBT4026; the cistron BCD2 is used in the strain CBT4028; the cistron GFP is used in the strain CBT4029; the cistron Lucifierase (luciferase) is used in the strain CBT4030.
  • the results showed that IL-10 production could be regulated by selecting different cistrons.
  • Table 16 The genotypes of engineered bacteria using different cistrons and the production of IL-10
  • IL-10 expressed in the medium supernatant and in the cells of the engineering bacteria knocking out different membrane proteins and the engineering bacteria overexpressing molecular chaperones after culturing in LB for 12 hours is shown in Table 17.
  • strains CBT4071, CBT4072, CBT4073, CBT4074, and CBT4020 were knocked out of the membrane proteins tolQ, tolR, tolA, pal, and lpp, respectively, and the control was the strain CBT4005 that did not modify the outer membrane.
  • the results showed that individually knocking out tolQ, tolR, tolA, pal and lpp could significantly increase the production and secretion efficiency of IL-10.
  • CBT4062 is an engineering strain that knocks out the membrane protein lpp and overexpresses molecular chaperones.
  • CBT4075 is an engineering strain that simultaneously knocks out lpp and mrcA and overexpresses molecular chaperones.
  • CBT4076 is an engineering strain that simultaneously knocks out lpp and ompT and overexpresses molecular chaperones. Combined knockout with mrcA or ompT further significantly increased IL-10 production and secretion levels.
  • CBT4077 which simultaneously knocked out lpp, mrcA and ompT and overexpressed molecular chaperones, did not show a higher IL-10 production.
  • CBT4112 is an engineering strain in which both pal and mrcA are knocked out. Compared with CBT4074 which only knocks out pal, the production of IL-10 has no significant change. Therefore, only when mrcA is knocked out in combination with lpp can the protein secretion efficiency be improved.
  • Table 17 The genotype and IL-10 production of engineered bacteria knocked out of different membrane proteins and engineered bacteria overexpressed chaperones
  • strain CBT4078 using an anaerobic promoter (EcN ⁇ maeB::PfnrS_BCD2_USP45_IL-10 ⁇ LPP ⁇ yicS/nepI::BBa_J23114_dsbA_dsbC_dnaK_dnaJ_grpE ⁇ yieN::BBa_J23114_groES_groEL_tig_fkpA_surA)
  • the expression levels of IL-10 in 1% w/v glucose medium were compared, and the results are shown in FIG. 2 .
  • the results showed that IL-10 production was significantly higher in anaerobic culture than in aerobic culture when anaerobic promoter was used.
  • the IL-22 coding sequence, signal peptide, promoter, ribosome binding site (RBS) and cistron were synthesized by GeneScript on a cloning plasmid (such as pUC57).
  • the IL-22 coding sequence, signal peptide, promoter, ribosome binding site (RBS) and cistron were amplified using the synthetic plasmid as a template, and the LHA and RHA at the insertion site were amplified using the EcN genome as a template.
  • the transcription terminator was amplified using plasmid pCBT003 as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected by overlapping PCR to obtain the expression cassettes of the donor gene fragments that are respectively connected to the LHA and RHA sequences on both sides.
  • the elements are arranged in the following order from 5' to 3': 5'-Promoter-Ribosomal Binding Site (RBS)-Cistron-Signal Peptide-IL-22 Coding Sequence-Terminator (each donor gene constructed
  • RBS 5'-Promoter-Ribosomal Binding Site
  • Cistron-Signal Peptide-IL-22 Coding Sequence-Terminator each donor gene constructed
  • SEQ ID NO: 157-163 The sequence structure of the fragment expression cassette is shown in SEQ ID NO: 157-163
  • the PCR product of the donor gene fragment expression cassette with LHA and RHA sequences on both sides and the pCBT003_kefB_sgRNA plasmid or pCBT003_maeB_sgRNA plasmid expressing sgRNA were transformed into competent cells containing pCBT001 at the same time, and the obtained single colony was used for the insertion site.
  • the primers were verified for amplification, and the clones with successful insertion of IL-22 into the genome were screened by the size of the amplified bands.
  • the production of IL-22 was detected by ELISA kit (Sino Biological, KIT13059).
  • the IL-22 coding sequences in this example are all sequences encoding wild-type human IL-22 (SEQ ID NO: 15); the inserted site maeB (CBT4038) or site kefB (CBT4041, CBT4041, CBT4042, CBT4043, CBT4039, CBT4040, CBT4016).
  • the sequence of the sgRNA targeting the maeB site is shown in SEQ ID NO: 194, and the sequences of homology arms on both sides of the maeB site are shown in SEQ ID NO: 195 and 196, respectively.
  • the sequence of the sgRNA targeting the kefB site is shown in SEQ ID NO: 197, and the homology arm sequences on both sides of the kefB site are shown in SEQ ID NO: 198 and 199, respectively.
  • sgRNA sequence targeting kefB site (SEQ ID NO:197):
  • LHA left homology arm sequence of the kefB site
  • RHA right homology arm sequence (SEQ ID NO: 199) of kefB site:
  • the yields of IL-22 expressed in culture medium supernatant and in cells of engineering bacteria using different promoters after 24 hours of culture in LB are shown in Table 19.
  • the BBa_J23119 promoter (inserted into the ⁇ maeB site) is used in the bacterial strain CBT4038; the BBa_J23101 promoter is used in the bacterial strain CBT4041; the BBa_J23102 promoter is used in the bacterial strain CBT4042; the BBa_J23108 promoter is used in the bacterial strain CBT4043; the BBa_J23110 is used in the bacterial strain CBT4039; used in BT4040 BBa_J23114 promoter.
  • the results show that the production of IL-22 can be regulated by selecting an appropriate promoter (such as BBa_J23119 promoter).
  • Table 19 The genotypes of engineered bacteria using different promoters and the production of IL-22
  • Table 20 The genotypes of engineering bacteria knocking out different membrane proteins and the production of IL-22
  • Amuc_1100 coding sequence, signal peptide, promoter, ribosome binding site (RBS) and cistron, etc. were synthesized by GeneScript on a cloning plasmid (such as pUC57).
  • Amuc_1100 coding sequence, signal peptide, promoter, ribosome binding site (RBS) and cistron, etc. were amplified using the synthetic plasmid as a template, and the LHA and RHA at the insertion site were amplified using the EcN genome as a template, and the transcription was terminated
  • the progeny was amplified using plasmid pCBT003 as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected by overlapping PCR to obtain the expression cassettes of the donor gene fragments that are respectively connected to the LHA and RHA sequences on both sides.
  • the elements are arranged in the following order from 5' to 3': 5'-Promoter-Ribosomal Binding Site (RBS)-Cistron-Signal Peptide-Amuc_1100 Coding Sequence-Terminator (expression of each donor gene fragment constructed
  • RBS 5'-Promoter-Ribosomal Binding Site
  • Peptide-Amuc_1100 Coding Sequence-Terminator expression of each donor gene fragment constructed
  • the sequence structure of the box is shown in SEQ ID NO: 139-143).
  • the obtained PCR product of the donor gene fragment expression cassette with LHA and RHA sequences on both sides and pCBT003_agaI/rsmI_sgRNA plasmid or pCBT003_malP/T_sgRNA plasmid or pCBT003_pflB_sgRNA plasmid or pCBT003_lldD_sgRNA plasmid or pCBT003_maeA_sgRNA plasmid expressing sgRNA were simultaneously transformed into pCBT003_lldD_sgRNA plasmid or pCBT003_maeA_sgRNA plasmid containing p Feelings of CBT001 In state cells, the obtained single colony was amplified with the verification primer of the insertion site, and the clones with Amuc_1100 successfully inserted into the genome were screened by the size of the amplified band.
  • the Amuc_1100 coding sequence in the present embodiment is coding wild-type Amuc_1100 (SEQ ID NO:5, shown in WT in table 21) or Y259A mutant Amuc_1100 (the numbering of the 259th is based on the sequence shown in SEQ ID NO:5 number, the sequence shown as Y259A in Table 21).
  • the inserted site is the agaI/rsmI site of the EcN genome; when inserting 2 copies of Amuc_1100, the inserted first and second sites are respectively the site agaI of the EcN genome /rsmI site and malP/T site; when inserting 3 copies of Amuc_1100, the inserted first, second and third sites were the sites agaI/rsmI, malP/T site and site pflB.
  • the sequence of the sgRNA targeting the agaI/rsmI site is shown in SEQ ID NO:200, and the sequences of the homology arms on both sides of the agaI/rsmI site are shown in SEQ ID NO:201 and 202, respectively.
  • the sequence of the sgRNA targeting the malP/T site is shown in SEQ ID NO: 203, and the sequences of homology arms on both sides of the malP/T site are shown in SEQ ID NO: 204 and 205, respectively.
  • the sequence of the sgRNA targeting the pflB site is shown in SEQ ID NO: 206, and the sequences of the homology arms on both sides of the pflB site are shown in SEQ ID NO: 207 and 208, respectively.
  • the sequence of the sgRNA targeting the lldD site is shown in SEQ ID NO:209, and the sequences of the homology arms on both sides of the lldD site are shown in SEQ ID NO:210 and 211, respectively.
  • the sequence of the sgRNA targeting the maeA site is shown in SEQ ID NO:212, and the sequences of homology arms on both sides of the maeA site are shown in SEQ ID NO:213 and 214, respectively.
  • the sgRNA sequence targeting the agaI/rsmI site (SEQ ID NO:200):
  • LHA left homology arm sequence (SEQ ID NO:201) of agaI/rsmI site:
  • RHA right homology arm sequence (SEQ ID NO:202) of agaI/rsmI site:
  • sgRNA sequence targeting malP/T site SEQ ID NO:203:
  • LHA Left homology arm sequence (SEQ ID NO:204) of malP/T site:
  • RHA Right homology arm sequence (SEQ ID NO:205) of malP/T site:
  • sgRNA sequence targeting pflB site SEQ ID NO:206:
  • LHA left homology arm sequence (SEQ ID NO:207) of pflB site:
  • RHA right homology arm sequence (SEQ ID NO:208) of pflB site:
  • the sgRNA sequence targeting the lldD site (SEQ ID NO:209):
  • LHA left homology arm sequence (SEQ ID NO:210) of lldD site:
  • RHA right homology arm sequence (SEQ ID NO:211) of lldD site:
  • LHA left homology arm sequence of the maeA site
  • RHA right homology arm sequence (SEQ ID NO:214) of maeA site:
  • the yield of Amuc_1100 was detected by Western Blot method.
  • the strain information is shown in Table 21.
  • the sequences of related elements are shown in Table 2-Table 9.
  • lanes A and B are bacterial strains CBT4101, which express a copy of Amuc_1100 (Y259A) using the BBA_J23101 promoter; lane C is bacterial strain CBT4107, which expresses a copy of Amuc_1100 (Y259A) using the BBA_J23110 promoter; lane D is the strain CBT4102, which expresses two copies of Amuc_1100 (Y259A) using the BBA_J23110 promoter; Lane E is bacterial strain CBT4103, which expresses three copies of Amuc_1100 (Y259A) using the BBA_J23101 promoter; Lane F is bacterial strain CBT4108, which expresses three Copies of Amuc_1100(Y259A), one of which uses the BBA_J23101 promoter, and the other two copies use the B
  • the preparation methods of IL-10, IL-22, and Amuc_1100 donor gene fragments are the same as those described in Examples 6.1, 6.2, and 6.3.
  • the construction of the two combination strains expressing IL-10 and IL-22 at the same time is to transfer the IL-22 donor gene fragment into the strain expressing IL-10; the construction of the two combination strains expressing IL-10 and Amuc_1100 at the same time is to The Amuc_1100 donor gene fragment was transferred into a strain expressing IL-10; the construction of two combined strains expressing IL-22 and Amuc_1100 at the same time was to transfer the Amuc_1100 donor gene fragment into a strain expressing IL-22; simultaneously expressing IL-22 -10, IL-22 and Amuc_1100 three-combination strain was constructed by transferring the Amuc_1100 donor gene fragment into the strain that expressed IL-10 and IL-22 simultaneously.
  • the growth curves of strains with single expression, double gene combination expression and triple gene combination expression are shown in Figure 4B.
  • CBT4070 expresses Amuc_1100 alone;
  • CBT4062 expresses IL-10 alone;
  • CBT4068 expresses IL-10 and Amuc_1100 at the same time;
  • CBT4066 expresses IL-22 alone;
  • CBT4069 expresses IL-22 and Amuc_1100 at the same time;
  • CBT4063 expresses IL-10 and IL-22 at the same time;
  • CBT4067 expresses IL-10, IL-22 and Amuc_1100 simultaneously.
  • the strain information is shown in Table 10.
  • Table 23 shows the results of the expression levels of IL-10 and IL-22 in the supernatant of strains expressing individually, in combination with two genes, and in combination with three genes.
  • CBT4062 expresses IL-10 alone
  • CBT4068 expresses IL-10 and Amuc_1100 at the same time
  • CBT4066 expresses IL-22 alone
  • CBT4069 expresses IL-22 and Amuc_1100 at the same time
  • CBT4070 expresses Amuc_1100 alone
  • CBT4063 expresses IL-10 and IL-22 at the same time
  • CBT4067 expresses IL-10, IL-22 and Amuc_1100 simultaneously.
  • the results showed that the expression of IL-10, IL-22 and Amuc_1100 in the combined expression engineering bacteria would not be reduced by the combination.
  • Table 23 The genotypes of the combined strains and the expression levels of IL-10, IL-22 and Amuc_1100
  • the sample to be detected was diluted to an appropriate concentration with whole cell culture medium, and 20 ⁇ L of the diluted sample and IL-10 standard were added to a flat-bottomed 96-well plate, and the EcN sample was used as a negative control.
  • a cell suspension of ⁇ 280,000 cells/mL in HEK-Blue TM IL-10 Cells (InvivoGen, Cat#hkb-il10) was prepared and 180 ⁇ L of this suspension ( ⁇ 50,000 cells) was quickly added to the wells to which the sample had been added.
  • the cell activity of IL-10 secreted in the supernatant of strains expressing alone, in combination with two genes, and in combination with three genes is shown in Figure 4C.
  • the negative control was EcN supernatant that did not express IL-10;
  • the concentration of IL-10 standard was 30 ng/mL;
  • CBT4062 expressed IL-10 alone, and the sample concentration was diluted to 21 ng/mL;
  • CBT4068 simultaneously expressed IL-10 and Amuc_1100 , the sample concentration was diluted to 28ng/mL;
  • CBT4063 simultaneously expressed IL-10 and IL-22, and the sample concentration was diluted to 21ng/mL;
  • CBT4067 simultaneously expressed IL-10, IL-22 and Amuc_1100, and the sample concentration was diluted to 29ng/mL.
  • the sample to be detected was diluted to an appropriate concentration with whole cell culture medium, and 20 ⁇ L of the diluted sample and IL-22 standard were added to a flat-bottomed 96-well plate, and the EcN sample was used as a negative control.
  • a cell suspension of ⁇ 280,000 cells/mL of HEK-Blue TM IL-22 Cells (InvivoGen, Cat#hkb-il22) was prepared, and 180 ⁇ L of this suspension ( ⁇ 50,000 cells) was quickly added to the wells to which the sample had been added.
  • the cell activity of IL-22 secreted in the supernatant of strains expressing alone, in combination with two genes, and in combination with three genes is shown in Figure 4D.
  • the negative control is EcN supernatant that does not express IL-22;
  • CBT4066 expresses IL-22 alone,
  • CBT4069 expresses IL-22 and Amuc_1100 at the same time;
  • CBT4063 expresses IL-22 and IL-10 at the same time;
  • CBT4067 expresses IL-10,
  • concentrations of IL-22 and Amuc_1100; IL-22 standard and samples of each strain were diluted to 0.33ng/mL, 1ng/mL and 3ng/mL.
  • the sample to be detected was diluted to an appropriate concentration with whole cell culture medium, and 20 ⁇ L of the diluted sample and the Amuc_1100 standard were added to a flat-bottomed 96-well plate, and the EcN sample was used as a negative control.
  • a cell suspension of ⁇ 280,000 cells/mL of HEK-Blue TM hTLR2 Cells (InvivoGen, Cat#hkb-htlr2) was prepared, and 180 ⁇ L of this suspension ( ⁇ 50,000 cells) was quickly added to the wells to which the samples had been added.
  • the strain CBT4080 was selected as the measurement object, and the activity of Amuc_1100 secreted in its supernatant was detected, and the results are shown in Figure 4E.
  • the negative control was the purified product of EcN supernatant that did not express Amuc_1100; the standard concentration of Amuc_1100 was 40 ⁇ g/mL; the purity of CBT4080 purified sample was about 70%, and the concentration was 28.8 ⁇ g/mL.
  • the results showed that the Amuc_1100 expressed and secreted by the engineered bacteria had biological activity and its activity was comparable to that of the standard product.
  • Example 8 Construction of anaerobically induced strains expressing IL-10, IL-22 and/or Amuc_1100 combinations
  • the preparation methods of IL-10, IL-22 and Amuc_1100 donor gene fragments are the same as those described in 6.1, 6.2 and 6.3 of Example 6.
  • the construction of the two combination strains expressing IL-10 and IL-22 at the same time is to transfer the IL-22 donor gene fragment into the strain expressing IL-10; the construction of the two combination strains expressing IL-10 and Amuc_1100 at the same time is to The Amuc_1100 donor gene fragment was transferred into a strain expressing IL-10; the construction of two combined strains expressing IL-22 and Amuc_1100 at the same time was to transfer the Amuc_1100 donor gene fragment into a strain expressing IL-22; simultaneously expressing IL-22 -10, IL-22 and Amuc_1100 three-combination strain was constructed by transferring the Amuc_1100 donor gene fragment into the strain that expressed IL-10 and IL-22 simultaneously.
  • the combined strains obtained in this example and the information of the insertion elements and knockout elements therein
  • Table 25 The expression levels of the strains IL-10, IL-22, and Amuc_1100 used in the experiment
  • the 5'-USP45_IL-10_rrnB_T1_T7Te-3' sequence of the IL-10 expression cassette without a promoter was amplified using the CBT4084 strain genome as a template, Psal_sTRSV-HHRz_RBS and PlacIQ_NahRAM_ter were amplified using a synthetic plasmid as a template, and the LHA of the insertion site maeB and RHA was amplified using the EcN genome as a template, the sgRNA at the maeB site was amplified using the plasmid pCBT003_maeB_sgRNA, and the plasmid backbone was amplified using pCBT010 as a template.
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected using the kit ClonExpress Ultra One Step Cloning Kit (Vazyme) to form an expression cassette containing one end connected to LHA 5'-Psal- sTRSV HHRz_RBS_USP45_IL-10_rrnB_T1_T7Te-3', the expression cassette 5'-PlacIQ-NahRAM-ter-3' (sequence shown in SEQ ID NO: 243) connected to RHA at the other end and the plasmid pCBT010_maeB_Psal_IL-10 of the sgRNA at the maeB site.
  • Vazyme ClonExpress Ultra One Step Cloning Kit
  • the plasmid pCBT010_maeB_Psal_IL-10 was transformed into competent cells containing pCBT001, and the obtained single colony was amplified with the verification primers of the insertion site, and the clones with IL-10 successfully inserted into the genome were screened by the size of the amplified band.
  • the production of IL-10 was detected by ELISA kit (Sino Biological, KIT10947A).
  • PlacIQ-NahRAM-ter sequence (SEQ ID NO: 243)
  • IL-12p40_linker1_IL-23p19 IL-17A
  • IL-19 IL-35
  • EBI3_linker2_IL-12p35 IL -37
  • TGF- ⁇ construct engineered bacteria in order to try to study their effects in vivo.
  • IL-23 (IL-12p40_linker1_IL-23p19), IL-17A, IL-19, IL-35 (EBI3_linker2_IL-12p35), IL-37, TGF- ⁇ coding sequence was amplified using the synthetic plasmid as a template , including the salicylic acid-inducible promoter Psal, ribozyme (sTRSV-HHRz), ribosome binding site (RBS), the 5'-Psal_sTRSV-HHRz_RBS_USP45-3' sequence of the secretion signal peptide USP45, including the transcription terminator rrnB_T1_T7Te and water
  • the plasmid backbone sequence of the 5'-rrnB_T1_T7Te_PlacIQ_NahRAM_ter-3' of the ylic acid regulatory element PlacIQ-NahRAM-ter was amplified using the plasmid pCBT010_m
  • the PCR primers used to amplify these fragments have 15-20bp homologous sequences to each other, so they can be connected using the kit ClonExpress Ultra One Step Cloning Kit (Vazyme) to form an expression cassette containing 5'-Psal-sTRSV HHRz_RBS_USP45_cytokine _rrnB_T1_T7Te-3', and expression cassettes pCBT010_Psal_IL-23, pCBT010_Psal_IL-17A, pCBT010_Psal_IL-19, pCBT010_Psal_IL-35, pCBT010_Psal_IL-37, pCBT010_Psal_TGF- ⁇ .
  • plasmids were respectively transformed into competent cells in Chassis bacteria CBT4114 (see Table XX for genotype), and the obtained single colonies were amplified with the verification primers of the plasmids, and the successful transfer of the plasmids was verified by the size of the amplified bands.
  • the production of cytokines was detected with ELISA kits, and the kits used in this example are shown in Table 27.
  • the bacterial strains containing these cytokine expression plasmids were cultured aerobically in LB medium for 4 hours and supplemented with 100 ⁇ M sodium salicylate for 2 hours.
  • the contents of cytokines in the supernatant and cells were shown in Table 28.
  • Example 11 Pharmacodynamic study of mouse enteritis (IBD) model induced by T cell transplantation
  • Enteritis in mice used in this experiment was induced by injecting purified CD4 + CD45RB high cells (without regulatory T cells) into severe combined immunodeficiency (SCID) mice (lacking T cells and B cells).
  • SCID severe combined immunodeficiency
  • Balb/C mice (20 g, 8-10 weeks old) were used to generate CD4 + CD45RB low and CD4 + CD45RB high cells
  • RAG1 -/- mice (20 g, 8-10 weeks old) were used to receive T cell transfer .
  • RAG1 -/- mice were randomly divided into 11 groups, 10 mice in each group.
  • Group 1 is the healthy group, receiving CD4 + CD45RB low cells
  • groups 2-11 are disease groups, receiving CD4 + CD45RB high cells.
  • the induction day of T cell transplantation was defined as day 0, and administration began on day 14.
  • EcN, CBT4084, CBT4095, CBT4096, CBT4088, CBT4098, CBT4110, and CBT4111 cultured aerobically in LB for 4 hours were concentrated and resuspended in 0.2M sodium bicarbonate + 1% w/v glucose aqueous solution.
  • the second group is the positive administration group. From the 14th to the 37th day, the TNF- ⁇ antibody was injected intraperitoneally once every 7 days, and at the same time, 100 ⁇ L of EcN bacteria suspension was intragastrically administered every day.
  • Embodiment 12 DSS-induced mouse enteritis (IBD) model drug efficacy study
  • mice (20g, 50 C57/B6 mice, 8 weeks old, 18-20g, female. were randomly divided into 10 model groups, with 5 mice in each group, and began to drink 2.5 %DSS, 4 days into normal water for 3 days, a total of 4 rounds of DSS induction.
  • the first group is the model control group, no drug administration
  • the second group is the positive drug group, from the 0th day to the 27th day by intragastric administration
  • the dosage is 25mpk.
  • 100 ⁇ L bacterial suspensions of EcN, CBT4096, CBT4088, CBT4098, and CBT4110 were intragastrically administered to groups 3-10, once a day, with a dose of 2.5 ⁇ 10 9 CFU per mouse. At the end of 27 days, the 28th day was the end point of the experiment, the mice were killed and the length of the intestines was measured.
  • Figure 7 shows the body weight change, DAI score (Disease activity index, which is also a commonly used index for evaluating enteritis) and colon length of the DSS-induced mouse enteritis model.
  • DAI score Disease activity index, which is also a commonly used index for evaluating enteritis
  • Figure 7C the colon lengths of the engineered bacteria CBT4096, CBT4088, CBT4098, and CBT4110 groups were all significantly increased (p ⁇ 0.05) (Figure 7C), among which in maintaining body weight (Figure 7A) and reducing the DAI score ( Figure 7B) , CBT4110 has a more significant advantage (p ⁇ 0.001).
  • Embodiment 13 GvHD model pharmacodynamic study
  • BALB/c(H-2d) mice and C57BL/6(H-2b) mice (6-8 weeks old) were used as donors and recipients, respectively.
  • the day of bone marrow transplantation (BMT) was day 0, and the day of radiation
  • the day when the photos were taken was Day -1.
  • Group 1 had 7 syngeneically transplanted mice
  • Groups 2-10 had 9 syngeneically transplanted mice
  • Group 11 had 7 irradiated non-grafted mice.
  • EcN, CBT4084, CBT4095, CBT4096, CBT4088, CBT4098, CBT4110, and CBT4111 cultured aerobically in LB for 4 hours were concentrated and resuspended in 0.2M sodium bicarbonate + 1% w/v glucose aqueous solution.
  • mice in group 1, group 2, group 10 and group 11 were intragastrically administered with 100 ⁇ L EcN cell suspension containing 2.5 ⁇ 10 9 CFU, and at the same time the mice in group 10
  • the mice in the group were given daily injections of 20 mg/kg prednisone, and 100 ⁇ L of bacterial suspensions of CBT4084, CBT4095, CBT4096, CBT4088, CBT4098, CBT4110, and CBT4111 were administered orally to groups 3-9, and the survival rate of the mice was recorded every day .
  • the survival rate of the GvHD animal model is shown in FIG. 8 .
  • the results showed that at day 20, CBT4096 expressing Amuc_1100 alone, CBT4098 expressing IL-22 and Amuc_1100 simultaneously, CBT4110 expressing IL-10 and IL-22 simultaneously, and CBT4111 expressing IL-10, IL-22 and Amuc_1100 simultaneously All the mice had the same or even higher survival rate than the positive drug group.
  • mice in the group all had a higher survival rate than the mice in the CBT4084 group expressing IL-10 alone, the CBT1095 group expressing IL-22 alone, and the CBT4096 group expressing Amuc_1100 alone, indicating that the engineering bacteria expressed in combination in the GvHD model were different. The degree is better than that of engineering bacteria expressed alone.
  • Embodiment 14 SLE model pharmacodynamic research
  • the experimental mice were divided into 6 groups, the first group was the negative control group, including 5 MRL/MpJ mice, the second group was the positive administration group, including 10 MRL/MpJ-Faslpr mice, and administered intragastrically every day 9mg/kg prednisone and 100 ⁇ L EcN bacterial suspension.
  • Groups 3-6 are drug administration groups, each containing 10 MRL/MpJ-Faslpr mice, which were given 100 ⁇ L of EcN, CBT4084, CBT4096, and CBT4088 bacterial suspension by intragastric administration every day, and the dosage was 2.5 ⁇ 10 9 per mouse CFU.
  • the kidneys were taken to make slices to evaluate the damage of renal tubules.
  • the injury of renal tubules is shown in Figure 9A.
  • the engineering bacteria CBT4084 expressing IL-10 alone and the engineering bacteria CBT4096 expressing Amuc_1100 alone could not significantly reduce the injury of renal tubules, but the simultaneous expression of IL-10 and Amuc_1100
  • the engineered bacteria CBT4088 can significantly reduce the damage of renal tubules, indicating that in the SLE model, the simultaneous expression of IL-10 and Amuc_1100 produced a synergistic effect.
  • the therapeutic effects of the engineered bacteria (CBT4111) expressing IL-10 and Amuc_1100 (CBT4088), IL-10 and IL-22 (CBT4110), and IL-10, IL-22 and Amuc_1100 at the same time were further compared.
  • the experimental mice were divided into 5 groups, the first group was the negative control group, including 6 MRL/MpJ mice, and the 2nd to 5th groups were the administration groups, each including 10 MRL/MpJ-Faslpr mice , orally administered 100 ⁇ L of EcN, CBT4088, CBT4110, and CBT4111 bacterial suspension every day, and the dosage was 1.0 ⁇ 10 10 CFU per mouse.
  • the concentration of anti-double-stranded DNA antibody IgG in serum and the concentration of albumin in urine were detected, and kidney sections were taken for PAS staining to evaluate glomerular damage.
  • Embodiment 15 CIA model pharmacodynamic research
  • Collagen-induced arthritis is an experimental autoimmune disease that can be induced by immunizing susceptible strains of rodents (rats and mice) with type II collagen. A spontaneous immune-mediated polyarthritis can develop in immunized animals.
  • the CIA model uses DBA/1 mice. On day 0, after all DBA/1 mice were anesthetized with 2%-5% isoflurane, 50 ⁇ L of collagen emulsion was subcutaneously injected at the base of the tail 2-3 cm away from the body. Three weeks later, that is, on the 21st day, the same volume of collagen emulsion was injected at the base of the tail to challenge.
  • mice On the day of the second collagen emulsion challenge, the modeled animals were randomly divided into 6 groups according to body weight, with 5 or 10 mice in each group, and the administration began on the day of grouping.
  • Groups 1-5 each consisted of 10 mice, and were given 100 ⁇ L of EcN, CBT4088, CBT4098, CBT4110, and CBT4111 bacterial suspension by intragastric administration every day, and the dosage was 1.5 ⁇ 10 10 cells per mouse.
  • the drug group including 5 mice, was orally administered with 100 ⁇ L EcN and 0.2 mg/kg dexamethasone every day.
  • the administration time was 36 days, and the incidence of arthritis in the limbs of animals in each group was observed and scored twice a week, and the thickness of the front and rear foot pads was measured at the same time.
  • the thickness of the front and rear footpads of the mice and the incidence of arthritis in the limbs are shown in Figure 10.
  • the engineering bacteria CBT4110 and IL-22 and the engineering bacteria CBT4111 expressing IL-10, IL-22 and Amuc_1100 at the same time can reduce the severity of the disease to a certain extent. ( FIG. 10A ) and disease score ( FIG. 10B ) improved most significantly (p ⁇ 0.05).
  • Asthma is a chronic inflammatory disease of the airways, characterized by an increase in the number of T helper 2 cells (Th2), eosinophils, and airway inflammation. It is accompanied by high levels of serum immunoglobulin E (IgE), and intrapulmonary production of interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 5 (IL-5) by allergen-specific Th2 cells. IL-13. Airway inflammation is associated with infiltration of eosinophils, neutrophils, and T and B lymphocytes in airway and lung tissue.
  • OVA ovalbumin
  • mice were randomly divided into 7 groups according to body weight, and the administration period was 31 days.
  • EcN, CBT4088, CBT4098, CBT4110, and CBT4111 cultured aerobically in LB for 4 hours were concentrated and resuspended in 0.2M sodium bicarbonate + 1% w/v glucose aqueous solution.
  • 10 mice in groups 1-5 each were given 100 ⁇ L of EcN, CBT4088, CBT4098, CBT4110, and CBT4111 bacterial suspension by intragastric administration every day, and the dosage was 1.5 ⁇ 10 10 cells per mouse.
  • Group 6 is the positive administration group, including 6 mice, which were given 100 ⁇ L of EcN bacterial suspension by intragastric administration every day, and 1 mg/kg of the positive drug dexamethasone was additionally given from the 27th to the 31st day.
  • Group 7 is the non-induced sensitization control group, including 5 mice, which are given 100 ⁇ L of EcN bacterial suspension by intragastric administration every day.
  • the mice in groups 1-6 were intraperitoneally injected with 100 ⁇ L sensitization solution (containing 20 ⁇ g ovalbumin and 2 mg alum) on the first day and the 14th day, and the mice in group 7 were injected with 100 ⁇ L PBS solution as a control.
  • CBT4110 which expresses both IL-10 and IL-22
  • CBT4111 which expresses IL-10, IL-22, and Amuc_1100

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Abstract

一种经遗传修饰的微生物及其应用。所述经遗传修饰的微生物表达至少一种、两种或三种外源基因(Amuc_1100 多肽、IL-10 和/或IL-22)。经遗传修饰的微生物在整合了外源目的基因后仍然能够稳定存活、在肠道内高效表达和分泌这些外源基因所编码的蛋白质或多肽，并且所述表达分泌的外源基因编码的蛋白质或多肽仍然能够具有较好的活性，从而用于炎症性疾病或自身免疫类疾病的治疗(例如，肠炎、关节炎)。

Description

一种经遗传修饰的微生物及其应用
本申请要求申请日为2021/11/26的中国专利申请2021114207638的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，具体地涉及一种经遗传修饰的微生物及其应用。
背景技术
炎症是机体清除体内致病源或受损细胞来帮助组织修复的正常反应。免疫系统紊乱常导致异常炎症，涉及免疫系统的炎症性疾病通常是一大类人类疾病的基础。
自身免疫疾病是一种对正常身体部位有异常免疫反应的疾病。仅在美国就有至少80种自身免疫性疾病影响着2400万人。目前的治疗方法包括非甾体抗炎药(NSAIDs)和免疫抑制剂。然而，这些治疗只能够缓解症状，而无法彻底治愈。而且，这些治疗通常会引起严重的副作用。
自身免疫疾病是多个致病信号通路导致的复杂疾病。目前，已经批准的针对单个信号通路的药物表现出有限的治疗效果，这提示调节多信号通路应该能有更好的治疗效果。然而，对于多信号通路的调节的研究十分有限。首先，尚不知调节哪些多个信号通路有效。其次，尚不知怎样的药物方法学能更有效地调节多个信号通路。由于缺乏有效的治疗方法，工业界在发现和开发治疗自身免疫疾病的新方法方面付出了巨大的努力，却尚未获得显著进展。
因此，本领域迫切需要开发出一种更加高效的治疗炎症或自身免疫性疾病的方法。
发明内容
在一个方面，本公开提供一种经遗传修饰的微生物，其包含至少两种分别编码选自下组的多肽的外源基因：a)Amuc_1100多肽；b)IL-10多肽；以及c)IL-22多肽。
在一些实施方式中，所述经遗传修饰的微生物包含分别编码下述多肽的外源基因：a)Amuc_1100多肽和IL-10多肽；b)IL-10多肽和IL-22多肽；或者c)Amuc_1100多肽和IL-22多肽。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在另一方面，本公开提供了经遗传修饰的微生物组合，其中所述经遗传修饰的微生物组合中包含至少两种不同的经遗传修饰的微生物，其中每种经遗传修饰的微生物各自分别 表达至少一种不同的外源基因；其中所述外源基因分别编码选自下组的多肽：a)Amuc_1100多肽；b)IL-10多肽；以及c)IL-22多肽。
在一些实施方式中，其中所述外源基因编码的多肽能够被所述微生物表达和/或在表达后能够被分泌到细胞外/微生物体外。
在一些实施方式中，所述微生物能够在人或者动物的肠道中表达和/或分泌所述外源基因编码的多肽。
在一些实施方式中，所述分泌是通过所述微生物的原生或非原生的分泌系统实现的。
在一些实施方式中，所述Amuc_1100多肽、IL-10多肽和/或IL-22多肽是去除了自身信号肽的多肽。
在一些实施方式中，所述去除了自身信号肽的Amuc_1100多肽、IL-10多肽和/或IL-22多肽还分别信号肽连接，例如Amuc_1100多肽、IL-10多肽和/或IL-22多肽分别与第一信号肽、第二信号肽或第三信号肽连接，并且所述第一信号肽、第二信号肽和第三信号肽能够将所述多肽分泌到微生物体外。
在另一方面，本公开提供一种包含至少一个重组表达盒的核苷酸序列，其包含i)至少一种或至少两种分别编码Amuc_1100多肽、IL-10多肽和/或IL-22多肽的外源基因，以及ii)可操作地连接所述至少一种或至少两种外源基因的一个或多个调控元件。
在一些实施方式中，所述核苷酸序列包含分别编码Amuc_1100多肽和IL-10多肽的外源基因、分别编码IL-10多肽和IL-22多肽的外源基因，或分别编码Amuc_1100多肽和IL-22多肽的外源基因。
在一些实施方式中，所述核苷酸序列包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在另一方面，本公开提供一种组合物，其中所述组合物包含：(a)作为活性成分的本公开所述的经遗传修饰的微生物；和(b)生理学上或药理学上可接受的载体。
在一些实施方式中，所述组合物是药物组合物。
在一些实施方式中，所述组合物为口服制剂。
在另一方面，本公开提供一种本公开所述的经遗传修饰的微生物或组合物在用于制备治疗或预防炎症性疾病或自身免疫疾病的药物中的用途。
在一些实施方式中，所述的自身免疫疾病选自下组：炎症性肠病、移植物抗宿主病(GvHD)、系统性红斑狼疮(SLE)、关节炎、哮喘，或其组合。
在另一方面，本公开提供一种本公开所述的经遗传修饰的微生物或组合物在制备用于改善炎症性疾病或自身免疫疾病治疗药物的治疗效果的药物中的用途。
在另一方面，本公开提供一种本公开经遗传修饰的微生物的制备方法，其中所述的制备方法包括步骤：向微生物中引入本公开所述的核苷酸序列，以使得所述核苷酸序列中的外源基因可以在所述微生物中表达。
在另一方面，本公开提供一种在有需要的个体中治疗或预防炎症性疾病或自身免疫疾病的方法，其包括：向所述个体施用有效量的本公开所述的经遗传修饰的微生物，或本公开所述的组合物。
在另一方面，本公开提供一种在正在接受药物治疗的个体中改善药物治疗效果的方法，所述药物包括治疗炎症性疾病或自身免疫疾病的药物，所述方法包括：向所述个体施用有效量的本公开所述的经遗传修饰的微生物，或本公开所述的组合物。
在另一方面，本公开提供一种经遗传修饰的微生物在制备用于治疗炎症性疾病或自身免疫疾病的药物中的用途，其中所述的炎症性疾病或自身免疫疾病选自下组：炎症性肠病(IBD)、移植物抗宿主病(GvHD)、系统性红斑狼疮(SLE)、关节炎和哮喘；其中所述经遗传修饰的微生物包含至少一种、至少两种或至少三种分别编码选自下组的多肽的外源基因：a)Amuc_1100多肽；b)IL-10多肽；和c)IL-22多肽。
附图说明
图1展示了外源基因表达盒插入EcN基因组的示意图。
图2有氧与厌氧条件对菌株CBT4078所表达的IL-10的产量的比较结果。
图3展示了不同拷贝数的Amuc_1100(Y259A)表达量的比较结果(A图)、通过改造细胞外膜以提高Amuc_1100的稳定性的比较结果(B图)。其中，A图中的A栏和B栏代表只有一个Amuc_1100(Y259A)拷贝在启动子BBA_J23101控制下的菌株CBT4101，C栏代表只有一个Amuc_1100(Y259A)拷贝在启动子BBA_J23110控制下的菌株CBT4107，D栏代表有两个Amuc_1100(Y259A)拷贝在启动子BBA_J23110控制下的菌株CBT4102，E栏代表有三个Amuc_1100(Y259A)拷贝在启动子BBA_J23101控制下的菌株CBT4103，F栏代表有一个在启动子BBA_J23101控制下的Amuc_1100(Y259A)拷贝和两个启动子BBA_J23110控制下的Amuc_1100(Y259A)拷贝的菌株CBT4108，G栏代表有三个Amuc_1100(Y259A)拷贝在启动子BBA_J23110控制下的菌株CBT4109。
图4A展示了表达IL-10、IL-22和Amuc_1100组合的EcN染色体谱图；
图4B展示了单独表达、双基因组合表达、三基因组合表达菌株的生长状态；
图4C展示了分泌的IL-10的生物活性；
图4D展示了分泌的IL-22的生物活性；
图4E展示了分泌的Amuc_1100的生物活性；
图中的“标品(40μg/mL)”代表Amuc_1100标准品，其浓度为40μg/mL，“EcN”代表菌株EcN分泌的Amuc_1100，“CBT4080(28.8μg/mL)”代表菌株CBT4080的Amuc_1100，其浓度为28.8μg/mL。
图5展示了添加不同浓度水杨酸钠继续培养1-4小时后培养基上清中IL-10的表达量。
图6A展示了T细胞移植诱导的小鼠肠炎模型中，使用不同菌株的药效终点结肠长度的比较结果；
图6B展示了T细胞移植诱导的小鼠肠炎模型中，使用不同菌株的结肠组织病理切片HE染色结果。
图7A展示了DSS诱导的小鼠肠炎模型体重变化；
图7B展示了DSS诱导的小鼠肠炎模型DAI评分；
图7C展示了DSS诱导的小鼠肠炎模型药效终点结肠长度。
图8展示了GvHD动物模型中各实验组小鼠的存活率。
图9A展示了SLE动物模型中各实验组小鼠肾小管损伤情况评分；
图9B展示了SLE动物模型中各实验组小鼠血清中抗双链DNA抗体IgG浓度；
图9C展示了SLE动物模型中各实验组小鼠尿液中白蛋白浓度；
图9D展示了SLE动物模型中各实验组小鼠肾脏组织病理切片PAS染色结果所显示的肾小球损伤情况。
图10A展示了CIA动物模型中各实验组小鼠足垫厚度。
图10B展示了CIA动物模型中各实验组小鼠疾病评分。
图11A展示了哮喘动物模型中各实验组小鼠的肺泡灌洗液(BALF)中的细胞总数计数结果；
图11B展示了哮喘动物模型中各实验组小鼠的肺泡灌洗液(BALF)中的嗜酸性粒细胞计数结果；
图11C展示了哮喘动物模型中各实验组小鼠的肺泡灌洗液(BALF)中的巨噬细胞计数结果；
图11D展示了哮喘动物模型中各实验组小鼠的肺泡灌洗液(BALF)中的中性粒细胞计数结果；
图11E展示了哮喘动物模型中各实验组小鼠的肺泡灌洗液(BALF)中的淋巴细胞计数结果。
图12展示了表达Cas9蛋白的pCBT001质粒所具有的核苷酸序列(SEQ ID NO:136)。
图13展示了pCBT003质粒所具有的核苷酸序列(SEQ ID NO:137)。
图14展示了pMUT2-kana质粒所具有的核苷酸序列(SEQ ID NO:138)。
图15展示了pCBT012质粒所具有的核苷酸序列(SEQ ID NO:215)。
图16展示了pCBT013质粒所具有的核苷酸序列(SEQ ID NO:216)。
具体实施方式
以下对本公开的描述仅打算说明本公开的各种实施方式。因此，所论述的具体修改不应被解释为对本公开范围的限制。所属领域的技术人员将显而易见，可在不脱离本公开的范围的情况下得到各种等效物、进行改变和修改，且应理解所述等效实施例将包含在本文中。本文引用的所有参考文献，包含出版物、专利和专利申请，均以全文引用的方式并入本文中。
I.定义
如本文所用，“一”和“所述”在本文中用于指一个(种)或超过一个(种)(即，至少一个(种))所述冠词的语法对象。举例来说，“一蛋白”意指一种蛋白或多于一种蛋白。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
如本文所用，术语“氨基酸”是指含有胺(-NH 2)和羧基(-COOH)官能团以及每种氨基酸特有的侧链的有机化合物。氨基酸的名称在本公开中也以标准的单字母或三字母代码表示，其概述如表1所示。
表1 氨基酸名称及代码
名称 三字母代码 单字母代码
丙氨酸 Ala A
精氨酸 Arg R
天冬酰胺 Asn N
天冬氨酸 Asp D
半胱氨酸 Cys C
谷氨酸 Glu E
谷氨酰胺 Gln Q
甘氨酸 Gly G
组氨酸 His H
异亮氨酸 Ile I
亮氨酸 Leu L
赖氨酸 Lys K
甲硫氨酸 Met M
苯丙氨酸 Phe F
脯氨酸 Pro P
丝氨酸 Ser S
苏氨酸 Thr T
名称 三字母代码 单字母代码
色氨酸 Trp W
酪氨酸 Tyr Y
缬氨酸 Val V
术语“多肽”、“肽”和“蛋白质”在本文中可互换地用以指氨基酸残基的聚合物。所述术语还适用于其中一个或多个氨基酸残基是对应天然存在的氨基酸的人工化学模拟物的氨基酸聚合物，并且适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。
与氨基酸序列有关的“保守取代”是指氨基酸残基被含具有类似物理化学特性的侧链的不同氨基酸残基置换。举例来说，可在具有疏水性侧链的氨基酸残基(例如Met、Ala、Val、Leu和Ile)间、具有中性亲水性侧链的氨基酸残基(例如Cys、Ser、Thr、Asn和Gln)间、具有酸性侧链的氨基酸残基(例如Asp、Glu)间、具有碱性侧链的氨基酸残基(例如His、Lys和Arg)间、或具有芳香族侧链的氨基酸残基(例如Trp、Tyr和Phe)间进行保守取代。如所属领域中已知，保守取代通常不会引起蛋白质构象结构的显著变化，并且因此可保留蛋白质的生物活性。
如本文所用，术语“突变体”是指与亲本序列具有至少70％序列一致性的多肽或多核苷酸。突变体可以与亲本序列相差一个或多个氨基酸残基或一个或多个核苷酸。举例来说，突变体可具有亲本序列的一个或多个氨基酸残基或一个或多个核苷酸的取代(包含但不限于保守取代)、添加、缺失、插入或截短或其任何组合。例如，在某些实施方式中，突变Amuc_1100与天然存在的对应物具有至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列一致性。
如本文所用，术语“片段”是指任何长度的亲本多肽或亲本多核苷酸的部分序列。片段仍可以保持亲本序列的至少部分功能。
当用于氨基酸序列(例如肽、多肽或蛋白质)时，术语“融合(fusion/fused)”是指例如通过化学键结或重组方式将两个或更多个氨基酸序列组合成非天然存在的单一氨基酸序列。融合氨基酸序列可以通过两个编码多核苷酸序列的遗传重组产生，并且可以通过将含有重组多核苷酸的构建体引入到微生物中的方法表达。
如本文中关于多肽或多核苷酸所用的术语“衍生物”是指化学修饰的多肽或多核苷酸，其中一个或多个明确定义数目的取代基已经共价连接于多肽的一个或多个特定氨基酸残基或多核苷酸的一个或多个特定核苷酸。对多肽的示范性化学修饰可以是例如一个或多个氨基酸的烷基化、酰化、酯化、酰胺化、磷酸化、糖基化、标记、甲基化或与一个或多个部分结合。对多核苷酸的示范性化学修饰可以是(a)末端修饰，例如5'端修饰或3'端修饰，(b)核碱基(或“碱基”)修饰，包含碱基的置换或去除，(c)糖修饰，包含在2'、3'和/或4'位置处的修饰，和(d)主链修饰，包含磷酸二酯键的修饰或置换。
根据本文的教导，这些片段、变异体、衍生物、突变体和类似物属于本领域熟练技术人员公知的范围。其中，所述的保守性取代的类似物优选根据表2进行氨基酸替换而产生。
表2 类似氨基酸总结表
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
如本文所用，术语“同源”是指在最好比对时，与另一序列具有至少60％(例如至少65％、70％、75％、80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的核酸序列(或其互补股)或氨基酸序列。
关于氨基酸序列(或核酸序列)的术语“％序列一致性”定义为在比对序列并在必要时引入空位以实现最大数目的相同氨基酸(或核酸)后，候选序列中与参考序列中的氨基酸(或核酸)残基相同的氨基酸(或核酸)残基的百分比。换句话说，氨基酸序列(或核酸序列)的序列一致性百分比(％)可以通过将相对于其比较的参考序列相同的氨基酸残基(或碱基)的数目除以候选序列或参考序列中氨基酸残基(或碱基)的总数(以较短者为准)来计算。氨基酸残基的保守取代可视为或可不视为相同残基。出于确定氨基酸(或核酸)序列一致性百分比的目的进行的比对可例如使用以下实现：可公开获得的工具，如BLASTN、BLASTp(可见于美国国家生物技术信息中心(U.S.National Center for Biotechnology Information；NCBI)的网站，另外参见Altschul S.F.等人，《分子生物学杂志》,215:403-410(1990)；Stephen F.等人，《核酸研究(Nucleic Acids Res.)》,25:3389-3402(1997))、ClustalW2(可见于欧洲生物信息研究所(European Bioinformatics Institute)网站，另外参见Higgins D.G.等人，《酶学方法(Methods in Enzymology)》,266:383-402(1996)；Larkin  M.A.等人，《生物信息学(Bioinformatics)》(英格兰牛津(Oxford,England)),23(21):2947-8(2007))和ALIGN或Megalign(DNASTAR)软件。所属领域的技术人员可使用所述工具所提供的默认参数，或可视需要例如通过选择适合算法来自定义比对的参数。
如本文所用，术语“核苷酸序列”、“核酸”或“多核苷酸”包含寡核苷酸(即短多核苷酸)。其还指合成的和/或非天然存在的核酸分子(例如，包含核苷酸类似物或经修饰的主链残基或键)。所述术语还指单股或双股形式的脱氧核糖核苷酸或核糖核苷酸寡核苷酸。所述术语涵盖含有天然核苷酸类似物的核酸。所述术语还涵盖具有合成主链的核酸样结构。除非另外指示，否则特定多核苷酸序列还隐含地涵盖其保守修饰变异体(例如简并密码子取代)、等位基因、直系同源物、SNP和互补序列，以及明确指示的序列。具体来说，可以通过产生其中一个或多个选定(或所有)密码子的第三位置被混合碱基和/或脱氧肌苷残基取代的序列实现简并密码子取代(参见Batzer等人，《核酸研究(Nucleic Acid Res.)》19:5081(1991)；Ohtsuka等人，《生物化学杂志(J.Biol.Chem.)》260:2605-2608(1985)；和Rossolini等人，《分子细胞探针(Mol.Cell.Probes)》8:91-98(1994))。
如本文所用，术语“编码(encoded/encoding)”是指能够转录成mRNA和/或翻译成肽或蛋白质。术语“编码序列”或“基因”是指编码肽或蛋白质的多核苷酸序列。这两个术语可以在本公开中互换使用。在一些实施方式中，编码序列是由信使RNA(mRNA)逆转录的互补DNA(cDNA)序列。在一些实施方式中，编码序列是mRNA。
术语“可操作地连接”是指两个或更多个所关注的生物序列在存在或不存在间隔子或连接子的情况下的并接，使得它们处于允许它们以预期方式起作用的关系中。当关于多肽使用时，其意指多肽序列以允许连接的产物具有预期的生物学功能的方式连接。例如，抗体可变区可以可操作地连接于恒定区，以提供具有抗原结合活性的稳定产物。所述术语还可以关于多核苷酸使用。举例来说，当编码多肽的多核苷酸可操作地连接于调节序列(例如启动子、增强子、沉默子序列等)时，其意指多核苷酸序列以允许调节多肽由多核苷酸的表达的方式连接。
如本文所用，术语“载体”是指一种运载体，可将遗传元件可操作地插入其中，以实现所述遗传元件的表达，从而产生由所述遗传元件编码的蛋白质、RNA或DNA，或复制所述遗传元件。载体可用于转化、转导或转染宿主细胞(如，微生物)，以使其携带的遗传元件在宿主细胞内表达。不同载体可适合于不同宿主细胞。载体的实例包含质粒；噬菌粒；粘粒；人工染色体，如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)；噬菌体，如λ噬菌体或M13噬菌体；和动物病毒。载体可以含有多种用于控制表达的元件，包含启动子序列、转录起始序列、增强子序列、可选元件和报告基因。另外，载体可以含有复制起点。载体还可以包含有助于其进入细胞的材料，包含但不 限于病毒粒子、脂质体或蛋白质包衣。载体可以是表达载体或克隆载体。
II.经遗传修饰的微生物
在一方面，本申请提供了一种经遗传修饰的微生物，其包含至少一种编码选自下组的外源基因：编码表达Amuc_1100多肽的基因、或编码表达IL-10多肽的基因、或编码表达IL-22多肽的基因、或以上任意组合。
在另一方面，本申请提供了一种包含至少两种分别编码选自下组多肽的外源基因的经遗传修饰的微生物：(i)Amuc_1100多肽，(ii)IL-10多肽，和(iii)IL-22多肽。
在某些实施方式中，所述经遗传修饰的微生物包含：a)分别编码Amuc_1100多肽和IL-10多肽的外源基因；b)分别编码IL-10多肽和IL-22多肽的外源基因；或者c)分别编码Amuc_1100多肽和IL-22多肽的外源基因。
在某些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在某些实施方式中，所述经遗传修饰的微生物包含编码Amuc_1100多肽、IL-10多肽或者IL-22多肽的外源基因。
如本文所用，“外源基因”是在所述微生物中通过遗传修饰引入的基因。外源基因可以包括在所述微生物中原本不表达的异源基因，也可以包括通过遗传修饰引入的原本在所述微生物中也有表达的内源基因(例如为提高表达量的目的)。
在本申请中，所述外源基因在所述微生物中表达的多肽也称为外源多肽。在一些实施方式中，所述外源多肽选自：Amuc_1100多肽、IL-10多肽，或IL-22多肽。在一些实施方式中，所述外源多肽是在所述微生物中原本不表达的异源多肽，例如，人IL-10多肽、人IL-22多肽等。当使用的微生物内源不表达Amuc_1100时，Amuc_1100也可以是异源多肽。
可用于本发明的微生物包含细菌、古细菌、真菌(例如酵母、丝状真菌)和藻类。
在一些实施方式中，本申请提供的所述微生物包含益生微生物或非病原性微生物。
所本文所述，“非病原性微生物体”是指不能在宿主中引起疾病或有害反应的微生物体。在一些实施方式中，非病原性微生物体不含脂多糖(LPS)。在一些实施方式中，非病原性微生物体为共生细菌。在一些实施方式中，非病原性微生物体为减毒病原性细菌。
在一些实施方式中，本发明中使用的微生物是益生微生物体。所本文所述，“益生微生物体”是指当以有效量施用时，对个体的健康或康乐提供有益作用，包括例如与改善人类或动物微生物群的平衡相关的健康益处，和/或用于恢复正常的微生物群。按照定义，所有益生微生物体都具有经证实的非病原性特征。一般来说，益生菌帮助肠道微生物群保持(或重新找到)其平衡、完整性和多样性。益生菌的作用可以是菌株依赖性的。
在一些实施方式中，益生微生物体包括益生微生物细胞(例如活的微生物细胞)的制 剂。
在一些实施方式中，益生微生物体为益生细菌或益生酵母。
在一些实施方式中，益生细菌选自由以下组成的群组：拟杆菌、双歧杆菌(例如两歧双歧杆菌)、梭菌、埃希氏菌、乳杆菌(例如嗜酸乳杆菌、保加利亚乳杆菌、副干酪乳杆菌、胚芽乳杆菌)和乳球菌。在一些实施方式中，所述益生细菌属于埃希氏菌属。在一些实施方式中，所述益生细菌属于物种大肠杆菌的菌株Nissle 1917(EcN)。在一些实施方式中，益生酵母选自由以下组成的群组：酿酒酵母、产朊假丝酵母、乳酸克鲁维酵母和卡氏酵母。在一些实施例中，益生微生物体为大肠杆菌的菌株Nissle 1917(EcN)。
大肠杆菌Escherichia coli作为研究微生物遗传、生理和代谢的模式菌株，由于遗传操作工具多样以及遗传背景清晰等优势成为重要的底盘细菌或宿主细菌之一。
在一些实施方式中，所述益生细菌是人类肠道正常存在的细菌。
在一些实施方式中，所述益生酵母选自由以下组成的群组：酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)、乳酸克鲁维酵母(Kluyveromyces lactis)和卡氏酵母(Saccharomyces carlsbergensis)。
在本申请中，术语“遗传修饰”是指通过人工干预的方式在细胞中(例如微生物体)引入在DNA和/或RNA中的修改或修饰或者引入外源的DNA或RNA。通常，所述修改或修饰可以通过重组核酸表达载体的方式引入，或者通过突变的方式引入，或者通过基因编辑的方式引入。
如本文所用，术语“经遗传修饰的微生物”是指其中引入了外源基因或外源性表达盒(包含含有表达盒的载体)的微生物。“经遗传修饰的微生物”也可以和“基因工程微生物”、“工程微生物”、“基因改造的微生物”或者“经基因工程改造的微生物”互换使用。在本申请中，所述外源性表达盒包含编码目的多肽或目的蛋白的多核苷酸，并且可以允许其表达。将表达盒引入到微生物中可以通过磷酸钙转染、DEAE-葡聚糖介导的转染或电穿孔实现(Davis,L.、Dibner,M.、Battey,I.,《基本分子生物学方法(Basic Methods in Molecular Biology)》(1986))，也可以通过基因编辑技术(例如，CRISPR技术)实现。
经遗传修饰的微生物还涵盖了本申请提供的微生物或其衍生物的任何后代。应理解，所有后代可能与母细胞不同，因为可能存在在复制期间发生的突变。
本申请还提供了一种包含至少一个重组表达盒的核苷酸序列，其包含i)至少一种或至少两种分别编码Amuc_1100多肽、IL-10多肽和/或IL-22多肽的外源基因，以及ii)可操作地连接所述至少一种或至少两种外源基因的一个或多个调控元件。
在一些实施方式中，本公开还提供了经遗传修饰的微生物的组合，其包括本公开所述的包含至少一种(例如包含至少一种、至少两种，或至少三种)外源基因的经遗传修饰的 微生物的组合。
在一些实施方式中，在所述的经遗传修饰的微生物组合中，包含两种不同的经遗传修饰的微生物，其中每种经遗传修饰的微生物各自分别表达一种不同的外源基因；其中，所述外源基因编码的多肽分别为：a)Amuc_1100多肽和IL-10多肽；b)IL-10多肽和IL-22多肽；或者c)Amuc_1100多肽和IL-22多肽。
在一些实施方式中，在所述的经遗传修饰的微生物组合中包含三种不同的经遗传修饰的微生物，其中每种经遗传修饰的微生物各自分别表达一种不同的外源基因；或所述经遗传修饰的微生物组合包含两种不同的经遗传修饰的微生物，其中所述两种经遗传修饰的微生物分别表达一种外源基因和另外两种外源基因；其中，所述外源基因编码的多肽分别为：Amuc_1100多肽、IL-10多肽和IL-22多肽。
A.Amuc_1100
Amuc_1100已知为嗜粘蛋白阿克曼氏菌(“A.muciniphila”)细菌种中存在的外膜蛋白。连同两个其它成员Amuc_1099和Amuc_1101，Amuc_1100位于涉及IV型菌毛样形成的基因簇内(Ottman等人，公共科学图书馆·综合(PLoS One),2017)。Amuc_1100的示范性氨基酸序列公开于GenBank：ACD04926.1中。在本公开中，术语“Amuc_1100”和“AMUC_1100”可互换地使用。
如本文所用，术语“Amuc_1100”广泛涵盖Amuc_1100多肽，以及Amuc_1100多核苷酸，例如编码Amuc_1100多肽的DNA或RNA序列。如本文所用，术语“Amuc_1100”进一步涵盖野生型Amuc_1100，以及与野生型Amuc_1100多肽在功能上等效的功能等效物。
在本申请中，当术语“野生型”用于描述多肽或多核苷酸时，是指所述多肽或多核苷酸的序列与自然界中发现的那些序列相同。野生型的多肽或多核苷酸可为原生或自然界中存在的多肽或多核苷酸序列，并且也广泛地包括其片段，即使所述片段本身可能未于自然界中发现。
在本申请中，当术语“功能等效物”用于描述多肽或多核苷酸时，是尽管氨基酸序列或多核苷酸序列或化学结构与参比序列相比具有差异，但仍至少部分保留参比序列的一种或多种生物功能的任何变异体。
在本申请中，当术语“变异体”用于描述多肽或多核苷酸时，涵盖所有种类的不同形式的所述多肽或多核苷酸，包含但不限于天然存在的多肽或多核苷酸的片段、突变体、融合物、衍生物、模拟物或其任何组合。
在本申请中，术语“野生型Amuc_1100”是指Amuc_1100多肽或多核苷酸的序列与自然界中发现的序列或那些序列相同。野生型的Amuc_1100可为原生或自然界中存在的 Amuc_1100序列，及其片段，即使所述片段本身可能未于自然界中发现。野生型Amuc_1100还可包含天然存在的变异体，如在不同细菌菌株中发现的突变体或同功异构物或不同原生序列。野生型的全长Amuc_1100多肽具有317个氨基酸残基的长度。野生型的Amuc_1100的示范性氨基酸序列包含但不限于氨基酸序列如SEQ ID NO:1所示的Amuc_1100(1-317)、如SEQ ID NO:1所示的氨基酸序列中的第31-317位所示的Amuc_1100(31-317)和如SEQ ID NO:1所示的氨基酸序列中的第81-317位所示的Amuc_1100(81-317)。
Amuc_1100(1-317)氨基酸序列(SEQ ID NO:1)：
Figure PCTCN2022134672-appb-000001
在一些实施方式中，Amuc_1100多肽包含野生型的Amuc_1100多肽的功能等效物。
在本申请中，野生型Amuc_1100的功能等效物是指，尽管氨基酸序列或多核苷酸序列或化学结构具有差异，但仍至少部分保留野生型Amuc_1100的一种或多种生物功能的任何Amuc_1100变异体。野生型Amuc_1100的生物功能包含但不限于a)调节和/或促进哺乳动物的肠道免疫系统功能，b)维持、恢复和/或增加哺乳动物的肠粘膜屏障的物理完整性，c)激活TLR2，d)提高哺乳动物中对癌症免疫疗法(例如免疫检查点调节剂)的免疫反应，和e)降低、延迟和/或预防哺乳动物中对一种或多种免疫检查点调节剂的耐药性。
在某些实施方式中，Amuc_1100的功能等效物保持亲本分子的实质生物活性。在某些实施例中，本文所述的Amuc_1100的功能等效物仍保持与野生型Amuc_1100实质上类似的功能，例如，Amuc_1100的功能等效物可保持野生型Amuc_1100的至少部分(例如，至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)例如调节肠道免疫性和/或活化TLR2的活性。
Amuc_1100多肽的功能等效物可包含野生型的Amuc_1100的突变体、片段、融合物、衍生物或其任何组合。Amuc_1100的功能等效物可包含对野生型的Amuc_1100进行人工改造产生的变异体，如通过重组方法或化学合成获得的人工多肽序列。Amuc_1100的功能等效物可在活性不受影响的前提下包含非天然存在的氨基酸残基。适合的非天然氨基酸包含例如β-氟丙氨酸、1-甲基组氨酸、γ-亚甲基谷氨酸、α-甲基亮氨酸、4,5-脱氢赖氨酸、羟基脯氨酸、3-氟苯丙氨酸、3-氨基酪氨酸、4-甲基色氨酸等。
在本申请中，Amuc_1100变异体可以涵盖所有种类的不同形式的Amuc_1100，包含但 不限于野生型Amuc_1100的片段、突变体、融合物、衍生物、模拟物或其任何组合。
尽管不希望受任何理论束缚，但某些野生型Amuc_1100片段(例如Amuc_1100(31-317))可以比全长对应物更高的亲和力结合和活化TLR2。
在一些实施方式中，Amuc_1100片段具有如SEQ ID NO:2所示的氨基酸序列。
Figure PCTCN2022134672-appb-000002
在一些实施方式中，Amuc_1100片段具有如SEQ ID NO:3所示的氨基酸序列。
Figure PCTCN2022134672-appb-000003
在一些实施方式中，所述Amuc_1100多肽包含如SEQ ID NO:5所示的序列，或包含与SEQ ID NO:5所示的序列具有至少80％序列一致性且仍保持调节肠道免疫和/或活化toll样受体2(TLR2)的活性的氨基酸序列。在一些实施方式中，所述Amuc_1100多肽包含与如SEQ ID NO:5所示序列具有至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性且仍保持调节肠道免疫和/或活化toll样受体2(TLR2)的活性的氨基酸序列。在一些实施方式中，编码SEQ ID NO:5所示氨基酸序列的核苷酸序列如SEQ ID NO:4所示。
在某些实施方式中，Amuc_1100多肽在第259位具有Y259A或Y259S突变，所述第259位的编号是基于如SEQ ID NO:5所示序列的编号。在一些实施方式中，具有Y259A突变的Amuc_1100多肽具有如SEQ ID NO:7所示的氨基酸序列。在一些实施方式中，编码如SEQ ID NO:7所示氨基酸序列的核苷酸序列如SEQ ID NO:6所示。在一些实施方式中，具有Y259S突变的Amuc_1100多肽具有如SEQ ID NO:9所示的氨基酸序列。在一些实施方式中，编码SEQ ID NO:9所示氨基酸序列的核苷酸序列如SEQ ID NO:8所示。
在某些实施方式中，Amuc_1100多肽进一步在N端或C端处包含标签或氨基酸延伸。标签可用于Amuc_1100多肽的纯化。氨基酸延伸可用于提高稳定性或减少清除率。可以使用任何适合的标签或延伸，例如His-标签(例如6×His标签)、蛋白质A、lacZ(β-gal)、麦芽糖结合蛋白质(MBP)、钙调蛋白结合肽(CBP)、内含肽-甲壳质结合结构域(内含肽-CBD)标签、基于链霉亲和素/生物素的标签、串联亲和纯化(TAP)标签、表位标签、报告基因标签等。
在某些实施例中，Amuc_1100多肽进一步包含将标签与Amuc_1100多肽的其余部分 分隔开的酶消化位点。酶消化位点可用于在需要时去除标签。适合酶消化位点的实例包含肠激酶识别位点(例如DDDDK)、因子Xa识别位点、吉能酶(genenase)I识别位点、弗林蛋白酶(furin)识别位点等。
Amuc_1100DNA序列(SEQ ID NO:4)
Figure PCTCN2022134672-appb-000004
Amuc_1100氨基酸序列(SEQ ID NO:5)
Figure PCTCN2022134672-appb-000005
Amuc_1100(Y259A)DNA序列(SEQ ID NO:6)
Figure PCTCN2022134672-appb-000006
Amuc_1100(Y259A)氨基酸序列(SEQ ID NO:7)
Figure PCTCN2022134672-appb-000007
Amuc_1100(Y259S)DNA序列(SEQ ID NO:8)
Figure PCTCN2022134672-appb-000008
Amuc_1100(Y259S)氨基酸序列(SEQ ID NO:9)
Figure PCTCN2022134672-appb-000009
此外，由于Amuc_1100是膜蛋白，其N端的信号肽序列将会促使Amuc_1100多肽进行膜定位，因而为了实现本公开的目的，即实现Amuc_1100多肽的分泌，需要将Amuc_1100的自身信号肽替换为本公开中能够将Amuc_1100多肽分泌到微生物体外的其它信号肽，例如USP45信号肽。
B.细胞因子
本公开所提供的经遗传修饰的微生物还可以表达和分泌一种或多种细胞因子。
在一些实施方式中，所述细胞因子为IL-10和IL-22。
在一些实施方式中，所述细胞因子还可以是IL-17A、IL-19、IL-23、IL-35、IL-37或TGF-beta。
白细胞介素-10(IL-10)
IL-10是一种维持免疫应答平衡的抗炎性细胞因子，由多种类型的细胞合成，包括B细胞、单核细胞、树突细胞、自然杀伤细胞和T细胞。IL-10由造血细胞表达的特异性受体识别，属于II类细胞因子。IL-10通过两个受体IL-10R1和IL-10R2及其下游的JAK/STAT通路传导信号，最终激活抗炎反应基因的表达。IL-10可抑制巨噬细胞和树突细胞的活性，间接抑制T细胞的活化和效应器的功能。IL-10对炎症性肠病、过敏反应等有保护作用。IL-10和/或其受体的缺陷与IBD和肠敏感性相关(Nielsen,2014)。
白介素-10(IL-10)是由几类细胞产生的多效细胞因子(pleiotropiccytokine)，所述细胞类型如巨噬细胞、单核细胞、Th2型和调节性T细胞以及B细胞。IL-10是具有免疫抑制特性和抗炎特性的细胞因子；其调节许多髓样细胞和淋巴样细胞的活性，并直接抑制T细胞和NK(天然杀伤)细胞产生几种炎性细胞因子。
IL-10最初描述为Th2细胞所产生的细胞因子合成抑制因子(cytokine synthesis inhibitory factor，CSIF)，Th2细胞抑制Th1细胞产生促炎细胞因子，如γ-干扰素(IFN-γ)、白介素-1-α(IL-1α)、白介素-1-β(IL-1β)、白介素-2(IL-2)以及肿瘤坏死因子α(TNF-α)。已经证明，除了抑制促炎细胞因子产生之外，IL-10可以抑制抗原特异性Th1细胞增殖，由此通过使这些细胞中主要组织相容性复合体(majorhistocompatibility complex，MHC)-II的表达失调节来降低单核细胞抗原呈递能力。
IL-10对Th1细胞的激活以及对促炎细胞因子的产生具有强烈的抑制作用这一发现，产生了IL-10是细胞介导的免疫应答的强免疫抑制剂这一假说。其他的作者已经提议使用这种细胞因子治疗急性和慢性炎症以及治疗自身免疫病。基于这些原因，这种细胞因子已经用于几种自身免疫病中，如牛皮癣、类风湿性关节炎和克罗恩氏病(Crohn′s disease)。然而，在其他疾病，如感染性过程或癌症中，因为其阻碍了益于治疗的Th1应答的诱导，因而具有副作用。这些过程的实例包括，麻疯病、肺结核、利什曼病和病毒感染。因此，IL-10在丙型肝炎病毒导致的慢性感染中大量表达，已有记载。作为用HCV抗原刺激的结果，这种细胞因子可以由Th2细胞产生。其也可以由抑制Th1型抗病毒效应细胞形成的调节性T细胞(D4和CD8)产生。最后，与HCV蛋白接触的感染的树突细胞(DC)或单核细胞，比非感染的细胞产生更大量的IL-10，这有利于Th2应答的发生，同时阻碍病毒的消除。
在一些实施方式中，本公开所提供的经遗传修饰的微生物可以表达和分泌IL-10。
如本文所用，术语“IL-10”广泛涵盖IL-10，以及IL-10多核苷酸，例如编码IL-10的DNA或RNA序列。如本文所用，术语“IL-10”进一步涵盖野生型IL-10，以及与野生型的IL-10在功能上等效的功能等效物。在本申请中，野生型IL-10的功能等效物是指尽管氨基酸序列或多核苷酸序列或化学结构具有差异，但仍至少部分保留野生型IL-10的一种 或多种生物功能的任何IL-10变异体。所述野生型IL-10的一种或多种生物功能包括但不限于，能够结合IL-10R1/IL-10R2，并通过受体-JAK-STAT信号通路发挥功能。
在一些实施方式中，所述IL-10包含SEQ ID NO:11所示的氨基酸序列，或包含与SEQ ID NO:11所示序列具有至少80％、至少85％、至少90％、至少95％或至少99％序列一致性且仍保持调节免疫细胞(例如，巨噬细胞、树突细胞)的活性的氨基酸序列。在一些实施方式中，所述IL-10包含与SEQ ID NO:11所示序列具有至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性且仍保持调节免疫细胞(例如，巨噬细胞、树突细胞)的活性的氨基酸序列。
在一些实施方式中，所述IL-10包含P2A的突变，其中编号相对于SEQ ID NO:13；即，具有所述的SEQ ID NO:13所示的氨基酸序列。
在本申请中，IL-10的变异体涵盖所有种类的不同形式的IL-10，包含但不限于IL-10的片段、突变体、融合物、衍生物、模拟物或其任何组合。
在一些实施方式中，本公开所提供的经遗传修饰的微生物可以包含编码IL-10(例如，人类IL-10)的任何合适的基因。在一些实施方式中，编码IL-10的基因包含SEQ ID NO:10所示的核苷酸序列，或包含与SEQ ID NO:10所示序列具有至少80％、至少85％、至少90％、至少95％或至少99％序列一致性且其编码蛋白仍保持调节免疫细胞(例如，巨噬细胞、树突细胞)的活性的核苷酸序列。在一些实施方式中，编码IL-10的基因包含与SEQ ID NO:10所示序列具有至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性且其编码蛋白仍保持调节免疫细胞(例如，巨噬细胞、树突细胞)的活性的核苷酸序列。
编码IL-10的基因可以包括修饰和/或突变，例如，以在诱导条件下增强稳定性、增加IL-10的表达和/或增加抗炎效力。在一些实施方式中，编码IL-10的基因包含SEQ ID NO:12所示的核苷酸序列。
在一些实施方式中，本公开所提供的经遗传修饰的微生物能够在诱导条件下(例如，在炎症组织中微环境因子诱导的条件下)产生IL-10。在一些实施方式中，所述经遗传修饰的微生物能够在低氧或厌氧条件下产生IL-l0。在一些实施方式中，所述经遗传修饰的微生物能够从1×10 9CFU的微生物中分泌至少300ng、至少350ng、至少400ng、至少450ng、至少500ng、至少550ng、至少600ng或至少650ng的IL-10。
IL-10DNA序列(SEQ ID NO:10)
Figure PCTCN2022134672-appb-000010
IL-10氨基酸序列(SEQ ID NO:11)
Figure PCTCN2022134672-appb-000011
IL-10(P2A)DNA序列(SEQ ID NO:12)
Figure PCTCN2022134672-appb-000012
IL-10(P2A)氨基酸序列(SEQ ID NO:13)
Figure PCTCN2022134672-appb-000013
白介素-22(IL-22)
如本文所用，“白介素-22”或“IL-22”是一种T细胞分泌的糖蛋白，其在发现时被命名为IL-10相关的T细胞衍生的可诱导因子(IL-10-related T cell-derived inducible factor，IL-TIF)，由于其功能的独特性现已成为研究最深入的IL-10家族成员之一。
IL-22主要由适应性免疫细胞(CD4阳性T细胞、CD8阳性T细胞等)和固有免疫细胞(LTi细胞、NK细胞等)分泌。已发现众多转录因子如信号转导和转录激活3(STAT3)、维甲酸相关孤核受体γτ(RORγτ)、芳香烃受体(AhR)等，以及细胞因子如IL-23、IL-6、IL-17、TNF-α、TNF-β等都能影响IL-22的表达。
IL-22通过结合IL-22RI受体和IL-10R2受体，发挥生物学功能。与其他免疫性细胞因子主要作用于造血源性细胞不同，IL-22主要在组织内的非血源性细胞如内皮细胞、基质细胞、成纤维细胞等发挥功能，并且IL-22广泛分布于很多组织中，如肺、肝脏、肾脏、胸 腺、乳腺、肠、皮肤及滑膜等组织。
IL-22通过激活STAT信号转导通路引起下游的增值及抗凋亡效应参与的黏膜屏障维护和修复功能，尽管IL-22能增强上皮细胞的损伤后修复并通过抗凋亡促增值提高上皮细胞的存活，但IL-22的持续高表达在某些疾病如肿瘤、自身免疫性疾病等条件下反而引起组织损伤和慢性炎症等致病性效应。另外，IL-22也可以诱导产生抗菌肽从而行使抵抗微生物及寄生虫侵袭的作用。正是由于IL-22如此广泛复杂的保护性和致病性功能，近年来许多研究都证实IL-22在感染、自身免疫性疾病及肿瘤等疾病中发挥独特的作用。
在一些实施方式中，本公开所提供的经遗传修饰的微生物表达和分泌IL-22。
如本文所用，所述IL-22包括具有SEQ ID NO：15的多肽及其功能等效物。如本文所用，术语“IL-22”广泛涵盖IL-22，以及IL-22多核苷酸，例如编码IL-22的DNA或RNA序列。如本文所用，术语“IL-22”进一步涵盖野生型IL-22，以及与野生型IL-22在功能上等效的功能等效物。在本申请中，野生型IL-22的功能等效物是指，尽管氨基酸序列或多核苷酸序列或化学结构具有差异，但仍至少部分保留野生型IL-22的一种或多种生物功能的任何IL-22变异体。所述野生型IL-22的一种或多种生物功能包括但不限于，能够结合IL-22R1/IL-10R2，通过Janus激酶(与IL-22R亚单位相关)和STAT分子发出信号的实质的活性。
在一些实施方式中，其中所述IL-22包含SEQ ID NO:15所示的序列，或包含与SEQ ID NO:15所示序列具有至少80％序列一致性且仍保持结合IL-22受体并调节IL-22受体表达细胞的活性的氨基酸序列。在一些实施方式中，所述IL-22包含与SEQ ID NO:15所示序列具有至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列一致性且仍保持结合IL-22受体并调节IL-22受体表达细胞的活性的氨基酸序列。
在本申请中，IL-22的变异体涵盖所有种类的不同形式的IL-22，包含但不限于IL-22的片段、突变体、融合物、衍生物、模拟物或其任何组合。
IL-22 DNA序列(SEQ ID NO:14)
Figure PCTCN2022134672-appb-000014
IL-22氨基酸序列(SEQ ID NO:15)
Figure PCTCN2022134672-appb-000015
白介素-17A(IL-17A)
如本文所用，“白介素-17”或“IL-17A”由活化的T细胞产生，并与普遍表达于所有细胞类型的IL-17R结合介导炎症反应。然而，IL-17A信号传递的确切机制仍未完全阐明。在包括RA的慢性炎症中，IL-17A可通过直接使基质降解，或间接使活化的炎性细胞增多并诱导其它致炎细胞因子包括IL-1b和TNF-a进入炎性组织，引起组织损害。IL-17A活化MAPKs(丝裂原活化蛋白激酶)所有的三个成员，即细胞外信号调节激酶(ERK1和ERK2)p44和p42、Jun蛋白N-端激酶(JNK)和p38。在人的纤维母细胞、肠上皮细胞和培养的软骨细胞中可见IL-17A诱导NF-kb的活化，该作用可能与TNF受体相关因子(TRAF)-6有关。在人单核白血病细胞系U937中，IL-17A可诱导Janus激酶(JAN)以及信号转导和转录激活蛋白(STAT)途径的某些成员发生酪氨酸磷酸化，其中包括Tyk2、JAK1，2和3、STAT1，2，3和4，提示JAK/STAT途径可能参与调节IL-17A的生物学效应。
IL-17A DNA序列(SEQ ID NO:217)
Figure PCTCN2022134672-appb-000016
IL-17A氨基酸序列(SEQ ID NO:218)
Figure PCTCN2022134672-appb-000017
白介素-19(IL-19)
如本文所用，“白介素-19”或“IL-19”主要是通过IL-20R1/R2这一对受体复合物传导生物信号而起生物学作用。IL19是细胞因子IL-10家族的成员，但IL-19的作用还不十分明确，对于IL-19是一个促炎性或抗炎性的细胞因子仍然存在争议和不确定性。有研究认为IL-19可能和IL-10一样具有抗炎效应，并且可能存在诱导淋巴细胞从Th1向Th2转变的表型。也有研究认为在单核细胞IL-19上调IL-6和TNF-α中产生，显示IL-19有促炎症的特点。尽管没有检测到IL-20R1在免疫细胞群的表达，但还是有很多研究清楚地显示出IL-19对这些细胞的效应。大多数的研究表明IL-19是Th2系统的一部分。有研究表明， IL-19在心肌炎中发挥抗炎效应。在炎症性肠道中内源性IL-19呈现出保护性作用，有学者建立了IL-19小鼠DSS大肠炎模型，并且表明这些小鼠相比于那些免疫完整的小鼠更易感大肠炎。活动性克恩病中，IL-19表达缺陷及对其反应的缺少有利于疾病炎症的发展。此外，腺病毒介导的IL-19融合基因转入大鼠后能有效减少损伤，并且激活MAPK；进一步发现一些细胞炎性因子能诱导IL-19的产生。而IL-19能通过减低编码炎症性蛋白的mRNA种属的稳定性从而减少血管平滑肌的炎症反应。一些体外实验的研究表明IL-19能诱导CD4+T细胞产生Th2细胞因子，在周围血单核细胞IL-19增加IL-10的产生，长时间暴露于IL-19的T细胞会下调IFN-γ，上调IL-4和IL-13。但是也存在一些争议的研究发现在单核细胞IL-19上调促炎因子IL-6和TNF-α的产生，在银屑病患者皮肤发现IL-19及其2条受体链IL-20R1/IL-20R2的表达，进一步研究发现减少IL-19水平的治疗是有效的。
IL-19 DNA序列(SEQ ID NO:219)
Figure PCTCN2022134672-appb-000018
IL-19氨基酸序列(SEQ ID NO:220)
Figure PCTCN2022134672-appb-000019
白介素-23(IL-23)
如本文所用，“白介素-23”或“IL-23”是一种由IL-12p40亚基和IL-23p19亚基组成的异二聚体细胞因子，其中p40亚基是IL-23与IL-12共用的。IL-23的功能性受体已被鉴定，由IL-12Rβ1和IL-23R组成。IL-23在1型极化T细胞的免疫应答中起作用。虽然IL-12可强有力激活幼稚T细胞，但是最初的报道认为IL-23可优先作用于记忆T细胞，促进其分泌IFN-γ和增殖，这表明IL-23在控制细菌感染上有重要作用。IL-23被进一步描述为是一种控制周围组织炎症的关键细胞因子。p19的过表达与多个器官以及包含皮肤在内的上皮组织的炎症有关。另外，IL-23还参与中枢神经系统和多种自身免疫性疾病的炎症。
IL-23(IL-12p40亚基)DNA序列(SEQ ID NO:221)
Figure PCTCN2022134672-appb-000020
Figure PCTCN2022134672-appb-000021
IL-23(IL-12p40亚基)氨基酸序列(SEQ ID NO:222)
Figure PCTCN2022134672-appb-000022
IL-23(IL-23p19亚基)DNA序列(SEQ ID NO:223)
Figure PCTCN2022134672-appb-000023
IL-23(IL-23p19亚基)氨基酸序列(SEQ ID NO:224)
Figure PCTCN2022134672-appb-000024
白介素-35(IL-35)
如本文所用，“白介素-35”或“IL-35”是由EB病毒诱导基因3(Epstein-Barr virus-induced gene 3,EBI3)蛋白和IL-12p35(IL-12A)亚基组成的异源二聚体，与IL-12、IL-23和IL-27共同组成了IL-12细胞因子家族，在T细胞增殖活化和细胞因子产生方面发挥重要的调节作用。研究证实，IL-35主要由调节性T细胞(regulatory T cells,Treg)分泌，是Treg发挥免疫负调控的主要细胞因子之一，在多种疾病(如实验性结肠炎、胶原诱导性关节炎、自身免疫性脱髓鞘炎、慢性肝炎、糖尿病、肿瘤等)中参与炎症的免疫调节，与疾 病的发生、发展密切相关。
IL-35(EBI3蛋白)DNA序列(SEQ ID NO:225)
Figure PCTCN2022134672-appb-000025
IL-35(EBI3蛋白)氨基酸序列(SEQ ID NO:226)
Figure PCTCN2022134672-appb-000026
IL-35(IL-12p35亚基)DNA序列(SEQ ID NO:227)
Figure PCTCN2022134672-appb-000027
IL-35(IL-12p35亚基)氨基酸序列(SEQ ID NO:228)
Figure PCTCN2022134672-appb-000028
白介素-37(IL-37)
如本文所用，“白介素-37”或“IL-37”共有五种不同亚型(IL-37a-e)，目前研究表明只有IL-37b具有生物学功能并在多种疾病中具有抗炎作用。IL-37既可以分泌到细胞外作为细胞因子与受体结合调节细胞活性，也可以进入细胞核作为转录调节因子。IL-37与L-18具有较高的同源性，可以结合IL-18结合蛋白(IL-18BP)对IL-18信号通路有一定的抑 制作用。
IL-37 DNA序列(SEQ ID NO:229)
Figure PCTCN2022134672-appb-000029
IL-37氨基酸序列(SEQ ID NO:230)
Figure PCTCN2022134672-appb-000030
TGF-β
如本文所用，“转化生长因子β”或“TGF-β”是一种具有多功能的细胞激素，在人体内，众多类型的细胞皆具有产生并分泌此种细胞激素的能力。TGF-β参与许多疾病的机制，扮演抑制或促进疾病的双重角色，包括伤口愈合、组织纤维化、粥状动脉硬化、癌症的发生与转移、自体免疫疾病、糖尿病并发症，以及阿兹海默症造成的神经损伤等，皆和TGF-β息息相关。人体存在两种免疫反应，第一型帮助型T细胞(TH1)媒介的正常免疫防御反应：负责感染性微生物的免疫防御机转。若TH1细胞功能过度旺盛，引起发炎反应的细胞激素分泌，例如介白素-2(Interleukin-2,IL-2)和干扰素γ(Interferon-γ,IFN-γ)等，提升细胞性免疫反应，攻击人体的特定组织或特殊细胞，造成该人体某些组织或器官的长期的伤害，尤其是自体免疫疾病及器官移植排斥。第二型帮助型T细胞(TH2)媒介的过敏免疫防御反应：负责寄生虫、叮咬虫类、过敏原与刺激物对障壁层器官的免疫防御机转。若TH2细胞功能过度旺盛，导致TH2细胞激素分泌量过高，促使B细胞产生大量过敏抗体IgE，IgE会诱发肥大细胞或嗜碱性白血球细胞释出发炎物质，如组织胺、介白素、细胞激素、血小板活化因子等，作用在细胞或血管上，造成血管舒张及平滑肌收缩，导致过敏性气喘、过敏性鼻炎、异位性皮肤炎等过敏症状产生。此两种免疫力在人体内是以天秤式的平衡来呈现，TH1及TH2两者互相平衡，且共同受到调节型细胞(Treg)与免疫调节因子(TGF-β)的调控，让身体免疫防御系统TH1及TH2维持平衡，即可达到预防自体免疫疾病及过敏相关疾病的作用。常见的遗传性过敏疾病，如：异位性皮肤炎、气喘、过敏性鼻炎等，体内均缺少TGF-β免疫调节因子。有研究表明，长期补充TGF-β可增加体内浓度，改善体质、减少发炎、减缓过敏指数、修复组织、延长母乳对婴幼童的保护力，提升身体对食物 耐受性、维持消化道机能。
TGF-βDNA序列(SEQ ID NO:231)
Figure PCTCN2022134672-appb-000031
TGF-β氨基酸序列(SEQ ID NO:232)
Figure PCTCN2022134672-appb-000032
C.表达盒
如本文所用，术语“表达盒”是指能够引导特定核苷酸序列在适当的微生物中表达的DNA序列，包含可操作地连接于所关注的核苷酸序列的启动子，所述所关注的核苷酸序列可操作地连接于终止信号。其通常还包含核苷酸序列恰当翻译所需的序列。编码区通常编码所关注的蛋白质，但也可以在反义方向的意义上编码所关注的功能RNA，例如反义RNA或非翻译RNA。包含所关注的核苷酸序列的表达盒可以是嵌合的，这意味着其组件中的至少一个相对于其其它组件中的至少一个是异源的。
如本文所用，术语“重组”是指在体外合成或以其它方式操纵的多核苷酸(例如，“重组表达盒”)，指使用重组多核苷酸或重组表达盒在细胞或其它生物系统中产生产物的方法，或指由重组多核苷酸编码的多肽。“重组表达盒”涵盖接合到表达盒或载体中的来自不同来源的核酸分子，以表达例如融合蛋白；或通过诱导型或组成型表达多肽产生的蛋白质。重组表达盒涵盖可操作地连接于一个或多个调节元件的重组多核苷酸。
在一些实施方式中，在本申请所述的经遗传修饰的微生物中，所述至少两种外源基因包含在(至少一个)外源性表达盒中。
在一些实施方式中，本申请所述的经遗传修饰的微生物中包含至少一种、至少两种或者全部三种选自以下的外源性表达盒：第一外源性表达盒(在本公开中也可以被称为“Amuc_1100表达盒”，“外源性表达盒A”或者“表达盒A”，上述名称在本公开中可互换使用，具有相同含义)，包含编码Amuc_1100多肽的核苷酸序列；第二外源性表达盒(在本公开中也可以被称为“IL-10表达盒”，“外源性表达盒B”或者“表达盒B”，上述名称在本公开中可互换使用，具有相同含义)，包含编码IL-10的核苷酸序列；和第三外源性表达盒(在本公开中也可以被称为“IL-22表达盒”，“外源性表达盒C”或者“表达盒C”，上述名称在本公开中可互换使用，具有相同含义)，其包含编码IL-22的核苷酸序列。
在一些实施方式中，所述至少一个外源性表达盒包含一个或多个调控元件(或者“表达调控元件”，二者在本文中具有相同含义)，其可操作地连接于所述外源基因。
在一些实施方式中，所述调节元件包含一个或多个选自由以下组成的群组的元件：启动子、核糖体结合位点(RBS)、顺反子、终止子和其任何组合。
i.启动子
如本文所用，术语“启动子”是指含有RNA聚合酶的结合位点并且起始DNA转录的通常在编码区上游的非翻译DNA序列。启动子区还可包含充当基因表达的调控因子的其它元件。在一些实施方式中，启动子适合于起始经遗传修饰的微生物中的编码Amuc_1100多肽、IL-10和/或IL-22的多核苷酸。在一些实施方式中，启动子为组成型启动子或诱导型启动子。
术语“组成型启动子”是指能够促进在其控制下和/或与其可操作地连接的编码序列或基因的连续转录的启动子。组成型启动子和变异体是所属领域中众所周知的，并且包含但不限于BBa_J23119、BBa_J23101、BBa_J23102、BBa_J23103、BBa_J23109、BBa_J23110、BBa_J23114、BBa_J23117、USP45_启动子、Amuc_1102_启动子、OmpA_启动子、BBa_J23100、BBa_J23104、BBa_J23105、BBa_I14018、BBa_J45992、BBa_J23118、BBa_J23116、BBa_J23115、BBa_J23113、BBa_J23112、BBa_J23111、BBa_J23108、BBa_J23107、BBa_J23106、BBa_I14033、BBa_K256002、BBa_K1330002、BBa_J44002、BBa_J23150、BBa_I14034、Oxb19、oxb20、BBa_K088007、Ptet、Ptrc、PlacUV5、BBa_K292001、BBa_K292000、BBa_K137031和BBa_K137029。示范性组成型启动子的核苷酸序列包含选自由以下组成的群组的核苷酸序列：如表3中所示的SEQ ID NO:16-56，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。在一些实施方式中，组成型启动子包含SEQ ID NO:16-56中任一所示的核苷酸序列。在一些实施方式中，所述启动子在体外，例如在培养、扩增和/或制造条件下具有活性。在一些实施方式中，所述启动子在体内，例如在体内环境，例如肠道和/或炎症微环境中存在的条件下具有活性。示例性的组成型启动子的核苷酸序列如表3所示。
表3 组成型启动子
Figure PCTCN2022134672-appb-000033
Figure PCTCN2022134672-appb-000034
如本文所用，术语“诱导型启动子”是指可以通过外部刺激，如化学、光、激素、应激或病原体，在一种或多种细胞类型中启用的受调控的启动子。诱导型启动子和变异体为所属领域中众所周知的，并且包含但不限于PLteto1、galP1、PLlacO1、Pfnrs、Psal、Pvan。在一些实施方式中，示范性诱导型启动子的核苷酸序列包含选自由以下组成的群组的核苷酸序列：如表4中所示的SEQ ID NO:57-61，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。
表4 诱导型启动子
Figure PCTCN2022134672-appb-000035
在一些实施方式中，启动子为内源启动子或外源启动子。外源启动子是指与编码区可操作组合的启动子，其中所述启动子不是生物体基因组中与所述编码区天然相关的启动子。基因组中与编码区天然相关或相连的启动子被称为所述编码区的内源启动子。在一些实施方式中，启动子可操作地连接于目的基因的转录起始位点的上游或下游。
在一些实施方式中，启动子选自：BBa_J23101、BBa_J23108、BBa_J23110、PfnrS、Psal、Pvan、BBa_J23119、BBa_J23102，或BBa_J23114。
ii.核糖体结合位点
如本文所用，术语“核糖体结合位点”(“RBS”)是指起始蛋白质翻译时核糖体结合于的mRNA上的序列。RBS为大约35个核苷酸长，并且含有三个离散结构域：(1)Shine-Dalgarno(SD)序列，(2)间隔区，和(3)编码序列(CDS)的前五到六个密码子。RBS和变异体在所属领域中众所周知，并且包含但不限于USP45、合成型(Synthesized)、Amuc_1102、OmpA。示范性RBS的核苷酸序列包含选自由以下组成的群组的核苷酸序列：如表5中所示的SEQ ID NO:62-65，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。在某些实施方式中，核糖体结合位点可操作性地连接于目的基因编码区域的上游8-13核苷酸处。
表5 核糖体结合位点(RBS)
RBS 序列 SEQ ID NO:
USP45 ggaggaaaaattaaaaaagaac 62
合成型 aaagaggagaaa 63
Amuc_1102 agggaa 64
OmpA taacgagg 65
iii.顺反子
如本文所用，术语“顺反子”是指经转录且编码多肽的核酸序列的区段。顺反子和变异体在所属领域中众所周知，并且包含但不限于GFP、BCD2、荧光素酶、MBP。示范性顺反子的核苷酸序列包含选自由以下组成的群组的核苷酸序列：如表6中所示的SEQ ID NO:66-75，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。在某些实施方式中，顺反子可操作性地连接于目的基因编码区域的N端。
表6 顺反子
Figure PCTCN2022134672-appb-000036
iv.终止子
如本文所用，术语“终止子”是指提供RNA聚合酶转录终止信号的核苷酸序列，其防止核苷酸后续并入到所得多核苷酸链，并且由此中断聚合酶介导的延伸。在一些实施方式中，本公开中使用的终止子为T7终止子。在一些实施方式中，终止子包含选自由以下组成的群组的核苷酸序列：如表7中所示的SEQ ID NO:76-79，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。在某些实施方式中，终止子可操作性地连接于编码基因的3'末端。在一些实施方式中，本公开中使用的终止子为rrnB_T1_T7Te_终止子，所述rrnB_T1_T7Te_终止子包括rrnB_T1_终止子和T7Te_终止子串联所得的序列，具体序列如下：caaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcacc ttcgggtgggcctttctgcg(SEQ ID NO:242)。
表7 转录终止子
Figure PCTCN2022134672-appb-000037
D.微生物中的分泌系统和/或输出路径
在某些实施方式中，在本文所提供的外源性表达盒中，编码Amuc_1100多肽、IL-10和/或IL-22的多核苷酸序列可操作地连接于信号肽。
如本文所用，术语“信号肽”或“信号序列”是指可以用于将异源多肽分泌到经培养细菌的周质或培养基中或将Amuc_1100多肽、IL-10和/或IL22分泌到周质中的肽。异源多肽的信号可以与细菌同源，或其可以是异源的，包含对于细菌中所产生的多肽原生的信号。对于Amuc_1100、IL-10和/或IL22，信号序列通常为细菌细胞内源性的，但其不必是内源性的，只要其对于其目的有效即可。分泌肽的非限制性实例包含USP45,OppA(ECOLIN_07295)、OmpA、OmpF、cvaC、TorA、fdnG、dmsA、PelB、HlyA、粘附素(ECOLIN_19880)、DsbA(ECOLIN_21525)、Gltl(ECOLIN_03430)、GspD(ECOLIN_16495)、HdeB(ECOLIN_19410)、MalE(ECOLIN_22540)、PhoA(ECOLIN_02255)、PpiA(ECOLIN_18620)、TolB、tort、mglB和lamB。在一些实施方式中，所述的分泌肽是USP45。
在某些实施方式中，本文提供的用于Amuc_1100、IL-10和/或IL22分泌的信号肽包含USP45、OmpA、DsbA、pelB、Cel-CD、sat或Amuc_1100、IL-10和/或IL22的内源信号肽(或者叫做“自身信号肽”，在本文中，“内源信号肽”与“自身信号肽”具有相同含义，都表示野生型多肽序列中原始具有或者天然存在的信号肽序列)。示范性信号肽的核苷酸序列包含选自由以下组成的群组的核苷酸序列：如表9中所示的SEQ ID NO:123-135，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。在一些实施方式中，信号肽包含选自由以下组成的群组的氨基酸序列：如表8中所示的SEQ ID NO:80-122，和其具有至少80％(例如至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)序列一致性的同源序列。在一些实施方式中，信号肽可操作地连接于表达目标蛋白的N端，在一些实施方式中，目标蛋白(例如Amuc_1100、IL-10和/或IL-22)的自身信号肽序列被去除，然后在N端与 选定的信号肽(如本文中所述的第一信号肽，第二信号肽或者第三信号肽，例如USP45信号肽)连接(即自身信号肽被替换为选定的信号肽)，用于蛋白的分泌。
在一些实施方式中，表达盒包含编码可操作地连接于信号肽和自转运蛋白结构域的Amuc_1100、IL-10和/或IL-22的核苷酸序列，其中信号肽和自转运蛋白结构域可操作地连接于Amuc_1100、IL-10和/或IL-22的相对端，例如信号肽可操作地连接于Amuc_1100、IL-10和/或IL-22的N端，并且自转运蛋白结构域可操作地连接于Amuc_1100、IL-10和/或IL-22的C端。所述构建体可用于V型自分泌介导的分泌，其中在通过原生分泌系统(如Sec系统)将前体蛋白质从细胞质易位到周质隔室中时，去除N端信号肽，并且另外，一旦自分泌物易位跨越外膜，可以通过自催化或蛋白酶催化的例如OmpT裂解去除C端自转运蛋白结构域，从而将成熟蛋白质(例如Amuc_1100多肽、IL-10和/或IL-22)释放到细胞外环境中。
表8 信号肽氨基酸序列
Figure PCTCN2022134672-appb-000038
Figure PCTCN2022134672-appb-000039
表9 部分信号肽DNA序列
Figure PCTCN2022134672-appb-000040
Figure PCTCN2022134672-appb-000041
在一些实施例中，信号肽可以通过存在于经遗传修饰的微生物(例如经遗传修饰的细菌)中的输出路径和/或分泌系统加工。信号肽通常在输出的前体蛋白质的N端，并且可以将前体蛋白质引导到细菌细胞质膜中的输出路径。分泌系统能够在从经工程改造的微生物(如细菌)分泌成熟蛋白质之前从前体蛋白质去除信号肽。
如本文所用，术语“分泌系统”在本文中与“输出系统”和“输出路径”可互换地使用，是指能够从微生物(例如细菌细胞质)分泌或输出所表达的多肽产物(例如Amuc_1100多肽)的原生或非原生分泌机制。如大肠杆菌的革兰氏阴性细菌中从外向内，存在外膜(OM)、肽聚糖细胞壁、周质、内膜(IM)和细胞质隔室。OM为极有效且选择性的渗透性屏障。OM为由内叶处的磷脂和外叶处的糖脂以及脂蛋白和β桶形蛋白组成的脂质双层。OM通过称为Lpp的脂蛋白固定到底层肽聚糖。周质密集地填充有蛋白质，且其比细胞质更粘稠。IM为磷脂双层并且主要位置容纳在能量产生、脂质生物合成、蛋白质分泌和转运中起作用的膜蛋白。
在细菌中，存在两种常见蛋白质输出路径，包含普遍存在的一般分泌(或Sec)路径和双精氨酸易位(或Tat)路径。通常，Sec路径处理未折叠状态的较高分子量前体蛋白质，其中信号肽将底物蛋白质靶向到膜结合的Sec移位酶。前体目标蛋白质递送到移位酶并且通过SecA、SecD和SecF穿过SecYEG孔。伴侣蛋白SecB、GroEL-GroES、DnaK-DnaJ-GrpE辅助目标蛋白运输。Tat路径通常运输完全折叠或甚至寡聚状态的蛋白质，且革兰氏阴性和阳性细菌中都由组分TatA、TatB和TatC组成。在膜易位之后，通过信号肽酶去除信号肽并且分泌成熟蛋白质。
对于革兰氏阴性细菌，其具有专用单步分泌系统，并且分泌系统的非限制性实例包含Sat分泌系统、I型分泌系统(T1SS)、II型分泌系统(T2SS)、III型分泌系统(T3SS)、IV型分泌系统(T4SS)、V型分泌系统(T5SS)、VI型分泌系统(T6SS)和多药物流出泵的耐药性-结瘤-分裂(resistance-nodulation-division，RND)家族、各种单膜分泌系统。
在一些实施例中，分泌系统对于经遗传修饰的微生物是原生或非原生的系统。对于微生物“原生”意思是分泌系统通常存在于微生物中，而对于微生物“非原生”意思是分泌系统不通常存在于微生物中，例如额外分泌系统，如来自细菌或病毒的不同物种、菌株或亚菌株的分泌系统，或与来自同一亚型的微生物的未经修饰的分泌系统相比经修饰和/或突 变的分泌系统。
在一些实施方式中，本公开的经遗传修饰的微生物进一步包含微生物基因组中的一种或多种基因改造。在一些实施方式中，其中所述一种或多种基因组改造包括对分泌系统的工程改造和/或优化以使得至少一种外膜蛋白编码基因被删除、失活或抑制。
在一些实施方式中，分泌系统经工程改造和/或优化以使得经遗传修饰的微生物中的至少一种外膜蛋白编码基因被删除、失活或抑制。在一些实施例中，所述外膜蛋白选自由以下组成的群组：OmpC、OmpA、OmpF、OmpT、pldA、pagP、tolA、Pal、To1B、degS、mrcA和lpp。
为了制造能够分泌所需治疗性蛋白质或肽的革兰氏阴性细菌(例如EcN)，需要遗传修饰细菌宿主并且使其具有“漏”或去稳定化的外膜，但不影响宿主细菌化学物理活性。基因，例如lpp、ompC、ompA、ompF、ompT、pldA、pagP、tolA、to1B、pal、degS、degP和nlpI可以被删除或诱发突变以产生“漏”的外膜。Lpp为细菌细胞中最丰富的多肽，且充当细菌细胞壁与肽聚糖的主要“订书针”。lpp的缺失对细菌生长具有极小影响，但增加Amuc_1100的分泌，例如增加至少一倍。诱导型启动子可以用于替换所选择的一种或多种基因的内源启动子以使对细胞存活率的负面影响最小化。
基因或编码区的“删除/缺失”或“失活”或“抑制”意指使由所述基因或编码区编码的酶或蛋白质不产生，或以非活性形式产生于微生物中，或以低于在相同或类似生长条件下在微生物的野生型形式中发现的含量在微生物中产生。这可以通过例如以下方法中的一种或多种实现：(1)同源重组，(2)基于RNA干扰的技术，(3)ZFN和TALEN，(4)CRISPR/Cas系统。
在一些实施方式中，分泌系统经工程改造以使得至少一种伴侣蛋白编码基因被扩增、过表达或活化。
伴侣蛋白涉及许多重要的生物过程，如寡聚蛋白复合物的蛋白质折叠和聚集，使蛋白质前体维持在未折叠状态以促进蛋白质跨膜运输，并使变性的蛋白质能够解聚和修复。其主要是辅助其它肽维持正常构象，以形成正确的寡聚结构，从而发挥正常的生理功能。各种伴侣蛋白是所属领域中众所周知的。在一些实施方式中，伴侣蛋白选自由以下组成的群组：dsbA、dsbC、dnaK、dnaJ、grpE、groES、groEL、tig、fkpA、surA、skp、PpiD和DegP。在一些实施例中，伴侣蛋白选自由以下组成的群组：来自70kDa热休克蛋白(Hsp70)的胞质SSA亚家族的Ssa1p、Ssa2p、Ssa3p和Ssa4p，BiP、Kar2、Lhs1、Sil1、Sec63、蛋白二硫键异构酶Pdi1p，在一些实施例中，伴侣蛋白由dsbA、dsbC、dnaK、dnaJ、grpE、groES、groEL、tig、fkpA和surA组成。
基因或编码区的“过表达(overexpressed/overexpression)”意指使由所述基因或编码区 编码的酶或蛋白质以高于在相同或类似生长条件下在微生物的野生型形式中发现的含量的含量在微生物中产生。这可以通过例如以下方法中的一种或多种实现：(1)设置更强的启动子，(2)设置更强的核糖体结合位点，如5'-AGGAGG的DNA序列，位于翻译起始密码子上游约四到十个碱基处，(3)设置终止子或更强的终止子，(4)改进在编码区中的一个或多个位点处的密码子的选择，(5)提高mRNA稳定性，和(6)通过在染色体中引入多个拷贝或将盒置于多拷贝质粒上来增加基因的拷贝数。由过表达的基因产生的酶或蛋白质称为“过产生”的。“过表达”的基因或“过产生”的蛋白质可以对于微生物是原生的，或其可以通过遗传修饰方法从不同生物体移植到微生物中，在后一种情况下，酶或蛋白质和编码酶或蛋白质的基因或编码区称为“外来”或“异源”的。外来或异源基因和蛋白质为过表达和过产生的，因为其不存在于未工程改造的微生物中。
对于酵母，分泌系统主要包含初生蛋白质易位、内质网(ER)中的蛋白质折叠、糖基化、蛋白质分选和运输。翻译期间信号肽一从核糖体出现，新合成的膜或分泌蛋白质就由信号识别颗粒(SRP)识别，随后靶向到穿过Sec61或Ssh1易位子孔的翻译后分泌。已实施各种方法以改进酵母中的异源蛋白质产生：(1)工程改造信号肽并且提高启动子强度和质粒拷贝数；(2)工程改造宿主菌株的翻译后路径，例如过表达ER折叠伴侣蛋白和囊泡运输组件，和减少细胞间和细胞外蛋白水解。异源蛋白质的分泌的示范性酵母分泌信号肽包含酿酒酵母α-交配因子(α-Mating Factor，α-MF)肽原前体(pre pro-peptide)、来自马克斯克鲁维酵母(Kluyveromyces marxianus)的菊粉酶的信号肽、来自普通菜豆凝集素(Phaseolus vulgaris agglutinin)的PHAE的信号肽、病毒毒素原前体(prepro toxin)的信号肽、米根霉(Rhizopus oryzae)淀粉酶或来自真菌里氏木霉(Trichoderma reesei)的疏水蛋白信号序列的信号肽。
E.基因组整合位点
在一些实施方式中，外源性表达盒整合于经遗传修饰的微生物的基因组中。
在一些实施方式中，所述外源性表达盒包含在质粒中，且所述质粒被引入所述微生物中并且适合在所述微生物中表达。
在一些实施方式中，本申请所述的经遗传修饰的微生物能够稳定生长、稳定表达非天然遗传物质，例如编码Amuc_1100、IL-10和/或IL-22的基因，或表达这些多肽/蛋白质的外源表达盒。在一些实施方式中，所述非天然遗传物质被导入到微生物的基因组中或在染色体外质粒上进行复制，从而使得该非天然遗传物质被保留、转录和表达。
在一些实施方式中，稳定的微生物(如，细菌)可以是包含表达且分泌Amuc_1100、IL-10和/或IL-22的经遗传修饰的微生物，其中携带编码Amuc_1100、IL-10和/或IL-22的外源表达盒的质粒或染色体被稳定地维持在该经遗传修饰的微生物中，使得所述外源表达 盒可以在所述经遗传修饰的微生物中被表达，且所述经遗传修饰的微生物能够在体外和/或在体内存活和/或生长。稳定的微生物能够在体外(例如，在培养基中)或在体内(例如，在消化道中)存活并生长。
在一些实施方式中，拷贝数影响非天然遗传物质的表达的稳定性。在一些实施方式中，拷贝数影响非天然遗传物质的表达的水平。在其中所述经遗传修饰的微生物包含一个或更多个基因序列和一个或更多个基因盒的实施方式中，基因序列可以存在于一个或更多个质粒上，并且基因盒可以存在于微生物染色体中，且反之亦然。另外，任何基因、基因盒或调节区域的多个拷贝(例如一个、两个、三个、四个、五个、六个或更多个)可以存在于微生物中，其中基因、基因盒或调节区域的一个或更多个拷贝可以如本文描述被突变或以其他方式改变。在一些实施方式中，将经遗传修饰的微生物工程化为包含相同基因、基因盒或调节区域的多个拷贝以增强拷贝数。在一些实施方式中，将经遗传修饰的微生物工程化为包含进行多种不同功能的基因盒的多种不同组件。在一些实施方式中，将经遗传修饰的微生物工程化为包含不同基因、基因盒或调节区域的一个或更多个拷贝，以产生表达多于一种治疗性分子和/或进行多于一种功能的经遗传修饰的微生物。在一些实施方式中，拷贝数影响非天然遗传物质的表达的稳定性。在一些实施方式中，拷贝数影响非天然遗传物质的表达的水平。
在一些实施方式中，两个或更多个基因序列为相同基因的多个拷贝。在一些实施方式中，两个或更多个基因序列为编码不同基因的序列。在一些实施方式中，两个或更多个基因序列为编码一个或更多个不同基因的多个拷贝的序列。在一些实施方式中，经遗传修饰的微生物包含用于治疗或预防炎症性疾病或自身免疫疾病的分子的一个或更多个基因盒。例如，经遗传修饰的微生物可以包含用于治疗或预防炎症性疾病或自身免疫疾病的分子的两个或更多个基因盒。在一些实施方式中，两个或更多个基因盒为相同基因盒的多个拷贝。在一些实施方式中，两个或更多个基因盒为用于产生相同或不同治疗或预防炎症性疾病或自身免疫疾病的分子的不同基因盒。在一些实施方式中，两个或更多个基因盒为用于分子的多个拷贝的基因盒。
另外，任何调节区域、启动子、基因、和/或基因盒的多个拷贝可以存在于经遗传修饰的微生物中，其中调节区域、启动子、基因、和/或基因盒的一个或更多个拷贝可以如本文描述被突变或以其他方式改变。在一些实施方式中，将经遗传修饰的微生物工程化为包含相同调节区域、启动子、基因、和/或基因盒的多个拷贝以增强拷贝数或者以包含进行多种不同功能的基因盒的多种不同组件或者以包含不同调节区域、启动子、基因、和/或基因盒的一个或更多个拷贝以产生表达多于一个治疗性分子和/或进行多于一种功能的经遗传修饰的微生物。
在一些实施方式中，外源性表达盒通过CRISPR-Cas基因组编辑系统整合于经遗传修饰的微生物的基因组中。本文提供的任何适合的微生物都可以经工程改造，以使得外源性表达盒整合到基因组中。
在一些实施方式中，经遗传修饰的微生物为大肠杆菌的菌株Nissle 1917(EcN)，并且外源性表达盒整合到EcN基因组中的位点中。在一些实施方式中，EcN基因组中的适合整合位点为整合位点agaI/rsmI、lacZ、kefB、malP/T、yicS/nepl、rhtB/C、maeB、malE/K、yieN、lldD、maeA、pflB或araB/C。用于本发明中的Amuc_1100、IL-10和/或IL-22整合于EcN基因组位点中。不希望受任何理论束缚，但认为本公开的EcN的基因组位点对于插入Amuc_1100、IL-10和/或IL-22的表达盒，通过以下特征中的至少一个是有利的：(1)所述位点的工程改造所影响的一种或多种细菌基因不是EcN生长所必需的，并且不改变宿主细菌的生物化学和生理活性，(2)所述位点可以容易地被编辑，和(3)所述位点中的Amuc_1100基因盒、IL-10和/或IL-22基因盒可以被转录。
F.营养缺陷体
在一些实施方式中，本公开的微生物进一步包含营养缺陷相关基因中的至少一个失活或缺失。
为了产生环境友好型细菌，可以通过基因编辑使细菌细胞生存所必需的一些必需基因被删除或失活，使经工程改造的细菌成为营养缺陷体。如本文所用，术语“营养缺陷体”是指微生物(例如微生物体菌株)的生长需要特定代谢物的外部来源，所述代谢物由于后天基因缺陷而不能合成。
如本文所用，术语“营养缺陷相关基因”是指微生物(例如微生物体，如细菌)生存所需的基因。营养缺陷相关基因可以是微生物体产生生存或生长所必需的营养素所必需的，或者可以是检测环境中调节转录因子活性的信号所必需的，其中不存在所述信号将引起细胞死亡。
在一些实施方式中，营养缺陷型修饰意图使微生物体在缺乏外源添加的生存或生长所必需的营养素时死亡，因为所述微生物体缺乏产生必需营养素所必需的一种或多种基因。在一些实施方式中，本文所述的经遗传修饰的细菌中的任一种还包含细胞生存和/或生长所需基因的缺失或突变。
细菌中的各种营养缺陷相关基因是所属领域中众所周知的。示范性营养缺陷相关基因包含但不限于：thyA、cysE、glnA、ilvD、leuB、lysA、serA、metA、glyA、hisB、ilvA、pheA、proA、thrC、trpC、tyrA、uraA、dapF、flhD、metB、metC、proAB、yhbV、yagG、hemB、secD、secF、ribD、ribE、thiL、dxs、ispA、dnaX、adk、hemH、IpxH、cysS、fold、rplT、infC、thrS、nadE、gapA、yeaZ、aspS、argS、pgsA、yeflA、metG、folE、yejM、gyrA、 nrdA、nrdB、folC、accD、fabB、gltX、ligA、zipA、dapE、dapA、der、hisS、ispG、suhB、tadA、acpS、era、rnc、fisB、eno、pyrG、chpR、Igt、fbaA、pgk、yqgD、metK、yqgF、plsC、ygiT、pare、ribB、cca、ygjD、tdcF、yraL、yihA、ftsN、murl、murB、birA、secE、nusG、rplJ、rplL、rpoB、rpoC、ubiA、plsB、lexA、dnaB、ssb、alsK、groS、psd、orn、yjeE、rpsR、chpS、ppa、valS、yjgP、yjgQ、dnaC、ribF、IspA、ispH、dapB、folA、imp、yabQ、flsL、flsl、murE、murF、mraY、murD、ftsW、murG、murC、ftsQ、ftsA、ftsZ、IpxC、secM、secA、can、folK、hemL、yadR、dapD、map、rpsB、infB、nusA、ftsH、obgE、rpmA、rplU、ispB、murA、yrbB、yrbK、yhbN、rpsl、rplM、degS、mreD、mreC、mreB、accB、accC、yrdC、def、fint、rplQ、rpoA、rpsD、rpsK、rpsM、entD、mrdB、mrdA、nadD、hlepB、rpoE、pssA、yfiO、rplS、trmD、rpsP、ffh、grpE、yfjB、csrA、ispF、ispD、rplW、rplD、rplC、rpsJ、fusA、rpsG、rpsL、trpS、yrfF、asd、rpoH、ftsX、ftsE、ftsY、frr、dxr、ispU、rfaK、kdtA、coaD、rpmB、djp、dut、gmk、spot、gyrB、dnaN、dnaA、rpmH、rnpA、yidC、tnaB、glmS、glmU、wzyE、hemD、hemC、yigP、ubiB、ubiD、hemG、secY、rplO、rpmD、rpsE、rplR、rplF、rpsH、rpsN、rplE、rplX、rplN、rpsQ、rpmC、rplP、rpsC、rplV、rpsS、rplB、cdsA、yaeL、yaeT、lpxD、fabZ、IpxA、IpxB、dnaE、accA、tilS、proS、yafF、tsf、pyrH、olA、rlpB、leuS、Int、glnS、fldA、cydA、infA、cydC、ftsK、lolA、serS、rpsA、msbA、IpxK、kdsB、mukF、mukE、mukB、asnS、fabA、mviN、rne、yceQ、fabD、fabG、acpP、tmk、holB、lolC、lolD、lolE、purB、ymflC、minE、mind、pth、rsA、ispE、lolB、hemA、prfA、prmC、kdsA、topA、ribA、fabi、racR、dicA、yd B、tyrS、ribC、ydiL、pheT、pheS、yhhQ、bcsB、glyQ、yibJ和gpsA。
在一种修饰中，必需基因thyA被删除或被另一基因替代，使得经遗传修饰的细菌依赖于外源胸腺嘧啶而生长或生存。向生长培养基添加胸腺嘧啶或天然具有高胸腺嘧啶含量的人体肠道可以支持thyA营养缺陷型细菌的生长和生存。这种修饰是为了确保经遗传修饰的细菌不能在肠道外或在缺乏营养缺陷基因产物的环境中生长和生存。
在一些实施方式中，微生物是一种或多种选自由以下组成的群组的物质的营养缺陷体：尿嘧啶、胸腺嘧啶、二氨基庚二酸、亮氨酸、组氨酸、色氨酸、赖氨酸、甲硫氨酸、腺嘌呤和非天然存在的氨基酸。在一些实施方式中，非天然存在的氨基酸选自由以下组成的群组：l-4,4′-联苯丙氨酸、对乙酰基-l-苯丙氨酸、对碘-l-苯丙氨酸和对叠氮基-l-苯丙氨酸。
在一些实施方式中，微生物包含变构调节的转录因子，所述转录因子能够检测调节所述转录因子的活性的环境中的信号，其中不存在所述信号将引起细胞死亡。所述“信号传导分子-转录因子”对可以包含选自由以下组成的群组的任一种或多种：色氨酸-TrpR、IPTG-LacI、苯甲酸酯衍生物-XylS、ATc-TetR、半乳糖-GalR、雌二醇-雌激素受体杂合蛋白、纤 维二糖-CelR和高丝氨酸内酯-luxR。
G.内源质粒的缺失
在一些实施方式中，经遗传修饰的细菌具有一种或多种内源质粒的缺失。
一些底盘细菌宿主包含一种或多种内源质粒，其为其转录消耗相当多的资源。不受任何理论束缚，认为删除这些内源质粒以释放可以更好地用于异源基因表达的资源。
一种或多种内源质粒可以通过所属领域中已知的方法从目标微生物去除。举例来说，EcN包含两种内源质粒pMUT1和pMUT2，其可以通过任何适当的方法从EcN去除。一种示例的方式是通过CRISPR-Cas9介导的DNA双链切割。简而言之，可以在EcN中导入特异性结合pMUT1或pMUT2的向导RNA(gRNA或单链向导RNA，即sgRNA)和表达Cas9蛋白的核酸序列(例如质粒)，从而在EcN中通过Cas9蛋白介导的DNA双链切割破坏pMUT1或pMUT2。作为另一个实施方式，也可以先将EcN中的pMUT2替换为表达特定抗性基因(例如卡那霉素抗性基因)的重组pMUT2质粒，例如通过筛选具有抗性的重组EcN。然后再导入特异性结合该抗性基因的gRNA或sgRNA和表达Cas9蛋白的核酸序列(例如质粒)，从而在EcN中Cas9蛋白介导的DNA双链切割破坏替换后的重组pMUT2质粒。
本申请的发明人惊讶地发现，本公开所述的经遗传修饰的微生物(如，细菌)不仅可以稳定地生长并保持活力，而且可以在其基因组中同时整合至少两种外源基因并仍然稳定高效地表达和分泌这些至少两种外源基因所编码的多肽/蛋白。更重要的是，所述表达分泌的全新外源多肽组合(Amuc_1100/IL-10/IL-22之至少两者组合)的微生物通过肠道施用，在动物模型展现出了对多种炎症性疾病或自身免疫性疾病(如，炎症性肠病)的治疗效果甚至是意料不到的协同作用，因此本公开的工程微生物作为活体药物具有广阔的临床应用前景。
本发明还提供了所述的经遗传修饰的微生物的制备方法，其中所述的制备方法包括步骤：向微生物中引入可外源性表达本发明的Amuc_1100、IL-10和/或IL-22的核苷酸序列，以使得所述核苷酸序列中的外源基因可以在所述微生物中表达，从而获得所述的经遗传修饰的微生物。
在一些实施方式中，本发明的示例性的优化的Amuc_1100表达盒具有以下序列SEQ ID NO:139至SEQ ID NO:143中任一所示的结构。IL-10表达盒具有以下序列SEQ ID NO:144至SEQ ID NO:156和235中任一所示的结构。IL-22表达盒具有以下序列SEQ ID NO:157至SEQ ID NO:163中任一所示的结构。
含有不同调控元件的Amuc_1100序列示例
在一些实施方式中，本申请提供了多种含有不同调控元件(例如不同启动子、不同信 号肽、不同顺反子等)的Amuc_1100具体的表达盒的序列，具体如下所示。在一些实施方式中，本申请还提供了包含任一所述Amuc_1100具体表达盒序列的经遗传修饰的微生物(例如EcN)。本申请提供的Amuc_1100具体表达盒的序列的示例如下所示：
BBa_J23101_USP45_Amuc_1100(Y259A)_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4101、CBT4103和CBT4108的相应表达盒，SEQ ID NO:139)
Figure PCTCN2022134672-appb-000042
BBa_J23110_USP45_Amuc_1100(Y259A)_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4102、CBT4107、CBT4108和CBT4109的相应表达盒，SEQ ID NO:140)
Figure PCTCN2022134672-appb-000043
Figure PCTCN2022134672-appb-000044
BBa_J23101_USP45_Amuc_1100(WT)_rrnB_T1(突变体)_T7Te(包含于本公开实施例菌株CBT4068、CBT4069、CBT4067和CBT4070的相应表达盒，SEQ ID NO:141)
Figure PCTCN2022134672-appb-000045
BBa_J23110_USP45_Amuc_1100(WT)_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4105、CBT4106、CBT4068、CBT4069、CBT4067和CBT4070的相应表达盒，SEQ ID NO:142)
Figure PCTCN2022134672-appb-000046
Figure PCTCN2022134672-appb-000047
PfnrS_USP45_Amuc_1100(WT)_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4080、CBT4096、CBT4088、CBT4098和CBT4111的相应表达盒，SEQ ID NO:143)
Figure PCTCN2022134672-appb-000048
含有调控元件的IL-10序列示例
在一些实施方式中，本申请提供了多种含有不同调控元件(例如不同启动子、不同信号肽、不同顺反子)的IL-10具体的表达盒的序列，具体如下所示。在一些实施方式中，本申请还提供了包含任一所述IL-10具体表达盒序列的经遗传修饰的微生物(例如EcN)。本申请提供的IL-10具体表达盒的序列的示例如下所示：
BBa_J23101_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4003的相应表达盒，SEQ ID NO:144)
Figure PCTCN2022134672-appb-000049
Figure PCTCN2022134672-appb-000050
BBa_J23110_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4005、CBT4071、CBT4072、CBT4073、CBT4074、CBT4112、CBT4020、CBT4075、CBT4076和CBT4077的相应表达盒，SEQ ID NO:145)
Figure PCTCN2022134672-appb-000051
BBa_J23108_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4004的相应表达盒，SEQ ID NO:146)
Figure PCTCN2022134672-appb-000052
BBa_J23101_DsbA_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4007的相应表达盒，SEQ ID NO:147)
Figure PCTCN2022134672-appb-000053
BBa_J23101_OmpA_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4009的相应表达盒，SEQ ID NO:148)
Figure PCTCN2022134672-appb-000054
BBa_J23101_PelB_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4011的相应表达盒，SEQ ID NO:149)
Figure PCTCN2022134672-appb-000055
Figure PCTCN2022134672-appb-000056
BBa_J23101_YebF_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4013的相应表达盒，SEQ ID NO:150)
Figure PCTCN2022134672-appb-000057
BBa_J23110_T7g10_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4026的相应表达盒，SEQ ID NO:151)
Figure PCTCN2022134672-appb-000058
BBa_J23110_BCD2_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4028、CBT4067、CBT4068、CBT4063和CBT4062的相应表达盒，SEQ ID NO:152)
Figure PCTCN2022134672-appb-000059
Figure PCTCN2022134672-appb-000060
BBa_J23110_GFP_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4029的相应表达盒，SEQ ID NO:153)
Figure PCTCN2022134672-appb-000061
BBa_J23110_荧光素酶_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4030的相应表达盒，SEQ ID NO:154)
Figure PCTCN2022134672-appb-000062
Figure PCTCN2022134672-appb-000063
PfnrS_BCD2_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4078的相应表达盒，SEQ ID NO:155)
Figure PCTCN2022134672-appb-000064
PfnrS_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4084、CBT4088、CBT4110和CBT4111的相应表达盒，SEQ ID NO:156)
Figure PCTCN2022134672-appb-000065
Psal_sTRSV-HHRz_USP45_IL-10_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4113的相应表达盒，SEQ ID NO:235)
Figure PCTCN2022134672-appb-000066
Figure PCTCN2022134672-appb-000067
含有不同调控元件的IL-22序列示例
在一些实施方式中，本申请提供了多种含有不同调控元件(例如不同启动子)的IL-22具体的表达盒的序列，具体如下所示。在一些实施方式中，本申请还提供了包含任一所述IL-22具体表达盒序列的经遗传修饰的微生物(例如EcN)。本申请提供的IL-22具体表达盒的序列的示例如下所示：
BBa_J23119_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4038的相应表达盒，SEQ ID NO:157)
Figure PCTCN2022134672-appb-000068
BBa_J23101_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4041的相应表达盒，SEQ ID NO:158)
Figure PCTCN2022134672-appb-000069
Figure PCTCN2022134672-appb-000070
BBa_J23102_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4042的相应表达盒，SEQ ID NO:159)
Figure PCTCN2022134672-appb-000071
BBa_J23108_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4043的相应表达盒，SEQ ID NO:160)
Figure PCTCN2022134672-appb-000072
BBa_J23110_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4039、CBT4016、CBT4110和CBT4111的相应表达盒，SEQ ID NO:161)
Figure PCTCN2022134672-appb-000073
Figure PCTCN2022134672-appb-000074
BBa_J23114_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4040、CBT4066、CBT4069、CBT4063和CBT4067的相应表达盒，SEQ ID NO:162)
Figure PCTCN2022134672-appb-000075
PfnrS_USP45_IL-22_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4095和CBT4098的相应表达盒，SEQ ID NO:163)
Figure PCTCN2022134672-appb-000076
含有不同调控元件的IL-17A序列示例
本申请提供的IL-17A具体表达盒的序列的示例如下所示：
Psal_USP45_IL-17A_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4114的相应表达盒，SEQ ID NO:236)
Figure PCTCN2022134672-appb-000077
含有不同调控元件的IL-19序列示例
本申请提供的IL-19具体表达盒的序列的示例如下所示：
Psal_USP45_IL-19_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4114的相应表达盒，SEQ ID NO:237)
Figure PCTCN2022134672-appb-000078
含有不同调控元件的IL-23序列示例
本申请提供的IL-23具体表达盒的序列的示例如下所示：
Psal_USP45_IL-12p40_linker1_IL-23p19_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4114的相应表达盒，SEQ ID NO:238)
Figure PCTCN2022134672-appb-000079
Figure PCTCN2022134672-appb-000080
含有不同调控元件的IL-35序列示例
本申请提供的IL-35具体表达盒的序列的示例如下所示：
Psal_USP45_EBI3_linker2_IL-12p35_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4114的相应表达盒，SEQ ID NO:239)
Figure PCTCN2022134672-appb-000081
Figure PCTCN2022134672-appb-000082
含有不同调控元件的IL-37序列示例
本申请提供的IL-37具体表达盒的序列的示例如下所示：
Psal_USP45_IL-37_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4114的相应表达盒，SEQ ID NO:240)
Figure PCTCN2022134672-appb-000083
含有不同调控元件的TGF-β序列示例
本申请提供的TGF-β具体表达盒的序列的示例如下所示：
Psal_sTRSV-HHRz_USP45_TGF-β_rrnB_T1_T7Te(包含于本公开实施例菌株CBT4114的相应表达盒，SEQ ID NO:241)
Figure PCTCN2022134672-appb-000084
在一些实施方式中，本申请还提供了多种经遗传修饰的EcN菌株，其具有如下表10所示的基因型。如表10所示，每种菌株以基因型名称来命名，其中EcN代表大肠杆菌E.Coli Nissle 1918，ΔmaeB代表所述菌株中的maeB位点进行插入，下划线部分代表本申请所述的具体表达盒名称，例如 BBa_J23101_USP45_IL-10代表该表达盒中具有BBa_J23101启动子，其可操作地连接于USP45信号肽的编码序列以及IL-10多肽的编码序列。具体表达盒的核酸序列如本申请的序列号SEQ ID NO:139到SEQ ID NO:163所示。一些菌株还缺失了一个或多个外膜蛋白，在其基因型名称体现为，例如ΔLPP，其代表缺失LPP外膜蛋白。一些菌株还过表达了一个或多个分子伴侣，在其基因型名称体现为，例如ΔyicS/nepI::BBa_J23114_dsbA_dsbC_dnaK_dnaJ_grpE，其代表在yicS/nepI位点处插入了表达分子伴侣_dsbA_dsbC_dnaK_dnaJ_grpE的表达盒，该表达盒中具有可操作地连接的BBa_J23114启动子序列。本领域技术人员根据本申请的描述，结合表10中所列出的基因型名称，能够知道每种菌株中具有的特定遗传修饰，并且能够确定上述特定遗传修饰，例如，通过对特定的插入序列或插入位点处的序列设计引物或探针，以进行检测和鉴定。
表10 本文所述的菌株基因型
Figure PCTCN2022134672-appb-000085
Figure PCTCN2022134672-appb-000086
Figure PCTCN2022134672-appb-000087
Figure PCTCN2022134672-appb-000088
III.组合物和试剂盒
在另一方面，本发明提供一种组合物，其包含：(a)包含本发明的经遗传修饰的微生物；和(b)生理学上可接受的载体。
如本文使用的，“组合物”指本发明的遗传工程化的细菌与其他组分诸如生理上合适的载体的制剂。可以互换使用的表达方式“生理上可接受的载体”指不会对生物体引起显著刺激并且不会消除所施用的经遗传修饰的微生物的生物学活性和特性的载体或稀释剂。
细菌可以约10 4到约10 13个菌落形成单位(CFU)范围内的量存在于组合物中。举例来说，微生物的有效量可以是约10 5CFU到约10 13CFU，优选地约10 6CFU到约10 13CFU，优选地约10 7CFU到约10 12CFU，更优选地约10 8CFU到约10 12CFU的量。微生物可以是活细胞或可以是死细胞。微生物的有效性与本文所提供的Amuc_1100、IL-10和/或IL-22的存在相关。
在一些实施方式中，本文所公开的组合物可以经配制用于口服施用，并且可以是营养或滋养组合物，例如食品、食品增补剂、饲料或饲料增补剂，如乳制品，例如发酵乳制品，如酸奶或酸奶饮料。在此情况下，组合物可包含营养学上可接受的载剂，其可为适合的食物基。所属领域的技术人员知道可涵盖活或死微生物体且可呈现为食品增补剂(例如，丸剂、片剂等)或功能性食品(例如饮料、发酵酸奶等)的多种配方。本文所公开的组合物还可以在胶囊、丸剂、液体溶液中配制为药剂，例如配制为囊封的冻干细菌等。在一些实施方式中，所述的组合物是益生菌组合物。
本文所公开的组合物可以被配制成单次施用或多次施用而对给定个体有效。举例来说，单次施用实质上有效降低施用组合物的哺乳动物个体的靶向疾病病状的监测症状。
在一些实施例中，配制组合物以使得单一口服剂量含有至少或至少约1×10 4CFU的细菌实体和/或真菌实体，且单一口服剂量将通常含有约或至少1×10 4、1×10 5、1×10 6、1×10 7、1×10 8、1×10 9、1×10 10、1×10 11、1×10 12、1×10 13的细菌实体和/或真菌实体。如果已知，例如给定菌株的细胞的浓度或所有菌株的合计浓度为例如每克组合物或每施用剂量1×10 4、1×10 5、1×10 6、1×10 7、1×10 8、1×10 9、1×10 10、1×10 11、1×10 12、1×10 13个活细菌实体(例如CFU)。在一些实施方式中，所述组合物中包含1×10 8–1×10 12CFU的本发明所述的经遗传修饰的微生物。
在一些配方中，按质量计，组合物含有至少约0.5％、1％、2％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％或大于90％本公开的微生物。在一些配方中，按质量计，所施用的剂量不超过200、300、400、500、600、700、800、900毫克或1、1.1、1.2、 1.3、1.4、1.5、1.6、1.7、1.8或1.9克的本申请提供的微生物。
用于本文所公开的组合物的生理学上可接受的载剂可包含例如生理学上可接受的液体、凝胶或固体载剂、水性媒剂、非水性媒剂、抗微生物剂、等渗剂、缓冲剂、抗氧化剂、悬浮剂/分散剂、螯合剂(sequestering/chelating agent)、稀释剂、佐剂、赋形剂或无毒辅助物质、所属领域中已知的其它组分或其各种组合。在一些实施方式中，本公开所述的组合物为液态制剂、固态制剂、半固态制剂。在一些实施方式中，所述的液态制剂选自下组：溶液制品或悬浮液制品。
为了进一步说明，水性媒剂可包含如氯化钠注射液、林格注射液、等渗右旋糖注射液、无菌水注射液或右旋糖和乳酸林格注射液；非水性媒剂可包含如植物来源的不挥发性油、棉籽油、玉米油、芝麻油或花生油；抗微生物剂可为抑细菌或抑真菌浓度，和/或可以添加到在多剂量容器中的组合物中，所述容器包含苯酚或甲酚、汞剂、苯甲醇、氯丁醇、甲基和丙基对羟基苯甲酸酯、硫柳汞、苯扎氯铵和苄索氯铵。等渗剂可以包含如氯化钠或右旋糖；缓冲剂可以包含如磷酸盐或柠檬酸盐缓冲剂；抗氧化剂可以包含如硫酸氢钠；悬浮剂和分散剂可以包含如羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮；螯合剂可以包含如乙二胺四乙酸(EDTA)或乙二醇四乙酸(EGTA)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。适合的赋形剂可包含例如水、生理盐水、右旋糖、甘油或乙醇。适合的无毒辅助物质可包含例如润湿剂或乳化剂、pH缓冲剂、稳定剂、溶解性增强剂或如乙酸钠、脱水山梨糖醇单月桂酸酯、三乙醇胺油酸酯或环糊精的试剂。
在一些实施方式中，本文所提供的组合物可为药物组合物。在一些实施方式中，本文所提供的组合物可以是食品增补剂。本文所提供的组合物除本文所提供的经遗传修饰的微生物之外可以包含药学上、营养学上(nutritionally/alimentarily)或生理学上可接受的载剂。优选形式将取决于预期施用模式和(治疗性)应用。载剂可以是适合于将本文提供的经遗传修饰的微生物递送到哺乳动物(例如人类)的胃肠道，优选地哺乳动物的肠粘膜屏障(更优选地结肠粘膜屏障)附近或内的任何相容性生理学上可接受的无毒物质。在一些实施方式中，所述药物组合物的剂型选自下组：粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
组合物可为液体溶液、悬浮液、乳液、丸剂、胶囊、片剂、持续释放制剂或散剂。口服制剂可包含标准载剂，如药物级甘露糖醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯烷酮、糖精钠、纤维素、碳酸镁等。在另一方面，本发明提供了一种试剂盒，其包含本发明所述的组合物。
必要时，所述试剂盒可进一步包含各种常规药物试剂盒组分中的一种或多种，例如具有一种或多种药学上可接受的载剂的容器、额外容器等，如对所属领域的技术人员将显而 易见。试剂盒中还可以包含作为插页或标签的说明书，指示应施用的组分的量、施用指南和/或混合组分的指南。
IV.适应症和治疗方法
另一方面，本发明提供了一种治疗或预防有需要的个体的炎症性疾病或自身免疫疾病的方法，所述方法包括：向所述个体施用有效量的本发明所述的经遗传修饰的微生物，或本发明所述的组合物。在某些实施方式中，所述经遗传修饰的微生物表达至少一种选自下组的外源基因：编码表达Amuc_1100多肽的外源基因、或编码表达IL-10多肽的外源基因、或编码表达IL-22多肽的外源基因、或以上任意组合的外源基因。
在一方面，本发明还提供了一种在正在接受药物治疗的个体中改善药物治疗效果的方法，所述药物包括治疗炎症性疾病或自身免疫疾病的药物，所述方法包括：向所述个体施用有效量的本发明所述的经遗传修饰的微生物，或本发明所述的组合物。在某些实施方式中，所述经遗传修饰的微生物表达至少一种选自下组的外源基因：编码表达Amuc_1100多肽的外源基因、或编码表达IL-10多肽的外源基因、或编码表达IL-22多肽的外源基因、或以上任意组合的外源基因。
在本发明的另一方面，还提供了如本发明所述的经遗传修饰的微生物或本发明组合物在用于制备治疗或预防炎症性疾病或自身免疫疾病的药物中的用途。
在一些实施方式中，所述炎症性疾病或自身免疫疾病是炎症性肠病(IBD)。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-10多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码IL-10多肽和IL-22多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-10多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码Amuc_1100多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在一些实施方式中，所述炎症性疾病或自身免疫疾病是系统性红斑狼疮(SLE)。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-10多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码IL-10多肽和IL-22多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-10多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码Amuc_1100多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-22多肽的 外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在一些实施方式中，所述炎症性疾病或自身免疫疾病是关节炎。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-10多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码IL-10多肽和IL-22多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-10多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码Amuc_1100多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在一些实施方式中，所述炎症性疾病或自身免疫疾病是哮喘。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-10多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码IL-10多肽和IL-22多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-10多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码Amuc_1100多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在一些实施方式中，所述炎症性疾病或自身免疫疾病是移植物抗宿主病(GvHD)。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-10多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码IL-10多肽和IL-22多肽的外源基因。在一些实施方式中，所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-10多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码Amuc_1100多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含编码IL-22多肽的外源基因。在一些实施方式中，所述的经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
在一些实施方式中，所述的受试者具有炎症性疾病或自身免疫疾病的病症/疾病(例如，本文描述的那些)或紊乱，或者具有所述疾病/病症的诱因。其宗旨是治疗、治愈、减轻、解除、改变、矫正、缓解、改善或影响疾病/病症，或引起该疾病/病症的诱因。
如本文所用，疾病、病症或病状的“治疗”包含预防或缓解疾病、病症或病状，减缓疾病、病症或病状的发作或发展速率，降低罹患疾病、病症或病状的风险，预防或延缓与疾病、病症或病状相关的症状的发展，减少或结束与疾病、病症或病状相关的症状，使疾病、病症或病状完全或部分消退，治愈疾病、病症或病状，或其某一组合。
如本文所用，术语“有效量”是指产生所需结果所必需的一种或多种药剂的量和/或剂量和/或剂量方案，例如足以在个体中缓和与个体正在接受疗法的疾病病状相关的一种或多种症状的量，或足以减轻个体中疾病病状的严重程度或延缓其进展的量(例如治疗有效量)，足以降低个体中疾病病状的风险或延缓其发作，和/或降低其最终严重程度的量(例如预防有效量)。有效量根据以下因素有所不同：被治疗的病症的严重程度、个体患者参数、包括年龄、身体状况、身高、性别和体重、治疗的持续时间、所述并行治疗的性质(如果有的话)、以及全科医生或其他医生的知识和经验范围内的其他因素。这些因素是在本领域中公知的，并且可以通过不超出常规的实验来确定。它通常优选的以合理的医学判断得出的安全的、其使用的各个组分或组合的最大剂量。但是如本领域的普通技术人员可理解的，患者可能跟据医疗原因、心理原因或几乎任何其他原因坚持要求较低剂量或可耐受的剂量。
如本文所用，术语“个体”包含人类和非人类动物。非人类动物包含所有脊椎动物，例如哺乳动物和非哺乳动物，如非人类灵长类动物、小鼠、大鼠、猫、兔、羊、狗、牛、鸡、两栖动物和爬行动物。除指出的以外，术语“患者”或“个体”在本文中可互换使用。
如本文所用，术语“炎症性疾病或自身免疫疾病”包括但不限于，炎症性疾病和自身免疫疾病。所述炎症性疾病可以包括自身免疫疾病，如炎症性肠病。“炎症性肠病”或“IBD”在本文中可互换使用，指与消化道炎症相关的一类疾病，以在胃肠道中通常由T细胞和活化的巨噬细胞驱动的显著局部炎症以及受损的上皮屏障功能为特征(Ghishan等，2014)，包括但不限于，克罗恩病、溃疡性结肠炎、白塞氏疾病(Behcet's disease)、淋巴细胞性结肠炎、胶原性结肠炎、转移性结肠炎和不确定性结肠炎。
本文所述的“自身免疫疾病”还包括移植物抗宿主病(GvHD)、系统性红斑狼疮、关节炎(如类风湿性关节炎、骨关节炎、银屑病性关节炎或青少年特发性关节炎)、哮喘(如过敏性哮喘或中性粒细胞性哮喘)。
本文所用的术语“移植物抗宿主”或“GVH”是指移植(供体)细胞对抗微生物和组织产生免疫反应。本文所用的术语“移植物抗宿主病”或“GvHD”是指一种由于由GVH所产生的移植(移植物)细胞对微生物和组织的影响所引起的非正常状态(包括急性和慢性)。例如，从他人获得血液或骨髓移植的患者具有患急性GvHD的风险。急性移植物抗宿主病(GvHD)是一种由供体免疫细胞引起的在移植有同种异体骨髓或血液细胞的患者 中的病症。肠表皮和肝脏是经常受到影响的组织，严重时，GvHD可以引起皮肤起泡或过度腹泻和消瘦。由供体免疫细胞引起的在肝脏中的炎症可以导致引起黄疸的阻塞。其它组织例如肺和胸腺也可能受到影响。对GvHD的诊断通常可以通过使用显微镜查看一小片皮肤、肝、胃或肠以对特定炎性特征进行观察来证实。
系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种具有高度异质性的自身免疫性疾病(autoimmune disease，AID)，发作和缓解贯穿其中，临床表现复杂多样，血清中出现以抗核抗体为代表的多种自身抗体和多系统受累是主要的临床特征。SLE的症状因人而异，可能从轻到重。常见症状包括关节疼痛和肿胀、发烧、胸痛、脱发、口腔溃疡、淋巴结肿大、感觉疲劳，还有一种红疹，最常见于面部。通常会有疾病发作期和症状较少的缓解期。SLE的病因尚不清楚。它被认为与遗传和环境因素有关。在同卵双胞胎中，如果一个受到影响，另一个也有24％的可能性。女性性激素、阳光、吸烟、维生素D缺乏和某些感染也被认为会增加风险。SLE的诊断具有一定的困难，目前主要是基于症状和实验室测试的组合来进行诊断的。
类风湿性关节炎(RA)是一种长期的自身免疫性疾病，其主要影响关节。RA通常会导致关节发热、肿胀和疼痛，并且在休息后疼痛和僵硬往往会恶化。最常见的是手腕和手部受累，身体两侧的关节通常相同。RA也可能影响身体的其他部位，包括皮肤、眼睛、肺、心脏、神经和血液。这可能导致红细胞计数低、肺部周围发炎和心脏周围发炎，也可能出现发烧和低能。通常，症状会在数周到数月内逐渐出现。虽然类风湿性关节炎的病因尚不清楚，但据信它涉及遗传和环境因素的组合。潜在的发病机制涉及人体免疫系统攻击关节，这导致关节囊发炎和增厚，也会影响潜在的骨骼和软骨。RA的诊断主要基于患者的体征和症状。在具体的诊断中，还可能结合X光和实验室检查以支持诊断或排除具有类似症状的其他疾病。
在一些实施方式中，所述的自身免疫疾病选自下组：炎症性肠病(例如克罗恩病或溃疡性结肠炎)、移植物抗宿主病(GvHD)、系统性红斑狼疮、关节炎(如类风湿性关节炎、骨关节炎、银屑病性关节炎或青少年特发性关节炎)、哮喘(如过敏性哮喘或中性粒细胞性哮喘)，或其组合。在一些实施方式中，其中所述自身免疫疾病包括移植物抗宿主病(GvHD)。在一些实施方式中，其中所述自身免疫疾病包括炎症性肠病(如克罗恩病或溃疡性结肠炎)、系统性红斑狼疮、哮喘、多发性硬化和/或风湿性关节炎。
大约70％的人体免疫系统在胃肠道。肠道自身免疫疾病(如炎症性肠病)与非肠道自身免疫疾病(如多发性硬化症)之间存在许多相关性和共同的疾病信号，包括1)细胞因子信号改变，2)免疫细胞活性改变，3)肠道菌群失调。无论在动物模型还是人体中，肠道菌群失调都与自身免疫疾病的发病机制密切相关。例如，在类风湿性关节炎和炎症性肠 病的早期阶段可以发现微生物成分的变化。一些独立的研究指出，西班牙和中国的SLE患者都存在肠道微生物群失调。肠道细菌的平衡对免疫系统的调节和发育至关重要。同时，炎症状态下肠道屏障功能受损，促使细菌从粘膜层泄漏到血管，从而刺激局部和全身的自身免疫途径。
肠道炎症信号通路、先天免疫系统和适应性免疫系统中的多个靶点已成为利用小分子、抗体、基因治疗或肠道微生物治疗方法开发新药物的研究热点。其中，以转基因细菌为原料的细菌载体基因治疗的潜力最近被业界所认可。然而，虽然这些经遗传修饰的微生物已经在一些临床前模型中显示出功效，但尚未观察到对患者的功效。
不希望受任何已有理论束缚，为了解决上述尚未满足的临床需求，本申请的发明人通过构建工程微生物(例如工程大肠杆菌EcN)作为活体药物，在肠道施用以导入外源多肽因子或其组合，由于工程菌能够在肠道内持续表达分泌外源多肽因子，就可以在一段时间内连续作用于肠道细胞，从而增强肠道屏障和/或提高肠道免疫，进而改善机体免疫疾病的症状。如果病灶位于肠道，则本公开经遗传修饰的微生物预期可以分布在病灶附近，其分泌的多肽因子甚至可直接作用于病灶部位的细胞，从而发挥治疗效果。而这样的作用机制是多肽类药物所无法达到的，这是因为：如果口服多肽类药物，则多肽很容易在肠道内被降解从而导致疗效不佳。即使通过使用更好的递送载体能一定程度上改善口服多肽的效果，但这仍然难以满足治疗需求并且导致高成本的问题；对于血清注射的方式，患者依从性问题很严重，并且由于细胞因子类药物直接进入血液循环，可能会带来更多的副作用。
发明人首先筛选了适合微生物表达和分泌、并能够在肠道的局部递送中起到治疗效果的外源基因组合。需要说明的是，活体药物需要保持微生物的活力从而能够正常生长并在体内发挥治疗作用，但是微生物持续表达外源多肽，特别是两种甚至更多种外源多肽则势必对细胞稳定带来一定压力，因此这就首先要求微生物表达的外源多肽不能对微生物自身活力产生过大不利影响，否则，微生物状态不佳将影响其表达和分泌治疗性外源多肽的能力，甚至微生物可能会通过内部的应激机制调低甚至关闭外源基因的表达，从而导致工程微生物构建失败。不过，哪些外源基因适合治疗性工程微生物的表达仍然是未知的且难以预期的，在非活体药物领域很多看似适合在微生物中表达的外源基因，构建用于治疗目的的工程菌时反而无法成功。发明人在筛选过程中确实发现了一些外源因子不适合利用本公开的工程微生物进行表达分泌，于是最终放弃(例如IL-19、IL-23、IL-37等)。发明人在确定了Amuc_1100、IL-10和/或IL-22多肽作为组合的基础后，进一步对这些多肽的信号肽(替换了各自自身的信号肽)和表达盒中的表达调控元件(例如选择合适的启动子、顺反子、核糖体结合位点等)以及底盘细菌的结构(选择敲除特 定的膜蛋白以提高膜的通透性)等进行了全面优化，从而使得工程菌能够高效稳定的表达上述全新的外源基因组合，在多个动物疾病模型中也证实了表达外源因子组合的全部或部分工程菌对多种炎症性疾病或自免性疾病产生了预料不到的疗效甚至表现出协同作用，具有广阔的临床应用前景。
提供以下实施例以更好地说明要求保护的发明，并且不应将其解释为限制本发明的范围。下文全部或部分描述的所有具体组合物，材料和方法均落入本发明的范围内。这些特定的组成，材料和方法无意于限制本发明，而仅是为了说明落入本发明范围内的特定实施例。本领域技术人员可以开发等同的组合物，材料和方法，而无需行使发明的能力并且不脱离本发明的范围。将理解的是，可以在本文描述的过程中做出许多变化，同时仍然保持在本发明的范围内。发明人的意图是，这样的变化包括在本发明的范围内。
实施例
实施例1：细菌基因组的编辑方案
1.1 sgRNA设计
在目标整合位点序列的两条链上寻找连接NGG PAM序列的20bp序列(N20NGG)并且针对EnN基因组进行blast处理。选择独特的20bp序列作为目标整合位点的sgRNA。选择sgRNA上游和下游的300-500bp序列作为左同源臂(LHA)和右同源臂(RHA)。
1.2 sgRNA质粒构建
将sgRNA序列添加到gRNA骨架反向引物的5’端，通过PCR扩增并用限制性内切酶PstI和SpeI进行酶切，将酶切后的PCR产物片段与同样酶切的质粒pCBT003(SEQ ID NO：137)连接，形成pCBT003_sgRNA质粒。
1.3供体基因片段的制备
待整合到EcN基因组的外源基因由金斯瑞(GeneScript)在克隆质粒(例如pUC57)上合成。外源基因以合成的质粒为模板进行扩增，所选整合位点的LHA和RHA以EcN的基因组为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起来，得到的包含LHA-外源基因-RHA的PCR产物用作供体基因片段。
实施例2：质粒转化方案
2.1 EcN/pCBT001电转感受态细胞的制备
将100-200ng pCBT001质粒(表达Cas9蛋白的质粒，SEQ ID NO:136)添加到100μL EcN电转感受态细胞中，将混合物转移到2mm电转杯中，电转条件设置为2.5kV、25μF、200Ω。电转后将细胞悬浮于1mL SOC，30℃下震荡(220rpm)培养两小时。之后将 所有细胞涂布于添加有50μg/mL壮观霉和链霉素的LB琼脂板上并且在30℃下培养过夜。板上生长的菌落为具有pCBT001质粒的EcN(EcN/pCBT001)。
将具有抗性的LB琼脂板上的单一EcN/pCBT001菌落接种到3mL添加有50μg/mL壮观霉素和链霉素的LB液体培养基中，在30℃下震荡(220rpm)培养过夜，之后将300μL此过夜培养物接种到30mL添加有50μg/mL壮观霉素和链霉素的LB液体培养基中，在30℃下在震荡(220rpm)培养。1小时后将IPTG以1mM的浓度添加到培养物中。当OD 600达到约0.6时，将细胞用20mL、10mL和5mL 10％的甘油(4℃)洗涤3次，并且最终再悬浮于300μL 10％甘油(4℃)中，并且以每管100μL分装到1.5mL离心管中。
2.2 pCBT003-sgRNA质粒及供体基因片段的转化
将大约2μg含有LHA-外源基因-RHA的供体基因片段PCR产物以及100-200ng表达整合位点的sgRNA的pCBT003_sgRNA转化到EcN/pCBT001的电转感受态细胞中。电转后将细胞悬浮于1mL SOC，30℃下震荡(220rpm)培养两小时。之后将所有细胞涂布于添加有50μg/mL壮观霉素、链霉素以及100μg/mL氨苄青霉素的LB琼脂板上。外源基因表达盒插入EcN基因组的示意图如图1所示。
实施例3：去除多余质粒的方案
3.1外源基因整合的验证
用于验证的引物设计在LHA的上游和RHA的下游。选取转化板上生长的单一菌落用验证引物进行菌落PCR，通过PCR产物的大小选择外源基因正确整合到基因组中的克隆。
3.2 pCBT003_sgRNA和pCBT001质粒的去除
将挑选出的正确克隆接种到3mL添加有50μg/mL壮观霉素、链霉素和10mM阿拉伯糖的LB液体培养基中，在30℃下震荡(220rpm)培养过夜，随后将培养物稀释10 6倍，取100μL涂布于添加有50μg/mL壮观霉素和链霉素的LB琼脂板上，30℃下培养过夜。随后挑选单菌落同时在添加有50μg/mL壮观霉素、链霉素和100μg/mL氨苄青霉素的LB琼脂板和添加有50μg/mL壮观霉素和链霉素的LB琼脂板上点样。仅生长在添加有50μg/mL壮观霉素和链霉素的LB琼脂板上的菌落是pCBT003_sgRNA质粒消除的菌落。
将pCBT003_sgRNA消除的克隆接种在LB液体培养基中，42℃下震荡(220rpm)培养过夜，随后将培养物稀释10 6倍并且取100μL涂布于LB琼脂板上。挑选单菌落同时在LB琼脂板和添加有50μg/mL壮观霉素和链霉素的LB琼脂板上点样。仅生长在LB琼脂板上的菌落是pCBT001消除的菌落。
3.3内源质粒的去除
3.3.1 pMUT1的消除
将表达pMUT1的sgRNA的质粒pCBT003_pMUT1_sgRNA转化进EcN/pCBT001中。 pMUT1的消除验证引物特异性地同源于pMUT1而不与EcN的基因组同源，用pMUT1的验证引物进行PCR，无法得到PCR产物的菌落为pMUT1消除菌落。
3.3.2 pMUT2的消除
将卡那霉素抗性基因连接到pMUT2上，得到质粒pMUT2-kana，其核苷酸序列如SEQ ID NO:138所示。将质粒pMUT2-kana转化到EcNΔpMUT1中，将转化后的细胞涂布于添加有10μg/mL卡那霉素的LB琼脂板上，长出的菌落为含有质粒pMUT2-kana的EcNΔpMUT1。EcNΔpMUT1/pMUT2-kana在添加有10μg/mL卡那霉素的LB液体培养基中传代数次之后，提取质粒，并通过电泳条带判断pMUT2是否全部被pMUT2-kana取代。之后将pCBT001转入pMUT2被全部取代的EcNΔpMUT1/pMUT2-kana中，再将表达卡那霉素抗性基因sgRNA的质粒pCBT003_Kana-sgRNA转入EcNΔpMUT1/pMUT2-kana/pCBT001中。得到的菌落用与卡那霉素抗性基因特异性的引物进行PCR，无法得到PCR产物的菌落为pMUT2-kana消除菌落。
本实施例中靶向pMUT1的sgRNA的序列如SEQ ID NO:164所示；靶向卡那霉素抗性基因的sgRNA的序列如SEQ ID NO:165所示
靶向pMUT1的sgRNA序列(SEQ ID NO:164)：
Figure PCTCN2022134672-appb-000089
靶向卡那霉素抗性基因的sgRNA序列(SEQ ID NO:165)：
Figure PCTCN2022134672-appb-000090
实施例4：膜蛋白的敲除方案
需要敲除的目标膜蛋白sgRNA的设计方法和sgRNA质粒的构建方法同实施例1里的1.1、1.2所述的方法。选择目标膜蛋白编码基因上游和下游的300-500bp序列作为左同源臂(LHA)和右同源臂(RHA)。所选敲除位点的LHA和RHA以EcN的基因组为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起来，得到的包含LHA-RHA的PCR产物用作供体片段。将得到的供体片段的PCR产物和表达敲除位点sgRNA的pCBT003_sgRNA质粒同时转化入含有pCBT001的感受态细胞中，得到的单菌落用敲除位点的验证引物进行扩增，通过扩增条带的大小筛选目标蛋白成功从基因组敲除的克隆。
本实施例中所敲除的膜蛋白包括tolQ、tolR、tolA、pal、lpp、mrcA和ompT。tolQ敲除位点的sgRNA的序列如SEQ ID NO:166所示，该tolQ敲除位点的两侧同源臂序列分别如SEQ ID NO:167和168所示；tolR敲除位点的sgRNA的序列如SEQ ID NO:169所示，该tolR敲除位点的两侧同源臂序列分别如SEQ ID NO:170和171所示；tolA敲除位点的sgRNA的序列如SEQ ID NO:172所示，该tolA敲除位点的两侧同源臂序列分别如SEQ ID  NO:173和174所示；pal敲除位点的sgRNA的序列如SEQ ID NO:175所示，该pal敲除位点的两侧同源臂序列分别如SEQ ID NO:176和177所示；lpp敲除位点的sgRNA的序列如SEQ ID NO:178所示，该lpp敲除位点的两侧同源臂序列分别如SEQ ID NO:179和180所示；mrcA敲除位点的sgRNA的序列如SEQ ID NO:181所示，该mrcA敲除位点的两侧同源臂序列分别如SEQ ID NO:182和183所示；ompT敲除位点的sgRNA的序列如SEQ ID NO:184所示，该ompT敲除位点的两侧同源臂序列分别如SEQ ID NO:185和186所示。
靶向tolQ位点的sgRNA序列(SEQ ID NO:166)：
Figure PCTCN2022134672-appb-000091
tolQ位点的左侧同源臂(LHA)序列(SEQ ID NO:167)：
Figure PCTCN2022134672-appb-000092
tolQ位点的右侧同源臂(RHA)序列(SEQ ID NO:168)：
Figure PCTCN2022134672-appb-000093
靶向tolR位点的sgRNA序列(SEQ ID NO:169)：
Figure PCTCN2022134672-appb-000094
tolR位点的左侧同源臂(LHA)序列(SEQ ID NO:170)：
Figure PCTCN2022134672-appb-000095
tolR位点的右侧同源臂(RHA)序列(SEQ ID NO:171)：
Figure PCTCN2022134672-appb-000096
Figure PCTCN2022134672-appb-000097
靶向tolA位点的sgRNA序列(SEQ ID NO:172)：
Figure PCTCN2022134672-appb-000098
tolA点的左侧同源臂(LHA)序列(SEQ ID NO:173)：
Figure PCTCN2022134672-appb-000099
tolA位点的右侧同源臂(RHA)序列(SEQ ID NO:174)：
Figure PCTCN2022134672-appb-000100
靶向pal位点的sgRNA序列(SEQ ID NO:175)：
Figure PCTCN2022134672-appb-000101
pal位点的左侧同源臂(LHA)序列(SEQ ID NO:176)：
Figure PCTCN2022134672-appb-000102
pal位点的右侧同源臂(RHA)序列(SEQ ID NO:177)：
Figure PCTCN2022134672-appb-000103
Figure PCTCN2022134672-appb-000104
靶向lpp位点的sgRNA序列(SEQ ID NO:178)：
Figure PCTCN2022134672-appb-000105
lpp位点的左侧同源臂(LHA)序列(SEQ ID NO:179)：
Figure PCTCN2022134672-appb-000106
lpp位点的右侧同源臂(RHA)序列(SEQ ID NO:180)：
Figure PCTCN2022134672-appb-000107
靶向mrcA位点的sgRNA序列(SEQ ID NO:181)：
Figure PCTCN2022134672-appb-000108
mrcA位点的左侧同源臂(LHA)序列(SEQ ID NO:182)：
Figure PCTCN2022134672-appb-000109
mrcA位点的右侧同源臂(RHA)序列(SEQ ID NO:183)：
Figure PCTCN2022134672-appb-000110
Figure PCTCN2022134672-appb-000111
靶向ompT位点的sgRNA序列(SEQ ID NO:184)：
Figure PCTCN2022134672-appb-000112
ompT位点的左侧同源臂(LHA)序列(SEQ ID NO:185)：
Figure PCTCN2022134672-appb-000113
ompT位点的右侧同源臂(RHA)序列(SEQ ID NO:186)：
Figure PCTCN2022134672-appb-000114
实施例5：过表达分子伴侣的方案
5.1分子伴侣的选择
利用质粒在工程菌CBT4020中表达不同分子伴侣组合，工程菌在LB中培养24小时后表达的IL-10在培养基上清及胞内的产量如表11所示。结果显示，可以通过表达分子伴侣提高IL-10的产量，同时表达分子伴侣dsbA、dsbC、dnaK、dnaJ、grpE、groES、groEL、tig、fkpA、surA的效果最好。
表11 不同分子伴侣组合及IL-10的产量
Figure PCTCN2022134672-appb-000115
Figure PCTCN2022134672-appb-000116
5.2分子伴侣质粒的构建
5.2.1质粒pCBT012的构建
启动子BBa_J23114、核糖体结合位点(RBS)以合成质粒为模板进行扩增，groES、groEL、tig编码序列以质粒pG-TF2为模板进行扩增，fkpA、surA编码序列以及插入位点yieN的LHA和RHA以EcN的基因组为模板扩增，转录终止子以质粒pCBT003为模板扩增，靶向yieN位点的sgRNA表达序列以sgRNA表达质粒pCBT003_yieN_sgRNA为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起，得到两侧分别与LHA和RHA-yieN_sgRNA序列连接的供体基因片段表达盒，分子伴侣表达盒上各元件从5’到3’按如下顺序排列：5’-启动子-核糖体结合位点(RBS)-groES-groEL-tig-fkpA-surA编码序列-终止子。pCBT012质粒骨架以质粒pCBT010为模板进行扩增，用于扩增pCBT012质粒骨架的PCR引物与分子伴侣表达盒具有15-20bp的同源序列，因此可以使用试剂盒ClonExpress Ultra One Step Cloning Kit(Vazyme)与分子伴侣表达盒连接起来，形成质粒pCBT012(SEQ ID NO:215)。
质粒pCBT010序列(SEQ ID NO:187)：
Figure PCTCN2022134672-appb-000117
Figure PCTCN2022134672-appb-000118
5.2.2质粒pCBT013的构建
启动子BBa_J23114、核糖体结合位点(RBS)以合成质粒为模板进行扩增，dnaK、dnaJ、grpE编码序列以质粒pKJE7为模板进行扩增，dsbA、dsbC编码序列以及插入位点yicS/nepI的LHA和RHA以EcN的基因组为模板扩增，转录终止子以质粒pCBT003为模板扩增，靶向yicS/nepI位点的sgRNA表达序列以sgRNA表达质粒pCBT003_yicS/nepI_sgRNA为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起，得到两侧分别与LHA和RHA-yicS/nepI_sgRNA序列连接的供体基因片段表达盒，分子伴侣表达盒上各元件从5’到3’按如下顺序排列：5’-启动子-核糖体结合位点(RBS)-dsbA-dsbC-dnaK-dnaJ-grpE编码序列-终止子。pCBT013质粒骨架以质粒pCBT010为模板进行扩增，用于扩增pCBT013质粒骨架的PCR引物与分子伴侣表达盒具有15-20bp的同源序列，因此可以使用试剂盒ClonExpress Ultra One Step Cloning Kit(Vazyme)与分子伴侣表达盒连接起来，形成质粒pCBT013(SEQ ID NO:216)。
5.3分子伴侣的基因组整合
将表达分子伴侣的质粒pCBT012、pCBT013分别转化入含有pCBT001的感受态细胞中，得到的单菌落用插入位点的验证引物进行扩增，通过扩增条带的大小筛选分子伴侣表达盒成功插入基因组的克隆。各个分子伴侣的序列登录号如表12所示。
表12 分子伴侣
分子伴侣 Uniprot ID
dsbA P0AEG4
dsbC P0AEG6
dnaK P0A6Y8
dnaJ P08622
grpE P09372
groES P0A6F9
groEL P0A6F5
tig P0A850
fkpA P45523
surA P0ABZ6
靶向yieN插入位点的sgRNA的序列如SEQ ID NO:188所示，该yieN插入位点的两侧同源臂序列分别如SEQ ID NO:189和190所示。靶向yicS/nepI插入位点的sgRNA的序列如SEQ ID NO:191所示，该yicS/nepI插入位点的两侧同源臂序列分别如SEQ ID NO:192和193所示。
靶向yieN位点的sgRNA序列(SEQ ID NO:188)：
Figure PCTCN2022134672-appb-000119
yieN位点的左侧同源臂(LHA)序列(SEQ ID NO:189)：
Figure PCTCN2022134672-appb-000120
yieN位点的右侧同源臂(RHA)序列(SEQ ID NO:190)：
Figure PCTCN2022134672-appb-000121
靶向yicS/nepI位点的sgRNA序列(SEQ ID NO:191)：
Figure PCTCN2022134672-appb-000122
yicS/NepI位点的左侧同源臂(LHA)序列(SEQ ID NO:192)：
Figure PCTCN2022134672-appb-000123
yicS/NepI位点的右侧同源臂(RHA)序列(SEQ ID NO:193)：
Figure PCTCN2022134672-appb-000124
实施例6：菌株的构建
6.1表达IL-10的菌株构建
6.1.1 IL-10供体基因片段表达盒的构建步骤
IL-10编码序列、信号肽、启动子、核糖体结合位点(RBS)以及顺反子等由金斯瑞(GeneScript)在克隆质粒(例如pUC57)上合成。IL-10编码序列、信号肽、启动子、核糖体结合位点(RBS)以及顺反子等以合成质粒为模板进行扩增，插入位点的LHA和RHA以EcN的基因组为模板扩增，转录终止子以质粒pCBT003为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起，得到两侧分别与LHA和RHA序列连接的供体基因片段表达盒，表达盒上各元件从5’到3’按如下顺序排列：5’-启动子-核糖体结合位点(RBS)-顺反子-信号肽-IL-10编码序列-终止子(所构建的各供体基因片段表达盒的序列结构如SEQ ID NO:144-156、235所示)。将得到的两侧带有LHA和RHA序列的供体基因片段表达盒的PCR产物和表达sgRNA的pCBT003_maeB_sgRNA质粒同时转化入含有pCBT001的感受态细胞中，得到的单菌落用插入位点的验证引物进行扩增，通过扩增条带的大小筛选IL-10成功插入基因组的克隆。IL-10的产量用ELISA试剂盒(义翘神州，KIT10947A)检测。
本实施例中的IL-10编码序列均为编码野生型人IL-10(SEQ ID NO:11)的序列；所插入的位点为EcN基因组的maeB，靶向maeB位点的sgRNA的序列如SEQ ID NO:194所示，该maeB位点的两侧同源臂序列分别如SEQ ID NO:195和196所示。
靶向maeB位点的sgRNA序列(SEQ ID NO:194)：
Figure PCTCN2022134672-appb-000125
maeB位点的左侧同源臂(LHA)序列(SEQ ID NO:195)：
Figure PCTCN2022134672-appb-000126
maeB位点的右侧同源臂(RHA)序列(SEQ ID NO:196)：
Figure PCTCN2022134672-appb-000127
本实施例中所得到的各表达IL-10的菌株及其中的插入元件和敲除元件信息如下表13所示。相关元件的序列如表2-表9的序列所示。
表13 IL-10表达菌株的各元件信息
Figure PCTCN2022134672-appb-000128
6.1.2 IL-10表达条件的优化
使用不同信号肽的工程菌在LB中培养24小时后表达的IL-10在培养基上清及胞内的产量如表14所示。其中，菌株CBT4007中使用了dsbA信号肽；菌株CBT4009中使用了ompA信号肽；菌株CBT4011中使用了pelB信号肽；菌株CBT4013中使用了yebF信号肽，菌株CBT4003中使用了USP45信号肽。在同样的稀释度下，使用OmpA、PelB、YebF信号肽的工程菌样品中IL-10的浓度低于检测下线，使用USP45信号肽时，IL-10的产量显著高于DsbA信号肽。
表14 使用不同信号肽的工程菌基因型及IL-10的产量
Figure PCTCN2022134672-appb-000129
*：“-”表示低于检测下限
使用不同启动子的工程菌在LB中培养24小时后表达的IL-10在培养基上清及胞内的产量如表15所示。其中，菌株CBT4003中使用BBa_J23101启动子；菌株CBT4004中使用BBa_J23108启动子；菌株CBT4005中使用BBa_J23110启动子。结果显示，可以通过选用合适的启动子(例如BBa_J23101启动子)来调节IL-10的产量。
表15 使用不同启动子的工程菌基因型及IL-10的产量
Figure PCTCN2022134672-appb-000130
使用不同顺反子的工程菌在LB中培养24小时后表达的IL-10在培养基上清中的产量如表16所示。其中，菌株CBT4026中使用顺反子T7g10；菌株CBT4028中使用顺反子BCD2；菌株CBT4029中使用顺反子GFP；菌株CBT4030中使用顺反子Lucifierase(荧光素酶)。结果显示，可以通过选择不同的顺反子来调节IL-10的产量。
表16 使用不同顺反子的工程菌基因型及IL-10的产量
Figure PCTCN2022134672-appb-000131
敲除不同膜蛋白的工程菌以及过表达分子伴侣的工程菌在LB中培养12小时后表达 的IL-10在培养基上清及胞内的产量如表17所示。其中，菌株CBT4071、CBT4072、CBT4073、CBT4074、CBT4020分别敲除了膜蛋白tolQ、tolR、tolA、pal和lpp，对照为未改造外膜的菌株CBT4005。结果显示，单独敲除tolQ、tolR、tolA、pal和lpp可以显著提高IL-10的产量与分泌效率。
CBT4062为敲除膜蛋白lpp并过表达分子伴侣的工程菌株。分子伴侣的过表达通过在菌株yicS/nepI位点插入BBa_J23114启动子启动的dsbA、dsbC、dnaK、dnaJ、和grpE，和在yieN位点插入BBa_J23114启动子启动的groES、groEL、tig、fkpA和surA，各个分子伴侣的序列登录号如表12所示。与只敲除lpp但不过表达分子伴侣的对照菌株CBT4028比，CBT4062中分子伴侣的过表达可以显著提高IL-10的产量。CBT4075为同时敲除lpp和mrcA并过表达分子伴侣的工程菌株，CBT4076为同时敲除lpp和ompT并过表达分子伴侣的工程菌株，与单独敲除lpp并过表达分子伴侣的CBT4062相比，lpp与mrcA或ompT的组合敲除进一步显著提高了IL-10的产量与分泌水平。而lpp、mrcA、ompT三个膜蛋白同时敲除并过表达分子伴侣的菌株CBT4077并没有显示出更高的IL-10产量。CBT4112为同时敲除pal和mrcA的工程菌株，与只敲除pal的CBT4074相比，IL-10的产量没有显著变化。因此，mrcA只有在和lpp组合敲除时才能提高蛋白的分泌效率。
表17 敲除不同膜蛋白的工程菌以及过表达分子伴侣的工程菌基因型及IL-10的产量
Figure PCTCN2022134672-appb-000132
针对使用厌氧启动子的菌株CBT4078(EcNΔmaeB::PfnrS_BCD2_USP45_IL-10ΔLPPΔyicS/nepI::BBa_J23114_dsbA_dsbC_dnaK_dnaJ_grpEΔyieN::BBa_J23114_groES_gr oEL_tig_fkpA_surA)，对有氧和厌氧条件下在LB(磷酸盐柠檬酸缓冲体系调pH＝8.0)+1％w/v葡萄糖培养基中的IL-10表达量进行了比较，结果如图2所示。结果显示，使用厌氧启动子时，厌氧培养得到的IL-10产量显著高于有氧培养。
6.2表达IL-22的菌株构建
6.2.1 IL-22供体基因片段表达盒的构建步骤
IL-22编码序列、信号肽、启动子、核糖体结合位点(RBS)以及顺反子等由金斯瑞(GeneScript)在克隆质粒(例如pUC57)上合成。IL-22编码序列、信号肽、启动子、核糖体结合位点(RBS)以及顺反子等以合成质粒为模板进行扩增，插入位点的LHA和RHA以EcN的基因组为模板扩增，转录终止子以质粒pCBT003为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起，得到两侧分别与LHA和RHA序列连接的供体基因片段表达盒，表达盒上各元件从5’到3’按如下顺序排列：5’-启动子-核糖体结合位点(RBS)-顺反子-信号肽-IL-22编码序列-终止子(所构建的各供体基因片段表达盒的序列结构如SEQ ID NO:157-163所示)。将得到的两侧带有LHA和RHA序列的供体基因片段表达盒的PCR产物和表达sgRNA的pCBT003_kefB_sgRNA质粒或pCBT003_maeB_sgRNA质粒同时转化入含有pCBT001的感受态细胞中，得到的单菌落用插入位点的验证引物进行扩增，通过扩增条带的大小筛选IL-22成功插入基因组的克隆。IL-22的产量用ELISA试剂盒(义翘神州，KIT13059)检测。
本实施例中的IL-22编码序列均为编码野生型人IL-22(SEQ ID NO:15)的序列；所插入的位点EcN基因组的位点maeB(CBT4038)或位点kefB(CBT4041、CBT4042、CBT4043、CBT4039、CBT4040、CBT4016)。靶向maeB位点的sgRNA的序列如SEQ ID NO:194所示，该maeB位点的两侧同源臂序列分别如SEQ ID NO:195和196所示。靶向kefB位点的sgRNA的序列如SEQ ID NO:197所示，该kefB位点的两侧同源臂序列分别如SEQ ID NO:198和199所示。
靶向kefB位点的sgRNA序列(SEQ ID NO:197)：
Figure PCTCN2022134672-appb-000133
kefB位点的左侧同源臂(LHA)序列(SEQ ID NO:198)：
Figure PCTCN2022134672-appb-000134
kefB位点的右侧同源臂(RHA)序列(SEQ ID NO:199)：
Figure PCTCN2022134672-appb-000135
本实施例中所涉及的各表达IL-22的菌株的具体信息如下表18所示，相关元件的序列如表2-表9的序列所示。
表18 IL-22表达菌株的各元件信息
Figure PCTCN2022134672-appb-000136
6.2.2 IL-22表达条件的优化
使用不同启动子的工程菌在LB中培养24小时后表达的IL-22在培养基上清及胞内的产量如表19所示。其中，菌株CBT4038中使用BBa_J23119启动子(插入ΔmaeB位点)；菌株CBT4041中使用BBa_J23101启动子；菌株CBT4042中使用BBa_J23102启动子；菌株CBT4043中使用BBa_J23108启动子；菌株CBT4039中使用BBa_J23110；菌株CBT4040中使用BBa_J23114启动子。结果显示，可以通过选用合适的启动子(例如BBa_J23119启动子)来调节IL-22的产量。
表19 使用不同启动子的工程菌基因型及IL-22的产量
Figure PCTCN2022134672-appb-000137
敲除不同膜蛋白的工程菌以及过表达分子伴侣的工程菌在LB中培养24小时后表达 的IL-22在培养基上清及胞内的产量如表20所示。结果表明，敲除了膜蛋白LPP的菌株CBT4016的IL-22分泌率显著高于未敲除LPP的菌株CBT4039。
表20 敲除不同膜蛋白的工程菌基因型及IL-22的产量
Figure PCTCN2022134672-appb-000138
6.3表达Amuc_1100的菌株构建
6.3.1 Amuc_1100供体基因片段表达盒的构建步骤
Amuc_1100编码序列、信号肽、启动子、核糖体结合位点(RBS)以及顺反子等由金斯瑞(GeneScript)在克隆质粒(例如pUC57)上合成。Amuc_1100编码序列、信号肽、启动子、核糖体结合位点(RBS)以及顺反子等以合成质粒为模板进行扩增，插入位点的LHA和RHA以EcN的基因组为模板扩增，转录终止子以质粒pCBT003为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此它们可以通过重叠PCR连接起，得到两侧分别与LHA和RHA序列连接的供体基因片段表达盒，表达盒上各元件从5’到3’按如下顺序排列：5’-启动子-核糖体结合位点(RBS)-顺反子-信号肽-Amuc_1100编码序列-终止子(所构建的各供体基因片段表达盒的序列结构如SEQ ID NO:139-143所示)。将得到的两侧带有LHA和RHA序列的供体基因片段表达盒的PCR产物和表达sgRNA的pCBT003_agaI/rsmI_sgRNA质粒或pCBT003_malP/T_sgRNA质粒或pCBT003_pflB_sgRNA质粒或pCBT003_lldD_sgRNA质粒或pCBT003_maeA_sgRNA质粒同时转化入含有pCBT001的感受态细胞中，得到的单菌落用插入位点的验证引物进行扩增，通过扩增条带的大小筛选Amuc_1100成功插入基因组的克隆。
本实施例中的Amuc_1100编码序列为编码野生型Amuc_1100(SEQ ID NO:5，表21中以WT所示)或Y259A突变型Amuc_1100(第259位的编号是基于SEQ ID NO:5所示序列的编号，表21中以Y259A所示)的序列。当插入1个拷贝的Amuc_1100时，所插入的位点EcN基因组的agaI/rsmI位点；当插入2个拷贝的Amuc_1100时，所插入的第一和第二位点分别为EcN基因组的位点agaI/rsmI位点和malP/T位点；当插入3个拷贝的Amuc_1100时，所插入的第一、第二和第三位点分别为EcN基因组的位点agaI/rsmI、malP/T位点和位点pflB。
靶向agaI/rsmI位点的sgRNA的序列如SEQ ID NO:200所示，该agaI/rsmI位点的两侧同源臂序列分别如SEQ ID NO:201和202所示。靶向malP/T位点的sgRNA的序列如SEQ ID NO:203所示，该malP/T位点的两侧同源臂序列分别如SEQ ID NO:204和205所示。靶向pflB位点的sgRNA的序列如SEQ ID NO:206所示，该pflB位点的两侧同源臂 序列分别如SEQ ID NO:207和208所示。靶向lldD位点的sgRNA的序列如SEQ ID NO:209所示，该lldD位点的两侧同源臂序列分别如SEQ ID NO:210和211所示。靶向maeA位点的sgRNA的序列如SEQ ID NO:212所示，该maeA位点的两侧同源臂序列分别如SEQ ID NO:213和214所示。
靶向agaI/rsmI位点的sgRNA序列(SEQ ID NO:200)：
Figure PCTCN2022134672-appb-000139
agaI/rsmI位点的左侧同源臂(LHA)序列(SEQ ID NO:201)：
Figure PCTCN2022134672-appb-000140
agaI/rsmI位点的右侧同源臂(RHA)序列(SEQ ID NO:202)：
Figure PCTCN2022134672-appb-000141
靶向malP/T位点的sgRNA序列(SEQ ID NO:203)：
Figure PCTCN2022134672-appb-000142
malP/T位点的左侧同源臂(LHA)序列(SEQ ID NO:204)：
Figure PCTCN2022134672-appb-000143
malP/T位点的右侧同源臂(RHA)序列(SEQ ID NO:205)：
Figure PCTCN2022134672-appb-000144
Figure PCTCN2022134672-appb-000145
靶向pflB位点的sgRNA序列(SEQ ID NO:206)：
Figure PCTCN2022134672-appb-000146
pflB位点的左侧同源臂(LHA)序列(SEQ ID NO:207)：
Figure PCTCN2022134672-appb-000147
pflB位点的右侧同源臂(RHA)序列(SEQ ID NO:208)：
Figure PCTCN2022134672-appb-000148
靶向lldD位点的sgRNA序列(SEQ ID NO:209)：
Figure PCTCN2022134672-appb-000149
lldD位点的左侧同源臂(LHA)序列(SEQ ID NO:210)：
Figure PCTCN2022134672-appb-000150
lldD位点的右侧同源臂(RHA)序列(SEQ ID NO:211)：
Figure PCTCN2022134672-appb-000151
靶向maeA位点的sgRNA序列(SEQ ID NO:212)：
Figure PCTCN2022134672-appb-000152
maeA位点的左侧同源臂(LHA)序列(SEQ ID NO:213)：
Figure PCTCN2022134672-appb-000153
maeA位点的右侧同源臂(RHA)序列(SEQ ID NO:214)：
Figure PCTCN2022134672-appb-000154
Amuc_1100的产量用Western Blot方法检测。菌株信息如表21所示。相关元件的序列如表2-表9的序列所示。
表21 Amuc_1100表达菌株
Figure PCTCN2022134672-appb-000155
Figure PCTCN2022134672-appb-000156
6.3.2 Amuc_1100表达条件的优化
对不同拷贝数的Amuc_1100(Y259A)表达量进行了比较，结果如图3A所示。其中，泳道A和B为菌株CBT4101，其表达一个拷贝的使用BBA_J23101启动子的Amuc_1100(Y259A)；泳道C为菌株CBT4107，其表达一个拷贝的使用BBA_J23110启动子的Amuc_1100(Y259A)；泳道D为菌株CBT4102，其表达两个拷贝的使用BBA_J23110启动子的Amuc_1100(Y259A)；泳道E为菌株CBT4103，其表达三个拷贝的使用BBA_J23101启动子的Amuc_1100(Y259A)；泳道F为菌株CBT4108，其表达三个拷贝的Amuc_1100(Y259A)，其中一个拷贝使用BBA_J23101启动子，另外两个拷贝使用BBA_J23110启动子；泳道G为菌株CBT4109，其表达三个拷贝的使用BBA_J23110启动子的Amuc_1100(Y259A)。该结果显示，可以通过增加拷贝数来提高Amuc_1100的表达量。
通过改造细胞外膜提高Amuc_1100的稳定性，并对此进行了测定和比较，结果如图3B所示。其中，菌株CBT4106中敲除了膜蛋白LPP和ompT；菌株CBT4105只敲除了膜蛋白LPP。该结果显示，敲除ompT可以减少上清液中Amuc_1100的降解。
6.4表达IL-10、IL-22和/或Amuc_1100组合的菌株构建
IL-10、IL-22、Amuc_1100供体基因片段的制备方法同本实施例6.1、6.2和6.3所述的 方法。同时表达IL-10和IL-22的两组合菌株的构建是将IL-22供体基因片段转入已表达IL-10的菌株中；同时表达IL-10和Amuc_1100的两组合菌株的构建是将Amuc_1100供体基因片段转入已表达IL-10的菌株中；同时表达IL-22和Amuc_1100的两组合菌株的构建是将Amuc_1100供体基因片段转入已表达IL-22的菌株中；同时表达IL-10、IL-22和Amuc_1100的三组合菌株的构建是将Amuc_1100供体基因片段转入已同时表达IL-10和IL-22的菌株中。本实施例中所得到的各组合菌株及其中的插入元件和敲除元件信息如下表21所示，相关元件的序列如表2-表9的序列所示。
表21 组合表达菌株中的各元件信息
Figure PCTCN2022134672-appb-000157
Figure PCTCN2022134672-appb-000158
其中，同时表达IL-10、IL-22和Amuc_1100的三组合菌株中，各基因在EcN染色体上的位置的示意图如图4A所示。
6.5菌株生长状态的测定
单独表达、双基因组合表达、三基因组合表达菌株的生长曲线如图4B所示。其中，CBT4070单独表达Amuc_1100；CBT4062单独表达IL-10；CBT4068同时表达IL-10和Amuc_1100；CBT4066单独表达IL-22；CBT4069同时表达IL-22和Amuc_1100；CBT4063同时表达IL-10和IL-22；CBT4067同时表达IL-10、IL-22和Amuc_1100。菌株信息如表10所示。
结果表明，所构建的单独表达、双基因组合表达、三基因组合表达的工程菌的生长未受到显著影响。
6.6外源基因表达量的测定
单独表达、双基因组合表达、三基因组合表达菌株上清液中IL-10和IL-22的表达量结果如表23所示。其中，CBT4062单独表达IL-10；CBT4068同时表达IL-10和Amuc_1100；CBT4066单独表达IL-22；CBT4069同时表达IL-22和Amuc_1100；CBT4070单独表达Amuc_1100；CBT4063同时表达IL-10和IL-22；CBT4067同时表达IL-10、IL-22和Amuc_1100。该结果显示，组合表达工程菌IL-10、IL-22以及Amuc_1100的表达不会因组合而降低。
表23 组合菌株基因型及IL-10、IL-22和Amuc_1100的表达量
Figure PCTCN2022134672-appb-000159
Figure PCTCN2022134672-appb-000160
实施例7：表达蛋白的活性检测
7.1 IL-10活性检测
将所要检测的样品用全细胞培养液稀释到合适的浓度，将20μL稀释好的样品以及IL-10标准品分别加入到平底96孔板中，其中EcN的样品用作阴性对照。制备～280,000细胞/mL的HEK-Blue TM IL-10Cells(InvivoGen,Cat#hkb-il10)细胞悬液，并将180μL该悬液(～50,000细胞)迅速加入到已添加有样品的孔中。将96孔板在5％CO 2下37℃培养22小时，之后取20μL上清液加入到新的平底96孔板中，并在每孔中加入180μL QUANTI-Blue试剂，37℃孵育一小时后读取A 620的数值。
单独表达、双基因组合表达、三基因组合表达菌株上清液中分泌的IL-10的细胞活性如图4C所示。其中，阴性对照为不表达IL-10的EcN上清液；IL-10标品浓度为30ng/mL；CBT4062单独表达IL-10，样品浓度稀释为21ng/mL；CBT4068同时表达IL-10和Amuc_1100，样品浓度稀释为28ng/mL；CBT4063同时表达IL-10和IL-22，样品浓度稀释为21ng/mL；CBT4067同时表达IL-10、IL-22和Amuc_1100，样品浓度稀释为29ng/mL。
结果显示，组合表达工程菌表达分泌的IL-10具有生物活性且其活性与标准品具有可比性。
7.2 IL-22活性检测
将所要检测的样品用全细胞培养液稀释到合适的浓度，将20μL稀释好的样品以及IL-22标准品分别加入到平底96孔板中，其中EcN的样品用作阴性对照。制备～280,000cells/mL的HEK-Blue TM IL-22Cells(InvivoGen,Cat#hkb-il22)细胞悬液，并将180μL该悬液(～50,000cells)迅速加入到已添加有样品的孔中。将96孔板在5％CO 2下37℃培养22小时，之后取20μL上清液加入到新的平底96孔板中，并在每孔中加入180μL QUANTI-Blue试剂， 37℃孵育一小时后读取A 620的数值。
单独表达、双基因组合表达、三基因组合表达菌株上清液中分泌的IL-22的细胞活性如图4D所示。其中，阴性对照为不表达IL-22的EcN上清液；CBT4066单独表达IL-22，CBT4069同时表达IL-22和Amuc_1100；CBT4063同时表达IL-22和IL-10；CBT4067同时表达IL-10、IL-22和Amuc_1100；IL-22标品及各菌株样品浓度皆稀释为0.33ng/mL、1ng/mL以及3ng/mL。
结果显示，组合表达工程菌表达分泌的IL-22具有生物活性且其活性与标准品具有可比性
7.3 Amuc_1100活性检测
将所要检测的样品用全细胞培养液稀释到合适的浓度，将20μL稀释好的样品以及Amuc_1100标准品分别加入到平底96孔板中，其中EcN的样品用作阴性对照。制备～280,000细胞/mL的HEK-Blue TM hTLR2Cells(InvivoGen,Cat#hkb-htlr2)细胞悬液，并将180μL该悬液(～50,000cells)迅速加入到已添加有样品的孔中。将96孔板在5％CO 2下37℃培养22小时，之后取20μL上清液加入到新的平底96孔板中，并在每孔中加入180μL QUANTI-Blue试剂，37℃孵育一小时后读取A 620的数值。
选取菌株CBT4080作为测定对象，对其上清液中分泌的Amuc_1100的活性进行了检测，结果如图4E所示。其中，阴性对照为不表达Amuc_1100的EcN上清液纯化产物；Amuc_1100标品浓度为40μg/mL；CBT4080纯化样品纯度约为70％，浓度为28.8μg/mL。该结果显示，工程菌表达分泌的Amuc_1100具有生物活性且其活性与标准品具有可比性。
实施例8：厌氧诱导表达IL-10、IL-22和/或Amuc_1100组合的菌株构建
IL-10、IL-22、Amuc_1100供体基因片段的制备方法同实施例6里的6.1、6.2和6.3所述的方法。同时表达IL-10和IL-22的两组合菌株的构建是将IL-22供体基因片段转入已表达IL-10的菌株中；同时表达IL-10和Amuc_1100的两组合菌株的构建是将Amuc_1100供体基因片段转入已表达IL-10的菌株中；同时表达IL-22和Amuc_1100的两组合菌株的构建是将Amuc_1100供体基因片段转入已表达IL-22的菌株中；同时表达IL-10、IL-22和Amuc_1100的三组合菌株的构建是将Amuc_1100供体基因片段转入已同时表达IL-10和IL-22的菌株中。本实施例中所得到的各组合菌株及其中的插入元件和敲除元件信息如下表24所示，相关元件的序列如表2-表9的序列所示。
表24 实施例8所用组合表达菌株的各元件信息
Figure PCTCN2022134672-appb-000161
Figure PCTCN2022134672-appb-000162
实验所用菌株在LB(磷酸盐柠檬酸缓冲体系调pH＝8.0)+1％w/v葡萄糖培养基中有氧培养4小时厌氧培养2小时后培养基上清中IL-10、IL-22、Amuc_1100的表达量如表25所示。
表25 实验所用菌株IL-10、IL-22、Amuc_1100的表达量
Figure PCTCN2022134672-appb-000163
Figure PCTCN2022134672-appb-000164
实施例9：水杨酸诱导表达IL-10的质粒构建
水杨酸诱导启动子Psal、核酶(sTRSV-HHRz)、核糖体结合位点(RBS)、水杨酸诱导启动子的抑制子表达盒5’-PlacIQ-NahRAM-ter-3’等由金斯瑞(GeneScript)在克隆质粒(例如pUC57)上合成。不含有启动子的IL-10表达盒5’-USP45_IL-10_rrnB_T1_T7Te-3’序列以CBT4084菌株基因组为模板进行扩增，Psal_sTRSV-HHRz_RBS和PlacIQ_NahRAM_ter以合成质粒为模板扩增，插入位点maeB的LHA和RHA以EcN的基因组为模板扩增，maeB位点的sgRNA以质粒pCBT003_maeB_sgRNA为模板扩增，质粒骨架以pCBT010为模板扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此可以使用试剂盒ClonExpress Ultra One Step Cloning Kit(Vazyme)连接起来，形成包含一端与LHA连接的表达盒5’-Psal-sTRSV HHRz_RBS_USP45_IL-10_rrnB_T1_T7Te-3’、另一端与RHA连接的表达盒5’-PlacIQ-NahRAM-ter-3’(序列如SEQ ID NO:243所示)以及maeB位点sgRNA的质粒pCBT010_maeB_Psal_IL-10。将质粒pCBT010_maeB_Psal_IL-10转化入含有pCBT001的感受态细胞中，得到的单菌落用插入位点的验证引物进行扩增，通过扩增条带的大小筛选IL-10成功插入基因组的克隆。IL-10的产量用ELISA试剂盒(义翘神州，KIT10947A)检测。
PlacIQ-NahRAM-ter序列(SEQ ID NO:243)
Figure PCTCN2022134672-appb-000165
Figure PCTCN2022134672-appb-000166
表26 实施例9所用表达菌株的各元件信息
Figure PCTCN2022134672-appb-000167
菌株CBT4113在LB培养基中有氧培养3小时后添加不同浓度水杨酸钠继续培养1-4小时后培养基上清中IL-10的表达量如图5所示。结果显示通过调节诱导剂的浓度和诱导时间可以实现对IL-10表达量的精准调控。
实施例10：其他细胞因子的表达
此外，申请人还尝试选择了其他多种同样具有免疫调节作用的细胞因子，例如IL-23(IL-12p40_linker1_IL-23p19)、IL-17A、IL-19、IL-35(EBI3_linker2_IL-12p35)、IL-37和TGF-β，并构建工程菌以期尝试研究其体内效果。具体过程如下：、IL-23(IL-12p40_linker1_IL-23p19)、IL-17A、IL-19、IL-35(EBI3_linker2_IL-12p35)、IL-37、TGF-β编码序列以合成的质粒为模板扩增，包含水杨酸诱导启动子Psal、核酶(sTRSV-HHRz)、核糖体结合位点(RBS)、分泌信号肽USP45的5’-Psal_sTRSV-HHRz_RBS_USP45-3’序列、包含转录终止子rrnB_T1_T7Te以及水杨酸调控元件PlacIQ-NahRAM-ter的5’-rrnB_T1_T7Te_PlacIQ_NahRAM_ter-3’的质粒骨架序列以质粒pCBT010_maeB_Psal_IL-10为模板进行扩增。用于扩增这些片段的PCR引物彼此具有15-20bp的同源序列，因此可以使用试剂盒ClonExpress Ultra One Step Cloning Kit(Vazyme)连接起来，形成包含表达盒5’-Psal-sTRSV HHRz_RBS_USP45_细胞因子_rrnB_T1_T7Te-3’、以及表达盒pCBT010_Psal_IL-23、pCBT010_Psal_IL-17A、pCBT010_Psal_IL-19、pCBT010_Psal_IL-35、pCBT010_Psal_IL-37、pCBT010_Psal_TGF-β。将这些质粒分别底盘菌CBT4114(基因型见表XX)中转化感受态细胞中，得到的单菌落用质粒的验证引物进行扩增，通过扩增条带的大小验证质粒的成功转入。细胞因子的产量用ELISA试剂盒检测，本实施例所使用的试剂盒如表27所示。
表27 细胞因子检测试剂盒
品牌 货号 名称
联科生物 EK1177-48 Human IL-17AF ELISA Kit
联科生物 EK123-48 Human IL-23 ELISA Kit
Abclonal RK00175 Human IL-19 ELISA kit
联科生物 EK135-48 Human IL-35 ELISA kit
Abclonal RK00117 Human IL-37 ELISA kit
联科生物 EK981-48 Human/Mouse/Rat TGF-β1 ELISA Kit
含有这些细胞因子表达质粒的菌株在LB培养基中有氧培养4小时添加100μM水杨酸钠继续培养2小时后上清和胞内的细胞因子含量如表28所示。
表28 各菌株在水杨酸诱导下的细胞因子产量
Figure PCTCN2022134672-appb-000168
结果意外的发现，上述细胞因子的表达分泌量都过低，难以达到起效浓度，推测EcN可能对这些蛋白的耐受力较差或者上述蛋白表达后并不稳定，因此上述因子不适合在EcN中进行表达分泌。
实施例11：T细胞移植诱导的小鼠肠炎(IBD)模型药效研究
本实验所用的小鼠肠炎通过向严重复合型免疫缺陷(SCID)小鼠(缺乏T细胞和B细胞)注射纯化的CD4 +CD45RB high细胞(不含有调节T细胞)诱导形成。Balb/C小鼠(20 g，8-10周龄)用于制备CD4 +CD45RB low和CD4 +CD45RB high细胞，RAG1 -/-小鼠(20g，8-10周龄)用于接收T细胞转移。RAG1 -/-小鼠被随机分为11组，每组10只。第1组为健康组，接受CD4 +CD45RB low细胞，第2-11组为疾病组，接受CD4 +CD45RB high细胞。T细胞移植诱导当天为第0天，从第14天开始给药。在LB中有氧培养4小时的EcN、CBT4084、CBT4095、CBT4096、CBT4088、CBT4098、CBT4110、CBT4111菌液浓缩重悬于0.2M碳酸氢钠+1％w/v葡萄糖水溶液中。第2组为阳性给药组，在第14-37天每7天腹腔注射TNF-α抗体一次，同时每天灌胃100μL EcN菌悬液。EcN、CBT4084、CBT4095、CBT4096、CBT4088、CBT4098、CBT4110、CBT4111的100μL菌悬液分别灌胃给第3-11组，每天一次，给药量为每只小鼠2.5×10 9CFU。第41天杀小鼠并测量肠子的长度，同时做HE染色病理分析。
在各实验组中，T细胞移植诱导的小鼠肠炎模型药效终点结肠长度如图6A所示。其中，CBT4096单独表达Amuc_1100；CBT4084单独表达IL-10；CBT4088同时表达IL-10和Amuc_1100；CBT4095单独表达IL-22；CBT4098同时表达IL-22和Amuc_1100；CBT4110同时表达IL-10和IL-22；CBT4111同时表达IL-10、IL-22和Amuc_1100。结果显示，无论单独表达还是组合表达的工程菌对与保持肠炎模型小鼠的结肠长度皆有效果，而统计分析显示，所有组合表达的工程菌的结肠长度均显著长于对照组(p<0.05)且与阳性对照药TNF-α抗体的效果有可比性，说明在T细胞移植诱导的小鼠肠炎模型中组合表达的工程菌治疗效果优于单独表达的工程菌。
EcN对照组、同时表达IL-10和IL-22的CBT4110小鼠结肠HE染色样本如图6B所示，CBT4110小鼠的结肠炎症情况显著好于对照组。
实施例12：DSS诱导的小鼠肠炎(IBD)模型药效研究
除了实施例11的小鼠肠道模型外，申请人还在DSS诱导的小鼠肠炎模型中进一步验证工程菌对小鼠肠炎的治疗效果。具体地，Balb/C小鼠(20g，C57/B6小鼠50只，8周龄，18-20g，雌性。随机分成10组模型组，每组5只小鼠，于第0天开始饮用2.5％DSS，4天换成正常水3天，共4轮DSS诱导。第1组为模型对照组，不给药，第2组为阳性药组，从第0天至第27天每天通过灌胃给阳性药CsA，给药量为25mpk。在LB中有氧培养4小时的EcN、CBT4096、CBT4088、CBT4098、CBT4110菌液浓缩重悬于0.2M碳酸氢钠+1％w/v葡萄糖水溶液中。EcN、CBT4096、CBT4088、CBT4098、CBT4110的100μL菌悬液分别灌胃给第3-10组，每天一次，给药量为每只小鼠2.5×10 9CFU，于第0天开始给药至第27天结束，第28天为实验终点，杀小鼠并测量肠子的长度。
DSS诱导的小鼠肠炎模型体重变化、DAI评分(Disease activity index，疾病活动指数，也是常用的评价肠炎的指标)及药效终点结肠长度如图7所示。与EcN对照组相比，工程 菌CBT4096、CBT4088、CBT4098、CBT4110组的结肠长度均显著增长(p<0.05)(图7C)，其中在维持体重(图7A)和降低DAI评分(图7B)方面，CBT4110具有更显著优势(p<0.001)。
实施例13：GvHD模型药效研究
BALB/c(H-2d)小鼠和C57BL/6(H-2b)小鼠(6-8周龄)分别用作供体和受体.骨髓移植(BMT)的当天为第0天，辐照的当天为第-1天。第1组有7只同源移植小鼠，第2-10组有9只同源移植小鼠，第11组有7只只辐照不移植的小鼠。在LB中有氧培养4小时的EcN、CBT4084、CBT4095、CBT4096、CBT4088、CBT4098、CBT4110、CBT4111菌液浓缩重悬于0.2M碳酸氢钠+1％w/v葡萄糖水溶液中。从第-1天开始到第30天，第1组、第2组、第10组和第11组的小鼠分别灌胃给药含有2.5×10 9CFU的100μL EcN细胞悬液，同时第10组的小鼠每天注射给药20mg/kg泼尼松，CBT4084、CBT4095、CBT4096、CBT4088、CBT4098、CBT4110、CBT4111的100μL菌悬液分别灌胃给第3-9组，每天记录小鼠的存活率。
GvHD动物模型的存活率如图8所示。结果表明，在第20天时，单独表达Amuc_1100的CBT4096、同时表达IL-22和Amuc_1100的CBT4098、同时表达IL-10和IL-22的CBT4110、同时表达IL-10、IL-22和Amuc_1100的CBT4111组的小鼠都与阳性药组有相同甚至更高的存活率。在第25天时，同时表达IL-10和Amuc_1100的CBT4088、同时表达IL-22和Amuc_1100的CBT4098、同时表达IL-10和IL-22的CBT4110、同时表达IL-10、IL-22和Amuc_1100的CBT4111组的小鼠都比单独表达IL-10的CBT4084、单独表达IL-22的CBT1095、单独表达Amuc_1100的CBT4096组的小鼠有更高的存活率，说明在GvHD模型中组合表达的工程菌都不同程度的优于单独表达的工程菌。而在第30天实验结束时，同时表达IL-22和Amuc_1100的CBT4098组的存活率仍然接近阳性药组。由此可见，表达IL-22和Amuc_1100的工程菌对GvHD具有最显著的治疗作用。
实施例14：SLE模型药效研究
首先评价了同时表达两种蛋白的工程菌与分别单独表达一种蛋白的工程菌在SLE模型中的治疗效果，具体地，SLE模型采用MRL/MpJ-Faslpr小鼠，MRL/MpJ小鼠作为阴性对照，给药时间为8周。在LB中有氧培养4小时的EcN、CBT4084、CBT4096、CBT4088菌液浓缩重悬于0.2M碳酸氢钠+1％w/v葡萄糖水溶液中。实验小鼠共分为6组，第1组为阴性对照组，包含5只MRL/MpJ小鼠，第2组为阳性给药组，包含10只MRL/MpJ-Faslpr小鼠，每天灌胃给9mg/kg泼尼松以及100μL EcN菌悬液。第3-6组为给药组，各包含10只MRL/MpJ-Faslpr小鼠，每天灌胃给100μL EcN、CBT4084、CBT4096、CBT4088菌悬液，给药量为每只小鼠2.5×10 9CFU。实验结束后，取肾脏制作切片，评估肾小管损伤情况。
肾小管损伤情况如图9A所示，与对照EcN相比，单独表达IL-10的工程菌CBT4084和单独表达Amuc_1100的工程菌CBT4096并不能显著减轻肾小管的损伤，但是同时表达IL-10和Amuc_1100的工程菌CBT4088能够显著减轻肾小管的损伤，说明在SLE模型中，同时表达IL-10和Amuc_1100产生了协同作用。
接下来，进一步比较了同时表达IL-10和Amuc_1100(CBT4088)、IL-10和IL-22(CBT4110)、以及同时表达IL-10、IL-22和Amuc_1100的工程菌(CBT4111)的治疗效果。具体地，实验小鼠共分为5组，第1组为阴性对照组，包含6只MRL/MpJ小鼠，第2-5组为给药组，各包含10只MRL/MpJ-Faslpr小鼠，每天灌胃给100μL EcN、CBT4088、CBT4110、CBT4111菌悬液，给药量为每只小鼠1.0×10 10CFU。实验终点检测血清中抗双链DNA抗体IgG浓度，尿液中白蛋白浓度，并取肾脏制作切片进行PAS染色，评估肾小球损伤情况。
首先，检测了SLE的血清学标志物抗双链DNA抗体IgG在小鼠血清中的浓度变化，结果如图9B所示，CBT4088和CBT4110均可以显著降低模型小鼠血清中抗双链DNA抗体IgG浓度(p<0.01)，而CBT4088的降幅最大。肾脏PAS染色显示EcN组中疾病小鼠肾小球毛细血管内皮细胞和系膜细胞显著增生，球囊腔变窄或闭塞，肾小球功能丧失，而CBT4088以及CBT4111的病理切片显示有明显好转(图9D)。不过仅CBT4088能够显著降低尿液中白蛋白浓度(肾脏损伤程度的指标)(p<0.01)(图9C)。以上结果表明，同时表达IL-10和Amuc_1100的工程菌CBT4088在治疗SLE方面具有最佳的效果。
实施例15：CIA模型药效研究
胶原蛋白诱导的关节炎(collagen-induced athritis，CIA)是一种实验性自发性免疫疾病，用二型胶原蛋白免疫啮齿动物易感品系(大鼠和小鼠)可以诱导出CIA。经免疫的动物可出现一种自发性免疫介导的多发性关节炎。CIA模型采用DBA/1小鼠。在第0天当天，所有DBA/1小鼠用2％-5％异氟烷麻醉后，在距离身体2-3cm尾根部皮下注射50μL胶原乳剂。三周后，即第21天，于尾根部同法注射相同体积胶原乳剂攻击。第二次胶原乳剂攻击当天将造模的动物按照体重随机分成6组，每组5或10只小鼠，分组当天开始给药。在LB中有氧培养4小时的EcN、CBT4088、CBT4098、CBT4110、CBT4111菌液浓缩重悬于0.2M碳酸氢钠+1％w/v葡萄糖水溶液中。第1-5组各包含10只小鼠，每天灌胃给100μL EcN、CBT4088、CBT4098、CBT4110、CBT4111菌悬液，给药量为每只小鼠1.5×10 10细胞，第6组为阳性给药组，包含5只小鼠，每天灌胃给100μL EcN以及0.2mg/kg地塞米松。给药时间为36天，每周两次观察每组动物四肢的关节炎发病情况并进行评分，同时测量前后足垫的厚度。
小鼠前后足垫的厚度以及四肢的关节炎发病情况评分如图10所示，同时表达IL-10和 Amuc_1100的工程菌CBT4088、同时表达IL-22和Amuc_1100的工程菌CBT4098、同时表达IL-10和IL-22的工程菌CBT4110以及同时表达IL-10、IL-22和Amuc_1100的工程菌CBT4111都可以一定程度的减轻疾病的严重程度，其中，同时表达IL-10和Amuc_1100的CBT4088对于足垫厚度(图10A)以及疾病评分(图10B)的改善最为显著(p<0.05)。
实施例16：OVA诱导的哮喘模型药效研究
哮喘是气道的慢性炎症性疾病，其特征是二型T辅助淋巴细胞(T helper 2cell，Th2)、嗜酸性粒细胞数量的增加以及气道炎症的出现。它伴有高水平的血清免疫球蛋白E(IgE)，以及由过敏原特异性Th2细胞在肺内产生的白细胞介素4(IL-4)、白细胞介素5(IL-5)和白细胞介素13(IL-13)。气道炎症与气道和肺组织中嗜酸性粒细胞、中性粒细胞以及T和B淋巴细胞的浸润有关。
在这项研究中，卵清蛋白(OVA)诱导的哮喘模型采用BALB/c雌性小鼠。将小鼠按体重随机分为7组，给药周期为31天。在LB中有氧培养4小时的EcN、CBT4088、CBT4098、CBT4110、CBT4111菌液浓缩重悬于0.2M碳酸氢钠+1％w/v葡萄糖水溶液中。第1-5组各10只小鼠，每天灌胃给100μL EcN、CBT4088、CBT4098、CBT4110、CBT4111菌悬液，给药量为每只小鼠1.5×10 10细胞。第6组为阳性给药组，包含6只小鼠，每天灌胃给100μL EcN菌悬液，其中第27至31天额外给阳性药地塞米松1mg/kg。第7组为未诱导致敏对照组，包含5只小鼠，每天灌胃给100μL EcN菌悬液。第1-6组小鼠在第1天和第14天腹腔注射100μL致敏溶液(含有20μg卵清蛋白和2mg明矾)，作为对照第7组小鼠注射100μL PBS溶液。在第28、29、30天，第1-6组的小鼠用溶于PBS(pH＝7.2)的1％OVA溶液攻击30分钟，作为对照第7组的小鼠用PBS(pH＝7.2)溶液进行攻击。
在第32天，所有动物都被安乐死。肺通过气管插管用PBS(pH＝7.2)溶液进行灌洗。肺泡灌洗液(BALF)中的细胞总数由血细胞计数器计数。使用细胞离心涂片和瑞氏-吉姆萨染色后，从细胞离心制备物中进行嗜酸性粒细胞、巨噬细胞、中性粒细胞、淋巴细胞计数。
肺泡灌洗液(BALF)中的细胞计数结果如图11A～11E所示，与第1组只给EcN的对照相比，所有给药组肺泡灌洗液(BALF)中的细胞总数、嗜酸性粒细胞数和巨噬细胞数都有所下降，其中同时表达IL-22和Amuc_1100的CBT4098可以显著降低细胞总数(p<0.01)、嗜酸性粒细胞数(p<0.05)和巨噬细胞数(p<0.05)。而同时表达IL-10和IL-22的CBT4110以及同时表达IL-10、IL-22和Amuc_1100的CBT4111也可以显著降低细胞总数和巨噬细胞数。
虽然已经参考特定实施例(其中一些是优选实施例)具体示出和描述了本公开，本领域技术人员应当理解，在不脱离本文公开的精神和范围的情况下，可以在形式和细节上进 行各种改变。

Claims (78)

  1. 一种经遗传修饰的微生物，其包含至少两种分别编码选自下组的多肽的外源基因：
    a)Amuc_1100多肽；
    b)IL-10多肽；以及
    c)IL-22多肽。
  2. 如权利要求1所述的经遗传修饰的微生物，其包含：
    a)分别编码Amuc_1100多肽和IL-10多肽的外源基因；
    b)分别编码IL-10多肽和IL-22多肽的外源基因；或者
    c)分别编码Amuc_1100多肽和IL-22多肽的外源基因。
  3. 如权利要求1所述的经遗传修饰的微生物，其包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
  4. 一种经遗传修饰的微生物，其包含编码Amuc_1100多肽、IL-10多肽或者IL-22多肽的外源基因。
  5. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述Amuc_1100多肽是野生型Amuc_1100多肽，或与野生型Amuc_1100多肽在功能上等效的功能等效物。
  6. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述IL-10多肽是野生型IL-10多肽，或与野生型IL-10多肽在功能上等效的功能等效物。
  7. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述IL-22多肽是野生型IL-22多肽，或与野生型IL-22多肽在功能上等效的功能等效物。
  8. 如权利要求5所述的经遗传修饰的微生物，其中所述功能等效物包含野生型Amuc_1100多肽的突变体、片段、融合物、衍生物或其任何组合，并至少部分保留野生型Amuc_1100的一种或多种生物功能，可选地，所述生物功能选自下组：a)调节和/或促进哺乳动物的肠道免疫系统功能，b)维持、恢复和/或增加哺乳动物的肠粘膜屏障的物理完整性，和c)激活TLR2。
  9. 如权利要求8所述的经遗传修饰的微生物，其中所述野生型Amuc_1100多肽的片段具有：
    (a)如SEQ ID NO:2所示的氨基酸序列，或具有与SEQ ID NO:2所示的氨基酸序列具有至少80％序列一致性的氨基酸序列，或
    (b)如SEQ ID NO:3所示的氨基酸序列，或与SEQ ID NO:3所示的氨基酸序列具有至少80％序列一致性的氨基酸序列。
  10. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述Amuc_1100多肽包含 SEQ ID NO:5所示的序列，或包含与SEQ ID NO:5所示的序列具有至少80％序列一致性且仍保持调节肠道免疫和/或活化toll样受体2(TLR2)的活性的氨基酸序列。
  11. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述Amuc_1100多肽在第259位的Y具有突变，所述第259位的编号是基于SEQ ID NO:5所示序列的编号；优选地，所述Amuc_1100多肽在第259位具有Y259A或Y259S突变，所述第259位的编号是基于SEQ ID NO:5所示序列的编号。
  12. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述IL-10多肽包含SEQ ID NO:11所示的序列，或包含与SEQ ID NO:11所示序列具有至少80％序列一致性且仍保持调节免疫细胞的活性的氨基酸序列。
  13. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述IL-22多肽包含SEQ ID NO:15所示的序列，或包含与SEQ ID NO:15所示序列具有至少80％序列一致性且仍保持结合IL-22受体并调节IL-22受体表达细胞的活性的氨基酸序列。
  14. 如前述任一项权利要求所述的经遗传修饰的微生物，其中所述微生物能够表达和/或分泌所述外源基因编码的多肽；或者
    所述微生物能够在人或动物的肠道中表达和/或分泌所述外源基因编码的多肽。
  15. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述外源基因包含在外源性表达盒中。
  16. 如权利要求15所述的经遗传修饰的微生物，其中所述外源性表达盒包含在质粒中，且所述质粒被引入所述微生物中并且适合在所述微生物中表达。
  17. 如权利要求15所述的经遗传修饰的微生物，其中所述外源性表达盒整合于所述经遗传修饰的微生物的基因组中。
  18. 如权利要求15所述的经遗传修饰的微生物，其中，所述微生物包含：第一外源性表达盒，其含有编码Amuc_1100多肽的核苷酸序列，以及第二外源性表达盒，其含有编码IL-10多肽的核苷酸序列；或者
    所述微生物包含：第二外源性表达盒，其含有编码IL-10多肽的核苷酸序列，以及第三外源性表达盒，其含有编码IL-22多肽的核苷酸序列；或者
    所述微生物包含：第一外源性表达盒，其含有编码Amuc_1100多肽的核苷酸序列，以及第三外源性表达盒，其含有编码IL-22多肽的核苷酸序列；或者
    所述微生物包含：第一外源性表达盒，其含有编码Amuc_1100多肽的核苷酸序列，第二外源性表达盒，其含有编码IL-10多肽的核苷酸序列，以及第三外源性表达盒，其含有编码IL-22多肽的核苷酸序列；或者
    所述微生物包含第一外源性表达盒，其含有编码Amuc_1100多肽的核苷酸序列；或者
    所述微生物包含第二外源性表达盒，其含有编码IL-10多肽的核苷酸序列；或者
    所述微生物包含第三外源性表达盒，其含有编码IL-22多肽的核苷酸序列。
  19. 如权利要求15-18中任一项所述的经遗传修饰的微生物，其中所述外源性表达盒在基因组中存在一个或更多个拷贝(例如两个、三个、四个、五个或六个等)。
  20. 如前述任一项权利要求所述的经遗传修饰的微生物，其中所述Amuc_1100多肽、IL-10多肽和/或IL-22多肽是去除了自身信号肽序列的多肽。
  21. 如权利要求20所述的经遗传修饰的微生物，所述Amuc_1100多肽在N端与第一信号肽连接，所述IL-10多肽在N端与第二信号肽连接，和/或所述IL-22多肽在N端与第三信号肽连接，优选地，所述第一信号肽、第二信号肽和/或第三信号肽能够将所述Amuc_1100多肽、所述IL-10多肽和/或所述IL-22多肽分泌到细胞外。
  22. 如权利要求20所述的经遗传修饰的微生物，其中所述第一信号肽、第二信号肽、第三信号肽可以相同或不同，可以分别包含选自由SEQ ID NO:80-122所示序列组成的氨基酸序列，或包含与所述氨基酸序列具有至少80％序列一致性的同源序列；
    优选地，其中第一信号肽、第二信号肽、和/或第三信号肽是USP45信号肽，并且所述USP45信号肽具有如SEQ ID NO:114所示的氨基酸序列或者与SEQ ID NO:114具有至少80％序列一致性的同源序列。
  23. 如权利要求21所述的经遗传修饰的微生物，其中所述第一信号肽、第二信号肽、和/或第三信号肽可以通过存在于所述微生物中的分泌系统加工；
    特别地，所述分泌系统对于所述微生物是原生或非原生的系统。
  24. 如权利要求15至23中任一项所述的经遗传修饰的微生物，其中所述外源基因与表达调控元件可操作性连接；优选地，所述表达调控元件包含启动子，更优选地，所述启动子选自下组：BBa_J23101、BBa_J23108、BBa_J23110、PfnrS、Psal、Pvan、BBa_J23119、BBa_J23102和BBa_J23114。
  25. 如权利要求24所述的经遗传修饰的微生物，其中所述表达调控元件包含核糖体结合位点(RBS)，优选地，所述核糖体结合位点的核苷酸序列如SEQ ID NO:63所示。
  26. 如权利要求24或25所述的经遗传修饰的微生物，其中所述表达调控元件包含顺反子，优选地，所述顺反子选自T7g10、BCD2、GFP和荧光素酶。
  27. 如权利要求24至26中任一项所述的经遗传修饰的微生物，其中，所述表达调控元件包含终止子，优选地，所述终止子为rrnB_T1_T7Te终止子，更优选地，所述rrnB_T1_T7Te终止子的序列如SEQ ID NO:242所示。
  28. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述经遗传修饰的微生物进一步包含对所述微生物基因组的一种或多种其它基因改造。
  29. 如权利要求28所述的经遗传修饰的微生物，其中所述一种或多种其它基因改造包括对分泌系统的工程改造和/或优化以使得至少一种外膜蛋白编码基因被删除、失活或抑制。
  30. 如权利要求29所述的经遗传修饰的微生物，其中所述外膜蛋白选自下组：lpp、mrcA、ompT、tolQ、tolR、tolA和pal。
  31. 如权利要求29所述的经遗传修饰的微生物，其中所述至少一种外膜蛋白包括：lpp、mrcA和/或ompT。
  32. 如权利要求29所述的经遗传修饰的微生物，其中所述至少一种外膜蛋白为lpp和mrcA。
  33. 如权利要求29至32中任一项所述的经遗传修饰的微生物，其中所述经遗传修饰的微生物进一步包含一种或多种过表达的分子伴侣。
  34. 如权利要求33所述的经遗传修饰的微生物，其中所述分子伴侣选自下组：dsbA、dsbC、dnaK、dnaJ、grpE、groES、groEL、tig、fkpA、surA或上述任意两种或更多种(例如三种、四种、五种、六种、七种、八种、九种或者十种)分子伴侣的组合。
  35. 如权利要求28所述的经遗传修饰的微生物，所述一种或多种其它基因改造包括至少一个营养缺陷相关基因的失活或缺失。
  36. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述微生物是一种或多种选自由以下组成的群组的物质的营养缺陷体：尿嘧啶、胸腺嘧啶、二氨基庚二酸、亮氨酸、组氨酸、色氨酸、赖氨酸、甲硫氨酸、腺嘌呤和非野生型氨基酸。
  37. 如权利要求36所述的经遗传修饰的微生物，其中所述非野生型氨基酸选自由以下组成的群组：l-4,4′-联苯丙氨酸、对乙酰基-l-苯丙氨酸、对碘-l-苯丙氨酸和对叠氮基-l-苯丙氨酸。
  38. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述微生物包含细菌、古细菌、真菌或藻类。
  39. 如权利要求38所述的经遗传修饰的微生物，其中所述真菌包括酵母或丝状真菌。
  40. 如前述任一权利要求所述的经遗传修饰的微生物，其中所述微生物包含益生微生物体或非病原性微生物体。
  41. 如权利要求40所述的经遗传修饰的微生物，其中所述益生微生物体为益生细菌或益生酵母。
  42. 如权利要求41所述的经遗传修饰的微生物，其中所述益生细菌选自由以下组成的群组：拟杆菌(Bacteroides)、双歧杆菌(Bifidobacterium)、梭菌(Clostridium)、埃希氏菌(Escherichia)、乳杆菌(Lactobacillus)和乳球菌(Lactococcus)。
  43. 如权利要求42所述的经遗传修饰的微生物，其中所述益生细菌属于埃希氏菌属。
  44. 如权利要求42所述的经遗传修饰的微生物，其中所述益生细菌是大肠杆菌(Escherichia coli)Nissle 1917菌株(EcN)。
  45. 如权利要求41所述的经遗传修饰的微生物，其中所述益生细菌是人类肠道正常存在的细菌。
  46. 如权利要求41所述的经遗传修饰的微生物，其中所述益生酵母选自由以下组成的群组：酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Candida utilis)、乳酸克鲁维酵母(Kluyveromyces lactis)和卡氏酵母(Saccharomyces carlsbergensis)。
  47. 一种经遗传修饰的微生物的组合，其包含至少两种(例如两种、三种或四种等)不同的如权利要求1-46中任一项所述的经遗传修饰的微生物。
  48. 一种包含至少一个重组表达盒的核苷酸序列，其包含i)至少一种或至少两种分别编码Amuc_1100多肽、IL-10多肽和/或IL-22多肽的外源基因，以及ii)可操作地连接所述至少一种或至少两种外源基因的一个或多个调控元件。
  49. 如权利要求48所述的核苷酸序列，其包含分别编码下述多肽的外源基因：a)Amuc_1100多肽和IL-10多肽；b)IL-10多肽和IL-22多肽，或c)Amuc_1100多肽和IL-22多肽。
  50. 如权利要求48所述的核苷酸序列，其包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
  51. 如权利要求48所述的核苷酸序列，其中所述核苷酸序列包含：第一重组表达盒，所述第一重组表达盒包含编码Amuc_1100多肽的核苷酸序列，以及与之可操作性连接的一个或多个调控元件；第二重组表达盒，所述第二重组表达盒包含编码IL-10多肽的核苷酸序列，以及与之可操作性连接的一个或多个调控元件；和/或第三重组表达盒，所述第三重组表达盒包含编码IL-22多肽的核苷酸序列，以及与之可操作性连接的一个或多个调控元件。
  52. 如权利要求51所述的核苷酸序列，其中所述编码Amuc_1100多肽的核苷酸序列编码自身信号肽被替换为第一信号肽的Amuc_1100多肽，所述编码IL-10的核苷酸序列编码自身信号肽被替换为第二信号肽的IL-10多肽，和/或所述编码IL-22多肽的核苷酸序列编码自身信号肽被替换为第三信号肽的IL-22多肽。
  53. 如权利要求52所述的核苷酸序列，其中所述第一信号肽、第二信号肽、第三信号肽可以相同或不同，可选地，所述第一信号肽、第二信号肽、和/或第三信号肽是USP45信号肽，并且所述USP45信号肽具有如SEQ ID NO:114所示的氨基酸序列，或与SEQ ID NO:114具有至少80％序列一致性的同源序列。
  54. 如权利要求48所述的核苷酸序列，其中所述一个或多个调控元件包含启动子、核糖体结合位点(RBS)、顺反子、终止子或其任何组合。
  55. 如权利要求54所述的核苷酸序列，其中，
    所述启动子选自：BBa_J23101、BBa_J23108、BBa_J23110、PfnrS、Psal、Pvan、BBa_J23119、BBa_J23102，或BBa_J23114；
    所述核糖体结合位点是合成型，其核苷酸序列如SEQ ID NO:63所示；
    所述顺反子选自下组：T7g10、BCD2、GFP、荧光素酶；和/或
    所述终止子为rrnB_T1_T7Te终止子，优选地，所述终止子的序列如SEQ ID NO:242所示。
  56. 如权利要求48所述的核苷酸序列，其中所述重组表达盒适合于在益生细菌或益生酵母中表达。
  57. 如权利要求48所述的核苷酸序列，其包含SEQ ID NO:139-163和235中任一所示的核苷酸序列，或其组合。
  58. 一种组合物，其中所述组合物包含：
    (a)作为活性成分的如权利要求1-46中任一项所述的经遗传修饰的微生物或如权利要求47所述的经遗传修饰的微生物的组合；和
    (b)生理学上或药理学上可接受的载体。
  59. 如权利要求58所述的组合物，其中所述的组合物为药物组合物。
  60. 如权利要求59所述的组合物，其中所述药物组合物为口服制剂。
  61. 如权利要求59所述的组合物，其中所述的药物组合物为液态制剂、固态制剂或半固态制剂。
  62. 如权利要求59所述的组合物，其中所述的药物组合物的剂型选自下组：粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂及舌下含片。
  63. 如权利要求60所述的组合物，其中所述的液态制剂选自下组：溶液制品或悬浮液制品。
  64. 如权利要求59所述的组合物，其中所述药物组合物包含1×10 8–1×10 12CFU的如权利要求1-46中任一项所述的经遗传修饰的微生物或如权利要求47所述的经遗传修饰的微生物的组合。
  65. 如权利要求58所述的组合物，其中所述组合物是可食用组合物。
  66. 如权利要求58所述的组合物，其中所述组合物是益生菌组合物。
  67. 如权利要求58所述的组合物，其中所述组合物是食品增补剂。
  68. 一种如权利要求1-46中任一所述的经遗传修饰的微生物、如权利要求47所述的 经遗传修饰的微生物的组合，或如权利要求58所述的组合物在用于制备治疗或预防炎症性疾病或自身免疫疾病的药物中的用途。
  69. 如权利要求68所述的用途，其中所述的自身免疫疾病选自下组：炎症性肠病、移植物抗宿主病(GvHD)、系统性红斑狼疮(SLE)、关节炎、哮喘，或其组合。
  70. 如权利要求69所述的用途，其中所述自身免疫疾病包括炎症性肠病包括克罗恩病或溃疡性结肠炎，和/或所述关节炎包括类风湿性关节炎、骨关节炎、银屑病性关节炎或青少年特发性关节炎，和/或所述哮喘包括过敏性哮喘或中性粒细胞性哮喘。
  71. 一种如权利要求1-46中任一项所述的经遗传修饰的微生物、或如权利要求47所述的经遗传修饰的微生物的组合，或如权利要求58-69中任一项所述的组合物在制备用于改善炎症性疾病或自身免疫疾病治疗药物的治疗效果的药物中的用途。
  72. 一种如权利要求1-46中任一项所述的经遗传修饰的微生物的制备方法，其中所述的制备方法包括步骤：
    向微生物中引入权利要求48-57中任一项所述的核苷酸序列，以使得所述核苷酸序列中的外源基因可以在所述微生物中表达。
  73. 一种在有需要的个体中治疗或预防炎症性疾病或自身免疫疾病的方法，其包括：
    向所述个体施用有效量的如权利要求1-46中任一项所述的经遗传修饰的微生物、或如权利要求47所述的经遗传修饰的微生物的组合，或如权利要求58-69中任一项所述的组合物。
  74. 如权利要求73所述的方法，其中所述自身免疫疾病选自下组：炎症性肠病、移植物抗宿主病(GvHD)、系统性红斑狼疮、关节炎、哮喘，或其组合。
  75. 如权利要求73所述的方法，所述自身免疫疾病包括炎症性肠病包括克罗恩病或溃疡性结肠炎，和/或所述关节炎包括类风湿性关节炎、骨关节炎、银屑病性关节炎或青少年特发性关节炎，和/或所述哮喘包括过敏性哮喘或中性粒细胞性哮喘。
  76. 一种在正在接受药物治疗的个体中改善药物治疗效果的方法，所述药物包括治疗炎症性疾病或自身免疫疾病的药物，所述方法包括：
    向所述个体施用有效量的如权利要求1-46中任一项所述的经遗传修饰的微生物、或如权利要求47所述的经遗传修饰的微生物的组合，或如权利要求58-67中任一项所述的组合物。
  77. 一种经遗传修饰的微生物在制备用于治疗炎症性疾病或自身免疫疾病的药物中的用途，其中所述的炎症性疾病或自身免疫疾病选自下组：炎症性肠病(IBD)、移植物抗宿主病(GvHD)、系统性红斑狼疮(SLE)、关节炎和哮喘；
    其中所述经遗传修饰的微生物包含至少一种、至少两种或至少三种分别编码选自下组 的多肽的外源基因：
    a)Amuc_1100多肽；
    b)IL-10多肽；和
    c)IL-22多肽。
  78. 如权利要求77所述的用途，其中：
    (1)所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-10多肽的外源基因；
    (2)所述经遗传修饰的微生物包含分别编码IL-10多肽和IL-22多肽的外源基因；
    (3)所述经遗传修饰的微生物包含分别编码Amuc_1100多肽和IL-22多肽的外源基因；
    (4)所述的经遗传修饰的微生物包含编码IL-10多肽的外源基因；
    (5)所述的经遗传修饰的微生物包含编码Amuc_1100多肽的外源基因；
    (6)所述的经遗传修饰的微生物包含编码IL-22多肽的外源基因；或
    (7)所述的经遗传修饰的微生物包含分别编码Amuc_1100多肽、IL-10多肽和IL-22多肽的外源基因。
PCT/CN2022/134672 2021-11-26 2022-11-28 一种经遗传修饰的微生物及其应用 Ceased WO2023093883A1 (zh)

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