WO2023153949A1

WO2023153949A1 - Protein carrier system based on cyanobacterial nano-sized extracellular vesicles

Info

Publication number: WO2023153949A1
Application number: PCT/PT2023/050003
Authority: WO
Inventors: Paulo Miguel NUNES DE OLIVEIRA; Jorge Alexandre FERREIRA MATINHA CARDOSO; Steeve DA SILVA LIMA; Cláudia Alexandra DOS REIS SERRA; Filipe FERNANDES COUTINHO; Aires Manuel PEREIRA DE OLIVA TELES
Original assignee: Ibmc - Instituto De Biologia Molecular E Celular; Universidade Do Porto; Ciimar - Centro Interdisciplinar De Investigação Marinha E Ambiental
Priority date: 2022-02-10
Filing date: 2023-02-10
Publication date: 2023-08-17
Also published as: PT117786A

Abstract

The present invention refers to a genetic construct for carrying a protein of interest in cyanobacterial extracellular vesicles (EVs) of Synechocystis sp. PCC6803. The present invention further relates to the derived transformed Synechocystis strain comprising the said construct. Cyanobacterium transformed with referred construct are, most surprisingly, further characterized by hypervesiculating EVs to larger extents than the wild-type and mutant strains known in the art. Cyanobacterium transformed with the referred construct secrete the protein of interest in EVs and to the extracellular medium thus, the present invention also refers to compositions comprising EVs derived from the said transformed Synechocystis strain, as well as compositions comprising the EV-free extracellular medium derived from the said transformed Synechocystis strain, for use in carrying proteins proteins/enzymes into animals, including mammals and fish. The present invention further refers to methods for transforming mammalian epithelial cells with EVs isolated from the said Synechocystis strain, which internalize the said EVs and display the protein of interest. Moreover, the present invention refers to methods by which cyanobacterial EVs can work as nanocarriers of custom proteins into fish, with prospective applications in modulation of nutritional status or stimulating specific adaptive immune responses. The construct, strain, compositions uses and methods in the present invention may be advantageously employed as a novel biotechnological tool for the specific delivery of biologically active proteins and enzymes in biotechnology in general and in aquaculture in specific.

Description

DESCRIPTION

PROTEIN CARRIER SYSTEM BASED ON CYANOBACTERIAL NANO-SIZED

EXTRACELLULAR VESICLES

Technical field of the invention

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.

State of the art

Production of fish in aquaculture has been one of the fastest growing food-producing sectors, now accounting for up to 50 percent of the world's fish that is used for food (FAO, 2020) . However, several constraints limit expansion of the sector, particularly consumers' quantity and quality demands, while there is a constant need to promote safe and effective fish-growth performance schemes, and to stimulate environmentally-f riendly solutions to protect fish against diseases outbreaks, and stressful rearing conditions. Therefore, different strategies and new "vaccination" approaches are needed to safeguard fish welfare, and feeding costs, reducing chemicals as prophylactic agents, and protect farmers and consumers' interests. The use of live microorganisms as probiotics is one of such approaches (Hai, 2015) , which has become popular in recent years as it reportedly improves feed value, contributes to enzymatic digestion of nutrients, inhibits the action of pathogenic microorganisms, shows anti- mutagenic and anti-carcinogenic activity, and potentiates immune responses (Pandiyan et al. , 2013) . With the development of biotechnology, new systems and techniques have been implemented for the expression of heterologous proteins and enzymes in probiotics cells (Yao et al. , 2020) , further potentiating their use. This approach is gaining considerable interest, as these proteins and enzymes can be carefully selected to meet a specific purpose, e.g. increasing adherence capability, trigger adaptive immune responses, or stimulate nutrient digestion (Yao et al. , 2020) . One example is the use of microbial photosynthetic systems towards a more sustainable development of the aquaculture sector, particularly by the use of microalgae or cyanobacteria as feed or dietary supplements (Abed et al. , 2009; Han et al. , 2019; Ma et al. , 2020; Morais Junior et al. , 2020) . Recently, through genetic modifications, microalgae have been engineered to express potential antigens for the development of an oral vaccination platform in fish (Kwon et al. , 2019) . Nevertheless, the choice of whole microorganisms to use in aquaculture remains far from consensual, mainly because the gastrointestinal microbiota of aquatic organisms has been poorly characterized and the effects of the probiotics agents remain to be extensively analyzed (Pandiyan et al. , 2013) . Thus, while the realization of probiotics as protein carrier and molecular display systems is yet to be fulfilled, other alternatives are being investigated .

In the context of carrying and delivering agents with a biological effect (bioactives) (Dezfooli et al. , 2019) , the use of bacterial extracellular vesicles (EVs) occupies a relevant position. 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) . 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) .

However, administration to higher animals of EVs isolated from Gramnegative bacteria is usually associated with pathological reactions, including systemic inflammatory responses (Park et al. , 2010) , mainly due to the presence of lipopolysaccharides (LPS) . This represents a serious limitation, which has prompted the search for alternatives to minimize the immunogenicity of bacterial EVs, such as: the use of chemical processes to decrease LPS content, the genetic engineering of EVs-producing bacterial strains for reduced and/or altered LPS content, and the choice of bacterial strains containing LPS with low immunogenicity .

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.

Summary of the Invention

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. In another embodiment of the present invention a cyanobacterial strain for hypervesiculating and carrying a protein of interest in

EVs is characterized by, comprising (through transient or stable transformation) the said construct, according to claim 2.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, compositions described above are suitable for use in carrying proteins of interest into an animal, according to claim 6.

Another embodiment of the present invention 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.

In another embodiment, 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.

In another embodiment, 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.

In one embodiment 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.

In another embodiment, 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.

In another embodiment, the said animal comprises zebrafish, european seabass, gilt-head bream, salmon, carp, catfish and other fish, according to claim 16.

In other embodiments, the said administration comprises immersion, oral administration or injection, according to claim 17.

Detailed Description

The present invention discloses that cyanobacterial-derived EVs are an effective vehicle for carrying and delivering proteins to mammalian cells and animals, including fish.

In particular, the present invention refers to the modulation of protein content in EVs derived from the unicellular, freshwater cyanobacterium Synechocystis sp . PCC 6803 (hereafter Synechocystis) .

To customize the protein content of Synechocystis EVs, the superfolder green fluorescent protein (sfGFP) is used here as reporter, and sfGFP is expressed in Synechocystis cells through a new construct pEV-trc-GFP, which is originally disclosed herein and is constructed for this purpose (SEQ ID NR:1) . 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.

Using the disclosed construct, it's possible to develop a method by which a cyanobacterium expressing the protein of interest in EVs is achieved for wild-type cells as background strain, comprising the step of transforming wild-type cells with the construct pEV-trc-GFP.

Through the said method, fully segregated cells (herein termed strain EV-trc-GFP) are cultivated in liquid medium, and a yellowish/greenish coloration of the extracellular medium can be observed (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) .

Occasionally, it is also possible to observe small fluorescent foci in the extracellular medium. Thus, 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) . Moreover, 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) .

To complement the characterization of sfGFP packed EVs, isolated samples are investigated by determining their size from the analysis of TEM micrographs (Figure 3) . Surprisingly, the results indicate that strain EV-trc-GFP releases approximately 3.5-fold more EVs than the wild-type strain and are characterized by hypervesiculating to larger extents than mutant strains known in the art. Moreover sfGFP- containing EVs are slightly smaller than EVs from any other strain investigated in this study (50% of the analyzed EVs had a diameter between 35 and 55 nm) .

Another aspect of the present invention refers to methods by which cyanobacterial EVs can work as nanocarriers of custom proteins into fish. For this purpose, 5 dpf zebrafish larvae are treated by immersion with isolated sfGFP loaded Synechocystis EVs for 24 h. Upon treatment, larvae observed by confocal microscopy show a clear fluorescence signal in the ventral region. Control larvae (in which no EVs is added) 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. This observation establishes that 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.

On the other hand, 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) .

The well-characterized unicellular cyanobacterium Synechocystis, and zebrafish (Danio rerio) are used here as model organisms and thus constitute non-limiting examples. On one hand, various 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.

In other embodiments, 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.

In other embodiments, 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.

In other embodiments, 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 .

In other embodiments, the system is compatible with mammalian cells, and allows introduction of heterologous proteins through internalization of EVs.

Brief Description of the Figures

Figure 1. pEV-trc-GFP Construct Map.

To construct pEV-trc-GFP, 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. To obtain the 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. In the case of the EV-trc-GFP mutant strain, medium is supplemented with antibiotics: 100 pg kanamycin mL-1 for the. For EVs isolation, 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 - Photograph of a liquid culture of EV-trc-GFP showing details of the yellow/green color found in the extracellular medium. B Confocal micrographs of EV-trc-GFP cells depicting the red autofluorescence signal from the photosynthetic pigments (Autofluorescence) , and the green signal from the sfGFP reporter (sfGFP) . A panel showing the result of merging the two micrographs is also presented (Merge) . Scale bar: 3 pm. C - sfGFP (upper panel) and autofluorescence (bottom panel) fluorescence intensity plots determined for 10 different EV-trc-GFP cells (a.u. , arbitrary units) . 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. This blot is representative of the results obtained from three independent biological replicates. E - Microscopic analyses of EVs isolated from EV-trc-GFP cells, including a confocal micrograph showing green fluorescent foci (scale bar: 5 pm) , and a transmission electron micrograph of immunogold labeled EVs. Black dots indicate the presence of sfGFP epitopes.

Figure 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-Q™ 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.

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.

Figure 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. The result of merging the DIC and GFP channel micrographs is presented on the lefthand side (Merge) . The white square in each "GFP channel" micrograph indicates the position where a larger magnification of the anatomic region corresponding to the gastrointestinal tract was obtained (right-hand side) .

For confocal microscopy analysis of 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. After the incubation period, 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. In addition, differential interference contrast (DIC) images are also acquired.

Figure 5. Synechocystis sp . PCC 6803 extracellular vesicles packaged with sfGFP are internalized by mammalian epithelial cells. Confocal micrographs of mammalian epithelial cells incubated for 24 h with vesicles isolated from Synechocystis strain EV-trc-GFP, and later fixed and stained with DAPI . The differential interference contrast (DIC) micrograph is shown in the middle panel, while the fluorescence signals detected in the GFP and DAPI channels are shown on the righthand panel. The result of merging the DIC and fluorescence signals micrographs is shown onto the left-hand side (Merge) .

SEQUENCE LISTING

SEQ ID NO : 1

CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGG GAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTA ATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGG AATTCGATTCTAAACTTACGGCATTGGCATCAACGGGAGCCACCGTCGGAGTAGGGGAAGTAACAAC GGGGGAACTGGTATTGGTGGGCAAAATTTTTACTAGCCATTGATTCCATTGGGGCAGATTGAACGCT C C C AAC C AC C AAAG T C C C C C T AAT AC AAC GAC AGAAC T AAAT AAC AG GAAAAAAG G AAT C C AAG GAG TTACCCCTCTTTTATGGGATGGAACTTCATCGACATTAAAGGTGGGAGGGGGAGGAGGCAATGGGGA CAATGGTGGTCCAGAAAAGGAAGGTGGCTCCGACGTCAAGGCAACGGGACATCCACAGGATTCACAG AAACGAACCTGGGGGCTAAGGCGGTTGCCACAATTAGTACAAAAAACGGGAGTAGTCATAGGTGAAA AC C C C GAC T AT AGAAT T AGAAAAAT T T AAC T T T T T AT C C GAAT T T T AT T C G T C C AT G T T C C C C AAAT AACTATCAAAATAATTGGAAAAATTAAATTATTTGGTCGTTGGTCACCGCTCCCTAAAGACCTGGCC ATTGTAAAGAGATTACACCCGTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCT GAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGA CCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATC TGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAAT GC T CT GCCAGT GT T ACAACCAAT TAAC CAAT T C T GAT TAGAAAAACT CAT C GAGCAT CAAAT GAAAC T G CAAT T T AT T CAT AT C AG GAT TAT CAAT AC C AT AT T T T T GAAAAAG CCGTTTCTG T AAT GAAG GAG AAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCC AAC AT CAAT AC AAC C T AT T AAT TTCCCCTCGT C AAAAAT AAG G T T AT C AAG T GAGAAAT C AC CAT GA GTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCC AGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTG AGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGC AG GAAC AC TGC C AG C G CAT C AAC AAT AT T T T C AC C T GAAT C AG GATATTCTTCTAATACCTG GAAT G CTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGAT GGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCA ACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTG TCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGA ATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTG T T TAT GTAAGCAGACAGT T T TAT T GT T CAT GAT GAT AT AT T T T TAT CT T GT GCAAT GTAACAT CAGA GATTTTGAGACACAACGTGGCTTTCGGGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTT CAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGCAATTGGCGGCCGCTTCTAGATCAT TTGTACAGTTCATCCATACCATGCGTGATGCCCGCTGCGGTTACGAACTCCAGCAGAACCATATGAT CGCGTTTCTCGTTCGGATCTTTAGACAGAACGCTTTGCGTGCTCAGATAGTGATTGTCTGGCAGCAG AACAGGACCATCACCGATTGGAGTGTTTTGCTGGTAGTGATCAGCCAGCTGCACGCTGCCATCCTCC ACGTTGTGGCGAATTTTAAAATTCGCTTTAATGCCATTTTTTTGTTTATCGGCGGTGATGTAAACAT TGTGGCTGTTAAAATTGTATTCCAGCTTATGGCCCAGGATATTGCCGTCTTCTTTAAAGTCAATGCC TTTCAGCTCAATGCGGTTTACCAGGGTATCGCCTTCAAATTTCACTTCCGCACGCGTTTTGTACGTG CCGTCATCCTTAAAGGAAATCGTGCGTTCCTGCACATAGCCTTCCGGCATGGCGGACTTGAAGAAGT CATGCTGCTTCATATGGTCCGGATAACGAGCAAAGCACTGAACACCATAAGTCAGCGTCGTTACCAG AGTCGGCCAAGGTACCGGCAGTTTACCAGTAGTACAGATGAACTTCAGCGTCAGTTTACCATTAGTT GCGTCACCTTCACCCTCGCCACGCACGGAAAACTTATGACCGTTGACATCACCATCCAGTTCCACCA GAATAGGGACGACACCAGTGAACAGCTCTTCGCCTTTACGCATCGTGCGGGACTGGGCAGAGGCCCG CCGGGGCAAATTAGCTACCACTAGGGCAGTGAGGGCGGTGCCGCCCACAAAAAAAGTCCGCCGGGAA ATCTTAGTTGTCATTTTCTCCTCTTTAATTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTA TACGAGCCGGATGATTAATTGTCAACAGCTCATTTCAGAAAATTGTGAGCGCTCACAATTCTGCAGT CCGGCAAAAAAACGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCG CGTTGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTC GTTTTATTTGATGCCTGGTACCGGGTGTAATCCATTGGGCACGAGAGTTAGTAAGGCAGTGGCAATT AATAGAGGCTTATGGTTGATTCGCATTGTTTTGCTCCTGAAATTTTCGGCAAATACAAATACTTCGC TCTTCTAGCCCTAT T AAC CAT T T T AAC GAC AAAT T GAT G G G G C AAC GAT T AAC AAAT AAT GAAT AAA T T T TAT GT T T T T CAAGAT GAAAAT T T GAAAAT T T GAT T T C C T TAT AT T T C T AC T AT AGAAGAC T AAT ACAAT TAGAT CTAAAAT T T GCAAGTAT AAAAAT CAGCAAATAGT T ATAT T GT TAAT AAT T CAAT GAC CCAATAACTCGTACTGTTATCTACGTGGTGAAAGCCAAAAAGACGAACAGTTTAGCCTCCTCCTCCT CGGCGATCGCCAAGCGAAATGTCATGGGAGATGTTCAGATTGAGCATTTTTTTCTAAAAGCCCTTGC TAAAACAAACCACATGTGCAGGGTGTCCCCGATGTTGACTAAATTCAGCGGCTCGACCATATGGGAG AGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATC ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCAC TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAG AGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA T T AT C AAAAAG GAT C T T C AC C T AGAT C C T T T T AAAT T AAAAAT GAAG T T T T AAAT C AAT C T AAAG TA TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTC TATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCC ATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAAC GATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAG AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCA GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAA AAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAAT TGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAA TAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTC CAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAA GCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGG CGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCT

GCGCGTAACCACCA

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Claims

CLAIMS 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. A cyanobacterial strain for hypervesiculating and carrying a protein of interest in EVs characterized by, comprising (through transient or stable transformation) the construct described in claim 1 The cyanobacterial strain according to the preceding claims, wherein the said cyanobacteria strain comprises Synechocystis PCC6803, Synechocystis tolC-mutant (AtolC) , Synechocystis fucS- mutant (AfucS) and Synechocystis double mutant (AtolC/Aspy) . Compositions of the extracellular medium of the cyanobacterial strain described in claims 2-3 characterized by, comprising the EVs isolated from the said extracellular medium. Compositions of the extracellular medium of the cyanobacterial strain described in claims 2-3 characterized by, comprising the EV-free fraction of the said extracellular medium. Compositions described in claims 4-5 for use in carrying proteins of interest into an animal. Use according to claim 6, wherein the said proteins 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. Uses according to claim 6, wherein the said animal comprises zebrafish, European seabass, gilt-head bream, salmon, carp, catfish and other fish. 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. . Method according to claim 9 wherein the said cyanobacteria comprise Synechocys tis PCC6803, Synechocystis tolC-mutant (AtolC) , Synechocystis fucS-mutant (AfucS) and Synechocystis double mutants (for example AtolC/Aspy) . . Method for obtaining a hypervesiculating Synechocystis strain carrying a protein of interest in EVs according to claims 9-10 characterized by, comprising the steps of : a) transforming a cyanobacteria with the construct described in claim 1. b) fully segregating mutant cells, by antibiotic selection. . 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. . Method according to claim 12, wherein the said preparation of compositions of the extracellular medium is characterized by, isolating EVs from the extracellular medium.

. Method according to claim 12, wherein the said preparation of compositions of the extracellular medium is characterized by, isolating the EV-free fraction of the extracellular medium. . Method according to claim 12, wherein 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. . Method according to claim 12 wherein the said animal comprises zebrafish, European seabass, gilt-head bream, salmon, carp, catfish and other fish. . Method according to claim 12 wherein the said administration comprises immersion, oral administration or in j ection .

Lisbon, 10^th February 2023