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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

• the present invention relates to the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in animals in general and including fish.
• EVs cyanobacterial extracellular vesicles
• microalgae or cyanobacteria as feed or dietary supplements
• microalgae or cyanobacteria as feed or dietary supplements
• microalgae have been engineered to express potential antigens for the development of an oral vaccination platform in fish (Kwon et al. , 2019) .
• EVs bacterial extracellular vesicles
• bioactives biological effect
• EVs are discrete and non-replicable proteoliposomal nanoparticles (Caruana et al. , 2020) , with a size between 20 and 500 nm in diameter (Zavan et al. , 2020) .
• These are bilayered nanostructures, derived from the bacterial cell envelope, containing membrane components as well as soluble products (Lima et al. , 2020) .
• Gram-negative bacteria in particular, have been extensively engineered to release EVs with customized cargo, aiming at fulfilling different purposes, namely for the delivery of chemotherapeutic agents (Kuerban et al. , 2020) , for carrying immunogenic antigens (Fantappie et al. , 2014) , and even for performing complex chemical reactions extracellularly otherwise difficult to implement in whole cells (Park et al. , 2014) .
• chemotherapeutic agents Kuerban et al. , 2020
• immunogenic antigens Fantappie et al. , 2014
• Park et al. , 2014 Park et al. , 2014
• Some key features of the vesicles have encouraged these applications, particularly the fact that cargo properties are maintained in EVs even in harsh conditions, and the capacity of EVs in protecting and trafficking cargo to inaccessible targets (Bennington and Kuehn, 2014) .
• Cyanobacteria are a remarkably diverse group of Gram-negative bacteria, with wide ecological distribution, and great metabolic plasticity. They are unique for being the only prokaryotes capable of performing oxygenic photosynthesis, and so, have minimal nutritional requirements. Thus, together with a wide range of genetic engineering tools available, cyanobacteria are increasingly regarded as promising, environmentally friendly, and highly sustainable microbial cell factories for the production of added value products (Heidorn et al. , 2011) . Nevertheless, the potential for using cyanobacterial EVs in biotechnological applications remains to be demonstrated and the specific tools for engineering heterologous protein expression and loading into EVs are lacking in prior art.
• the present invention refers to a genetic construct for hypervesiculating and carrying a protein of interest in extracellular vesicles (EVs) characterized by, comprising SEQ ID NR:1, according to claim 1.
• EVs extracellular vesicles
• EVs is characterized by, comprising (through transient or stable transformation) the said construct, according to claim 2.
• the said cyanobacterial strain comprises Synechocystis PCC6803, Synechocys ti s tolC-mutant (AtolC) , Synechocystis fucS-mutant (AfucS) and Synechocystis double mutant (AtolC/Aspy) , according to claim 3.
• compositions of the extracellular medium of the cyanobacterial strain described above are characterized by, comprising the EVs isolated from the said extracellular medium, according to claim 4.
• compositions of the extracellular medium of the cyanobacterial strain described above are characterized by, comprising the EV-free fraction of the said extracellular medium, according to claim 5.
• compositions described above are suitable for use in carrying proteins of interest into an animal, according to claim 6.
• the said proteins of interest are selected from the group consisting of proteins for fine- tuning metabolic functions in fish towards an improved nutritional status and proteins for modulating immune system to generate specific immune responses against selected antigens stimulating protection against a pathogenic agent, according to claim 7.
• the said animal comprises zebrafish, European seabass, gilt-head bream, salmon, carp, catfish and other fish, according to claim 8.
• Another embodiment of the present invention refers to a method for hypervesiculating and carrying a protein of interest in EVs characterized by, comprising the step of transforming a cyanobacteria with the construct with SEQ ID NR:1, according to claim 9.
• the said cyanobacteria comprise Synechocystis PCC6803, Synechocystis tolC-mutant (AtolC) , Synechocystis fucS- mutant (AfucS) and Synechocystis double mutants (for example AtolC/Aspy) , according to claim 10.
• Another embodiment refers to a method for obtaining the hypervesiculating Synechocystis strain carrying a protein of interest in EVs mentioned above which is characterized by, comprising the steps of : a) transforming a cyanobacteria with the construct described above . b) fully segregating mutant cells, by antibiotic selection, in accordance with claim 11.
• Another embodiment of the present invention refers to the method for carrying proteins of interest into an animal characterized by, comprising the steps of: a) preparation of compositions of the extracellular medium of a Synechocystis strain transformed with the construct of SEQ ID NR: 1. b) administration of the said compositions to an animal, in accordance with claim 12.
• the said preparation of compositions of the extracellular medium is characterized by, isolating EVs from the extracellular medium, according to claim 13. In another embodiment, the said preparation of compositions of the extracellular medium is characterized by, isolating the EV-free fraction of the extracellular medium, according to claim 14.
• the said proteins of interest are selected from the group consisting of proteins for fine-tuning metabolic functions in fish towards an improved nutritional status and proteins for modulating immune system to generate specific immune responses against selected antigens stimulating protection against a pathogenic agent, according to claim 15.
• the said animal comprises zebrafish, European seabass, gilt-head bream, salmon, carp, catfish and other fish, according to claim 16.
• the said administration comprises immersion, oral administration or injection, according to claim 17.
• cyanobacterial-derived EVs are an effective vehicle for carrying and delivering proteins to mammalian cells and animals, including fish.
• the present invention refers to the modulation of protein content in EVs derived from the unicellular, freshwater cyanobacterium Synechocystis sp .
• PCC 6803 (hereafter Synechocystis) .
• sfGFP superfolder green fluorescent protein
• the said construct is characterized by comprising a sequence targeting the protein of interest to the periplasm by the Tat dependent signal peptide of the Synechocystis periplasmic protein FutA2 and is further characterized by comprising a strong promoter Ptrc.x.lacI, in accordance to the sequence detailed in SEQ ID NO: 1 and to the construct map of Figure 1, and from heron termed plasmid pEV-trc-GFP.
• strain EV-trc-GFP fully segregated cells
• Figure 2A Cells are observed by confocal microscopy to evaluate sfGFP expression and cellular localization of the fluorescent signal
• Figure 2B The sfGFP dependent fluorescent signal is localized outside of the autofluorescence signal, which derives from the photosynthetic pigments located in the thylakoid membranes, indicating that most of the sfGFP protein is in the periplasm of cyanobacterial cells ( Figure 2C) .
• another aspect of the present invention refers to the extracellular medium of EV-trc-GFP cells, which can be collected, and the respective EVs isolated.
• the EV-free extracellular medium can also be further concentrated and analyzed.
• Western blotting analysis of the various fractions, using a GFP specific antibody determines that EV-trc-GFP cells indeed express sfGFP, in agreement with confocal microscopy results, and that the reporter successfully accumulates in the extracellular medium, both in isolated EVs fractions and in concentrated EV-free extracellular medium ( Figure 2D) .
• it is possible to quantify that approximately 90% of the sfGFP protein present in the extracellular medium is found soluble in the medium, with the remaining part present in the isolated EVs fraction ( Figure 2D) .
• Characterization of EVs obtained from the EV-trc-GFP strain is carried out by different microscopic methods. Confocal microscopy observations of isolated EVs preparations determined the existence of numerous, highly fluorescent foci in the sample ( Figure 2E) , consistent with possible sfGFP packed EVs.
• TEM analysis of negatively-stained samples indicated the presence of many spherical nanostructures, morphologically similar to EVs from the Synechocystis wild-type and mutant strains studied in this work. Moreover, TEM analysis of immunogold labeled EVs samples demonstrates the presence of sfGFP inside EVs ( Figure 2E) .
• Another aspect of the present invention refers to methods by which cyanobacterial EVs can work as nanocarriers of custom proteins into fish.
• 5 dpf zebrafish larvae are treated by immersion with isolated sfGFP loaded Synechocystis EVs for 24 h.
• larvae observed by confocal microscopy show a clear fluorescence signal in the ventral region.
• Control larvae do not show fluorescence signal ( Figure 4) .
• Closer inspection of the signal in respect to the zebrafish larvae anatomy indicates that it originates mainly from the gastrointestinal (GI) tract of the animal, particularly the intestine.
• GI gastrointestinal
• Synechocystis EVs with customized protein cargo i, can be suspended in the medium where zebrafish larvae are maintained; ii, are ingested by the animals; iii, accumulate in the GI tract; and iv, can help to maintain structure and activity of the heterologous protein, as sfGFP retained its fluorescence throughout the whole process.
• mammalian epithelial cells incubated for 24 h with EVs isolated from Synechocystis strain EV-trc-GFP internalize the said Synechocystis EVs and display sfGFP, as evidenced by confocal microscopy ( Figure 5) .
• Synechocystis mutant strains with different cell envelope/sur f ace properties and differential vesiculation capacity may be considered on other embodiments of the present invention, comprising the tolC-mutant known for its inability to secrete the structural component of the S-layer (protein S111951) and to release more EVs than the wild-type strain; the fucS-mutant known because of its truncated O-antigen portions of the LPS (Fisher et al. , 2013) or a novel Synechocystis double mutant strain (AtolC/Aspy) which shows higher vesiculation capacity than the tolC-mutant.
• tolC-mutant known for its inability to secrete the structural component of the S-layer (protein S111951) and to release more EVs than the wild-type strain
• fucS-mutant known because of its truncated O-antigen portions of the LPS (Fisher et al. , 2013) or a novel Syn
• this invention presents an innovative protein carrier system to fish, which, aside from zebrafish, may also comprise the European seabass, gilt-head bream, salmon, carp, catfish and others as non-limiting examples.
• the customized cyanobacterial EVs can be used as nanocarriers for the targeted delivery of specific proteins to fish, comprising proteins for fine-tuning metabolic functions in fish towards an improved nutritional status, or modulating its immune system to generate specific immune responses against selected antigens, stimulating protection against a given pathogenic agent.
• cyanobacterial EVs can be administered to fish in several ways, e.g. immersion, oral, or injection without compromising delivery efficacy and maximizing protein/ enzyme activity .
• the system is compatible with mammalian cells, and allows introduction of heterologous proteins through internalization of EVs.
• the synthetic promoter Ptrc.x.lacO the signal peptide sequence of the Synechocystis native protein FutA2 (amino acids 1 to 35) which determines its translocation to the periplasm, and the gene encoding the reporter protein super-folder Green Fluorescent Protein (sfGFP) are amplified by PCR using specific oligonucleotides. Assembly of the different DNA fragments is performed by sequential overlap-extension PCR.
• the 953 bp fragment is digested with PstI and Xbal, and ligated to variants of plasmid pSN15 previously digested with the same restriction enzymes, rendering plasmid pEV-trc-GFP. Identity of the fragment is determined by Sanger sequencing. Figure 2. Synechocystis sp . PCC 6803 extracellular vesicles loaded with the reporter sfGFP.
• the cyanobacterium Synechocystis sp. PCC 6803 wild-type (sub-strain GT-Kazusa; glucose tolerant, with S-layer, and non-motile) are maintained in liquid BG11 medium (Rippka et al. , 1979) in 100 mL Erlenmeyer flasks, kept on an orbital shaker (100 r.p.m. ) , under a 16h light (OSRAM Lumilux, 18W/865, Cool daylight) (30-40 pmol photons m-2 s-1) / 8 h dark regimen, at 28 °C.
• Synechocystis EV-trc-GFP strain expressing the heterologous protein sfGFP and further targeting it to extracellular vesicles
• Synechocystis wildtype cells are naturally transformed with pEV-trc-GFP, and fully segregated mutants are obtained.
• medium is supplemented with antibiotics: 100 pg kanamycin mL-1 for the.
• Synechocystis cultures are centrifuged at 4400 g for 10 min at room temperature to separate cells from the extracellular medium.
• a total volume of 3.6 L of extracellular medium is filtered through 0.45 pm pore size filters, and further concentrated by ultrafiltration using centrifugal filters with a molecular weight cut-off of 100 kDa (Millipore) .
• a final volume of approximately 15 mL of concentrated extracellular medium is then centrifuged at 100000 g for 3 h, at 4 °C, as previously described (Biller et al. , 2022) .
• the resulting EVs pellets are then suspended in sterile BG11 medium and stored at -80 °C until further analysis .
• a panel showing the result of merging the two micrographs is also presented (Merge) . Scale bar: 3 pm.
• Fluorescence intensity was quantified across the chosen cells as indicated by the white line drawn over one of the EV-trc-GFP cells in the merged fluorescence signal confocal micrograph, and plotted against line distance (in pm) .
• D - Western blot analysis for the detection of sfGFP protein in 10 pg of total cellular protein (TP) , and cell free concentrated extracellular media (EM) samples of EV- trc-GFP cells. From the whole extracellular medium sample, extracellular vesicles (EVs) were separated from the soluble part (sEM) , and also included for analysis. EM, sEM and EVs samples were normalized for initial cell-free culture volume concentrated, final volume of concentrated sample, and culture OD730.
• FIG. 3 Characterization of the vesicles released by the Synechocystis sp. PCC 6803 strain EV-trc-GFP.
• A Transmission electron micrograph representative of the morphological aspect of the vesicles released by EV-trc-GFP strain. Scale bar: 200 nm.
• B The Synechocystis wild-type (WT) and EV-trc-GFP strains were cultivated under standard conditions and their EVs isolated. Prior to loading, samples were normalized for cell culture density (OD730) , volume of cell-free culture medium concentrated and concentration factor.
• EVs samples were then separated by electrophoresis on 16% (w/v) SDS-polyacrylamide gels, and stained with the Pro-QTM Emerald 300 Lipopolysaccharide Gel Stain Kit (Life Technologies) for the detection of LPS .
• the gel is representative of 5 independent biological replicates, and the densitometry analysis and quantification is shown on to the right. ***, p ⁇ 0.001.
• C - Histogram showing particle size distribution of EVs samples from the EV-trc- GFP strain. EVs were separated in 5 nm size groups, distributed between 30 and 130 nm, and in groups comprising those with a size below 30 nm or higher than 130 nm.
• Vesicle diameter was determined by direct measurement from electron micrographs of three independent biological replicates. Abundance of each size group is presented as relative figures, i.e. number of vesicles with a given diameter range relative to the total number of vesicles measured.
• Synechocystis EVs To characterize Synechocystis EVs, various methods known in the art are used, namely for determining their amount and size (by nanoparticle tracking analysis and TEM) , their morphological aspect (by TEM, immunogold labelling and confocal microscopy) and their protein content by western blot, which is also used to analyse protein content of the EV-free extracellular medium.
• FIG. 4 Cyanobacterial extracellular vesicles loaded with sfGFP accumulate in the gastrointestinal tract of zebrafish larvae. Confocal micrographs of 5 dpf zebrafish larvae subjected for 24 hours to immersion trials without (Control - upper panels) or with 1000 pg mL-1 sfGFP-loaded EVs isolated from the cyanobacterial strain EV- trc-GFP (lower panels) . The differential interference contrast (DIC) micrographs are shown in the middle panels, while the fluorescence signal detected in the GFP channel (excitation: 488 nm; emission: 495 - 540 nm) are shown on the right hand panels.
• DIC differential interference contrast
• zebrafish larvae treated with sfGFP-loaded Synechocys tis EVs 5 dpf zebrafish larvae are subjected to immersion trials performed in 24-well flat-bottom plates (Sarstedt) incubated at 28 °C, in a final volume of 2 mL, for 24h.
• Three zebrafish larvae are treated with 0 (control) or 1000 pg mL-1 of sfGFP-loaded EVs in zebrafish embryo medium. Each experiment is performed three times.
• zebrafish larvae are euthanized by subjecting the larvae to a lethal dose of 300 mg L-l tricaine methanesulfonate (MS222) , immediately mounted on a glass slide, with 50% glycerol in PBS solution as mounting medium, and visualized by confocal microscopy (Leica TCS SP5 II) .
• the sfGFP signal is collected between 495 and 540 nm upon exposing the specimens to a laser beam of 488 nm, using the same acquisition settings for all tested conditions.
• differential interference contrast (DIC) images are also acquired.
• Pandiyan, P. Balaraman, D. , Thirunavukkarasu, R. , George, E.G.J. , Subaramaniyan, K. , Manikkam, S. , and Sadayappan, B. (2013) Probiotics in aquaculture, Drug Invention Today 5: 55-59.

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