Modified covalently cross-linked pili and recombinant bacteria comprising same

Abstract

The present disclosure provides a fusion polypeptide comprising a carrier protein and a polypeptide of interest wherein the polypeptide of interest is fused to or inserted into a terminal of the carrier protein, and wherein the carrier protein is a pilin from covalently crosslinked pili (CLP) of a microorganism. The present disclosure also provides a recombinant cell comprising the modified CLP, and the modified CLP, the modified CLP comprising the fusion polypeptide.

Description

Technical Field

[0001] The present disclosure relates to bioengineering. In particular, the present disclosure relates to engineered bacteria, such as Corynebacterium glutamicum, comprising modified covalently-linked pili (CLP). Background Art

[0002] Engineered living materials (ELMs) involve engineered living functional materials with unique "living" properties, such as autonomous growth, self-repair, and environmental responsiveness found only in natural living functional materials. A wide range of remarkable ELMs have been developed for applications in biosensors, bioremediation, biomedicine, biomanufacturing, wearable devices, and electronic devices. Depending on the source of their structural components, ELMs can be produced by simultaneously manufacturing materials using engineered cells and incorporating new functions into them (called self-assembled living functional materials or bio-ELMs) or by embedding living cells in organic or inorganic matrices (called hybrid living functional materials). Self-organizing living functional materials aim to reproduce the autonomous, adaptable, and multifunctional properties of natural living functional materials and provide opportunities for acquiring new capabilities using engineered biological systems.

[0003] Despite the progress made in ELM, the further development and application of self-organizing living functional materials remain challenging due to the lack of engineered chassis and the limited accessibility of programmable endogenous biopolymers in microorganisms, especially non-pathogenic bacteria. Currently, there are only model microbial systems such as Escherichia coli and Bacillus subtilis and their extracellular amyloid fibers, and several non-model systems, including K. rhaeticus, which produces bacterial cellulose, Caulobacter crescentus, which contains S-layer lattice proteins, and Pantoea agglomerans, a dominant bacterial component of fungal natural raw materials (Tang, T.-C. et al., Materials design by synthetic biology. Nat. Rev. Mater. 6, 332-350, 2021; Caro-Astorga, J. et al., Bacterial cellulose spheroids as building blocks for 3D and patterned living materials and for regeneration. Nat. Commun. 12, 1-9, 2021; Charrier, M. et al. Engineering the S-layer of Caulobacter crescentus as a foundation for stable, high-density, 2Dliving materials. ACS Synth. Biol. 8, 181-190, 2018; and Huang, J. et al. Programmable and printable Bacillus subtilis biofilms as engineered living materials. Nat. Chem. Biol. 15, 34-41, 2019).

[0004] Some Gram-positive bacteria include covalently cross-linked pili (CLP). Different from the non-covalently cross-linked pili produced in Gram-negative bacteria (Ramirez, NA et al., New paradigms of pilus assembly mechanisms in gram-positive actinobacteria. Trends Microbiol. 28, 999-1009, 2020). CLP monomer subunits are usually connected by intermolecular isopeptide bonds catalyzed by sortase (sortase), with huge tensile strength (McConnell, SA et al., Protein labeling via a specific lysine-isopeptide bond using the pilin polymerizing sortase from Corynebacterium diphtheriae. J. Am. Chem. Soc. 140, 8420-8423, 2018). In addition, CLP subunits contain autocatalytic intramolecular isopeptide bonds, which are less susceptible to proteolytic cleavage and can dissipate mechanical energy (Ramirez, NA et al., 2020), thus conferring CLP robustness. In addition, several pilin proteins in the CLP structure of different strains contain additional disulfide bonds, further enhancing stability (Kang, HJ et al., The Corynebacterium diphtheriae shaft pilin SpaAis built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds. Proc. Natl. Acad. Sci. USA 106, 16967-16971, 2009).

[0005] Therefore, there is still a need to develop new chassis for ELMs (e.g., self-organizing living functional materials), preferably CLP-forming bacteria. Summary of the Invention

[0006] Based on the discovery of the biosynthetic gene cluster (BGC) for covalently cross-linked fimbriae (CLP) fibers in the industrial workhorse Corynebacterium glutamicum, the inventors developed an integrated technology platform for ELM.

[0007] In a first aspect, the present disclosure provides a fusion polypeptide comprising a carrier protein and a polypeptide of interest, wherein the polypeptide of interest is fused to a terminus of the carrier protein or inserted into the carrier protein, and wherein the carrier protein is a pilus protein of covalently cross-linked pili (CLP) from a microorganism.

[0008] In some embodiments, the microorganism is a gram-positive bacterium, such as a bacterium selected from the group consisting of Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lacticaseibacillus paracasei, Bacillus thuringiensis, and Lactococcus paracasei; preferably Corynebacterium glutamicum. In some embodiments, the carrier protein is a major pilin.

[0009] In some embodiments, the polypeptide of interest is fused to the terminus of a carrier protein. In some embodiments, the polypeptide of interest is fused to the N-terminus of a carrier protein.

[0010] In some embodiments, the polypeptide of interest is inserted into a carrier protein. In some embodiments, the polypeptide of interest is inserted into a loop of a carrier protein.

[0011] In some embodiments, the carrier protein is a backbone pilin protein from Corynebacterium glutamicum. In some embodiments, the polypeptide of interest is inserted into the M domain of the backbone pilin protein. In some embodiments, the polypeptide of interest replaces the M domain of the backbone pilin protein or a portion thereof. In some embodiments, the carrier protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein, or inserted between the positions corresponding to G215 and L216 of SEQ ID NO: 1, between the positions corresponding to G236 and E237 of SEQ ID NO: 1, or between the positions corresponding to G336 and T337 of SEQ ID NO: 1.

[0012] In some embodiments, the carrier protein comprises amino acids 35-509 of SEQ ID NO: 1. In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein or inserted between G215 and L216, between G236 and E237, or between G336 and T337 of SEQ ID NO: 1.

[0013] In a second aspect, the present disclosure provides polynucleotides encoding the fusion polypeptides of the present disclosure, vectors comprising the polynucleotides, and host cells comprising the polypeptides, the polynucleotides, or the vectors of the present disclosure.

[0014] In a third aspect, the present disclosure provides a recombinant cell comprising a polynucleotide encoding a fusion polypeptide, wherein the fusion polypeptide comprises a carrier protein and a polypeptide of interest, wherein the polypeptide of interest is fused to a terminus of the carrier protein or inserted into the carrier protein, wherein the carrier protein is a pilin of a CLP, and wherein the recombinant cell is capable of expressing the polynucleotide and displaying a modified CLP comprising the fusion polypeptide.

[0015] In some embodiments, the recombinant cell is a Gram-positive bacterium, such as a bacterium selected from Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lactobacillus paracasei, Bacillus thuringiensis and Lactobacillus paracasei; preferably Corynebacterium glutamicum. In some embodiments, the carrier protein is a backbone pilin protein.

[0016] In some embodiments, the polypeptide of interest is fused to the terminus of a carrier protein. In some embodiments, the polypeptide of interest is fused to the N-terminus of a carrier protein.

[0017] In some embodiments, the polypeptide of interest is inserted into a carrier protein. In some embodiments, the polypeptide of interest is inserted into the turn region of a carrier protein.

[0018] In some embodiments, the carrier protein is a backbone pilin protein from Corynebacterium glutamicum. In some embodiments, the polypeptide of interest is inserted into the M domain of the backbone pilin protein. In some embodiments, the polypeptide of interest replaces the M domain of the backbone pilin protein or a portion thereof. In some embodiments, the carrier protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein, or inserted between the positions corresponding to G215 and L216 of SEQ ID NO: 1, between the positions corresponding to G236 and E237 of SEQ ID NO: 1, or between the positions corresponding to G336 and T337 of SEQ ID NO: 1.

[0019] In some embodiments, the carrier protein comprises amino acids 35-509 of SEQ ID NO: 1, and the polypeptide of interest is fused to the N-terminus of the carrier protein or inserted between G215 and L216, between G236 and E237, or between G336 and T337 of SEQ ID NO: 1.

[0020] In some embodiments, the recombinant cell comprises two or more polynucleotides, each encoding two or more fusion polypeptides, each fusion polypeptide comprising a different polypeptide of interest, and the modified CLP comprises these two or more polypeptides.

[0021] In a fourth aspect, the present disclosure provides a method for preparing the recombinant cell of the present disclosure, comprising introducing a polynucleotide encoding the fusion polypeptide of the present disclosure into a host cell derived from a microorganism having a CLP.

[0022] In some embodiments, the host cell's native backbone pilin protein is knocked out. In some embodiments, the method comprises the step of knocking out the native backbone pilin protein.

[0023] In a fifth aspect, the present disclosure provides a modified covalently cross-linked pilus (CLP) comprising a plurality of fusion polypeptides of the present disclosure.

[0024] In a sixth aspect, the present disclosure provides a method for preparing a modified CLP, comprising the following steps:

[0025] a) providing a fusion polypeptide of the present disclosure; and

[0026] b) providing sortase activity.

[0027] In some embodiments, the fusion polypeptide is provided by transcribing and / or translating a polynucleotide of the present disclosure. In some embodiments, the sortase activity is provided by transcribing and / or translating one or more polynucleotides encoding a sortase. In some embodiments, the sortase is encoded by a gene that is identified as being present in the same cluster as a gene encoding a carrier protein in nature. In some embodiments, the sortase is a Class C sortase, such as srtC1 and / or srtC2, preferably wherein srtC1 and srtC2 are encoded by genes from the same cluster. In some embodiments, the method is an in vitro method.

[0028] In a seventh aspect, the present disclosure provides a polynucleotide construct or combination of polynucleotide constructs comprising a polynucleotide of the present disclosure, and one or more polynucleotides encoding a sortase.

[0029] In some embodiments, the sortase is encoded by a gene that is identified as occurring in the same cluster as a gene encoding a carrier protein in nature. In some embodiments, the sortase is a class C sortase, such as srtC1 and / or srtC2, preferably wherein srtC1 and srtC2 are encoded by genes from the same cluster. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A map of plasmid pEK-spa2 is shown.

[0031] Figure 2 Shown is the workflow for constructing two expression cassettes in tandem.

[0032] Figure 3 A map of a plasmid containing two expression cassettes in tandem is shown.

[0033] Figure 4 A map of plasmid pZ9-dxs_crtEBI is shown.

[0034] Figure 5 A map of plasmid pET-28a-Spa2 is shown.

[0035] Figure 6 The sequence encoding the sortase genes srtC1 and srtC2 and the sortase-catalyzed fimbriae protein genes spa1, spa2 and spa3 is shown. Cg CLP biosynthetic gene cluster (BGC).

[0036] Figure 7 TEM and AFM images show that the skeletal pilin Spa2 is important for Cg The scale bars in the TEM and AFM images are 200 nm and 400 nm, respectively.

[0037] Figure 8 Figure 2 shows the composition of CLP in C. glutamicum identified by immunogold labeling. (a) Cartoon diagram showing Cg CLP fibers contain two minor pilin proteins (Spa1 and Spa3) and a skeletal pilin protein, Spa2. (b) Immunogold labeling and TEM images show Cg The composition and distribution of CLP fimbriae proteins indicate that Spa2 is a skeletal fimbriae protein. Cg CLP was subjected to single immunogold labeling; gold-modified goat anti-rabbit IgG was used as the secondary antibody to label the target pilin. Cg CLP was double immunogold labeled with 30 nm and 5 nm gold-modified goat anti-rabbit IgG for Spa1 and Spa2, respectively. Cg CLP was double-labeled with 15 nm and 5 nm gold-modified goat anti-rabbit IgG for Spa2 and Spa3, respectively. (c) Quantitative analysis by whole-cell filtration ELISA Cg CLP composition (detected with α-Spa1, α-Spa2 and α-Spa3 antibodies respectively). Quantitative results also showed that Spa2 isCg The main components of CLP. Each experiment was performed at least three times, and the standard error is shown. The scale bar in the TEM images represents 200 nm.

[0038] Figure 9 Deletion of the srtC1 and srtC2 genes abolished pili formation. Cg TEM images (detected with α-Spa2) (a), AFM images (b), and whole-cell filtration ELISA quantification (c) of CLP fibers. Scale bars in TEM (a) and AFM (b) images are 200 nm and 400 nm, respectively. For immunogold labeling, α-Spa2 was used as the primary antibody, and 10 nm gold-modified goat anti-rabbit IgG was used as the secondary antibody. Each ELISA experiment was performed at least three times, and standard errors are shown.

[0039] Figure 10 Shown for mass spectrometry analysis Cg Isolation of CLP fibers. Purification by nickel affinity chromatography Cg SDS-PAGE gel electrophoresis analysis of CLP fibers showed that the high molecular weight Cg CLP polymers were eluted at 100 mM imidazole.

[0040] Figure 11 Show Cg Identification of the intermolecular isopeptide bond of Spa2 monomer polymerization in CLP. m / z 832.9 is shown. 2+ The fragmentation spectrum of the parent ion at , which contains Spa2 i Lys194 (blue font) and Spa2 i+1 Intermolecular isopeptide bond (green font) between Thr477 (red font).

[0041] Figure 12 The signal peptide of Spa2 was identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). (a) Cartoon diagram showing Spa2 cut The amino acid sequence of the purified Spa2 was modified (residues 470-509 at the C-terminus of Spa2 were replaced with 6His), so that the Spa2 monomer would not aggregate but be secreted into the culture medium as a monomer. (b) SDS-PAGE gel electrophoresis showed that the purified Spa2 cut (c) LC-MS / MS identified residues 1-34 at the N-terminus of Spa2 as a signal peptide. This figure shows m / z 916.4538 generated by chymotrypsin digestion of Spa2. 2+ MS / MS spectrum of a peptide. Predicted b-type and y-type ions (not all included) are listed above and below the peptide sequence, respectively. Matching ions are labeled in the spectrum.

[0042] Figure 13 Shown is the measurement of Spa2 by quadrupole time-of-flight mass spectrometry cut The measured molecular weight is better than Spa2 cut The calculated value is ≈54.7 Da, indicating the presence of three intramolecular isopeptide bonds and two disulfide bonds in monomeric Spa2. The formation of an intramolecular isopeptide bond results in the loss of one amine molecule, ≈17 Da; the formation of a disulfide bond results in the loss of two hydrogen atoms, ≈2 Da.

[0043] Figure 14 The diffraction of the BL18U1 beamline at the Shanghai Synchrotron Radiation Facility (Shanghai, China) is shown. resolution of Spa2 crystals.

[0044] Figure 15 The X-ray crystal structure of Spa2 is shown, which consists of three tandem Ig-like domains: the N-domain (pink), the M-domain (blue), and the C-domain (green). The residues involved in the formation of three intramolecular isopeptide bonds (yellow) and two disulfide bonds (red) are shown as sticks.

[0045] Figure 16 Comparison of the crystal structure of Spa2 predicted by AlphaFold2 with the crystal structures of 3HR6 and 4HSS is shown. (a) Chain A (yellow) in the Spa2 crystal structure is superimposed on the Spa2 structure predicted by AlphaFold2 (blue) by PyMOL alignment. The structures are superimposed using the α-carbon (Cα) atoms of 410 residues, with a root mean square deviation (RMSD) of This shows that AlphaFold2 accurately predicts the folding of Spa2 for a single domain. Chain A in the Spa2 crystal structure (yellow) overlaps with the crystal structures of 3HR6 (pink) (b) and 4HSS (green) (c), with RMSD values ​​of (270 Cα atoms), (311 Cα atoms).

[0046] Figure 17 Shown are omit electron density maps, demonstrating the presence of internal covalent bonds in the Spa2 crystal structure. The 2mFo-DFc omit electron density map for three isopeptide bonds (a) and two disulfide bonds (b) is shown as a blue grid with contour lines at 1.0σ. The omit electron density map was generated using the Phenix composite omit map.

[0047] Figure 18Figure 2 shows the identification of disulfide and intramolecular isopeptide bond formation at appropriate sequence positions in Spa2 by LC-MS / MS analysis. (a) Cartoon illustration of key features in Spa2, including three intramolecular isopeptide bonds within a single domain, two disulfide bonds in the N-domain (C97-C128) and the C-domain (C380-C432), the pilin motif of YPKN in the N-domain, and the sortase cleavage sorting signal motif of LPLTG in the C-domain. (b) MS / MS spectrum of a peptide generated by pepsin digestion of Spa2 containing a disulfide bond between Cys97 and Cys128, m / z 1407.4 4+ (c) MS / MS spectrum of a peptide generated by pepsin digestion of Spa2 containing a disulfide bond between Cys380 and Cys432, with m / z 1583.7 2+ (d) MS / MS spectrum of a peptide generated by pepsin digestion of Spa2 containing an internal isopeptide bond between Lys57 and Asn195, with m / z 1326.9 4+ (e) MS / MS spectrum of a peptide generated by pepsin digestion of Spa2 containing an internal isopeptide bond between Lys203 and Asn318, with m / z 1324.6 3+ (f) MS / MS spectrum of a peptide generated by pepsin digestion of Spa2 containing an internal isopeptide bond between Lys355 and Asp466, m / z 754.6 4+ For (b)–(f), the predicted b-type and y-type ions (not all included) are listed above and below the peptide sequences, respectively; disulfide bonds and intramolecular isopeptide bonds are shown as red and yellow bars, respectively.

[0048] Figure 19 and 20 Δspa2 strains (expressing Spa2 or Spa2 variants K194A, LPLTG, 474LALAA478 , E158A, D246A, E435A, D246A / E435A, C97A, C380A, and C97A / C380A) to assess the key residues in Spa2 that promote the formation of inter- and intramolecular isopeptide and disulfide bonds by TEM bioimaging. Figure 19 ), and quantitative analysis by whole-cell filtration ELISA (detected by anti-Spa2 antibody) Cg The amount of CLP fibers ( Figure 20 ). Figure 20 The results are expressed as mean ± SD. Figure 20The P values ​​for the Spa2 mutant strain and the Spa2 strain, from left to right, are P < 0.0001, P < 0.0001, P = 0.4664, P = 0.8673, P = 0.7137, P = 0.0011, P = 0.0008, P = 0.0004, and P < 0.0001. Not significant (NS) P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical analysis was performed using the t-test. Figure 19 The scale bar in the figure is 200 nm.

[0049] Figure 21 Shown is Spa2 determined by quadrupole time-of-flight mass spectrometry cut The exact molecular weight of the mutant variant. E158A cut (a) D246A cut (b) E435A cut (c) and D246A / E435A cut The measured molecular weights of (d) are ≈54.9, 37.3, 21.4, and 4.0 Da smaller than the calculated values ​​for the relevant variants, respectively, indicating that three, two, one, and no intramolecular isopeptide bonds are retained in the corresponding monomeric mutants, respectively. cut Mutant variant E158A cut 、D246A cut 、E435A cut and D246A / E435A cut Δspa2 was expressed in and purified by nickel affinity chromatography.

[0050] Figure 22 The modular genetic design strategy is shown to Cg Rational engineering of the CLP protein backbone: Cartoon showing functionalized polymeric Spa2 backbone pilin proteins by incorporating proteins of interest (POIs) (e.g., mCherry, a fluorescent reporter protein) at candidate insertion sites, including Q35 (E1) at the N-terminus, and G215 (E2), G236 (E3), and G336 (E4) in the M-domain without disulfide bonds, based on structural validation.

[0051] Figure 23 The fluorescence intensity and Cg Quantification of CLP fiber amounts by whole-cell filtration ELISA (detected by anti-Spa2 antibody) (a); and confocal microscopy imaging (b) (Scale bar = 2 μm).

[0052] Figure 24TEM images of assembled mCherry-Spa2 fusion proteins associated with the cell surface based on immunogold labeling are shown. TEM images of Δspa2 cells (a), E1 cells (b), E2 cells (c), E3 cells (d), and E4 cells (e). TEM samples were collected from a Δspa2 strain containing a plasmid expressing various mCherry-Spa2 fusions under the native constitutive promoter of the spa2 gene. For immunogold labeling, α-Spa2 was the primary antibody and 10 nm gold-modified goat anti-rabbit IgG was the secondary antibody. Scale bar, 200 nm.

[0053] Figure 25 Figure 3 shows the extracellular secretion and assembly of R-Spa2 pilin onto the cell surface of engineered Corynebacterium glutamicum cells. Cg In CLP fibers: a series of R-Spa2 fusion protein constructs containing functional R peptides / proteins with different amino acid sequences.

[0054] Figure 26 Showing assembled R-Spa2 on the cell surface based on immunogold labeling and TEM imaging Cg Morphology of CLPs, scale bar = 200 nm.

[0055] Figure 27 Engineered with various fusion domains Cg Functional characterization of CLP. (a) TEM image showing Ni-NTA-modified AuNP anchored on 6His-Spa2 Cg (b) Confocal microscopy image showing SpyTag-Spa2 on CLP. Cg Green fluorescence emitted by CLP cells, SpyCatcher-EGFP protein binding partner binds to SpyTag-Spa2 through the Spytag-SpyCatcher interaction pair Cg (c) Confocal microscopy image showing SpyCatcher-Spa2 covalently linked to CLP cells. Cg CLP cells emit green fluorescence, and the SpyTag-EGFP protein binds to the SpyCatcher-Spa2 protein through the Spytag-SpyCatcher interaction pair. Cg CLP cells are covalently linked. (d) Confocal microscopy image showing Venus-Spa2 Cg Green fluorescence emitted by CLP cells. (e) Mfp3Spep-Spa2 CgFluorescence images of CLP cells and quantitative analysis of their immobilization capacity. Immobilized microspheres on a substrate (left panel) before (top panel) and after (bottom panel) impaction with a water jet at a constant discharge pressure of 5 psi. Quantitative analysis of the relative capacity of different cells to immobilize PS microspheres on a substrate (right panel). (f) Detection of CcEgl-Spa2 by 3,5-dinitrosalicylic acid (DNS) assay. Cg CLP cells degrade carboxymethyl cellulose into glucose. Each experiment was performed at least three times, and the standard error is shown. Scale bars in a: 200 nm, in b, c, and e: 2 μm, and in d: 100 μm.

[0056] Figure 28 Schematic diagram showing the simultaneous expression of two Spa2 fimbriae protein fusion proteins, N-Ven-Spa2 and C-Ven-Spa2 (N-Ven-Spa2+C-Ven-Spa2 strain), which contain the N-terminal (N-Ven) and C-terminal (C-Ven) modules of the split-Venus system, resulting in the coassembly of the split-Venus components into the final functional Cg CLP structure.

[0057] Figure 29 TEM images of assembled split-Venus components fused to Spa2 in association with the cell surface based on immunogold labeling are shown. N-Ven + C-Ven cells expressing the cosecreted split-Venus system (a); N-Ven-Spa2 cells expressing a Spa2 pilin fusion protein of N-Venus-Spa2 (b); C-Ven-Spa2 cells expressing a Spa2 pilin fusion protein of C-Venus-Spa2 (c); and N-Ven-Spa2 + C-Ven-Spa2 cells simultaneously expressing two Spa2 pilin fusion proteins, N-Ven-Spa2 and C-Ven-Spa2 (d). TEM samples were collected from a Δspa2 strain containing plasmids expressing various Spa2 fusion proteins under the native constitutive promoter of the spa2 gene. For immunogold labeling, α-Spa2 was the primary antibody and 10 nm gold-modified goat anti-rabbit IgG was the secondary antibody. Scale bar, 200 nm.

[0058] Figure 30 The split-Venus components are shown to co-assemble as CgCLP fibers lead to increased fluorescence intensity. (a) Engineered C. glutamicum cells exhibit enhanced fluorescence intensity only in the N-Ven-Spa2+C-Ven-Spa2 strain. (b) Confocal microscopy of C. glutamicum cells shows that the strongest Venus fluorescence signal occurs at the extracellular site of the N-Ven-Spa2+C-Ven-Spa2 strain (scale bar = 2 μm).

[0059] Figure 31 Schematic diagram showing the engineered Corynebacterium glutamicum living functional material converting cell biomass into lycopene value-added products by combining extracellular cellulose degradation and intracellular biotransformation capabilities. Specifically, for extracellular cellulose degradation (step 1), endo-1,4-β-glucanase (TrEgl) from Trichoderma reesei and β-glucosidase (SdBgl) from S. degradans were simultaneously fused to Spa2 pilin (TrEgl-Spa2+SdBgl-Spa2) and co-assembled into Cg The CLP structure likely forms the catalytic cascade for the extracellular degradation of cellulose to glucose. For intracellular conversion (step 2), glucose was used for lycopene production in the pathway-engineered Corynebacterium glutamicum strain C003 via induction with IPTG. G3P: glyceraldehyde-3-phosphate; IPP: isopentenyl phosphate.

[0060] Figure 32 Production of lycopene from biowaste using engineered C. glutamicum containing modified CLPs is shown. a, TEM image showing that C003 cells containing the P2 plasmid are able to coassemble TrEgl and SdBgl into Cg CLP structure, while C001, C002 and C004 cells cannot. Labeled with 10nm gold particles by immunogold labeling Cg CLP. Scale bar, 200 nm. b, Only when TrEgl and SdBgl are coassembled CgOnly when the CLP structure (TrEgl-Spa2 + SdBgl-Spa2, strain C003) was present did the ELM degrade CMC-Na in the culture medium from a viscous gel to a dilute solution, superior to the case when the free enzyme was secreted (TrEgl + SdBgl, strain C004). Δspa2Δdec (strain C001) served as the negative control. c, Degradation assay using CMC-Na as the substrate. The enzyme activity of strain C003 was fourfold higher than that of strain C004. d, Lycopene production was determined by HPLC in strain C003 cultured in M63 medium, using CMC-Na instead of glucose as the carbon source and induced by the addition of IPTG. Results are presented as mean ± SD. The P values ​​in c for strains C003, C004, and C001 were P < 0.0001 and P = 0.8629, respectively. Not significant (NS) P > 0.05, **** P < 0.0001. Statistical analysis was performed using the t-test. Each experiment was performed at least three times. Detailed Description of the Invention

[0062] 1. Definition

[0063] Unless otherwise indicated, all terms used herein have the same meanings as understood by one skilled in the art, and the practice of the present disclosure will employ conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of one skilled in the art.

[0064] As used herein, the term "covalently cross-linked pili" or "CLP" refers to pili in which monomers are interconnected by covalent bonds. The engineered living functional material herein refers to pili formed by engineered monomers, i.e., the fusion polypeptides of the present disclosure, or recombinant bacteria that form pili.

[0065] As an example of a CLP-forming bacterium, the Gram-positive bacterium Corynebacterium glutamicum is “generally recognized as safe” (GRAS); this bacterium provides a potential platform for various products such as amino acids and lycopene.

[0066] As used herein, the term "peptide" is interchangeable with "polypeptide" and "protein" to refer to a chain of at least two amino acids linked by peptide bonds, such as ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written from left to right, showing the direction from the amino terminus to the carboxyl terminus. "Peptide," "polypeptide," and "protein" may include, but are not limited to, enzymes, antibodies, hormones, ligands, receptors, and the like.

[0067] The term "amino acid" includes naturally occurring amino acids and non-natural amino acids in proteins. Conventional nomenclature (single-letter and three-letter) for naturally occurring amino acids in proteins is used, as can be seen in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0068]

[0069] As used herein, the term "fusion polypeptide" is a recombinant product comprising two or more peptide fragments that are not present in a single native polypeptide. The fragments can be fused directly or through a linker, such as a flexible linker, for example, a GS linker. In general, a fusion polypeptide can be produced by expressing a polynucleotide comprising a nucleotide sequence encoding the two or more peptide fragments and a linker (if present), arranged in a desired order.

[0070] As used herein, the term "polynucleotide" generally refers to a nucleic acid molecule (e.g., 100 bases in length and up to 30k nucleotides in length) and a sequence that is complementary (antisense) or identical (sense) to the sequence of a messenger RNA (mRNA) or miRNA fragment or molecule. The term can also refer to a transcribed or non-transcribed DNA or RNA molecule.

[0071] As used herein, the term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide that is isolated from a naturally occurring gene or modified to contain non-naturally occurring nucleic acid segments. When a polynucleotide construct contains the control sequences required for expression of a coding sequence of the present disclosure, the polynucleotide construct comprises an "expression cassette."

[0072] As used herein, the term "exogenous polynucleotide" refers to a nucleotide sequence that is not derived from the host in which it is located. It can be identical to or heterologous to the host's DNA. An example is a sequence of interest that is inserted into a vector. Such exogenous DNA sequences can be derived from a variety of sources, including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also encompass DNA sequences encoding antisense oligonucleotides.

[0073] As used herein, the term "expression cassette" refers to a polynucleotide fragment comprising a polynucleotide encoding a polypeptide operably linked to additional nucleotides (eg, control sequences) that provide for expression of the polynucleotide.

[0074] As used herein, the term "encoding" means that a polynucleotide directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which generally begins with the ATG start codon or other start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.

[0075] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0076] "Control sequences" include all elements necessary or beneficial for expression of a polynucleotide encoding a polypeptide of the present disclosure. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, an enhancer, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter and signals for termination of transcription and translation.

[0077] For example, the control sequence can be a suitable promoter sequence, a nucleotide sequence that is recognized by the host cell to express a polynucleotide encoding a polypeptide of the present disclosure. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the lac operon of Escherichia coli. Promoters also include mutant, truncated, and hybrid promoters that can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0078] As used herein, the term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence so that the control sequence directs the expression of the polypeptide coding sequence.

[0079] Various manipulations can be performed on the polynucleotide encoding the polypeptide of interest to enhance expression of the polypeptide. Depending on the expression vector or host, manipulation of the polynucleotide, such as codon optimization, may be desirable or necessary prior to insertion into a vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0080] As used herein, the term "recombinant" refers to a nucleic acid, vector, polypeptide or protein that is produced by DNA recombination (cloning) methods and can be distinguished from a native or wild-type nucleic acid, vector, polypeptide or protein.

[0081] As used herein, the term "hybridize" refers to nucleotide sequences that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other and typically remain hybridized to each other under given stringent hybridization and washing conditions.

[0082] For the present disclosure, to determine the percent identity between two amino acid sequences or two nucleotide sequences, the sequences are aligned for the purpose of optimal comparison (e.g., a gap can be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are the same length.

[0083] Those skilled in the art will be aware of various computer programs that can be used to determine the identity between two sequences.

[0084] "Percent identity" or "percent sequence identity" refers to a comparison between the amino acids of two polypeptides or between the nucleotides of two polynucleotides such that, when optimally aligned, the two polypeptides or polynucleotides have approximately the specified percentage of identical amino acids. For example, "95% identity" refers to a comparison between the amino acids of two polypeptides or between the nucleotides of two polynucleotides such that, when optimally aligned, 95% of the amino acids in the two polypeptides or 95% of the nucleotides in the two polynucleotides are identical.

[0085] Those skilled in the art are aware of various hybridization conditions, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0086] Of course, the polynucleotides disclosed herein do not include polynucleotides that hybridize only to a complementary segment of a poly A sequence (such as the 3' terminal poly (A) of an mRNA) or poly T (or U) residues.

[0087] As used herein, the term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as vector recipients. The term includes the progeny of the original cell that has been transduced. Thus, as used herein, "host cell" generally refers to a cell that has been transduced with an exogenous DNA sequence. It should be understood that due to natural, accidental, or artificial mutations, the progeny of a single parent cell may not necessarily be morphologically identical or complementary to the original parent in terms of genome or total DNA.

[0088] 2. Fusion peptide

[0089] Through genetic manipulation, bioimaging, and structural characterization, the Spa2 protein was identified as the backbone pilin of the CLP fiber structure. Using structure-guided design, the inventors developed a novel, engineered extracellular protein scaffold that can be genetically attached to multiple sites of the Spa2 protein for multiple functional peptides or proteins.

[0090] The present disclosure provides a fusion polypeptide comprising a carrier protein and a polypeptide of interest, wherein the polypeptide of interest is fused to a terminus of the carrier protein or inserted into the carrier protein, and wherein the carrier protein is a pilin of covalently cross-linked pili (CLP) from a microorganism.

[0091] In some embodiments, the microorganism is a Gram-positive bacterium, such as a bacterium selected from the group consisting of Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lactobacillus paracasei, Bacillus thuringiensis, and Lactobacillus paracasei; preferably Corynebacterium glutamicum. The bacteria may include, but are not limited to, bacteria selected from the group consisting of Corynebacterium glutamicum strain BE (GenBank assembly accession: GCA_013046805.1), Corynebacterium glutamicum ATCC 14067 (GenBank assembly accession: GCA_002243555.1), Corynebacterium glutamicum strain YI (GenBank assembly accession: GCA_001643035.1), Corynebacterium glutamicum strain ATCC 13869 (GenBank assembly accession: GCA_001687645.1), Corynebacterium glutamicum AJ1511 (GenBank assembly accession: GCA_002355675.1), Corynebacterium glutamicum strain XV (GenBank assembly accession: GCA_001936195.1), Corynebacterium glutamicum strain CP (GenBank assembly accession: GCA_001643035.1), Corynebacterium glutamicum strain ATCC 13869 (GenBank assembly accession: GCA_001687645.1), Corynebacterium glutamicum strain AJ1511 (GenBank assembly accession: GCA_002355675.1), Corynebacterium glutamicum strain XV (GenBank assembly accession: GCA_001936195.1), Corynebacterium glutamicum strain CP (GenBank assembly accession: GCA_001643035.1), accession: GCA_001447865.2), Corynebacterium glutamicum R (GenBank assembly accession: GCA_000010225.1), Corynebacterium glutamicum strain USDA-ARS-USMARC-56828 (GenBank assembly accession: GCA_001518935.2), Bifidobacterium breve strain LMC520 (GenBank assembly accession: GCA_001990225.1), Bifidobacterium breve strain BR3 (GenBank assembly accession: GCA_001281425.1), Bifidobacterium breve strain NRBB51 (GenBank assembly accession: GCA_002838405.1), Bifidobacterium breve strain NRBB09 (GenBank assembly accession: GCA_002838325.1), Bifidobacterium breve 12L ... accession: GCA_000568955.1), Bifidobacterium breve strain DRBB26 (GenBank assembly accession: GCA_002838225.1), Bifidobacterium breve strain 180W83 (GenBank assembly accession: GCA_002838525.1), Bifidobacterium breve strain JSRL01 (GenBank assembly accession: GCA_009498435.1), Bifidobacterium breve 689b (GenBank assembly accession: GCA_000569055.1), Bifidobacterium breve strain DRBB29 (GenBank assembly accession: GCA_002838705.1), Bifidobacterium breve strain DRBB27 (GenBank assembly accession: GCA_002838445.1), Bifidobacterium breve strain JR01 (GenBank assembly accession: GCA_009931415.1), Bifidobacterium breve S27 (GenBank assembly accession: GCA_000569055.1). accession: GCA_000569075.1), Bifidobacterium breve ACS-071-V-Sch8b (GenBank assembly accession: GCA_000213865.1), Bifidobacterium breve strain NRBB56 (GenBank assembly accession: GCA_002838425.1), Bifidobacterium breve DSM 20213=JCM 1192 (GenBank assembly accession: GCA_001025175.1), Bifidobacterium breve strain NRBB01 (GenBank assembly accession: GCA_002838245.1), Bifidobacterium breve strain FDAARGOS_561 (GenBank assembly accession: GCA_003813065.1), Bifidobacterium breve strain NCTC11815 (GenBank assembly accession: GCA_002838425.1). accession: GCA_900637145.1), Bifidobacterium breve strain NRBB52 (GenBank assembly accession: GCA_002838385.1), Bifidobacterium breve strain 082W48 (GenBank assembly accession: GCA_002838545.1), Bifidobacterium breve strain lw01 (GenBank assembly accession: GCA_003860285.1), and Bifidobacterium breve UCC2003 (GenBank assembly accession: GCA_000220135.1).1), Bifidobacterium breve strain NRBB11 (GenBank assembly accession: GCA_002838305.1), Bifidobacterium breve strain NRBB04 (GenBank assembly accession: GCA_002838285.1), Bifidobacterium breve NCFB 2258 (GenBank assembly accession: GCA_000569035.1), Bifidobacterium breve strain NRBB20 (GenBank assembly accession: GCA_002838645.1), Bifidobacterium breve strain NRBB27 (GenBank assembly accession: GCA_002838665.1), Bifidobacterium breve strain NRBB49 (GenBank assembly accession: GCA_002838685.1), Bifidobacterium breve strain NRBB18 (GenBank assembly accession: GCA_002838605.1), Bifidobacterium breve strain NRBB02 (GenBank assembly accession: GCA_002838645.1), accession: GCA_002838265.1), Bifidobacterium breve strain NRBB19 (GenBank assembly accession: GCA_002838625.1), Bifidobacterium breve strain 017W439 ​​(GenBank assembly accession: GCA_002838465.1), Bifidobacterium breve JCM 7017 (GenBank assembly accession: GCA_000568975.1), Bifidobacterium breve strain NRBB50 (GenBank assembly accession: GCA_002838365.1), Bifidobacterium breve strain 139W423 (GenBank assembly accession: GCA_002838565.1), Bifidobacterium breve strain DRBB28 (GenBank assembly accession: GCA_002838505.1), Bifidobacterium breve strain CNCM I-4321 (GenBank assembly accession: GCA_002838585.1), Bifidobacterium breve strain DRBB30 (GenBank assembly accession: GCA_002838725.1), and Bifidobacterium breve strain NRBB57 (GenBank assembly accession: GCA_002838345.1), Bifidobacterium breve strain 215W447a (GenBank assembly accession: GCA_002838485.1), Lactococcus lactis subsp. cremoris NZ9000 (GenBank assembly accession: GCA_000143205.1), Lactococcus lactis subsp. cremoris MG1363 (GenBank assembly accession: GCA_000009425.1), Lactococcus lactis subsp. cremoris A76 (GenBank assembly accession: GCA_000236475.1), Lactococcus lactis strain SRCM103457 (GenBank assembly accession: GCA_004194355.1), Lactococcus lactis strain CBA3619 (GenBank assembly accession: GCA_000143205.1), Lactococcus lactis subsp. cremoris MG1363 (GenBank assembly accession: GCA_000009425.1), Lactococcus lactis subsp. cremoris A76 (GenBank assembly accession: GCA_000236475.1), Lactococcus lactis strain SRCM103457 (GenBank assembly accession: GCA_004194355.1), Lactococcus lactis strain CBA3619 (GenBank assembly accession: GCA_007954765.1), Lactococcus lactis strain WiKim0098 (GenBank assembly accession: GCA_016406265.1), Lactococcus lactis strain K_LL005 (GenBank assembly accession: GCA_014334715.1), Lactococcus lactis subsp. lactis strain G121 (GenBank assembly accession: GCA_013395015.1), Lactococcus lactis strain N8 (GenBank assembly accession: GCA_014884605.1), Lactococcus lactis subsp. lactis 10-1 (GenBank assembly accession: GCA_000344575.1), Lactococcus lactis subsp. lactis strain F44 (GenBank assembly accession: GCA_014334715.1), Lactococcus lactis subsp. lactis strain G121 (GenBank assembly accession: GCA_013395015.1), Lactococcus lactis strain N8 (GenBank assembly accession: GCA_014884605.1), Lactococcus lactis subsp. lactis strain 10-1 (GenBank assembly accession: GCA_000344575.1), Lactococcus lactis subsp. lactis strain F44 (GenBank assembly accession: GCA_014334715.1), Lactococcus lactis subsp. lactis strain assembly accession: GCA_002804185.1), Lactococcus lactis subsp. lactis bv. diacetylactis strain S50 (GenBank assembly accession: GCA_003627395.2), Lactococcus lactis strain FDAARGOS_1064 (GenBank assembly accession: GCA_016127135.1), Lactococcus lactis strain FDAARGOS_887 (GenBank assembly accession: GCA_016027975.1), Lactococcus lactis subsp. lactis strain UC77 (GenBank assembly accession: GCA_002078615.2), Lactococcus lactis strain FDAARGOS_866 (GenBank assembly accession: GCA_016028815.1), Lactococcus lactis strain IL1403 (GenBank assembly accession: GCA_003722275.1), Lactococcus lactis strain FDAARGOS_865 (GenBank assembly accession: GCA_016028835.1), Lactococcus lactis subsp. cremoris IBB477 (GenBank assembly accession: GCA_001856165.1), Lactobacillus paracasei strain TD 062 (GenBank assembly accession: GCA_009834405.1), Lactobacillus paracasei strain HM1 (GenBank assembly accession: GCA_018064185.1), Bacillus thuringiensis strain FDAARGOS_794 (GenBank assembly accession: GCA_013267795.1), Bacillus thuringiensis strain XL6 (GenBank assembly accession: GCA_000774075.2), Bacillus thuringiensis strain Bt-GS57 (GenBank assembly accession: GCA_017751245.1), Bacillus thuringiensis strain HER1410 (GenBank assembly accession: GCA_013340745.1), Bacillus thuringiensis serovar tolworthi (GenBank assembly accession: GCA_013267795.1), Bacillus thuringiensis strain XL6 (GenBank assembly accession: GCA_000774075.2), Bacillus thuringiensis strain Bt-GS57 (GenBank assembly accession: GCA_017751245.1), Bacillus thuringiensis strain HER1410 (GenBank assembly accession: GCA_013340745.1), Bacillus thuringiensis serovar tolworthi (GenBank assembly accession: GCA_013267795.1), Bacillus thuringiensis strain XL6 (GenBank assembly accession: GCA_000774075.2), accession: GCA_001548175.1), Bacillus thuringiensis strain BT62 (GenBank assembly accession: GCA_003054785.2), and Bacillus thuringiensis strain HD12 (GenBank assembly accession: GCA_001598095.1), Bacillus thuringiensis serovar alesti strain BGSC 4C1 (GenBank assembly accession: GCA_001640965.1), Bacillus thuringiensis LM1212 (GenBank assembly accession: GCA_003546665.1), Lactobacillus paracasei strain 347-16 (GenBank assembly accession: GCA_012955485.1), Lactobacillus paracasei subsp. tolerans strain MGB0734 (GenBank assembly accession: GCA_015476135.1), Lactobacillus paracasei subsp. tolerans strain MGB0747 (GenBank assembly accession: GCA_015476175.1), Lactobacillus paracasei strain CBA3611 (GenBank assembly accession: GCA_012955485.1), Lactobacillus paracasei subsp. tolerans strain MGB0734 (GenBank assembly accession: GCA_015476135.1), Lactobacillus paracasei subsp. tolerans strain MGB0747 (GenBank assembly accession: GCA_015476175.1), Lactobacillus paracasei strain CBA3611 (GenBank assembly accession: GCA_012955485.1), Lactobacillus paracasei subsp. accession: GCA_007292115.1), Lactobacillus paracasei subsp. paracasei strain GR0548 (GenBank assembly accession: GCA_019175405.1), Lactobacillus paracasei subsp. paracasei strain IBB3423 (GenBank assembly accession: GCA_009739485.1), Lactobacillus paracasei strain NFFJ04 (GenBank assembly accession: GCA_014905075.1), Lactobacillus paracasei strain HL182 (GenBank assembly accession: GCA_017638905.1), Lactobacillus paracasei strain Lpc10 (GenBank assembly accession: GCA_003199005.1), Lactobacillus paracasei subsp. firmus strain AO356 (GenBank assembly accession: GCA_0031990 ... assembly accession: GCA_003957435.1), Lactobacillus paracasei subsp. firmus strain MGB0625 (GenBank assembly accession: GCA_015476155.1), Lactobacillus paracasei strain 10266 (GenBank assembly accession: GCA_008329845.1), Lactobacillus paracasei subsp. firmus strain S-NB (GenBank assembly accession: GCA_016757695.1), Lactobacillus paracasei strain Lp02 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain ZFM54 (GenBank assembly accession: GCA_003627255.1), Lactobacillus paracasei subsp. paracasei strain TMW 1.1434 (GenBank assembly accession: GCA_002813615.1), Lactobacillus paracasei strain SRCM103299 (GenBank assembly accession: GCA_004141835.1), Lactobacillus paracasei strain NJ (GenBank assembly accession: GCA_007637635.1), Lactobacillus paracasei strain EG9 ... strain TMW 1.1434 (GenBank assembly accession: GCA_002813615.1), Lactobacillus paracasei strain SRCM103299 (GenBank assembly accession: GCA accession: GCA_003177075.1), Lactobacillus paracasei strain TK-P4A (GenBank assembly accession: GCA_015377585.1), Lactobacillus paracasei subsp. paracasei strain BD5115 (GenBank assembly accession: GCA_018596415.1), and Lactobacillus paracasei subsp. paracasei JCM 8130 (GenBank assembly accession: GCA_000829035.1), preferably Corynebacterium glutamicum ATCC 14067.

[0092] In some embodiments, the carrier protein is a backbone pilin protein.

[0093] Preferably, the insertional fusion of the polypeptide of interest does not interfere with the formation of intermolecular isopeptide bonds, disulfide bonds, or intramolecular isopeptide bonds in the carrier protein.

[0094] In some embodiments, the polypeptide of interest is fused to the terminus of a carrier protein. In some embodiments, the polypeptide of interest is fused to the N-terminus of a carrier protein.

[0095] In some embodiments, the polypeptide of interest is inserted into a carrier protein. In some embodiments, the polypeptide of interest is inserted into the turn region of a carrier protein.

[0096] In some embodiments, the carrier protein is a backbone pilin protein (Spa2 protein) from Corynebacterium glutamicum. It was observed that the Spa2 protein (SEQ ID NO: 1) comprises three tandem Ig-like domains, including an N-domain (residues 36-197), an M-domain (residues 198-343) and a C-domain (residues 344-469), which is consistent with other backbone pilin proteins. It was also observed that the absence of the M-domain did not affect the formation of CLPs. In some embodiments, the polypeptide of interest is inserted into the M domain of the backbone pilin protein. In some embodiments, the polypeptide of interest replaces the M domain or a portion thereof of the backbone pilin protein.

[0097] The Spa2 proteins from different Corynebacterium glutamicum strains may differ in sequence. In some embodiments, the carrier protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 1, 2, 3, or 4. In some embodiments, the carrier protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, 2, 3 or 4, wherein the residues corresponding to residues C97, C128, K194, C380, C432 and LPLTG (474-478) and optionally E158, D246 and / or E435 of SEQ ID NO: 1 are unchanged. The carrier protein may be the mature form of SEQ ID NO: 1, 2, 3 or 4, i.e., lacking the signal peptide. In some embodiments, the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4. In some embodiments, the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4, wherein the amino acid sequence corresponding to SEQ ID NO: 1 is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4. Residues C97, C128, E158, K194, D246, C380, C432, E435 and LPLTGT(474-478) of NO: 1 and optionally residues E158, D246 and / or E435 are unchanged.

[0098] In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein or inserted between positions corresponding to G215 and L216 of SEQ ID NO: 1, between positions corresponding to G236 and E237 of SEQ ID NO: 1, or between positions corresponding to G336 and T337 of SEQ ID NO: 1.

[0099] In some embodiments, the carrier protein comprises amino acids 35-509 of SEQ ID NO: 1. In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein or inserted between G215 and L216, between G236 and E237, or between G336 and T337 of SEQ ID NO: 1.

[0100] In some embodiments, the polypeptide of interest is directly linked to the N-terminus of the carrier polypeptide. In some embodiments, the polypeptide of interest is linked to the N-terminus of the carrier polypeptide via a peptide linker, such as a flexible linker.

[0101] The peptide linker can generally be a short peptide having about 4-20 or more amino acids, such as a combination of Ser and Gly residues, which is a conventional flexible linker. In some embodiments, the peptide linker used in the present disclosure is (G4S)n, n=1-4. In some embodiments, the peptide linker used in the present disclosure is (G3S)n, n=1-4. In some embodiments, the peptide linker used in the present disclosure is (G4S)2, i.e., SEQ ID NO:22. In some embodiments, the peptide linker is the C10 linker of SEQ ID NO:23.

[0102] The polypeptide of interest can be selected based on the desired application of the fusion polypeptide.

[0103] In some embodiments, a fusion polypeptide is provided to bind, capture, or enrich a target molecule, and the polypeptide of interest is a polypeptide that can recognize the target peptide, including but not limited to a ligand, a receptor, an antigen, and an antibody such as a scFV and a nanobody. For example, a fusion polypeptide is provided to capture a protein comprising SpyTag (SEQ ID NO: 37), while the polypeptide of interest comprises SpyCatcher (SEQ ID NO: 15), or vice versa.

[0104] In some embodiments, the fusion polypeptide is provided as an adhesive and the polypeptide of interest is an adhesive peptide, eg, Mfp35 (SEQ ID NO: 38).

[0105] In some embodiments, fusion polypeptides are provided to catalyze chemical or biochemical reactions, and the polypeptide of interest is an enzyme. In some embodiments, fusion polypeptides are provided to degrade carbohydrates such as cellulose, and the polypeptide of interest can be an endo-1,4-β-glucanase, for example, from Trichoderma reesei (TrEgl, SEQ ID NO: 19), and / or a β-glucosidase, for example, from Saccharophagus degradans (SdBgl, SEQ ID NO: 21). In some embodiments, fusion polypeptides are provided to degrade refractory organic matter, such as plastics, and the polypeptide of interest is an enzyme responsible for degradation, such as PETase.

[0106] 3. Polynucleotides and Vectors

[0107] The present disclosure provides polynucleotides encoding the fusion polypeptides of the present disclosure.

[0108] The polynucleotides of the present disclosure can be amplified using cDNA, mRNA or genomic DNA as templates and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acids amplified as above can be cloned into appropriate vectors and characterized by DNA sequence analysis.

[0109] The polynucleotides of the present disclosure can be prepared by standard synthetic techniques, for example, by using an automated DNA synthesizer.

[0110] The present disclosure also relates to complementary strands of the nucleic acid molecules described herein.A nucleic acid molecule that is complementary to another nucleotide sequence is one that is sufficiently complementary to a nucleotide sequence such that it can hybridize to the other nucleotide sequence to form a stable duplex.

[0111] To express the fusion polypeptides of the present disclosure, polynucleotide constructs and vectors comprising the polynucleotides of the present disclosure, such as expression vectors, are also provided.

[0112] In some embodiments, the polynucleotides of the present disclosure are operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter, such as the natural promoter that drives the Spa2 gene in Corynebacterium glutamicum. In some embodiments, the promoter is an inducible promoter.

[0113] In some embodiments, the expression vector comprises a Lac operator.

[0114] The polynucleotide encoding the polypeptides of the present disclosure may be subjected to various manipulations to allow for polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide may be desirable or necessary prior to insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0115] In order to identify and select host cells containing the expression vectors of the present disclosure, the vectors of the present disclosure preferably contain one or more selectable markers that allow for easy selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene whose product confers biocide or viral resistance, heavy metal resistance, complements an auxotrophic deficiency, etc. For example, bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

[0116] Vector of the present disclosure can be integrated into the genome of host cell, or autonomously replicate in cell, and this is independent of genome.Be integrated into the host cell genome or the required element of autonomous replication and be known in the art (referring to, for example, Sambrook et al., 1989 mentioned above).

[0117] 4. Recombinant Cells

[0118] The present disclosure provides a recombinant cell comprising a polynucleotide encoding a fusion polypeptide, wherein the fusion polypeptide comprises a carrier protein and a polypeptide of interest, wherein the polypeptide of interest is fused to a terminus of the carrier protein or inserted into the carrier protein, wherein the carrier protein is a pilin of a CLP, and wherein the recombinant cell is capable of expressing the polynucleotide and displaying a modified CLP comprising the fusion polypeptide.

[0119] In some embodiments, the carrier protein in the fusion polypeptide is a native backbone pilus protein of the recombinant cell.

[0120] In some embodiments, the recombinant cell is a recombinant Gram-positive bacterium, such as a bacterium selected from Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lactobacillus paracasei, Bacillus thuringiensis, and Lactobacillus paracasei; preferably Corynebacterium glutamicum. The bacteria may include, but are not limited to, bacteria selected from the group consisting of Corynebacterium glutamicum strain BE (GenBank assembly accession: GCA_013046805.1), Corynebacterium glutamicum ATCC 14067 (GenBank assembly accession: GCA_002243555.1), Corynebacterium glutamicum strain YI (GenBank assembly accession: GCA_001643035.1), Corynebacterium glutamicum strain ATCC 13869 (GenBank assembly accession: GCA_001687645.1), Corynebacterium glutamicum AJ1511 (GenBank assembly accession: GCA_002355675.1), Corynebacterium glutamicum strain XV (GenBank assembly accession: GCA_001936195.1), Corynebacterium glutamicum strain CP (GenBank assembly accession: GCA_001643035.1), Corynebacterium glutamicum strain ATCC 13869 (GenBank assembly accession: GCA_001687645.1), Corynebacterium glutamicum strain AJ1511 (GenBank assembly accession: GCA_002355675.1), Corynebacterium glutamicum strain XV (GenBank assembly accession: GCA_001936195.1), Corynebacterium glutamicum strain CP (GenBank assembly accession: GCA_001643035.1), accession: GCA_001447865.2), Corynebacterium glutamicum R (GenBank assembly accession: GCA_000010225.1), Corynebacterium glutamicum strain USDA-ARS-USMARC-56828 (GenBank assembly accession: GCA_001518935.2), Bifidobacterium breve strain LMC520 (GenBank assembly accession: GCA_001990225.1), Bifidobacterium breve strain BR3 (GenBank assembly accession: GCA_001281425.1), Bifidobacterium breve strain NRBB51 (GenBank assembly accession: GCA_002838405.1), Bifidobacterium breve strain NRBB09 (GenBank assembly accession: GCA_002838325.1), Bifidobacterium breve 12L ... accession: GCA_000568955.1), Bifidobacterium breve strain DRBB26 (GenBank assembly accession: GCA_002838225.1), Bifidobacterium breve strain 180W83 (GenBank assembly accession: GCA_002838525.1), Bifidobacterium breve strain JSRL01 (GenBank assembly accession: GCA_009498435.1), Bifidobacterium breve 689b (GenBank assembly accession: GCA_000569055.1), Bifidobacterium breve strain DRBB29 (GenBank assembly accession: GCA_002838705.1), Bifidobacterium breve strain DRBB27 (GenBank assembly accession: GCA_002838445.1), Bifidobacterium breve strain JR01 (GenBank assembly accession: GCA_009931415.1), Bifidobacterium breve S27 (GenBank assembly accession: GCA_000569055.1). accession: GCA_000569075.1), Bifidobacterium breve ACS-071-V-Sch8b (GenBank assembly accession: GCA_000213865.1), Bifidobacterium breve strain NRBB56 (GenBank assembly accession: GCA_002838425.1), Bifidobacterium breve DSM 20213=JCM 1192 (GenBank assembly accession: GCA_001025175.1), Bifidobacterium breve strain NRBB01 (GenBank assembly accession: GCA_002838245.1), Bifidobacterium breve strain FDAARGOS_561 (GenBank assembly accession: GCA_003813065.1), Bifidobacterium breve strain NCTC11815 (GenBank assembly accession: GCA_002838425.1). accession: GCA_900637145.1), Bifidobacterium breve strain NRBB52 (GenBank assembly accession: GCA_002838385.1), Bifidobacterium breve strain 082W48 (GenBank assembly accession: GCA_002838545.1), Bifidobacterium breve strain lw01 (GenBank assembly accession: GCA_003860285.1), and Bifidobacterium breve UCC2003 (GenBank assembly accession: GCA_000220135.1).1), Bifidobacterium breve strain NRBB11 (GenBank assembly accession: GCA_002838305.1), Bifidobacterium breve strain NRBB04 (GenBank assembly accession: GCA_002838285.1), Bifidobacterium breve NCFB 2258 (GenBank assembly accession: GCA_000569035.1), Bifidobacterium breve strain NRBB20 (GenBank assembly accession: GCA_002838645.1), Bifidobacterium breve strain NRBB27 (GenBank assembly accession: GCA_002838665.1), Bifidobacterium breve strain NRBB49 (GenBank assembly accession: GCA_002838685.1), Bifidobacterium breve strain NRBB18 (GenBank assembly accession: GCA_002838605.1), Bifidobacterium breve strain NRBB02 (GenBank assembly accession: GCA_002838645.1), accession: GCA_002838265.1), Bifidobacterium breve NRBB19 (GenBank assembly accession: GCA_002838625.1), Bifidobacterium breve strain 017W439 ​​(GenBank assembly accession: GCA_002838465.1), Bifidobacterium breve JCM 7017 (GenBank assembly accession: GCA_000568975.1), Bifidobacterium breve strain NRBB50 (GenBank assembly accession: GCA_002838365.1), Bifidobacterium breve strain 139W423 (GenBank assembly accession: GCA_002838565.1), Bifidobacterium breve strain DRBB28 (GenBank assembly accession: GCA_002838505.1), Bifidobacterium breve strain CNCM I-4321 (GenBank assembly accession: GCA_002838 assembly accession: GCA_002838585.1), Bifidobacterium breve strain DRBB30 (GenBank assembly accession: GCA_002838725.1), and Bifidobacterium breve strain NRBB57 (GenBank assembly accession: GCA_002838345.1), Bifidobacterium breve strain 215W447a (GenBank assembly accession: GCA_002838485.1), Lactococcus lactis subsp. cremoris NZ9000 (GenBank assembly accession: GCA_000143205.1), Lactococcus lactis subsp. cremoris MG1363 (GenBank assembly accession: GCA_000009425.1), Lactococcus lactis subsp. cremoris A76 (GenBank assembly accession: GCA_000236475.1), Lactococcus lactis strain SRCM103457 (GenBank assembly accession: GCA_004194355.1), Lactococcus lactis strain CBA3619 (GenBank assembly accession: GCA_007954765.1), Lactococcus lactis strain WiKim0098 (GenBank assembly accession: GCA_000143205.1), Lactococcus lactis subsp. cremoris MG1363 (GenBank assembly accession: GCA_000009425.1), Lactococcus lactis subsp. cremoris A76 (GenBank assembly accession: GCA_000236475.1), Lactococcus lactis strain SRCM103457 (GenBank assembly accession: GCA_004194355.1), Lactococcus lactis strain CBA3619 (GenBank assembly accession: GCA_007954765.1), Lactococcus lactis strain WiKim0098 (GenBank assembly accession: GCA_016406265.1), Lactococcus lactis strain K_LL005 (GenBank assembly accession: GCA_014334715.1), Lactococcus lactis subsp. lactis strain G121 (GenBank assembly accession: GCA_013395015.1), Lactococcus lactis strain N8 (GenBank assembly accession: GCA_014884605.1), Lactococcus lactis subsp. lactis 10-1 (GenBank assembly accession: GCA_000344575.1), Lactococcus lactis subsp. lactis strain F44 (GenBank assembly accession: GCA_002804185.1), Lactococcus lactis subsp. lactis diacetyl strain S50 (GenBank assembly accession: GCA_003627395.2), Lactococcus lactis strain FDAARGOS_1064 (GenBank assembly accession: GCA_014884605.1), Lactococcus lactis subsp. lactis strain assembly accession: GCA_016127135.1), Lactococcus lactis strain FDAARGOS_887 (GenBank assembly accession: GCA_016027975.1), Lactococcus lactis subsp. lactis strain UC77 (GenBank assembly accession: GCA_002078615.2), Lactococcus lactis strain FDAARGOS_866 (GenBank assembly accession: GCA_016028815.1), Lactococcus lactis strain IL1403 (GenBank assembly accession: GCA_003722275.1), Lactococcus lactis strain FDAARGOS_865 (GenBank assembly accession: GCA_016028835.1), Lactococcus lactis subsp. cremoris IBB477 (GenBank assembly accession: GCA_001856165.1), Lactobacillus paracasei strain TD 062 (GenBank assembly accession: GCA_009834405.1), Lactobacillus paracasei strain HM1 (GenBank assembly accession: GCA_018064185.1), Bacillus thuringiensis strain FDAARGOS_794 (GenBank assembly accession: GCA_013267795.1), Bacillus thuringiensis strain XL6 (GenBank assembly accession: GCA_000774075.2), Bacillus thuringiensis strain Bt-GS57 (GenBank assembly accession: GCA_017751245.1), Bacillus thuringiensis strain HER1410 (GenBank assembly accession: GCA_013340745.1), Bacillus thuringiensis serovar tolworthi (GenBank assembly accession: GCA_001548175.1), Bacillus thuringiensis strain BT62 (GenBank assembly accession: GCA_003054785.2), Bacillus thuringiensis strain HD12 (GenBank assembly accession: GCA_001598095.1), Bacillus thuringiensis serovar alessi strain BGSC 4C1 (GenBank assembly accession: GCA_001548175.1), Bacillus thuringiensis strain BT62 (GenBank assembly accession: GCA_003054785.2), Bacillus thuringiensis strain HD12 (GenBank assembly accession: GCA_001598095.1), Bacillus thuringiensis serovar alessi strain BGSC 4C1 (GenBank assembly accession: GCA_001548175.1), assembly accession: GCA_001640965.1), Bacillus thuringiensis LM1212 (GenBank assembly accession: GCA_003546665.1), Lactobacillus paracasei strain 347-16 (GenBank assembly accession: GCA_012955485.1), Lactobacillus paracasei subsp. firmus strain MGB0734 (GenBank assembly accession: GCA_015476135.1), Lactobacillus paracasei subsp. firmus strain MGB0747 (GenBank assembly accession: GCA_015476175.1), Lactobacillus paracasei strain CBA3611 (GenBank assembly accession: GCA_007292115.1), Lactobacillus paracasei subsp. paracasei strain GR0548 (GenBank assembly accession: GCA_019175405.1), Lactobacillus paracasei subsp. paracasei strain IBB3423 (GenBank assembly accession: GCA_009739485.1), Lactobacillus paracasei strain NFFJ04 (GenBank assembly accession: GCA_014905075.1), Lactobacillus paracasei strain HL182 ... assembly accession: GCA_017638905.1), Lactobacillus paracasei strain Lpc10 (GenBank assembly accession: GCA_003199005.1), Lactobacillus paracasei subsp. firmus strain AO356 (GenBank assembly accession: GCA_003957435.1), Lactobacillus paracasei subsp. firmus strain MGB0625 (GenBank assembly accession: GCA_015476155.1), Lactobacillus paracasei strain 10266 (GenBank assembly accession: GCA_008329845.1), Lactobacillus paracasei subsp. firmus strain S-NB (GenBank assembly accession: GCA_016757695.1), Lactobacillus paracasei strain Lp02 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain ZFM54 ... 10266 (GenBank assembly accession: GCA_008329845.1), Lactobacillus paracasei subsp. firmus strain S-NB (GenBank assembly accession: GCA_016757695.1), Lactobacillus paracasei strain Lp02 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain ZFM54 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain accession: GCA_003627255.1), Lactobacillus paracasei subsp. paracasei strain TMW 1.1434 (GenBank assembly accession: GCA_002813615.1), and Lactobacillus paracasei strain SRCM103299 (GenBank assembly accession: GCA_004141835.1), Lactobacillus paracasei strain NJ (GenBank assembly accession: GCA_007637635.1), Lactobacillus paracasei strain EG9 (GenBank assembly accession: GCA_003177075.1), Lactobacillus paracasei strain TK-P4A (GenBank assembly accession: GCA_015377585.1), Lactobacillus paracasei subsp. paracasei strain BD5115 (GenBank assembly accession: GCA_018596415.1), and Lactobacillus paracasei subsp. paracasei JCM 8130 (GenBank assembly accession: GCA_000829035.1), preferably Corynebacterium glutamicum ATCC 14067.

[0121] In some embodiments, the carrier protein is a backbone pilus protein. In some embodiments, the carrier protein is a native backbone pilus protein of bacteria.

[0122] Preferably, the insertion and fusion of the polypeptide of interest does not affect the formation of intermolecular isopeptide bonds, disulfide bonds or intramolecular isopeptide bonds in the carrier protein.

[0123] In some embodiments, the polypeptide of interest is fused to the terminus of a carrier protein. In some embodiments, the polypeptide of interest is fused to the N-terminus of a carrier protein.

[0124] In some embodiments, the polypeptide of interest is inserted into a carrier protein. In some embodiments, the polypeptide of interest is inserted into the turn region of a carrier protein.

[0125] In some embodiments, the carrier protein is a backbone pilin protein (Spa2 protein) from Corynebacterium glutamicum. It was observed that the Spa2 protein (SEQ ID NO: 1) comprises three tandem Ig-like domains, including an N-domain (residues 36-197), an M-domain (residues 198-343) and a C-domain (residues 344-469), which is consistent with other backbone pilin proteins. It was also observed that the absence of the M-domain did not affect the formation of CLPs. In some embodiments, the polypeptide of interest is inserted into the M domain of the backbone pilin protein. In some embodiments, the polypeptide of interest replaces the M domain or a portion thereof of the backbone pilin protein.

[0126] The Spa2 proteins from different Corynebacterium glutamicum strains may differ in sequence. In some embodiments, the carrier protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 1, 2, 3, or 4. In some embodiments, the carrier protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 1, 2, 3 or 4, wherein the residues corresponding to residues C97, C128, K194, C380, C432 and LPLTG (474-478) and optionally E158, D246 and / or E435 of SEQ ID NO: 1 are unchanged.

[0127] The carrier protein can be the mature form of SEQ ID NO: 1, 2, 3 or 4, i.e., lacking the signal peptide. In some embodiments, the carrier protein comprises amino acids 35-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4. In some embodiments, the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4, or an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4, wherein the amino acid sequence corresponding to SEQ ID NO: 1 is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO: 4. Residues C97, C128, E158, K194, D246, C380, C432, E435 and LPLTGT(474-478) of NO: 1, as well as the residues of optionally present E158, D246 and / or E435, are unchanged.

[0128] In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein or inserted between positions corresponding to G215 and L216 of SEQ ID NO: 1, between positions corresponding to G236 and E237 of SEQ ID NO: 1, or between positions corresponding to G336 and T337 of SEQ ID NO: 1.

[0129] In some embodiments, the carrier protein comprises amino acids 35-509 of SEQ ID NO: 1. In some embodiments, the polypeptide of interest is fused to the N-terminus of the carrier protein or inserted between G215 and L216, between G236 and E237, or between G336 and T337 of SEQ ID NO: 1.

[0130] In some embodiments, the polypeptide of interest is directly linked to the N-terminus of the carrier polypeptide. In some embodiments, the polypeptide of interest is linked to the N-terminus of the carrier polypeptide via a peptide linker, such as a flexible linker.

[0131] The peptide linker can generally be a short peptide having about 4-20 or more amino acids, such as a combination of Ser and Gly residues, which is a conventional flexible linker. In some embodiments, the peptide linker used in the present disclosure is (G4S)n, n=1-4. In some embodiments, the peptide linker used in the present disclosure is (G3S)n, n=1-4. In some embodiments, the peptide linker used in the present disclosure is (G4S)2, i.e., SEQ ID NO:22. In some embodiments, the peptide linker is the C10 linker of SEQ ID NO:23.

[0132] The polypeptide of interest can be selected based on the desired application of the fusion polypeptide. In some embodiments, the fusion polypeptide is provided to degrade carbohydrates such as cellulose, and the polypeptide of interest can be an endo-1,4-β-glucanase from Trichoderma reesei (TrEgl, SEQ ID NO: 19) and / or a β-glucosidase from Saccharophagus degradans (SdBgl, SEQ ID NO: 21).

[0133] In some embodiments, the recombinant cell comprises two or more polynucleotides, each encoding two or more fusion polypeptides, each fusion polypeptide comprising a different polypeptide of interest, and the modified CLP comprises these two or more polypeptides.

[0134] In some embodiments, recombinant cells are provided to bind, capture, or enrich a target molecule, and the polypeptide of interest is a polypeptide that can recognize the target peptide, including but not limited to ligands, receptors, antigens, and antibodies such as scFV and nanobodies. For example, recombinant cells are provided to capture a protein comprising SpyTag (SEQ ID NO: 37), while the polypeptide of interest comprises SpyCatcher (SEQ ID NO: 15), or vice versa.

[0135] In some embodiments, the recombinant cell is provided as the binder and the polypeptide of interest is a binding peptide, eg, Mfp35 (SEQ ID NO: 38).

[0136] In some embodiments, recombinant cells are provided to catalyze a chemical or biochemical reaction, and the polypeptide of interest is an enzyme. In some embodiments, recombinant cells are provided to degrade carbohydrates such as cellulose, and the polypeptide of interest can be an endo-1,4-β-glucanase, such as from Trichoderma reesei (TrEgl, SEQ ID NO: 19), and / or a β-glucosidase, such as from Saccharophagus degradans (SdBgl, SEQ ID NO: 21). In some embodiments, recombinant cells are provided to degrade refractory organic matter, such as plastics, and the polypeptide of interest is an enzyme responsible for degradation, such as PETase.

[0137] The present disclosure provides a method for preparing the recombinant cell of the present disclosure, comprising introducing a polynucleotide encoding the fusion polypeptide of the present disclosure into a host cell.

[0138] In some embodiments, the carrier protein in the fusion polypeptide is a native backbone pilus protein of the host cell.

[0139] In some embodiments, the host cell is a gram-positive bacterium. In some embodiments, the host cell is a bacterium selected from the group consisting of Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lactobacillus paracasei, Bacillus thuringiensis and Lactobacillus paracasei; preferably Corynebacterium glutamicum. The bacteria may include, but are not limited to, bacteria selected from the group consisting of Corynebacterium glutamicum strain BE (GenBank assembly accession: GCA_013046805.1), Corynebacterium glutamicum ATCC 14067 (GenBank assembly accession: GCA_002243555.1), Corynebacterium glutamicum strain YI (GenBank assembly accession: GCA_001643035.1), Corynebacterium glutamicum strain ATCC 13869 (GenBank assembly accession: GCA_001687645.1), Corynebacterium glutamicum AJ1511 (GenBank assembly accession: GCA_002355675.1), Corynebacterium glutamicum strain XV (GenBank assembly accession: GCA_001936195.1), Corynebacterium glutamicum strain CP (GenBank assembly accession: GCA_001643035.1), Corynebacterium glutamicum strain ATCC 13869 (GenBank assembly accession: GCA_001687645.1), Corynebacterium glutamicum strain AJ1511 (GenBank assembly accession: GCA_002355675.1), Corynebacterium glutamicum strain XV (GenBank assembly accession: GCA_001936195.1), Corynebacterium glutamicum strain CP (GenBank assembly accession: GCA_001643035.1), accession: GCA_001447865.2), Corynebacterium glutamicum R (GenBank assembly accession: GCA_000010225.1), Corynebacterium glutamicum strain USDA-ARS-USMARC-56828 (GenBank assembly accession: GCA_001518935.2), Bifidobacterium breve strain LMC520 (GenBank assembly accession: GCA_001990225.1), Bifidobacterium breve strain BR3 (GenBank assembly accession: GCA_001281425.1), Bifidobacterium breve strain NRBB51 (GenBank assembly accession: GCA_002838405.1), Bifidobacterium breve strain NRBB09 (GenBank assembly accession: GCA_002838325.1), Bifidobacterium breve 12L ... accession: GCA_000568955.1), Bifidobacterium breve strain DRBB26 (GenBank assembly accession: GCA_002838225.1), Bifidobacterium breve strain 180W83 (GenBank assembly accession: GCA_002838525.1), Bifidobacterium breve strain JSRL01 (GenBank assembly accession: GCA_009498435.1), Bifidobacterium breve 689b (GenBank assembly accession: GCA_000569055.1), Bifidobacterium breve strain DRBB29 (GenBank assembly accession: GCA_002838705.1), Bifidobacterium breve strain DRBB27 (GenBank assembly accession: GCA_002838445.1), Bifidobacterium breve strain JR01 (GenBank assembly accession: GCA_009931415.1), Bifidobacterium breve S27 (GenBank assembly accession: GCA_000569055.1). accession: GCA_000569075.1), Bifidobacterium breve ACS-071-V-Sch8b (GenBank assembly accession: GCA_000213865.1), Bifidobacterium breve strain NRBB56 (GenBank assembly accession: GCA_002838425.1), Bifidobacterium breve DSM 20213=JCM 1192 (GenBank assembly accession: GCA_001025175.1), Bifidobacterium breve strain NRBB01 (GenBank assembly accession: GCA_002838245.1), Bifidobacterium breve strain FDAARGOS_561 (GenBank assembly accession: GCA_003813065.1), Bifidobacterium breve strain NCTC11815 (GenBank assembly accession: GCA_002838425.1). accession: GCA_900637145.1), Bifidobacterium breve strain NRBB52 (GenBank assembly accession: GCA_002838385.1), Bifidobacterium breve strain 082W48 (GenBank assembly accession: GCA_002838545.1), Bifidobacterium breve strain lw01 (GenBank assembly accession: GCA_003860285.1), and Bifidobacterium breve UCC2003 (GenBank assembly accession: GCA_000220135.1).1), Bifidobacterium breve strain NRBB11 (GenBank assembly accession: GCA_002838305.1), Bifidobacterium breve strain NRBB04 (GenBank assembly accession: GCA_002838285.1), Bifidobacterium breve NCFB 2258 (GenBank assembly accession: GCA_000569035.1), Bifidobacterium breve strain NRBB20 (GenBank assembly accession: GCA_002838645.1), Bifidobacterium breve strain NRBB27 (GenBank assembly accession: GCA_002838665.1), Bifidobacterium breve strain NRBB49 (GenBank assembly accession: GCA_002838685.1), Bifidobacterium breve strain NRBB18 (GenBank assembly accession: GCA_002838605.1), Bifidobacterium breve strain NRBB02 (GenBank assembly accession: GCA_002838645.1), accession: GCA_002838265.1), Bifidobacterium breve strain NRBB19 (GenBank assembly accession: GCA_002838625.1), Bifidobacterium breve strain 017W439 ​​(GenBank assembly accession: GCA_002838465.1), Bifidobacterium breve JCM 7017 (GenBank assembly accession: GCA_000568975.1), Bifidobacterium breve strain NRBB50 (GenBank assembly accession: GCA_002838365.1), Bifidobacterium breve strain 139W423 (GenBank assembly accession: GCA_002838565.1), Bifidobacterium breve strain DRBB28 (GenBank assembly accession: GCA_002838505.1), Bifidobacterium breve strain CNCM I-4321 (GenBank assembly accession: GCA_002838585.1), Bifidobacterium breve strain DRBB30 (GenBank assembly accession: GCA_002838725.1), and Bifidobacterium breve strain NRBB57 (GenBank assembly accession: GCA_002838345.1), Bifidobacterium breve strain 215W447a (GenBank assembly accession: GCA_002838485.1), Lactococcus lactis subsp. cremoris NZ9000 (GenBank assembly accession: GCA_000143205.1), Lactococcus lactis subsp. cremoris MG1363 (GenBank assembly accession: GCA_000009425.1), Lactococcus lactis subsp. cremoris A76 (GenBank assembly accession: GCA_000236475.1), Lactococcus lactis strain SRCM103457 (GenBank assembly accession: GCA_004194355.1), Lactococcus lactis strain CBA3619 (GenBank assembly accession: GCA_007954765.1), Lactococcus lactis strain WiKim0098 (GenBank assembly accession: GCA_000143205.1), Lactococcus lactis subsp. cremoris MG1363 (GenBank assembly accession: GCA_000009425.1), Lactococcus lactis subsp. cremoris A76 (GenBank assembly accession: GCA_000236475.1), Lactococcus lactis strain SRCM103457 (GenBank assembly accession: GCA_004194355.1), Lactococcus lactis strain CBA3619 (GenBank assembly accession: GCA_007954765.1), Lactococcus lactis strain WiKim0098 (GenBank assembly accession: GCA_016406265.1), Lactococcus lactis strain K_LL005 (GenBank assembly accession: GCA_014334715.1), Lactococcus lactis subsp. lactis strain G121 (GenBank assembly accession: GCA_013395015.1), Lactococcus lactis strain N8 (GenBank assembly accession: GCA_014884605.1), Lactococcus lactis subsp. lactis 10-1 (GenBank assembly accession: GCA_000344575.1), Lactococcus lactis subsp. lactis strain F44 (GenBank assembly accession: GCA_002804185.1), Lactococcus lactis subsp. lactis diacetyl strain S50 (GenBank assembly accession: GCA_003627395.2), Lactococcus lactis strain FDAARGOS_1064 (GenBank assembly accession: GCA_014884605.1), Lactococcus lactis subsp. lactis strain assembly accession: GCA_016127135.1), Lactococcus lactis strain FDAARGOS_887 (GenBank assembly accession: GCA_016027975.1), Lactococcus lactis subsp. lactis strain UC77 (GenBank assembly accession: GCA_002078615.2), Lactococcus lactis strain FDAARGOS_866 (GenBank assembly accession: GCA_016028815.1), Lactococcus lactis strain IL1403 (GenBank assembly accession: GCA_003722275.1), Lactococcus lactis strain FDAARGOS_865 (GenBank assembly accession: GCA_016028835.1), Lactococcus lactis subsp. cremoris IBB477 (GenBank assembly accession: GCA_001856165.1), Lactobacillus paracasei strain TD 062 (GenBank assembly accession: GCA_009834405.1), Lactobacillus paracasei strain HM1 (GenBank assembly accession: GCA_018064185.1), Bacillus thuringiensis strain FDAARGOS_794 (GenBank assembly accession: GCA_013267795.1), Bacillus thuringiensis strain XL6 (GenBank assembly accession: GCA_000774075.2), Bacillus thuringiensis strain Bt-GS57 (GenBank assembly accession: GCA_017751245.1), Bacillus thuringiensis strain HER1410 (GenBank assembly accession: GCA_013340745.1), Bacillus thuringiensis serovar tolworthi (GenBank assembly accession: GCA_001548175.1), Bacillus thuringiensis strain BT62 (GenBank assembly accession: GCA_003054785.2), Bacillus thuringiensis strain HD12 (GenBank assembly accession: GCA_001598095.1), Bacillus thuringiensis serovar alessi strain BGSC 4C1 (GenBank assembly accession: GCA_001548175.1), Bacillus thuringiensis strain BT62 (GenBank assembly accession: GCA_003054785.2), Bacillus thuringiensis strain HD12 (GenBank assembly accession: GCA_001598095.1), Bacillus thuringiensis serovar alessi strain BGSC 4C1 (GenBank assembly accession: GCA_001548175.1), assembly accession: GCA_001640965.1), Bacillus thuringiensis LM1212 (GenBank assembly accession: GCA_003546665.1), Lactobacillus paracasei strain 347-16 (GenBank assembly accession: GCA_012955485.1), Lactobacillus paracasei subsp. firmus strain MGB0734 (GenBank assembly accession: GCA_015476135.1), Lactobacillus paracasei subsp. firmus strain MGB0747 (GenBank assembly accession: GCA_015476175.1), Lactobacillus paracasei strain CBA3611 (GenBank assembly accession: GCA_007292115.1), Lactobacillus paracasei subsp. paracasei strain GR0548 (GenBank assembly accession: GCA_019175405.1), Lactobacillus paracasei subsp. paracasei strain IBB3423 (GenBank assembly accession: GCA_009739485.1), Lactobacillus paracasei strain NFFJ04 (GenBank assembly accession: GCA_014905075.1), Lactobacillus paracasei strain HL182 ... assembly accession: GCA_017638905.1), Lactobacillus paracasei strain Lpc10 (GenBank assembly accession: GCA_003199005.1), Lactobacillus paracasei subsp. firmus strain AO356 (GenBank assembly accession: GCA_003957435.1), Lactobacillus paracasei subsp. firmus strain MGB0625 (GenBank assembly accession: GCA_015476155.1), Lactobacillus paracasei strain 10266 (GenBank assembly accession: GCA_008329845.1), Lactobacillus paracasei subsp. firmus strain S-NB (GenBank assembly accession: GCA_016757695.1), Lactobacillus paracasei strain Lp02 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain ZFM54 ... 10266 (GenBank assembly accession: GCA_008329845.1), Lactobacillus paracasei subsp. firmus strain S-NB (GenBank assembly accession: GCA_016757695.1), Lactobacillus paracasei strain Lp02 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain ZFM54 (GenBank assembly accession: GCA_013307125.1), Lactobacillus paracasei strain accession: GCA_003627255.1), Lactobacillus paracasei subsp. paracasei strain TMW 1.1434 (GenBank assembly accession: GCA_002813615.1), and Lactobacillus paracasei strain SRCM103299 (GenBank assembly accession: GCA_004141835.1), Lactobacillus paracasei strain NJ (GenBank assembly accession: GCA_007637635.1), Lactobacillus paracasei strain EG9 (GenBank assembly accession: GCA_003177075.1), Lactobacillus paracasei strain TK-P4A (GenBank assembly accession: GCA_015377585.1), Lactobacillus paracasei subsp. paracasei strain BD5115 (GenBank assembly accession: GCA_018596415.1), and Lactobacillus paracasei subsp. paracasei JCM 8130 (GenBank assembly accession: GCA_000829035.1), preferably Corynebacterium glutamicum ATCC 14067.

[0140] In some embodiments, the host cell is modified to inactivate the native backbone pilin protein. In some embodiments, the method includes the step of knocking out the native backbone pilin protein. The endogenous polynucleotide encoding the backbone pilin protein can also be replaced by a polynucleotide encoding a fusion polypeptide by homologous recombination.

[0141] 5. Modified CLP

[0142] The present disclosure provides a modified covalently cross-linked pilus (CLP) comprising a plurality of fusion polypeptides of the present disclosure. In some embodiments, the modified CLP is cell-free.

[0143] The present disclosure further provides a method for preparing a modified CLP, comprising the steps of: a) providing a fusion polypeptide of the present disclosure; and b) providing sortase activity. In some embodiments, the modified CLP is cell-free.

[0144] In some embodiments, the fusion polypeptide is provided by transcribing and / or translating a polynucleotide of the present disclosure.In some embodiments, the sortase activity is provided by transcribing and / or translating one or more polynucleotides encoding a sortase.

[0145] In some embodiments, the sortase is encoded by a gene that is identified as occurring in the same cluster as a gene encoding a carrier protein in nature. In some embodiments, the method comprises contacting a fusion polypeptide of the present disclosure with a sortase protein. In some embodiments, the sortase is a Class C sortase, such as srtC1 and / or srtC2, preferably wherein srtC1 and srtC2 are encoded by genes from the same cluster. In some embodiments, the method is an in vitro method.

[0146] The present disclosure provides a polynucleotide construct or combination of polynucleotide constructs comprising a polynucleotide of the present disclosure, and one or more polynucleotides encoding a sortase.

[0147] In some embodiments, the sortase is encoded by a gene that is identified as occurring in the same cluster as a gene encoding a carrier protein in nature. In some embodiments, the sortase is a class C sortase, such as srtC1 and / or srtC2, preferably wherein srtC1 and srtC2 are encoded by genes from the same cluster.

[0148] benefit

[0149] The modified CLPs and recombinant cells enable enzyme cascade reactions and improve the catalytic efficiency of multi-enzyme systems. Immobilizing enzymes on CLPs and recombinant cells can achieve whole-cell catalysis. Example

[0150] Example 1. Materials and methods

[0151] Unless otherwise stated, the experiments in the examples are routine experiments in the art, and experiments using commercially available kits or reagents were performed according to the manufacturer's instructions.

[0152] 1.1. Strains, plasmids, and culture media

[0153] General approach

[0154] The original DNA sequence was completely synthesized (Genewiz, Nanjing, China) or PCR-generated. All PCR products were produced by KOD DNA polymerase (TOYOBO, Japan). All plasmid constructions were connected using T4 DNA ligase (New England BioLabs, Boston, MA) or assembled using NEB Builder HiFi DNA Assembly MasterMix (New England BioLabs, Boston, MA). All plasmids or unmarked strains were confirmed by DNA sequencing (GENEWIZ, Guangzhou, China). The primers used in the examples are listed in Table 1.

[0155] Table 1. Primers

[0156]

[0157]

[0158] Growth medium

[0159] Corynebacterium glutamicum ATCC140675 was provided by Dr. Zheng's research group at South China University of Technology. Corynebacterium glutamicum ATCC14067 was grown in BHI liquid medium and used for recovery at 30°C and 250 rpm (37 g L -1 Brain Heart Infusion (Becton, Dickinson and company) overnight. Cg CLP formation was achieved by inoculating Corynebacterium glutamicum ATCC14067 into M63 liquid culture medium (15.6 g L -1 The cells were cultured in M63 broth (Sangon Biotech, Guangzhou, China) supplemented with 1 mM MgSO4 and 0.2% (wt / vol) glucose and incubated at 30°C for 2-3 days without shaking. The antibiotic used for the culture of C. glutamicum was kanamycin (25 μg mL -1 ) and chloramphenicol (7.5 μg mL -1 ).

[0160] Gene expression was induced using 1 mM / 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) or 1 mM theophylline. Trans1-T1 (TransGen Biotech, Shenzhen, China) was used as a cloning host for plasmid manipulation, and Escherichia coli (E. coli) BL21 (DE3) (New England BioLabs, Boston, MA) was used for protein expression. E. coli was grown in Luria-Bertani medium (10 g L -1 Peptone, 5 g L -1 Yeast extract, 10g L -1 NaCl) at 37°C or 16°C (suitable for protein expression). The antibiotic for E. coli culture was kanamycin (50 μg mL -1 ) and chloramphenicol (30 μg mL -1 ).

[0161] Strain construction

[0162] The RecET-Cre / loxP system was used to achieve a marker-free deletion strain of Corynebacterium glutamicum ATCC 14067. Huang, Y. et al. (Recombineering using RecET in Corynebacterium glutamicum ATCC 14067 via a self-excisable cassette. Sci. Rep. 7, 1-8, 2017) described the detailed method of marker-free deletion.

[0163] Briefly, to create a CLP-deficient strain of Δclp, we first constructed the self-excisable cassette, Δclp-cassette. The primer pair ck-S / A was used to amplify the Cre-Kan cassette from the PBS-Cre-Kan plasmid. The primer pairs clpL-S / A and clpR-S / A were used to amplify an approximately 800-bp left and right homologous fragment from the genome of Corynebacterium glutamicum ATCC 14067. Finally, all dsDNA fragments, including the Cre-Kan cassette and left and right homologous fragments, were used in a subsequent fusion PCR reaction, generating a linear, self-excisable dsDNA cassette of approximately 4,385 bp using the primer pair clpL-S / clpR-A.

[0164] Similarly, to construct the Δspa1-cassette, primer pairs spa1L-S / A, spa1R-S / A, ck-S / A, and spa1L-S / spa1R-A were used to amplify the left and right homologous fragments, the Cre-Kan cassette, and the linear self-excisable dsDNA cassette, respectively.

[0165] For the Δspa2-cassette, primer pairs spa2L-S / A, spa2R-S / A, ck-S / A, and spa2L-S / spa2R-A were used to amplify the left and right homologous fragments, the Cre-Kan cassette, and the linear self-excisable dsDNA cassette, respectively.

[0166] For the Δspa3-cassette, primer pairs spa3L-S / A, spa3R-S / A, ck-S / A, and spa3L-S / spa3R-A were used to amplify the left and right homologous fragments, the Cre-Kan cassette, and the linear self-excisable dsDNA cassette, respectively.

[0167] For the ΔsrtC1ΔsrtC2-cassette, primer pairs srtC1L-S / A, srtC2R-S / A, ck-S / A, and srtC1L-S / srtC2R-A were used to amplify the left and right homologous fragments, the Cre-Kan cassette, and the linear self-excisable dsDNA cassette, respectively.

[0168] For the ΔsrtA-cassette, primer pairs srtAL-S / A, srtAR-S / A, ck-S / A, and srtAL-S / srtAR-A were used to amplify the left and right homologous fragments, the Cre-Kan cassette, and the linear self-excisable dsDNA cassette, respectively.

[0169] For the Δdec-cassette, primer pairs decL-S / A, decR-S / A, ck-S / A, and decL-S / decR-A were used to amplify the left and right homologous fragments, the Cre-Kan cassette, and the linear self-excisable dsDNA cassette, respectively.

[0170] Then, by electroporation, the dsDNA box that can self-excision for different gene no-mark deletion is transformed into the competent cell (Corynebacterium glutamicum ATCC 1406) expressing exonuclease-recombinase RecE / T, multiple Kan resistance bacterium colonies are produced on BHI agar plate. Specifically, cell-plasmid DNA / dsDNA mixture is transferred in ice-cold electric rotating cup (0.1cm electrode gap).Electroporation is carried out with Bio-Rad Micropulser, it is set to 3 times 1.8KV / cm (Ec1) pulses (referring to Huang et al., Recombineering using RecET in Corynebacterium glutamicumATCC14067 via a self-excisable cassette, Sci Rep 7,7916 (2017)).

[0171] The strain of the present invention is characterized in that: the 1mM theophylline of the present invention is induced to express in the presence of the Cre enzyme and the Cre / lox site-specific recombination is used to excise the selective marker.Finally, the PCR fragment from the mutant genome is sequenced to further identify.The gained mutant strain used in this research is called Corynebacterium glutamicum ATCC 14067Δclp (Δclp), Corynebacterium glutamicum ATCC 14067Δspa1 (Δspa1), Corynebacterium glutamicum ATCC 14067Δspa2 (Δspa2), Corynebacterium glutamicum ATCC 14067Δspa3 (Δspa3) and Corynebacterium glutamicum ATCC 14067ΔsrtC1ΔsrtC2 (ΔsrtC1ΔsrtC2).By transforming the Δspa3-box into the Δspa1 bacterial strain, Corynebacterium glutamicum ATCC 14067Δspa1Δspa3 (Δspa1Δspa3) mutant is constructed. As described above, Corynebacterium glutamicum ATCC 14067 Δspa2ΔsrtA (Δspa2ΔsrtA) and Corynebacterium glutamicum ATCC 14067 Δspa2Δdec (Δspa2Δdec) mutants were constructed by transforming the ΔsrtA-cassette and the Δdec-cassette into the Δspa2 strain, respectively.

[0172] Plasmid construction

[0173] i) Construction of constitutive expression plasmids for Spa2 pilin and different fusion proteins

[0174] The pEC-XK99E plasmid was used as the original plasmid. The pEC-XK99E backbone (GNENWIZ, China), the coding sequences of Spa2 or various recombinant Spa2 (SEQ ID NOs: 1, 5, 8-14, and 24, respectively), and the natural promoter of the spa2 gene (SEQ ID NO: 26) were amplified by PCR, and then all DNA fragments were assembled with NEB Builder HiFi DNA Assembly Master Mix to construct plasmids pEK-spa2, pEK-spa2cut, pEK-E1 / mCherry-spa2, pEK-E2 / mCherry-spa2, pEK-E3 / mCherry-spa2, pEK-E4 / mCherry-spa2, pEK-6his-spa2, pEK-SpyTagSpa2, pEK-Mfp3Spep-Spa2, pEK-N-mCherry-C (see, for example, Figure 1 ).

[0175] ii) Construction of pEK-SpyCatcher-Spa2, pEK-Venus-Spa2, pEK-CcEgl-Spa2, pEK-N-Ven_C-Ven, pEK-N-Ven-Spa2, pEK-C-Ven-Spa2, pEK-N-Ven-Spa2_C-Ven-Spa2, pEC-TrEgl_SdBgl, and pEC-TrEgl-Spa2_SdBgl-Spa2 plasmids

[0176] Based on the pEC-XK99E backbone, which has additional SmaI, XbaI, NcoI, BamHI, SpeI, and SalI restriction sites, two basic plasmids 203 and 204 were constructed by Gibson assembly using NEB Builder HiFi DNA Assembly Master Mix (see Figure 2 ). SmaI, XbaI and NcoI were used to fuse the protein with the Spa2 pilus protein, and SpeI and SalI (Takara) were used to insert another independent expression cassette of the fusion protein.

[0177] To create the pEK-SpyCatcher-Spa2, pEK-Venus-Spa2, pEK-CcEgl-Spa2, pEK-N-Ven-Spa2, and pEK-TrEgl-Spa2 plasmids, the coding sequences of SpyCatcher, Venus, CcEgl, N-Ven, and TrEgl (SEQ ID NOs: 15-19) were cloned into the SmaI and XbaI sites of 203 by ligation.

[0178] To construct pEK-N-Ven and pEK-TrEgl plasmids, the CDS of N-Ven and TrEgl (SEQ ID NOs: 18 and 19) were inserted into the linearized backbone of 203 (digested with SmaI and Spel, Takara) by Gibson assembly.

[0179] To create the pEK-C-Ven-Spa2 and pEK-SdBgl-Spa2 plasmids, the CDSs of C-Ven and SdBgl (SEQ ID NOs: 20 and 21) were cloned into the SmaI and XbaI sites in 204 by ligation.

[0180] To construct the pEK-C-Ven and pEK-SdBgl plasmids, the CDS of C-Ven and SdBgl (SEQ ID NOs: 20 and 21) were inserted into the linearized backbone of 204 (digested with SmaI and SalI, Takara) by Gibson assembly.

[0181] Finally, the C-Ven-Spa2 cassette was obtained by digesting pEK-C-Ven-Spa2 with SpeI and SalI, and then cloned into the pEK-N-Ven-Spa2 plasmid (digested with SpeI and SalI, Takara) to construct the tandem expression plasmid pEK-N-Ven-Spa2_C-Ven-Spa2 (see Figure 3 ).

[0182] Similar strategies were used to construct other tandem expression plasmids pEK-TrEgl-Spa2_SdBgl-Spa2, pEK-N-Ven_C-Ven, and pEK-TrEgl_SdBgl. pEC-TrEgl-Spa2_SdBgl-Spa2 and pEC-TrEgl_SdBgl were constructed by replacing kanamycin resistance with chloramphenicol resistance (see Figure 3 ).

[0183] iii) Construction of pZ9-dxs_crtEBI plasmid

[0184] The dxs (SEQ ID NO: 27) and crtEBI (crtE, SEQ ID NO: 28 and crtBI, SEQ ID NO: 29) gene fragments were amplified from the genome of Corynebacterium glutamicum ATCC 13032 using primer pairs dxs-A / dxs-S, crtE-S / crtE-A, and crtBI-S / crtBI-A, respectively; dxs and crtEBI driven by the Ptac promoter (SEQ ID NO: 31) were amplified using primer pair ptrc-S / ptrc-A; and the lacI fragment (SEQ ID NO: 30) was amplified from pEC-XK99E using primer pair lacI-S / lacI-A.

[0185] Then, the dxs, crtEBI, and lacI fragments were assembled into the pZ9 backbone (GENEWIZ, China) by Gibson assembly to construct the pZ9-dxs_crtEBI plasmid ( Figure 4 ).

[0186] iv) Construction of pET-28a-Spa2 plasmid

[0187] The coding sequence of Spa2 (SEQ ID NO: 6) was amplified from the genome of Corynebacterium glutamicum ATCC 14067 and then assembled into the pET-28a(+) backbone (Novagen, Madison, WI) by Gibson assembly (see Figure 5 ).

[0188] Transmission electron microscopy and immunogold labeling

[0189] Transmission Electron Microscopy Imaging: Corynebacterium glutamicum cells cultured in M63 medium for 2-3 days were harvested, washed twice in PBS buffer, and 20 μL of the M63 liquid culture (OD600 ≈ 1) was deposited onto a carbon-coated TEM grid for 5-10 minutes. The sample was washed twice with 50 μL of PBS buffer and three times with 20 μL of water, after which the excess solution was quickly removed with filter paper. The cells were deposited onto a copper wire grid, negatively stained with 15 μL of 2% w / v uranyl acetate solution for 1 minute, and dried under an infrared lamp for 10 minutes. The sample was examined in a JEOL JEM-1400 transmission electron microscope at an accelerating voltage of 120 kV.

[0190] Immunogold labeling: Spa1 (SEQ ID NO: 31, Spa1-Ab), Spa2 (SEQ ID NO: 32, Spa2ab) and Spa3 (SEQ ID NO: 33, Spa3-Ab) were CgPartial CDS of CLP fimbriae protein was expressed in Escherichia coli, purified and injected into rabbits to prepare specific polyclonal antibodies α-Spa1, α-Spa2 and α-Spa3, respectively (Your Bio-Tech Partner, Shanghai, China).

[0191] For immunogold labeling, 20 μ L of Corynebacterium glutamicum liquid culture (OD600≈1) in M63 are placed on carbon coating grid for 10min, washed twice with PBS buffer, washed three times with water.Sample is blocked 30min with PBS containing 1% bovine serum albumin (Sangon Biotech, A600332-0100).Blot solution with filter paper, the cells deposited on the copper wire net are used in the PBS containing 1% BSA at 1:200 dilution of fimbriae protein primary antibody (above-mentioned polyclonal antibody) dyeing 1h, then washed and blocked (PBS+1%BSA).Sample is used in the PBS containing 1% BSA at 1:50 dilution of 10nm gold-modified goat anti-rabbit IgG (Bioss, Beijing, China) dyeing 45min, then washed three times with PBS, washed five times with water.Then, carry out negative staining, drying and imaging as above. Double immunogold labeling was performed according to Budzik, JM et al. (Assembly of pili on the surface of Bacillus cereus vegetative cells. Mol. Microbiol. 66, 495-510, 2007) with some modifications. Briefly, after incubation with the primary antibody, the samples were incubated at room temperature for 2 h with PBS containing 3% paraformaldehyde and 2% glutaraldehyde. The samples were washed three times with PBS and incubated at room temperature for 10 min with 0.02 M glycine in PBS. The immunogold labeling process was performed with a second pilin antibody and gold-modified goat anti-rabbit IgG of varying sizes (5 nm, 15 nm, or 30 nm) (Bioss, Beijing, China), followed by negative staining, drying, and imaging.

[0192] Quantification of CLP by whole-cell filtration ELISA

[0193] The presence of extracellular amyloid was detected by whole cell filtration ELISA for the quantitative determination of CLP (see Nguyen, PQ et al., Programmable biofilm-based materials from engineered curlinanofibres. Nat. Commun. 5, 1-10, 2014). Briefly, Corynebacterium glutamicum was cultured in M63 liquid medium for 48 h, the culture was collected, washed and diluted on ice with Tris-buffered saline containing 0.1% Proclin™ 300 (Sigma, 48912-U) to an OD600 of 0.1. Then, 25 μL of the diluted culture was loaded into Multiscreen-GV96-well filter plates (0.22 mm pore size; EMD Millipore), followed by washing (TBST (Sangon Biotech, C520009-0005) + 0.1% Proclin™ 300), blocking (TBST + 0.1% Proclin™ 300 + 1% bovine serum albumin + 0.01% H2O2), incubation with α-Spa2 (diluted to 1:5,000 in TBST + 0.1% Proclin™ 300), washing and blocking as described above, and incubation with goat anti-rabbit HRP-conjugated secondary antibody (Sangon Biotech, Guangzhou, China; diluted to 1:5,000 in TBST + 0.1% Proclin™ 300). Subsequently, a color reaction was performed using Ultra-TMB (3,30,5,50-tetramethyl-benzidine, Thermo Fisher, 34028), and the reaction was terminated by adding 2M H2SO4. Finally, the absorbance of the product was measured at 450 nm (reference wavelength 650 nm) using a Cytation reader (BioTek).

[0194] AFM imaging

[0195] The culture in 2mL M63 liquid culture medium is incubated on mica surface for 2-4h, to allow sample deposition. Use pipette to suck away excess solution and wash twice with water. Then the sample is dried with nitrogen and collected immediately for AFM imaging. On Dimension FastScanTM AFM (Bruker), use silica cantilever (SANASYST-AIR, Bruker, K=0.4N / m,~70kHz) to carry out ScanAsyst mode AFM.

[0196] Expression and purification of recombinant Spa2

[0197] Recombinant Spa2 was expressed as an N-terminal His-tagged protein. E. coli BL21 (DE3) transformed with plasmid pET-28a-Spa2 (CaCl2 method) was grown overnight at 37°C to provide a starter culture for expression. 1% (v / v) of the starter culture was used to inoculate a 50 μg mL -1 A total of 1 L of culture medium containing kanamycin was added and grown at 37°C. When the OD600 reached 0.8, the culture temperature was lowered to 16°C, and IPTG was added to a final concentration of 0.5 mM to induce protein overexpression. After 16 hours, the cells were collected by centrifugation, and the cell pellet was suspended in buffer A (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) and lysed by high-pressure homogenization. The cell lysate was centrifuged at 12,000 rpm for 30 minutes at 4°C.

[0198] The resulting supernatant was loaded onto a nickel affinity column (5 mL, GE) pre-equilibrated with buffer A (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The His-tagged Spa2 protein was eluted with buffer A containing 50 mM imidazole. The His-tagged Spa2 protein was buffer-exchanged into buffer A and de-tagged with HRV3c (SEQ ID NO: 34, 1 mg / 50 mg Spa2) overnight at 4°C. The digestion product was loaded onto a 5-mL Ni-NTA column (GE) and eluted with a buffer A / buffer B (buffer A + 500 mM imidazole) gradient (5% buffer B, 10% buffer B, 20% buffer B, and 100% buffer B). The effluent from 10% buffer B was collected.

[0199] Further purification was performed by ion exchange chromatography (HiTrap Q HP, 5 mL & Cytiva) and size exclusion chromatography (Uniondex 75 pg 16 / 60, UNION-BIOTECH, China). The entire protein purification process was carried out at 4°C.

[0200] Protein crystallization and structure determination

[0201] The final purified protein was concentrated to 20 mg mL-1 in 10 mM Tris-HCl pH 8.0 and 50 mM NaCl for crystallization. The sitting-drop vapor diffusion technique (http: / / soft-matter.seas.harvard.edu / index.php / Vapor_Diffusion_Method) was used to crystallize the Spa2 protein. Crystals were obtained by mixing 4 μL of Spa2 protein with 4 μL of a stock solution (0.2 M sodium sulfate, 0.1 M Bis-Tris propane pH 7.5, 20% w / v PEG 3350) and incubating the mixture at 18°C ​​for 1-2 weeks. The crystals were immersed in a cryoprotectant solution consisting of the stock solution and 20% (v / v) glycerol and then rapidly frozen with liquid nitrogen. Diffraction data were collected using flash-frozen crystals (in a nitrogen stream at 100 K) at the BL18U1 beamline of the Shanghai Synchrotron Radiation Facility (Shanghai, China). The data were processed by XDS9 and then further processed using STARANISO10 (a server from Global Phasing Company).

[0202] The recombinant Spa2 crystal diffracted to Resolution( Figure 14 ), belongs to space group P212121, unit cell parameters α=β=γ=90.0°, with two molecules in the asymmetric unit. The structure was solved by molecular replacement using the Spa2 coordinates predicted by PHASER11 and Alphafold Colab12 as a template. Further manual modeling was performed using COOT13. The model was refined using PHENLX14. Data collection, phasing, and refinement statistics are given in Table 3. The structure was drawn using PyMOL 2.3.4 (https: / / pymol.org / 2 / ).

[0203] Fluorescence measurement

[0204] Microplate reader measurement: A colony of Corynebacterium glutamicum was inoculated into 10 mL of BHI and cultured for 12 h. The cells were then transferred to M63 with an initial OD600 of 0.1 at 30 ° C for 3 days without shaking. The cells were collected by centrifugation at 5,000 rpm, washed three times with PBS and diluted with PBS (OD600 ≈ 0.5). Accurately transfer 200 μL of sample to a flat-bottomed 96-well black plate and analyze on a Tecan Infinite Pro 200 microplate reader. The excitation / emission wavelengths for mCherry fluorescence intensity were 580 / 610 and for Venus fluorescence intensity were 510 / 545 nm. Fluorescence intensity divided by OD absorbance is the normalized fluorescence intensity.

[0205] Fluorescence (confocal) microscopy imaging: Cells prepared for microplate reader measurement were dropped onto glass slides and imaged using a Nikon TI2-E inverted microscope. Microscope light source power, detector gain, and image processing settings were kept consistent across samples.

[0206] Strains expressing SpyTag-Spa2, SpyCatcher-Spa2, and Spa2 (strain Δspa2 transformed with pEK-SpyTagSpa2, pEK-SpyCatcherSpa2, and pEK-spa2, respectively) were cultured in M63 on glass-bottomed culture dishes for 3 days. The dishes were then washed three times with PBS containing 0.5% Tween80 (PBST) and blocked with PBST containing 1% BSA for 1 hour. The SpyTag-Spa2 and Spa2 groups were incubated with purified GFP-SpyCatcher (SEQ ID NO: 35) at room temperature for 1 hour, and the SpyCatcher-Spa2 and Spa2 groups were incubated with purified GFP-SpyTag (SEQ ID NO: 36) at room temperature for 1 hour. All samples were washed three times with PBS buffer and imaged under a Nikon TI2-E inverted microscope.

[0207] Microsphere binding assay

[0208] The Spa2 strain or the Mfp3Spep-Spa2 strain was cultured in M63 medium (3 mL) supplemented with 200 μL of a green fluorescent PS microsphere solution in a 35-mm culture dish containing 2-3 glass slides at 30°C for 3 days without shaking. The settled glass slides were then removed and gently rinsed to remove unadhered microspheres. Water jets were applied at a constant discharge pressure of 5 psi for 15 s on a pressure-flow controller (PG-MFC-8CH, PreciGenome) to compare the binding capacity of the different samples. Fluorescence images were recorded before and after mechanical impact with water jets.

[0209] Mass spectrometry analysis

[0210] 1) Sample preparation

[0211] i) Preparation of Spa2cut and its mutant variants

[0212] As described above, the pEK-spa2cut plasmid was transferred into Δspa2 by electroporation to construct strain Δspa2-pEK-spa2cut for expressing a monomer of Spa2cut (SEQ ID NO: 5). The cells were inoculated in M63 medium containing 25 μg mL-1 kanamycin and cultured for 3 days. The supernatant (200 mL) was collected and concentrated to 1 mL, and then purified by nickel affinity chromatography as previously described in the section "Expression and purification of recombinant Spa2". Spa2cut was eluted with 100 mM imidazole. The final purified protein buffer was exchanged into 10 mM Tris-HCl, 100 mM NaCl, pH 8.0. A similar process was followed for the Spa2cut mutant variants E158Acut, D246Acut, E435Acut and D246A / E435Acut.

[0213] ii) Cg Isolation of CLP

[0214] The method of isolating SpaA pili of Corynebacterium diphtheriae was used to collect Cg CLP fibers (see Kang, HJ et al., The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds. Proc. Natl. Acad. Sci. USA 106, 16967-16971, 2009). Specifically, an engineered CLP fiber for polymer purification was generated by transforming the plasmid pEK-6his-spa2 into a strain lacking the spa2 gene and the housekeeping sortase encoding gene srtA. Cg CLP. Due to the lack of sortase A, the Δspa2ΔsrtA-pEK-6his-spa2 strain is able to express 6His- Cg CLP is secreted into the culture medium. To express 6His- Cg CLP polymer, Δspa2ΔsrtA-pEK-6his-spa2 cells were inoculated with 25 μg mL -1 Kanamycin-containing M63 medium was cultured for 3 days. Cg For CLP purification, 500 mL of supernatant was collected and concentrated to 5 mL in a buffer solution of 10 mM Tris-HCl, 100 mM NaCl, pH 8.0, and purified by nickel affinity chromatography. 6His- CgCLP polymer. Then the purified 6His- Cg CLP fibers were boiled in SDS sample buffer (6× protein loading buffer, TransGen Biotech, DL101-02) and subjected to SDS-PAGE gel analysis. High molecular weight proteins were cut from the Coomassie Brilliant Blue-stained SDS-PAGE gel. Cg CLP polymer bands are prepared for intermolecular peptide bond identification.

[0215] 2) Protein precipitation and digestion

[0216] i) Processing samples for signal peptide identification

[0217] The Spa2cut solution was precipitated with acetone (1:4) and the particles were dried with Speedvac (room temperature) for 1-2 min. The particles were then dissolved in 100 mM Tris-HCl (pH 8.5) supplemented with 8 M urea. 5 mM TCEP (ThermoScientific) for reduction and 10 mM iodoacetamide (Sigma) for alkylation were added and incubated at room temperature for 30 min. The protein mixture was diluted (1:4) and digested overnight with 1:40 (w / w) chymotrypsin. The protease-digested peptide solution was desalted using a MonoSpin™ C18 column (GLScience, Tokyo, Japan) and dried with Speedvac.

[0218] ii) Processing samples for intramolecular covalent bond identification

[0219] To identify intramolecular isopeptide bonds, Spa2cut samples were processed according to the same protocol as previously described for signal peptide identification. To identify disulfide bonds, Spa2cut samples were processed according to a similar protocol, except that pepsin (Promega) was intentionally added for digestion and 5 mM TCEP (Thermo Scientific) was avoided to ensure that disulfide bonds, if any, remained intact.

[0220] iii) Processing of samples for identification of intermolecular isopeptide bonds

[0221] Coomassie brilliant blue stained CgThe SDS-PAGE gel bands of CLP fibers were cut into small pieces and washed in water, then washed in 50% acetonitrile and 50mM NH4HCO3 in 100% acetonitrile. The samples were reduced with 10mM TCEP (Thermo Scientific) in 100mM NH4HCO3 at 55°C for 1 hour and alkylated with 55mM iodoacetamide (Sigma) in 100mM NH4HCO3 at 37°C in the dark for 30 minutes. The gel slices were then washed with 100mM NH4HCO3 and 100% acetonitrile and dried. The samples were first digested with 3μg trypsin (Promega) in 50mM NH4HCO3 at 37°C overnight, and then 1μg of Asp-N endoproteinase (Promega) was added and incubated again overnight. The digested peptides were extracted twice with 50% acetonitrile containing 5% formic acid.

[0222] 3) LC / tandem MS (MS / MS) analysis of peptides

[0223] Protease-digested peptides were analyzed by LCMS / MS using an Easy-nLC1200 nano HPLC (Thermo Scientific) hybrid system with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific). Peptides were separated on a 30 cm long pulled-tip analytical column (75 μm ID, packed with ReproSil-Pur C18-AQ 1.9 μm resin, Dr. Maisch GmbH) at 55°C using a 120 min linear gradient in 0.1% formic acid in water (buffer A) and 0.1% formic acid in 80% acetonitrile (buffer B) at a flow rate of 300 nl / min. One full scan MS spectrum (m / z 300-1800) was acquired, followed by the first 20 MS / MS events, which were generated sequentially from the first to the 20 most intense ions selected from the full MS spectrum at 30% normalized collision energy. Peptide validation for signal peptide identification was performed automatically in PEAKS ABv2.0 (Tran, NH et al. Complete de novo assembly of monoclonal antibody sequences. Sci. Rep. 6, 1-10, 2016). Peptides containing isopeptide bonds were identified using plink2 software (pFind Team, Beijing, China) (Lu, S. et al. Mapping native disulfide bonds at aproteome scale. Nat. Methods 12, 329-331, 2015). Peptides containing intermolecular isopeptide bonds were manually analyzed from MS / MS data based on the theoretical m / z of predicted peptides containing isopeptide bonds. Cg CLP-digested peptides.

[0224] 4) Accurate molecular weight determination

[0225] The precise molecular weights of Spa2cut and its variants were determined by quadrupole time-of-flight mass spectrometry (Agilent 6550iFunnel Q-TOF) using a linear gradient on an HPLC system. The raw MS data were deconvoluted using the BioConfirm algorithm integrated into MassHunter software.

[0226] Enzymatic activity assay

[0227] 3,5-Dinitrosalicylic acid (DNS) was used to determine the enzymatic activity of cellulase on carboxymethyl cellulose sodium salt (CMC-Na, Sigma, USA) (Dong, C. et al. Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production. Biotechnol. Biofuels 13, 1-9, 2020). 10OD of TrEgl-Spa2_SdBgl-Spa2 (C003 strain) and TrEgl_SdBgl (C004 strain) cells were concentrated to 500 μL and incubated at 50°C in 2mL 50mM acetic acid (pH 4.8) containing 1% (w / v) CMC-Na substrate for 30min. The reaction was terminated by adding DNS and boiling for 10min; reducing sugars were detected at 540nm. One unit of enzyme activity was defined as the amount of cells that released 1 μmol of glucose from cellulose within 1 min at 50°C.

[0228] Quantitative Analysis of Lycopene by HPLC

[0229] The lycopene-producing plasmid pZ9-dxs_crtEBI was transformed into the strain TrEgl_SdBgl to construct recombinant strains C003 and C004, which produced lycopene using cellulose. The C003 and C004 strains were inoculated with 25 μg mL -1 Kanamycin and 7.5 μg mL -1 The cells were cultured in 10 mL of BHI containing chloramphenicol at 30°C with a stirring speed of 200 rpm for 12 h. The cells were then transferred to 50 mL of modified M63 medium (15.6 g L -1 Cultures were grown in M63 broth supplemented with 1 mM MgSO4, 2% (wt / vol) CMC-Na) at an initial OD600 of 3 at 30°C for 2 days with or without the addition of 1 mM IPTG.

[0230] According to Li, C. et al. (Heterologous production of α-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method. Bioresour. Technol. 341, 125782, 2021), quantitative analysis of lycopene production was performed. IPTG-induced and uninduced cells (1 mL) were collected into 2 mL lysis matrix Y (MP Biomedicals) tubes by centrifugation at 12,000 rpm for 5 min. The particles were resuspended in a mixture of 60% hexane and 40% acetone and lysed using a FastPrep R-24 5G bead mill and lysis system (MP Biomedicals) for lycopene extraction. The lysis conditions were 30 s once, 1 min apart, for a total of 6 times.

[0231] The samples were centrifuged at 14,000 rpm for 10 min at 4°C, and the resulting supernatant was transferred to a brown 2 mL screw-cap glass vial (Agilent Technologies) and directly subjected to HPLC analysis. Lycopene quantification was performed on an Agilent 1260 Series HPLC system (Agilent Technologies) using a YMC Carotenoid (250 × 4.6 mm L.D., YMC) and detected by a diode array detector (DAD) at 450 nm. For separation, a binary gradient elution was used, changing the eluent from 100% eluent A of methanol / methyl tert-butyl ether / water (81 / 15 / 4) to 100% eluent B of methanol / methyl tert-butyl ether / water (7 / 90 / 3) over 90 min at a flow rate of 1.0 mL min at 20°C. The injection volume was 10 μL (eluent A for 2 min, eluent B for 2 min to 95 min, and eluent A for 95 min to 100 min).

[0232] Example 2. Exploring the molecular assembly of CLP structure in Corynebacterium glutamicum

[0233] This example is to study the CLP assembly in the industrial chassis host Corynebacterium glutamicum ATCC 14067 (called Cg CLP).

[0234] Identification of key building blocks for CLP assembly in Corynebacterium glutamicum

[0235] The industrial chassis host, Corynebacterium glutamicum, is a “generally recognized as safe” (GRAS) strain with well-established gene editing tools and is widely used in the industrial production of valuable products such as amino acids, diamines, terpenes, and other chemicals (Zhao, N. et al. Development of a Transcription Factor-Based Diamine Biosensor in Corynebacterium glutamicum. ACS Synth. Biol. 10, 3074-3083, 2021; and Xu, X. et al., Ledesma-Amaro, R. & Liu, L. Microbial chassis development for natural product biosynthesis. Trends Biotechnol. 38, 779-796, 2020).

[0236] In C. glutamicum, we predicted that the CLP BGC contains three pilin-encoding genes, spa1, spa2, and spa3, and two sortase-encoding genes, srtC1 and srtC2 ( Figure 6 ), which is similar to the CLP gene cluster of the SpaH type (a relatively little studied pilus type) in pathogenic Corynebacterium diphtheriae (Mandlik, A. et al., Pili in Gram-positive bacteria: assembly, involvement in colonization and biofilm development. Trends Microbiol. 16, 33-40, 2008).

[0237] In TEM and AFM imaging, there were no filamentous structures on the cell surface of C. glutamicum after CLP BGC deletion, while the filamentous structure phenotype was restored after CLP BGC supplementation ( Figure 7 ),show Cg CLP BGCs are responsible for fiber formation.

[0238] The polyclonal antibodies against Spa1, Spa2 and Spa3 were used to identify Cg Composition of CLP. Cg TEM images of CLP show that Cg CLP fibers contain two minor pilin proteins, Spa1 and Spa3, and a skeletal pilin protein, Spa2 ( Figure 8 ). was used to evaluate the effects of three pilin proteins on CgTEM and AFM imaging of the specific role of CLP assembly showed that cells deficient in Spa1 (Δspa1 strain), Spa3 (Δspa3 strain), or both (Δspa1Δspa3 strain) could still produce fibers ( Figure 7 In contrast, cells lacking Spa2 (Δspa2) failed to produce any fibers, and overexpression of Spa2 (Spa2) promoted the formation of numerous long fibers across the entire cell surface ( Figure 7 ).

[0239] TEM and AFM images also showed that cells lacking both SrtC1 and SrtC2 (ΔsrtC1ΔsrtC2) completely blocked fibril formation ( Figure 9 ).

[0240] In summary, it has been confirmed that the scaffold pilin Spa2 protein is catalyzed by sortase Cg The scaffolding pilin is an essential building block for CLP assembly and production, similar to the well-studied role of SpaA in the assembly of the pathogenic Corynebacterium diphtheriae pili. Despite this similarity, the large differences in size and sequence of scaffolding pilin proteins from different Gram-positive pathogens make it difficult to predict whether the structural principles underlying CLPs in other hosts also apply. Cg CLP becomes challenging.

[0241] 2.2. Cg Isopeptide bonds and disulfide bonds during CLP assembly

[0242] In determining whether the Spa2 scaffold pilin is Cg After that, experiments were conducted to determine the essential building blocks for CLP fiber generation. Cg The formation of intermolecular isopeptide bonds, disulfide bonds, or intramolecular isopeptide bonds during CLP assembly.

[0243] First, the purified Cg CLP polymers ( Figure 10 ), and then digested in gel with trypsin (Promega) and AspN endoproteinase (Promega). Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze the digestion products and verify the presence of intermolecular isopeptide bonds (bond formation leads to the elimination of water molecules, resulting in a slight decrease in molecular weight). Specifically, the peptide peak at m / z 832.92+ ( Figure 11 and Table 2 ) showed that the scaffold pilin Spa2 is cross-linked between K194 at the N-terminus of Spa2i and T477 at the C-terminus of Spa2i+1 (Lys194-Thr477).

[0244] Table 2. Product ions generated during MS / MS of peptides containing the intermolecular isopeptide bond between Lys194 and Thr477 of Spa2 at m / z 832.92+

[0245]

[0246]

[0247] aMonoisoptic mass of the observed ion

[0248] b Theoretical ion. Monoisotopic masses were calculated using the Fragment Ion Calculator.

[0249] (http: / / db.systemsbiology.net:8080 / proteomicsToolkit / FragIonServlet.html).

[0250] c Difference between observed ion mass and theoretical ion mass.

[0251] The recombinant variant of Spa2 secreted by Corynebacterium glutamicum cells (Spa2 cut , SEQ ID NO: 5) by quadrupole time-of-flight mass spectrometry ( Figure 12 ) indicates a molecular weight of 46,504.6Da ( Figure 13 ), than the secretion of Spa2 cut The expected value calculated from the amino acid sequence is approximately 54.7 Da less. This detected mass is consistent with the loss of three NH3 units and two H2 units, indicating the formation of three intramolecular isopeptide bonds (loss of one amine molecule, ≈17 Da) and two disulfide bonds (loss of two hydrogen atoms, ≈2 Da) in Spa2.

[0252] Structural characteristics of the skeletal pilin Spa2

[0253] Using the coordinates predicted by Alphafold Colab as a template, PHASER was used to obtain Spa2 by molecular replacement method. X-ray crystal structure at high resolution (PDB ID: 7WOI) ( Figure 15 and Table 3)( Figure 16 a).

[0254] Table 3. Data collection and refinement statistics

[0255]

[0256]

[0257] a The values ​​in brackets correspond to the outermost level of the data.

[0258] b R merge =ΣΣi|I(h)i-<I(h)> | / ΣΣi|I(h)i|, where<I(h)> is the average equivalent intensity.

[0259] c R work =Σ|Fo-Fc| / Σ|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.

[0260] d R free =Σ|Fo-Fc| / Σ|Fo|. This value is calculated using a test data set consisting of 5% of the total data randomly selected from the observed reflections.

[0261] Spa2 is arranged as three tandem Ig-like domains, including the N-domain (residues 36-197, pink), the M-domain (residues 198-343, blue), and the C-domain (residues 344-469, green), forming The elongated molecules ( Figure 15 The three tandem Ig-like domains of Spa2 are closely related to the backbone pilin protein SpaA from the human pathogen Corynebacterium diphtheriae (PDB ID: 3HR6, root mean square deviation (RMSD) More than 270 α-carbon (Cα) atoms, Figure 16 b) and SpaD (PDB ID: 4HSS, RMSD More than 311 Cα atoms, Figure 16 c) Similar (Kang, HJ et al., 2009above, and Kang, HJ et al. Aslow-forming isopeptide bond in the structure of the major pilinSpaD from Corynebacterium diphtheriae has implications for pilusassembly. Acta Crystallogr.D Biol. Crystallogr.70, 1190-1201, 2014). The crystals of Spa2 adopt head-to-tail stacking, which makes Spa2 i N-domain in Spa2 i+1 C-domain adjacency in ( Figure 15 ), which is similar to Spa2 monomer through Spa2 iN-terminal K194 and Spa2 i+1 The results of intermolecular isopeptide bond connection between T477 at the C-terminus are consistent with those of Figure 11 These results together indicate that Cg The bioassembly of CLP fibers occurs via head-to-tail polymerization of Spa2 monomers.

[0262] Furthermore, interpretation of the electron density map clearly revealed three common isopeptide bonds and two unique disulfide bonds in the Spa2 structure ( Figure 17 ). Pepsin-digested Spa2 cut LC-MS / MS analysis of the product also confirmed the formation of multiple covalent bonds ( Figure 18 Isopeptide bonds connect Lys57 and Asn195 to catalytic Glu158 in the N-domain; Lys203 and Asn318 to catalytic Asp246 in the M-domain; and Lys355 and Asn466 to catalytic Glu435 in the C-domain ( Figure 17 a). It is noteworthy that the presence of three intramolecular isopeptide bonds distributed in the three domains of the backbone pilin Spa2 in Corynebacterium glutamicum is similar to the characteristics of the backbone pilin SpaD from pathogenic Corynebacterium diphtheriae (Kang, HJ et al., 2014, supra), but is very different from the backbone pilin SpaA of pathogenic Corynebacterium diphtheriae, which lacks an isopeptide bond in the N-terminal domain (Kang, HJ et al., 2009, supra). In addition, two disulfide bonds are formed between Cys97 and Cys128 in the N-domain and between Cys380 and Cys432 in the C-domain, respectively ( Figure 17 b). Notably, the presence of two disulfide bonds in Spa2 is highly unique compared to other backbone pilin proteins from human pathogenic bacteria, such as Spy0128 from Streptococcus pyogenes 37 (PDB ID: 3B2M) and BcpA from Bacillus cereus 38 (PDB ID: 3KPT), which lack disulfide bonds, and SpaA and SpaD from Corynebacterium diphtheriae, which contain only one disulfide bond in the C-terminal domain (Kang, HJ et al., 2009 and 2014, supra).

[0263] Intermolecular polymerization between Spa2 monomers

[0264] Functional assays were performed on various Spa2 mutant variants expressed in ΔSpa2 to explore their in vivo Cg In fact, K194A and LPLTG 474LALAA478 Mutagenesis experiments with variants blocked CgCLP production, confirmed by Lys194 in the N-domain and LPLTG in the C-domain 474-478 All participate in the polymerization of Spa2 monomers ( Figure 19 and 20 ).

[0265] A series of Spa2 variants were generated to further test how the intramolecular isopeptide and disulfide bonds in the Spa2 monomer contribute to Cg Formation and stabilization of CLPs.

[0266] First, variants of Spa2 were constructed in the Δspa2 strain containing alanine substitutions (E158A, D246A, and E435A) at Glu158, Asp246, and Glu435, which originally catalyze the formation of Lys-Asn isopeptide bonds in each domain (the substitutions were introduced into the Spa2 coding sequence in pEK-Spa2, respectively). LC-MS / MS analysis, bioimaging characterization, and ELISA quantification showed that E158A, D246A, and E435A eliminated one or two intramolecular isopeptide bonds ( Figure 21 ac), none of these are correct Cg any significant impact on CLP generation ( Figure 19 and 20 Only the double mutation variant D246A / E435A eliminates all three intramolecular isopeptide bonds in Spa2 ( Figure 21 d), only 44.9% of the Cg CLP( Figure 20 ).

[0267] Secondly, variants C97A and C380A abolished disulfide bonds in the N- and C-domains of Spa2, respectively. TEM analysis showed that the mutations in Spa2 Cg Effects of CLP formation ( Figure 19 ). ELISA assay showed that Spa2 variants Cg The extent of CLP formation was significantly reduced ( Figure 20 ). We also found that in the C97A / C380A double mutation variant, Cg CLP formation was completely blocked ( Figure 19 and 20 ).

[0268] Taken together, these results suggest that both isopeptide bonds and disulfide bonds contribute to the formation of CLPs in C. glutamicum, with disulfide bonds being the most stable Cg The most important element of the CLP structure.

[0269] Example 3. Engineering of a programmable extracellular protein scaffold Cg CLP

[0270] CLP structures are attractive building blocks for a variety of applications because these extracellular fibers possess very high tensile strength due to their extensive inter- and intramolecular isopeptide bonds. Furthermore, as an extracellular matrix, CLP fibers can be conveniently and reliably positioned directly outside of cells. Finally, their proteinaceous nature makes them potentially amenable to elaborate processing using genetic engineering.

[0271] This example was performed to determine suitable fusion sites for adding peptides / proteins to Spa2. Based on the crystal structure of Spa2 observed in Example 2 and the characterization of specific functional domains within Spa2, fusion of the protein of interest (POI) was tested at four different locations, one site at the N-terminus of Spa2, three sites in the M-domain, and a lack of disulfide bonds ( Figure 22 ).

[0272] Transform the extracellular space with exogenous expression plasmids (pEK-E1 / mCherry-spa2, pEK-E2 / mCherry-spa2, pEK-E3 / mCherry-spa2 or pEK-E4 / mCherry-spa2) Cg The CLP-deficient strain Corynebacterium glutamicum ATCC 14067Δspa2 (Δspa2), in which CLP formation is abolished, was used for expression of the Spa2 fusion protein to test the restored Cg CLP fiber generation.

[0273] The fluorescent reporter protein mCherry was fused at the intterrogated positions to generate functional fusion proteins (SEQ ID NOs: 8-11) while retaining the sortase-catalyzed covalently cross-linked pilus formation ability of Spa2. Figure 22 As shown, four sites of mCherry addition / insertion were tested, including Q35 at the N-terminus of Spa2 (E1), G215 in the M-domain turn 1 (E2), G236 in the M-domain turn 2 (E3), and G336 in the M-domain β23-sheet (E4). Quantitative analysis (ELISA) showed that cells expressing each fusion protein emitted fluorescence and were able to form fibers ( Figure 23 a).

[0274] Confocal microscopy showed that all engineered variants had detected mCherry fluorescence, and the fluorescence at the extracellular site of Corynebacterium glutamicum cells was obvious ( Figure 23 b), consistent with the TEM imaging results, shows that mCherry functionalized Cg CLP fiber Figure 24Combining the results of ELISA, fluorescence intensity, confocal microscopy and TEM imaging, it can be concluded that E1 and E2 are more ideal sites for fusing functional POI, producing a large number of functionalized Cg CLP fiber.

[0275] The Δspa2 strains transformed with plasmids pEK-6his-spa2, pEK-SpyTagSpa2, pEK-Mfp3Spep-Spa2, pEK-SpyCatcher-Spa2, pEK-Venus-Spa2, and pEK-CcEgl-Spa2, respectively, expressed various Spa2 fusion proteins (six POIs, each fused to the E1 position via a linker of SEQ ID NO: 23) (see Figure 25 All these fusion proteins were successfully expressed, secreted and formed Cg CLP( Figure 26 ).

[0276] TEM images show that Ni-NTA modified AuNPs are anchored on 6His-Spa2 Cg CLP Figure 27 a). Confocal microscopy images showing SpyTag-Spa2 Cg Green fluorescence emitted by CLP cells, SpyCatcher-EGFP protein binding partner binds to SpyTag-Spa2 through the Spytag-SpyCatcher interaction pair Cg CLP cells are covalently linked ( Figure 27 b) Confocal microscopy images showing SpyCatcher-Spa2 Cg Green fluorescence emitted by CLP cells, SpyTag-EGFP protein binding partner through the Spytag-SpyCatcher interaction pair with SpyCatcher-Spa2 Cg CLP cells are covalently linked ( Figure 27 c) Confocal microscopy images showing Venus-Spa2 Cg Green fluorescence image of CLP cells ( Figure 27 d). Mfp3Spep-Spa2 Cg Fluorescence images and quantitative analysis of CLP cell immobilization capacity. Immobilized microspheres on substrates (left) before (top) and after (bottom) impaction with a water jet at a constant discharge pressure of 5 psi. Quantitative analysis of the relative capacity of different cells to be immobilized on PS microspheres on substrates (right). Figure 27 e). Detection of CcEgl-Spa2 by 3,5-dinitrosaloculoc acid (DNS) assay Cg CLP cells degrade carboxymethyl cellulose into glucose ( Figure 27 f).

[0277] These findings indicate that sortase-mediated polymerization is not disrupted by POI fusions to Spa2 monomers, particularly POI fusions within the Spa2 N-terminus and M-domain turn 1, and that proteins of various types and sizes can be engineered into Cg CLP is a universal programmable extracellular protein skeleton.

[0278] To evaluate the programmable Cg To investigate whether the CLP extracellular protein skeleton can support the co-assembly of multiple heterologous proteins, we used the well-established spilt-Venus system in the Δspa2 strain (see Figure 28 , and Kodama, Y. & Hu, C.-D. An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. BioTechniques 49, 793-805, 2010).

[0279] The Δspa2 strain was transformed with plasmids pEK-N-Ven-Spa2, pEK-C-Ven-Spa2 and pEK-N-Ven-Spa2_C-Ven-Spa2, respectively, and the Δspa2 strain transformed with pEK-N-Ven_C-Ven was used as a control.

[0280] As shown in TEM images of transformed cells, the co-assembly of two different proteins did not interfere with Cg CLP assembly ( Figure 29 Fluorescence intensity measurement and confocal microscopy imaging showed that the highest fluorescence intensity was observed in cells in which the split-Venus components were simultaneously fused with Spa2 ( Figure 30 ). When only N-Ven and C-Ven are secreted simultaneously but not anchored to Cg When the CLP skeleton was used, almost no fluorescence was detected ( Figure 30 These results suggest that the separated components can be Cg CLP backbone co-assembly.

[0281] Example 4. Engineered Living Functional Materials to Degrade Cellulosic Biomass into Valuable Chemicals

[0282] This example aims to demonstrate that multiple cellulases in Corynebacterium glutamicum ATCC 14067Δspa2 are co-assembled into a catalytic cascade to degrade cellulose into glucose extracellularly to support the production of a specific chemical of interest (e.g., lycopene) ( Figure 31).

[0283] Specifically, endo-1,4-β-glucanase from Trichoderma reesei (TrEgl, SEQ ID NO: 19) and β-glucosidase from Saccharophagus degradans (SdBgl, SEQ ID NO: 21) were co-assembled in Cg In CLP fibers, these two enzymes are known to work synergistically to degrade cellulose into glucose through an enzymatic cascade reaction.

[0284] Lycopene can be produced by engineered Corynebacterium glutamicum via the methylerythritol phosphate (MEP) pathway (Li, C. et al. Heterologous production of α-Carotene in Corynebacterium glutamicum using a multi-copy chromosomal integration method. Bioresour. Technol. 341, 125782, 2021). Cg The C001 chassis (Δspa2Δdec) was constructed by transforming strain C001 with the plasmid pZ9-dxs_crtEBI for IPTG-induced expression of the dxs gene and crtEBI gene cluster. The C002 strain was then transformed with the plasmids pEC-TrEgl-Spa2_SdBgl-Spa2 and pEC-TrEgl_SdBgl, respectively, to generate strains C003 and C004.

[0285] like Figure 32 As shown, strain C003 co-assembles TrEgl and SdBgl on the cell surface. Cg CLP fiber ( Figure 32 a) and degrade sodium carboxymethylcellulose (CMC-Na, an ether derivative of cellulose) in the culture medium, which is based on the transformation of the culture medium from a viscous gel to a dilute solution ( Figure 32 b) Only TrEgl and SdBgl are secreted simultaneously but not anchored toCg Strain C004 with the CLP backbone did not exhibit similar behavior.

[0286] The extracellular activity of cellulase assays showed that strain C003 produced 4 times more reducing sugars than strain C004 ( Figure 32 c). Figure 32 As shown in (d), after culturing in M63 medium with CMC-Na as the sole carbon source for 36 h, the lycopene production titer of strain C003 reached 0.83 mg / g dry cell weight (DCW).

[0287] sequence

[0288] SEQ ID NO: 1 Wild-type Spa2

[0289] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASA QQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLG LYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEG TDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAI ATGVSDAEGVVRWNDVTDPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0290] SEQ ID NO: 2 Wild-type Spa2

[0291] MNPTPKRISAAVLSLALGFTIAGAPILAPVVSAQTSITGGAASTVNLSAVSLTINKYDGLPVTNTDDLANLAPLDATFTIEKVLLNNKLNTLAGWQELSGYTPTTAPTDPDFTPISVTTDAETGRGVVRTTDDGTTIAGTNVVDPTFVVGAYVVTEVAKEGYSVAPPFLVTLPFTDSDTNDWNYDQVVHPKNQADVTVEKGVEDLGATIGSTLNYTISAPLPAGNLTSLEVLDNLPDELGAATNVVVSTRINGAPLVELPLIDTTPVTTGGTDAVPNNLSIVFGDEDRAELESRRVGNQGLEIVVKFSAVITSLPDNGTIVNHAVINLPGGLTYTTDHGNPDDGAQTHLGDLIIRNVNNNTPAELIEGGSATYELWRCQADGTGGFHVISQVSAATDPAGPAVDTFTTAGGTITLYGVQAIDWVNGATPSAEIQDLCVVELESPDGYELNPEPTQVEYVLPRDGYNMEADVIHLASDDVITLPATGGNGTMILIGAGVLVAAAGGAAAVRGNRARKN

[0292] SEQ ID NO:3 wild-type Spa2

[0293] MKKNHKRISAAVLSLALGFTIAGAPMLAPVASAQQVTITAGQASTVNENAAVTLTIQKRLGEVNNAPNGIADVPFQVQKLNTGSLATLAGWEDVLALQTKLQTTGLVAADFDPSYDEEITTNPSGNIQIVTGGANDAGGDFTVGAYLVTEKAFGNYTVADPFIVTLPHAEGNSWNYEQTVTPKNQLVEVDKTVSDAGVHLGANIAYTISASIPAEDLTSLAIIDDLPDELGAAQSIVVKTFGAIDPLDIELILNDTDYTPDPVDGDGNQLTIELTPAGLTKLNNLRPGNPNLVLQVTFSAKVVELPGNGVISNDVIINYPNQSIDTSDPNDPNDGSETRFGSLSITKTDQADDSELEGAVFELWRCQPAGTNGWAVIGDKLPIINDPAVVDSATYDNYVNNTHGGTVPTTFSTDVDGEATVYGVQTFDFVNGEYLPSGTDSQICLVEVEAPTGYNLIPEPIPVTDYVDSSHQSPDFYNMTANIENIKNDDLVNLPETGGNGTMAMIAAGVLVAAGGAAVRGNRARNK

[0294] SEQ ID NO:4 wild-type Spa2

[0295] MNPTPKRISAAVLSLALGFTIAGAPILAPVASAQITADAVSSIDLDADVSLTIHKRLGEPDPNDGDPTNAPGTALPNIQFRIEKVDLNNELDTLAGWSDLNTLQANAAGELDALIDAAENTGGKTTVATISTGTTGTADIQSTPDFGVVYLVTEIQNGNYTVAKPFLVTLPFADADGLWDYNQVVAPKNQVVSVSKDVADPGATIGSTINYTARASVPADDLRFIVIDNLPTVLAVPADTDVVVESPADIELTEGTDYTLTITGQEVRVEFLPA GLTNLEVLRANDSELEVLKFPSQIIGLPTDGIIRNDIEVLLPNDGRVTTDPTDPDNPGNPEDGAETHLGMLTINKVDDEDLPITADTASFQLWRCQDQAGRLQVTGSPLFGSNSLTIDDTPASQINEFITVDGVATYGVQVYNFVNGATPAVGVNDQLCVVETEAPEGYVLNPEPQAVSFDTDTATDPFMVVNLEDTLTGQLPATGGNGTMILIGAGLVAAAGGAAAVRGNRARKNA

[0296] SEQ ID NO:5 Spa2 cut

[0297] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKHHHHHH

[0298] SEQ ID NO:6 Recombinant Spa2

[0299] MGSSHHHHHHSSGLVPRGSLEVLFQGPMQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKK

[0300] SEQ ID NO:7 mCherry

[0301] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0302] SEQ ID NO:8 E1 / mCherry-spa2

[0303] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGGGSGGGGSQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0304] SEQ ID NO:9 E2 / mCherry-spa2

[0305] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGGGGSVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGGGSLTVGSVVEWTISQTVPALNDGEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0306] SEQ ID NO:10 E3 / mCherry-spa2

[0307] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGGGGSVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGGGSEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0308] SEQ ID NO:11 E4 / mCherry-spa2

[0309] MTSKSSAFRRRLTAAVGTVAVAGVISMGQVASAQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGGGSVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGE GEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGGGSTTPPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0310] SEQ ID NO:12 6his-spa2

[0311] MTSKSSAFRRRLTAAVGTVAVAGVISMGQVASAHHHHHHQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGTTARSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEGQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0312] SEQ ID NO:13 SpyTagSpa2

[0313] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAAHIVMVDAYKPTKQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEGQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0314] SEQ ID NO:14 Mfp3Spep-Spa2

[0315] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAGYDGYNWPYGYNGYRYGWNKGWNGYQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDDGDSCEPIDLANTNDW AQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEQYTSA TIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSWSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDIDNPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNA ENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAVYALRRRNEA

[0316] SEQ ID NO:15 SpyCatcher

[0317] MVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHID

[0318] SEQ ID NO:16 Venus

[0319] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYLTADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0320] SEQ ID NO:17 CcEgl

[0321] PGYDASLIPNLQIPQKNIPNNDGMNFVKGLRLGWNLGNTFDAFNGTNITNELDYETSWSGIKTTKQMIDAIKQKGFNTVRIPVSWHPHVSGSDYKISDVWMNRVQEVVNYCIDNKMYVILNTHHDVDKVKGYFPSSQYMASSKKYITSVWAQIAARFANYDEHLIFEGMNEPRLVGHANEWWPELTNSDVVDSINCINQLNQDFVNTVRATGGKNASRYLMCPGYVASPDGATNDYFRMPNDISGNNNKIIVSVHAYCPWNFAGLAMADGGTNAWNINDSKDQSEVTWFMDNIYNKYTSRGIPVIIGECGAVDKNNLKTRVEYMSYYVAQAKARGILCILWDNNNFSGTGELFGFFDRRSCQFKFPEIIDGMVKYAFEAKTDPDPVIVYGDYNNDGNVDALDFAGLKKYIMAADHAYVKNLDVNLDNEVNAFDLAILKKYLLGMVSKLPSNSR

[0322] SEQ ID NO:18 N-Ven

[0323] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYLTA

[0324] SEQ ID NO:19 TrEgl

[0325] QQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTSSGNSWTDTSLVSSCLARK

[0326] SEQ ID NO:20 C-Ven

[0327] DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0328] SEQ ID NO:21 SdBgl

[0329] MNRLTLPPSSRLRSKEFTFGVATSSYQIEGGIDSRLPCNWDTFCEQPNTIIDNTNGAIACDHINRWQDDIELIANLGVDAYRFSIAWGRVINLDGSLNNEGVTFYKNILTKLREKNLKAYITLYHWDLPQHLEDAGGWLNRDTAYKFRDYVNLITQALDDDVFCYTTLNEPFCSAYLGYEIGVHAPGIKDLASGRKAAHHLLLAHGLAMQVLRKNCPNSLSGIVLNMSPCYAGSNAQADIDAAKRADDLLFQWYAQPLLTGCYPDAINSLPDNAKPPICEGDMALISQPLDYLGLNYYTRAVFFADGNGGFTEQVPEGVELTDMGWEVYPQGLTDLLIDLNQRYTLPPLLITENGAAMVDELVNGEVNDIARINYFQTHLQAVHNAIEQGVDVRGYFAWSLMDNFEWALGYSKRFGITYVDYQTQKRTLKASGHAFAEFVSSRS

[0330] SEQ ID NO:22 Linker 1 (GS)

[0331] GGGGSGGGGS

[0332] SEQ ID NO:23 Linker 2 (C10)

[0333] AGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGAGAGAGPEGGS

[0334] SEQ ID NO:24 N-mCherry-C

[0335] MTSKSSAFRRLTAAVGTVAVAVAGVISMGQVASAQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVS IPLPHAQQNWLYDVHVYPKNQEVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVTVTVQDSSLQD GEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKTTPHTYWGQLTVNKGDTGMVNKLAGAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKKDGPNLPLTGGQGTLMMTIGGLALMLVGAGAVYALRRRNEA

[0336] SEQ ID NO:25 Spa2 promoter

[0337]

[0338] SEQ ID NO:26 right

[0339]

[0340] SEQ ID NO:27 crtE

[0341]

[0342] SEQ ID NO:28 crtBI

[0343] ATGACACACCAAAATTCGCCTCTCTTCCTTAAAAGTGCACTGAGACTTTACAATCGGGCCTCATTCAAGGCTTCACATAAAGTGATCGAAGAATATTCGACGAGCTTCAGTCTGTCTACGTGGTTGCTATCCCCACGCATACGAAATGACATACGAAATCTCTATGCAGTAGTTCGTATCGCCGATGAGATTGTCGACGGCACTGCACATGCCGCTGGTTGCTCAACTGCCAAAATCGAAGAGATTCTCGATGCCTATGAAATTGCGGTTCTTGCAGCACCACAACAACGCTTCAACACAGATCTTGTTTTACAAGCTTATGGTGAAACTGCCCGACGCTGTGATTTCGAACAAGAGCATGTAATAGCCTTCTTTGCATCAATGCGTAAGGACCTCAAAGCTAATACACACGACCCAGATAGCTTCACAACGTATGTCTATGGCTCCGCGGAAGTTATAGGCCTGCTTTGTCTCAGCGTTTTCAACCAAGGTAGAACGATTAGCAAAAAACGGCTAGAGATTATGCAAAACGGAGCCCGCTCATTGGGAGCGGCATTCCAGAAAATTAACTTTCTCCGTGACTTGGCAGAAGATCAGCAAAATTTGGGCCGATTTTATTTCCCCAAAACCAGCCAAGGAACTCTTACTAAAGAACAAAAAGAAGATCTCATCGCTGATATCCGTCAAGACCTAGCAATTGCCCACGATGCATTTCCAGAAATACCAGTGCAGGCTCGCATCGGAGTGATCTCTGCTTATTTGCTCTTTCAAAAACTCACTGACCGAATTGAGGCTACTCCTACCGCCGATTTATTGCGGGAGCGAATCAGAGTTCCACTTCATATCAAACTCTCTACACTCGCTAGAGCCACGATGAAAGGTCTATCTATGAGCATCTACAGAAAGAATTCGTGA

[0344] SEQ ID NO:29 lacI

[0345]

[0346] SEQ ID NO:30 Ptac promoter

[0347] ATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA

[0348] SEQ ID NO:31 Spa1-Ab

[0349] ADEETETNVEEVTSPDLTDTETEQDSEKVDDGIELPTTNPLDLIDTLLGEEPTAPYIHWDVRDTEGNLVPGATFKLEHRDGDEWVSGPGADQIEDCDEICAALKDQDQLDIDTTAGEILLENRDTGLGSRLEEGENYRLSQVEAPEGYSWVIEGENPQTIGDDDEDSAKWNGVDIHDFGTFEVQQGAPMAFRAARAGDSLTCEPGYVYGISATGQLQQVTNGSVTNIGRSAAGVASFNGLGIGDFGQTVYAYERQNNARTAKMYSFDPGTERWSDTGDSYNTANSPGFYQGTLVAGAVNLSNGKYYFGGFQASGSGQVFKIWEYDPEQSSQNRFSYKGQIQTSRFSNPTANGDMAFNAAGDLFVVRGSGSTTTVFSVTAENFEAANGGSITTSGSNNFTTMSNVNGVAFDSSGRAYLGSESQIRSYAMPG

[0350] SEQ ID NO:32 Spa2-Ab

[0351] MQQATAVGPDQPGAPTHGSLTVHKYVGQEGNAGTGEISVPGGQPLEGAEFTIWRLGTNDGDSCEPIDLANTNDWAQVPTGAAPRELSAVQNDFCLVDGGTARTTNSAGEYTFGNLDLGLYYVQETDAPANIVSRTAPFYVSIPLPHAQQNWLYDVHVYPKNQEVDAPTKTINSDSDQAGKGLTVGSVVEWTISQTVPALNDGEGQYTSATIWDVLNPAELEYAGTTSVSLNGTPLVEGTDYTIDAGVVSSLTEKKLAEIKAGDTIEVVFTTTVLAVTETGDINPGSEGPDKPGYGSEFNGGTTPGGTTPHTYWGQLTVNKGDAEFAVFNNAENGVCAPEAPETDAIATGVSDAEGVVRWNDVTPDNPLGLWIANSSDGEIANPNKDYCLYETKAPSGYVAGPVQKVNITPGTTAKLVVDFENTKK

[0352] SEQ ID NO:33 Spa3-Ab

[0353] RTITGNVSDVSFETISQSCINGISLEIQKQKHKPYQNAQPGNIPAGVISGLTFEALLINDVDITSDEGWNLANSLSVKQAEERGFSQRFTAETDVAGAAKFQKLLPVGLYLIREITPENPHKDYKTSEPFLITLPVGNVTGDAWQCDVVIKTKETPDPDPDPTPTPTSPQPPTSTETTTPLVPTPPITPPAEDITGSTEKDPS

[0354] SEQ ID NO:34 HRV3c

[0355] MGSSHHHHHSSGLVPRGSHMGPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ

[0356] SEQ ID NO:35 GFP-SpyCatcherMGVDTLSGLSSEQGQQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIDGGGGSGGGSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHHHH

[0357] SEQ ID NO:36 GFP-SpyTagMAHIVMVDAYKPTKGSGSGSMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYK TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH

[0358] SEQ ID NO:37 SpyTag

[0359] AHIVMVDAYKPTK

[0360] SEQ ID NO:38 Mfp35

[0361] GYDGYNWPYGYNGYRYGWNKGWNGY

Claims

1. A fusion polypeptide comprising a carrier protein and a polypeptide of interest, wherein the polypeptide of interest is fused to the end of the carrier protein or inserted into the carrier protein, and wherein the carrier protein is a pilin of covalently cross-linked pili (CLP) from a microorganism.

2. The fusion polypeptide of claim 1 , wherein the microorganism is a Gram-positive bacterium, such as a bacterium selected from the group consisting of Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lacticaseibacillus paracasei, Bacillus thuringiensis, and Lacticaseibacillus paracasei; preferably Corynebacterium glutamicum.

3. The fusion polypeptide of claim 1 or 2, wherein the carrier protein is a backbone pilin protein.

4. The fusion polypeptide of any one of claims 1 to 3, wherein the polypeptide of interest is fused to the N-terminus or C-terminus of the carrier protein.

5. The fusion polypeptide of claim 4, wherein the polypeptide of interest is fused to the N-terminus of the carrier protein.

6. The fusion polypeptide of any one of claims 1 to 3, wherein the polypeptide of interest is inserted into the carrier protein.

7. The fusion polypeptide of claim 6, wherein the polypeptide of interest is inserted into the turn region of the carrier protein.

8. The fusion polypeptide of claim 6, wherein the carrier protein is a backbone pilin protein from Corynebacterium glutamicum, and wherein the polypeptide of interest is inserted into the M domain of the backbone pilin protein.

9. The fusion polypeptide of any one of claims 1-8, wherein the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO:

4.

10. The fusion polypeptide of any one of claims 1 to 9, wherein the polypeptide of interest is fused to the N-terminus of a carrier protein, or inserted between positions corresponding to G215 and L216 of SEQ ID NO: 1, between positions corresponding to G236 and E237 of SEQ ID NO: 1, or between positions corresponding to G336 and T337 of SEQ ID NO:

1.

11. The fusion polypeptide of any one of claims 1 to 10, wherein the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, and wherein the polypeptide of interest is fused to the N-terminus of the carrier protein, or inserted between G215 and L216, between G236 and E237, or between G336 and T337 of SEQ ID NO:

1.

12. A polynucleotide encoding the fusion polypeptide of any one of claims 1 to 11.

13. A vector comprising the polynucleotide of claim 12.

14. A host cell comprising the polypeptide of any one of claims 1 to 11, the polynucleotide of claim 12 or the vector of claim 13.

15. A recombinant cell comprising a polynucleotide encoding a fusion polypeptide, wherein the fusion polypeptide comprises a carrier protein and a polypeptide of interest, wherein the polypeptide of interest is fused to a terminus of the carrier protein or inserted into the carrier protein, wherein the carrier protein is a pilin of a CLP, and wherein the recombinant cell is capable of expressing the polynucleotide and displaying a modified CLP comprising the fusion polypeptide.

16. The recombinant cell of claim 15, wherein the recombinant cell is a Gram-positive bacterium.

17. The recombinant cell of claim 15 or 16, wherein the bacterium is selected from the group consisting of Corynebacterium glutamicum, Bifidobacterium breve, Lactococcus lactis, Lactobacillus paracasei, Bacillus thuringiensis and Lactobacillus paracasei; preferably Corynebacterium glutamicum.

18. The recombinant cell of any one of claims 15-17, wherein the carrier protein is a backbone pilin protein.

19. The recombinant cell of any one of claims 15 to 18, wherein the polypeptide of interest is fused to the N- or C-terminus of a carrier protein.

20. The recombinant cell of claim 19, wherein the polypeptide of interest is fused to the N-terminus of a carrier protein.

21. The recombinant cell of any one of claims 15-18, wherein the polypeptide of interest is inserted into a carrier protein.

22. The recombinant cell of claim 21, wherein the polypeptide of interest is inserted into the turn region of a carrier protein.

23. The recombinant cell of claim 22, wherein the carrier protein is a backbone pilin protein from Corynebacterium glutamicum, and wherein the polypeptide of interest is inserted into the M domain of the backbone pilin protein.

24. The recombinant cell of any one of claims 15-23, wherein the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, amino acids 34-520 of SEQ ID NO: 2, amino acids 34-530 of SEQ ID NO: 3, or amino acids 34-519 of SEQ ID NO:

4.

25. The recombinant cell of any one of claims 15-24, wherein the polypeptide of interest is fused to the N-terminus of a carrier protein, or is inserted between positions corresponding to G215 and L216 of SEQ ID NO: 1, between positions corresponding to G236 and E237 of SEQ ID NO: 1, or between positions corresponding to G336 and T337 of SEQ ID NO:

1.

26. The recombinant cell of any one of claims 15-25, wherein the carrier protein comprises amino acids 36-509 of SEQ ID NO: 1, and wherein the polypeptide of interest is fused to the N-terminus of the carrier protein, or inserted between G215 and L216, between G236 and E237, or between G336 and T337 of SEQ ID NO:

1.

27. The recombinant cell of any one of claims 15-26, wherein the recombinant cell comprises two or more polynucleotides, each encoding two or more fusion polypeptides, each fusion polypeptide comprising a different polypeptide of interest, and the modified CLP comprises the two or more polypeptides.

28. A method for preparing the recombinant cell according to any one of claims 15 to 27, comprising introducing the polynucleotide according to claim 12 or the vector according to claim 13 into a host cell.

29. The method of claim 28, wherein the host cell is a bacterium having a native CLP.

30. The method of claim 28 or 29, wherein the host cell is a Gram-positive bacterium.

31. The method of any one of claims 28-30, wherein the method comprises the step of knocking out the native backbone pilin of the host cell.

32. A modified covalently cross-linked pilus (CLP) comprising a plurality of fusion polypeptides according to any one of claims 1 to 11.

33. A method for preparing a modified CLP, comprising the following steps: a) providing a fusion polypeptide according to any one of claims 1 to 11; as well as b) providing sortase activity.

34. The method of claim 33, wherein the sortase is encoded by a gene identified as occurring in the same cluster as a gene encoding a carrier protein in nature.

35. The method of claim 33 or 34, wherein the sortase is a class C sortase, such as srtCl and / or srtC2, preferably wherein srtCl and srtC2 are encoded by genes from the same cluster.

36. The method of any one of claims 33-35, wherein the method is an in vitro method.

37. A polynucleotide construct or combination of polynucleotide constructs comprising the polynucleotide of claim 12, and one or more polynucleotides encoding a sortase.

38. The polynucleotide construct or combination of polynucleotide constructs of claim 37, wherein the sortase is encoded by a gene identified as occurring in the same cluster as a gene encoding a carrier protein in nature.

39. The polynucleotide construct or combination of polynucleotide constructs according to claim 37 or 38, wherein the sortase is a class C sortase, such as srtC1 and / or srtC2, preferably wherein the srtC1 and srtC2 are encoded by genes from the same cluster.

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