Genetic tools for producing recombinant proteins in bacterial pathogens that affect fish farming in aquaculture

Abstract

The present invention provides a platform for producing complete proteins or small peptides in P. damselae and with the possibility of also being successfully used in other genera of marine bacteria. This invention contributes to the design of a new category of vectors for use in biotechnology, based on native MRB plasmids.

Description

[0001] Genetic tools for the production of recombinant proteins in bacterial pathogens that affect farmed fish in aquaculture Technical field The present invention belongs to the field of molecular biology and provides a platform for the production of complete proteins or small peptides in P. damselae (Photobacterium damselae) and with the potential to be also used successfully in other genera of marine bacteria. This invention contributes to the design of a new category of vectors for use in biotechnology, based on native MRB type plasmids ("Marine-RNA based"). Background One of the challenges to improve the currently marketed vaccines against bacterial pathogens that affect farmed fish in aquaculture is to produce specific antigens recombinantly, using microbial cell factories. The recombinant antigens would be used to design subunit vaccines.Currently, the cell factories used are always heterologous, meaning the antigenic proteins are never produced in the same bacterial species or strain in which the antigenic proteins were identified. Instead, they are produced from other species more commonly used in the laboratory, including Escherichia coli, Bacillus subtilis, and Pichia pastoris, among others. The use of a heterologous cell factory has many drawbacks, which are not always discussed. Some are not even considered because they are unsuspected or because they are ignored. They can lead to significant deficits in the proper functioning of the antigenic formulation. Specifically, we would highlight that the use of a heterologous microbial factory requires that the final antigenic protein possess the post-translational modifications that it would have in vivo in its autologous host. For example, glycosylations, acetylations, acylations, etc.and other post-translational modifications, can only be guaranteed to occur if the recombinant protein is produced in its autologous species. There are methodological limitations to producing recombinant proteins in autologous strains, especially when it comes to marine bacteria: 1. The ge etcas dspo bes tools are designed for laboratory species such as Escherichia coli, Bacillus, Pichia, Saccharomyces etc. Plasmid vectors for these species contain origins of replication derived respectively from those same species, but not from marine bacteria, in which many of these laboratory vectors do not replicate. 2 .Marine bacteria are not easily transformable by either electroporation or chemical transformation, so the introduction of recombinant DNA must almost necessarily be done by conjugation. 3. Original promoters and signal peptides from marine bacteria that allow the production (and, if possible, secretion into the extracellular medium) of recombinant peptides in marine bacteria are not available. 4. Therefore, a range of plasmid vectors is needed that combine the following characteristics: a. They must replicate efficiently in pathogenic marine bacteria in aquaculture (bacteria of the genera Photobacterium, Vibrio, Aeromonas, etc.). b. They must be able to be introduced by conjugation into marine bacteria (the vector must contain an origin of transfer or oriT). c. The expression vector must have a strong promoter that can be efficiently transcribed in marine bacterial species.That the vector has a signal peptide to encourage the recombinant protein to be secreted outside the bacteria, which would facilitate purification and. That the vector has a multi-cloning site for the introduction of additional genes of interest. The present invention provides a platform that combines all these characteristics. Brief description of the invention One aspect of the present invention relates to the expression vector or plasmid characterized in that it comprises:. a.An OV origin of the P. damselae subsp. piscicida (Pdp) and / or P. damselae subsp. damselae (Pdd) type; b. a transfer origin (oriT), preferably of the RK2 / RP4 type; c. an insertion site for inserting a gene of interest; d. a promoter operably linked to a sequence, preferably DNA, encoding a signal peptide and to a sequence, preferably DNA, encoding the gene of interest, wherein i) the sequence encoding the signal peptide is that of the signal peptide of the damselysin (Dly) gene of Photobacterium damselae subsp. damselae, and the promoter is that of the damselysin (Dly) gene of Photobacterium damselae subsp. damselae, or where ii) the sequence encoding the signal peptide is the signal peptide of the PirA gene of Photobacterium damselae subsp. damselae, and the promoter is that of the PirA gene of Photobacterium damselae subsp. damselae; e. a transcription termination region; and f.optionally or preferably one or more of any of the following elements selected from the list consisting of: positive selection genes such as antibiotic resistance genes and other molecules with antimicrobial activity; genes that solve auxotrophies; or metal resistance genes, negative selection genes (sacB, rpsL, ccdB), reporter genes (lacZ, luxCDABE, galK), toxin-antitoxin systems, restriction-modification systems, fluorescent protein genes (GFP, CFP, YFP, dsRed2, mCherry) and catabolic pathway genes; wherein preferably said expression vector or plasmid is additionally characterized by the absence of transposases genes and their inactivated versions (pseudogenes). In the context of the present invention, transposase is an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome by a cut-and-paste mechanism, or a replicative transposition mechanism.The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the transposon Tn3.1. Transposases are classified under EC Number: EC 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known. It is noted that a pseudogene is a segment of DNA that structurally resembles a gene, but is not capable of encoding a protein. Preferably, the expression vector or plasmid is characterized in that it consists essentially of, or consists of: a. An OriV origin of replication of the MRB type from P. damselae subsp. piscicida (Pdp) and / or P. damselae subsp. damselae (Pdd); b. an origin of transfer (oriT), preferably of the RK2 / RP4 type; c. an insertion site for inserting a gene of interest; d.a promoter operably linked to a sequence, preferably DNA, encoding a signal peptide and to a sequence, preferably DNA, encoding the gene of interest, wherein i) the sequence encoding the signal peptide is the signal peptide of the damselysin (Dly) gene of Photobacterium damselae subsp. damselae, and the promoter is that of the damselysin (Dly) gene of Photobacterium damselae subsp. damselae, or where ii) the sequence encoding the signal peptide is the signal peptide of the PirA gene of Photobacterium damselae subsp. damselae, and the promoter is that of the PirA gene of Photobacterium damselae subsp. damselae; and e. a transcription termination region; and f.optionally or preferably one or more of any of the following elements selected from the list consisting of: positive selection genes such as antibiotic resistance genes and other molecules with antimicrobial activity; genes that solve auxotrophies; or metal resistance genes, negative selection genes (sacB, rpsL, ccdB), reporter genes (lacZ, luxCDABE, galK), toxin-antitoxin systems, restriction-modification systems, fluorescent protein genes (GFP, CFP, YFP, dsRed2, mCherry) and catabolic pathway genes; In a preferred embodiment, the sequence encoding the signal peptide is the signal peptide of the damselysin (Dly) gene of Photobacterium damselae subsp. damselae, and the promoter is the damselysin (Dly) gene of Photobacterium damselae subsp. Damselae. In another preferred embodiment, the sequence encoding the signal peptide is the signal peptide of the PirA gene of Photobacterium damselae subsp.damselae, and the promoter is the PirA gene from Photobacterium damselae subsp. damselae. It is noted that these sequences are illustrated below: >PirA promoter (11Kda). The present invention includes sequences having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the promoter sequence indicated above as long as it is capable of promoting transcription of the gene of interest to be expressed. The present invention includes sequences having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the signal peptide sequence indicated above as long as it maintains its function. In another preferred embodiment, the MRB type origin of replication consists of SEQ ID No. 1 or a nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID No. 1 and which is capable of acting as an origin of replication (initial sequence with which the plasmic DNA will be replicated in the recipient cell).In another preferred embodiment, the MRB type origin of replication consists of SEQ ID No 3 or a nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID No 3 and that is capable of acting as an origin of replication (initial sequence with which the plasmic DNA will be replicated in the recipient cell). In another preferred embodiment, the MRB type origin of replication consists of any one of the sequences: SEQ ID No 1, 2, 3 or 6. In another preferred embodiment, the origin of transfer consists of SEQ ID NO 7 or a nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID No 7 and which is capable of functioning as a starting point for conjugative transfer.In another preferred embodiment, the promoter consists of SEQ ID NO 8 or a nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 8 and that is capable of promoting the transcription of the gene of interest to be expressed. In another preferred embodiment, the signal peptide consists of SEQ ID NO 9 or a nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 9 and that encodes a protein that maintains the function of the protein encoded by SEQ ID NO 9.In another preferred embodiment, the transcription termination region consists of SEQ ID NO 10 or any other nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 10 and maintains the transcription termination function. In another preferred embodiment, the signal peptide consists of SEQ ID NO 9, where the promoter consists of SEQ ID NO 8 and where the transfer origin consists of SEQ ID NO 7. In another preferred embodiment, the expression vector or plasmid additionally comprises a multiple cloning site (MCS). In another preferred embodiment, the expression vector or plasmid additionally comprises a coding sequence of a histidine tag at the N or C terminal position to the gene of interest.In another preferred embodiment, the expression vector or plasmid consists of the nucleotide sequence SEQ ID NO 17 or any other nucleotide sequence that has at least 99% sequence identity with the sequence SEQ ID NO 17. In another preferred embodiment, the expression vector or plasmid consists of the nucleotide sequence SEQ ID NO 18 or any other nucleotide sequence having at least 99% sequence identity with the sequence SEQ ID NO 18. An additional aspect of the present invention relates to a bacterial strain belonging to Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida or V. natriegens (Vibrio natriegens) comprising the plasmid or vector as defined in the previous aspect or in any of its preferred embodiments. Another additional aspect relates to a method for producing a recombinant protein comprising introducing the expression vector as defined in the previous aspect or in any of its preferred embodiments, into a host cell, culturing the host cell under conditions suitable for expressing the protein and recovering the protein.Preferably, the host cell is selected from the list consisting of Photobacteriumdamselae subsp. damselae, P. damselae subsp. piscicida or V. natriegens. Brief description of the figures Fig. 1. Schematic showing the structure of the pVibexp vector and the origin of each module. The vector is composed of 5 modules: an origin of replication (oriV), an origin of transfer (oriT), a promoter and a signal peptide for cloning GOIs, a His-Tag, an rrnB transcriptional terminator and a multiple cloning site (MCS) with numerous single-cut enzymes. Of these modules, the oriV and the selection marker (AbR) are interchangeable modules, flanked by NheI and BamHI, respectively. Fig. 2. (A) Schematic of the structure of plasmid pVibexp and the hybridization sites of the primers used in the PCR analysis. The expected amplicon sizes are specified in the table below.Agarose gel electrophoresis of PCR amplified products with the P1+P2 (B, C), P3+P4 (D), and P5+P6 (E) combinations of the transformant clones E. coli DH5α (D1), S-17-λ-pir (S1, S2, and S3), and β-3914 (β1), as well as of the Pdd transconjugants RM-71 (RM) and LD-07 (LD), of Pdp (PP3, AQV27.1, AQP18.1), and of V. natriegens CCUG16374 (VN). C-, negative control. L, 1 Kb molecular weight marker. Fig. 3. (A) Schematic showing the structure of the pVibexp+dly construct and the hybridization sites of the primers used in the PCR analysis. The expected amplicon sizes are specified in Table 4.2. (B) Photograph of E. coli DH5α transformant clones from plasmid pVibexp+dly on TSA-1-Cm20 plates supplemented with egg yolk (YH) (3%). Agarose gel electrophoresis of PCR amplified products from E. coli DH5α (D1) and β-3914 (β1, β2 and β3) transformant clones with the combinations (C) P1+P2, (D) P1+PC16, (E) P3+P4 and (F) P5+P6.C-, negative control. L, 1 Kb molecular weight marker. Fig. 4. (A) Representative photograph of the phospholipase activity of the Pdd, Pdp and V. natriegens transconjugant clones for pVibexp and pVibexp+dly on TSA-1-Cm5 plates supplemented with egg yolk (YH) (3%) after 24 h of incubation. Agarose gel electrophoresis of the PCR amplified products of the Pdd transconjugants A-162 (1) and LD-07 (2), of V. natriegens CCUG16374 (3), of Pdp AQV27.1 (4), AQP18.1 (5) with the combinations (B) P1+PC16, (C) P1+P2, (D) P3+P4 and (E) P+P6. The expected sizes are specified in Table X. C-, negative control. L, 1 Kb molecular weight marker. Fig. 5. Agarose gel electrophoresis of the transconjugant clones (1-4) of P. damselaesubsp. piscicida AQV27.1 (PDP66) and AQP18.1 (PDP72) for the pVibexp vector and for the pVibexp+dly vector amplified by PCR with primers specific for the aip56 toxin encoded in the virulence plasmid pPHDP10 (A), with combinations P3 and P4 (B) and combinations P1 and P2 (C). Fig. 6. Schematic of the construction of the pA-162-Vibexp vector. The pAVZ185 vector (pUC57-Vibexp) was marked with the cat gene, for chloramphenicol resistance, amplified from the pKD3 plasmid, in the MCS module (1). The sequence corresponding to the Vibexp-cat vector was released by digestion with NheI (2). By sequencing, the sequence of the MRB type plasmid identified in the Pdd A-162 strain was closed (3). The complete plasmid sequence was amplified by inverse PCR (4) and the cloning of the pVibexp modules into the p-A162 plasmid gave rise to the pA-162-Vibexp vector. To test the vector functionality, the tbpD gene was cloned as a cargo in the MCS while the P. citrea dly-like gene was cloned as both a GOI and a cargo in the MCS.Fig. 7.(A) Diagram of the pA-162-Vibexp vector showing the hybridization sites of the primers used for PCR analysis. Agarose gel electrophoresis of the PCR amplified products of the transformant clones of E. coli DH5α (D1) and β-3914 (β1) with the combinations (B) P1+P2, (C) P3+P4, (D) P5+P6. C-, negative control. L, 1 Kb molecular weight marker. Fig. 8. (A) Diagram of the pA-162-Vibexp+tbpD construct. The expected amplicon sizes for each primer pair are indicated on the right. Agarose gel electrophoresis of PCR products obtained after amplifying complemented ΔtbpD transconjugant clones (1, 2, and 3) with primers specific for the deleted region of the TbpD gene (B) and for different regions of the construct (C). Δ, deletion mutant AVZ159 clone for ΔtbpD. β1, E. coli clone Β-3914 (Le Roux, F., Binesse, J., Saulnier, D., & Mazel, D. (2007).Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector. Applied and Environmental Microbiology, 73(3), 777– 784) donor of the pA-162-Vibexp+TbpD construct. C-, negative control. L, 1 kB molecular weight marker. Fig. 9. (A) Schematic of the pA-162-Vibexp+dly-like construct showing the hybridization sites of the primers used for PCR analysis. The expected amplicon sizes for each primer pair are shown in the table below. (B) Photograph showing E. coli DH5α transformant clones of pA-162-Vibexp+dly-like on TSA-1-Cm20 plates supplemented with egg yolk (YH) (3%) and (C) of an LD-07 transconjugant carrying the empty plasmid and the pA-162-Vibexp+dly-like construct. Agarose gel electrophoresis of the PCR-amplified products of the E. coli transformant clones.coli DH5α (D1) and β-3914 (β1) and the transconjugant clones of strain LD-07 (1, 2 and 3) with the different primer combinations (D, E, F and G). C-, negative control. L, 1 Kb molecular weight marker. Fig. 10. (A) Schematic of the pA 162 Vibexp+rDly like construct showing the hybridization sites of the primers used in the PCR analysis. The expected amplicon sizes are specified in the table above left. (B) Photograph showing the transconjugant clones of Pdd LD-07 for the pA-162-Vibexp+rDly-like vector on TSA-1-Cm5 plates supplemented with egg yolk (YH) (3%). (C) and (D) Agarose gel electrophoresis of PCR amplified products from transformant clones of E. coli β-3914(β1) and transconjugant clones of LD-07 for the vector pA-162- Vibexp+rDly-like (1, 2, 3 and 4) with the combinations indicated in the table. C-, negative control. L, 1 Kb molecular weight marker. Fig. 11.Diagram of the construction of the pVibexp vector. The pAVZ185 vector (pUC57- Vibexp) was tagged with the chloramphenicol resistance gene cat, amplified from plasmid pKD3, in the MCS module (1). The sequence corresponding to the Vibexp-cat vector was released from pUC57 by digestion with NheI, this construct was ligated with the oriV gene, amplified from pPHDP10 of Pdp DI-21, giving rise to the final structure of the pVibexp vector. Fig. 12. Expression levels offered in FPKM units (Fragments per Kilobase per Millionfragments Mapped) for (A) the dly gene (marked with a green arrow) (Matanza & Osorio, 2018) and (B) for the pirA gel (marked with a blue arrow). Fig. 13. Dly and PirA proteins are secreted in large quantities via the T2SS (type 2 secretion system). SDS-PAGE profile of Pdd supernatants. Fig. 14. Expression of the dly and pirA genes (indicated by a red arrow) is modulated in response to changes in NaCl.(A) Differential expression levels (in Log2FoldChange) of induced (in orange) and repressed (in blue) genes at 1% NaCl versus 3% NaCl. (B) Comparative analysis of SDS-PAGE profiles of Pdd culture supernatants at 1% NaCl and 3% NaCl (Barca et al., 2023). Description of the invention The results obtained, set forth in Example 1 of the present invention, led to the construction of two vectors: the pVibexp vector and the pA-162-Vibexp vector. The pVibexpse vector was composed of 5 functional modules: (1) an origin of replication or oriV of the MRB type (obtained from the virule cap 0 plasmid of strain 1 of P. damselae subsp. piscicida (Photobacterium damselae subsp. piscicida Pdp), (2) an oriT transfer origin of the RK2 / RP4 type, (3) the promoter and signal peptide of the dly gene of strain RM-71 of Pdd (Photobacterium damselae subsp.damselae) for cloning genes of interest (GOIs), (4) the histidine tag (6-HisTag) and the T1 and T2 terminators of the rrnB gene and finally (5) the sequence corresponding to the multiple cloning site (MCS). The chloramphenicol resistance gene was selected as a selection marker, which was amplified from plasmid pKD3 and cloned into the MCS of the platform. Thus, the MCS sequence constitutes a "loading module" that, beyond serving as a cloning point for the resistance marker, attributes to this vector various uses such as, for example, for genetic complementation through cloning genes with a native promoter. The results obtained demonstrated that the pVibexp vector is replicative, selectable and mobilizable by conjugation to various strains of Pdd, Pdp and V. natriegens.Furthermore, to test the usefulness of pVibexp as a recombinant protein expression vector, we cloned the Pdd dly gene under the control of the vector promoter and signal peptide. The results obtained demonstrated that the pVibexp vector is a functional vector for the production and secretion of the Dly toxin in several Pdd and Pdp strains and allowed us to validate the primers designed for the cloning of GOIs in the platform. Furthermore, an in silico search identified, for the first time, an MRB-type plasmid in a Pdd strain. Cloning of all the modules of the pVibexp platform, except for oriV, into this native plasmid isolated from Pdd strain A-162, gave rise to the pA-162-Vibexp vector. The cloning of the tbpD gene in the MCS of this platform has allowed the successful genetic complementation of the ΔtbpD mutant of Pdd, obtained during the course of this invention. Likewise, the results obtained demonstrated that the pA-162-Vibexp vector is functional in P.damselae subsp. piscicida and in V. natriegens, so that its usefulness could be extended to other members of the Vibrionaceae family. Furthermore, the cloning of the Pseudoalteromonas citrea phospholipase, under the control of the promoter and the signal peptide of the dly gene, has shown that the pA-162-Vibexp platform allows the expression and secretion of this heterologous phospholipase in Pdd. These preliminary results demonstrate that the designed vector is a potential ethical vector for the production and secretion of recombinant proteins in Pdd. Therefore, in this invention we have designed and constructed two plasmid vectors that have proven to be replicative, selectable and easily mobilized by conjugation to both Pdd and other Vibrios, useful for the genetic complementation of Pdd. They constitute genetic tools with potential for the production of recombinant proteins in P. damselae as a factory organism.Therefore, a first aspect of the present invention relates to an or plasmid expression vector comprising: a) An origin of replication of the MRB type, preferably an OriV origin of replication derived from P. damselae subsp. piscicida (Pdp) and / or P. damselae subsp. damselae (Pdd); b) an origin of transfer (oriT), preferably of the RK2 / RP4 type; c) an insertion site for inserting a gene of interest; d) a promoter functionally linked to a sequence, preferably DNA, encoding a signal peptide and to a sequence, preferably DNA, encoding the gene of interest, wherein preferably the sequence encoding the signal peptide is the signal peptide of the damselysin (dly) gene of Photobacterium damselae subsp. damselae (Pdd), and the promoter is the damselysin (dly) gene of Photobacterium damselae subsp. damselae (Pdd); ye) a transcription termination region.a) Origin of replication of the MRB type In the context of the present invention, an origin of replication of the MRB type (“Marine-RNA based”) is preferably defined as a DNA sequence that codes for two RNAs (RNAI and RNAII) that are transcribed in opposite directions, contain complementary sequences and that regulate the replication of plasmids isolated from marine bacteria of the Vibrionaceae family. In the context of the present invention, this definition also includes SEQ ID NO 1. In a preferable embodiment of the invention, the oriV sequence of vector 1 (pVibexp) comprises or consists of the sequence SEQ ID NO 1 described below: [SEQ ID NO 1] ggtcaggatctttggatgatcatcggcttgagttatttcaagttggagagtagtatctttgcccagatgtgaaaaaaccagcctt. P the nucleotide sequence SEQ ID NO 1 having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO 1. In the present invention, "identity" or "sequence identity" is understood to mean the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two nucleotide / amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST [Altschul SF et al. Basic local alignment search tool. J Mol Biol.1990 Oct 5; 215 (3) :403-10], The BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechnology Information (NCBI). In the present invention, the term "functional variant" refers to amino acid or nucleotide sequences that have a percentage of identity with any one of the sequences of the invention and that maintain the functionality of the sequence from which they are derived. In another preferred embodiment of the invention, SEQ ID NO 1 or any functional variant of said nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90% or 95% sequence identity with the sequence SEQ ID NO 1, is flanked by sequences that represent restriction sites. As an example. For example, we represent SEQ ID NO 2 which is included as a preferred embodiment of the present invention: SE ID NO 2 oriV modules of both vectors. These flanking sequences were included with the idea of ​​being able to exchange the oriV sequence for any other version. In another preferable embodiment of the invention, the oriV sequence of the vector is the oriV sequence of vector 2 (pA-162-Vibexp) comprising or consisting of the sequence SEQ ID NO 3 described below: [SEQ ID NO 3] attaaaacagccttaaaagccgtttacgggcgttagaattttatctaaaacaccggcttcaacgttaattttcagcgaaca This The nucleic acid sequence presents two coding sequences (marked in bold and underlined) that correspond to the ORF1 (SEQ ID NO 4) and ORF2 (SEQ ID NO 5) sequences. It should be noted that these sequences are not the sequences that code for the oriV RNAs. They are sequences that are part of the native MRB vector of strain A-162. [SEQ ID NO 4] MGIKGKAIGSIGSLCVDWVMSNGITLIVALIGGGGMTYLSSISAALNQYGFLAWIAVGLVTFVIIA Preferably, this embodiment of the present invention comprises any functional variant of the nucleotide sequence SEQ ID NO 3 having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO 3. In another preferable embodiment of the invention, SEQ ID NO 3 or any functional variant of said nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 3, is flanked by sequences representing restriction sites. By way of example, we represent SEQ ID NO 6 which is included as a preferred embodiment of the present invention: [SEQ ID NO 6] the oriV modules of both vectors. These flanking sequences were included with the idea of ​​being able to exchange the oriV sequence for any other version. b) Origin of transfer (oriT). In a preferred embodiment, the origin of transfer of the vector or plasmid of the first aspect of the invention comprises or consists of SEQ ID NO 7 [SEQ ID NO 7] CTTTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTC or in any other functional variant of said nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID No. 7.c) and d) promoter and signal peptide In a preferred embodiment, the promoter comprises or consists of SEQ ID NO 8 [SEQ ID NO 8] ATCAATGGTCACAATATAGAGTATTCGATTAAGAAATATAATTATATTAGTTGAAAATTTCATATA o in any functional variant of said nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID No. 8. The promoter is functionally linked to a sequence, preferably DNA, that encodes a signal peptide and to a sequence, preferably DNA, that encodes the gene of interest, wherein preferably the sequence that encodes the signal peptide is the signal peptide of the damselysin gene (dly) of Photobacterium damselae subsp. damselae. It is noted that the cloning of the gene of interest (GOI, "Gene of Interest") under the control of both components (the promoter and the signal peptide), directs the secretion of the target protein to the outside of the cell, preferably to the culture supernatant, greatly simplifying the subsequent procedures for recovering the protein.Preferably, the sequence encoding the signal peptide comprises or consists of SEQ ID NO 9 [SEQ ID NO 9] ATGAAAATAAAAACGCTTACTATGTTAATTGTGTGCTGCTCCTCAAATGCATATGCTTTT. o e nany functional variant of said nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 9 and that codes for a protein that maintains the function of the protein encoded by SEQ ID NO 9 (MKIKTLTMLIVCCSSNAYAF (amino acid sequence of SEQ ID NO 9 represented here as SEQ ID NO 15)). That is, the cloning of a gene of interest (GOI, “Gene of Interest”) under the control of both components (the promoter and the signal peptide), directs the secretion of the target protein to the outside of the cell, preferably to the culture supernatant, greatly simplifying the subsequent procedures for recovering the protein. e) Transcription termination region.In a preferred embodiment, the transcription termination region comprises or consists of SEQ ID NO 10 [SEQ ID NO 10] AGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGAT. o in any functional variant of said nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID No. 10 and maintains the function of terminating transcription. Other components of the expression vector or plasmid. The expression vector or plasmid of the first aspect of the invention comprises at least the following components or elements (preferably in the order set out below): MRB (Origin of replication) - oriT (origin of transfer) - promoter - signal peptide - transcription terminator However, in a preferred embodiment of the invention, the expression vector or plasmid of the first aspect of the invention additionally comprises at least one multiple cloning site (MCS): MRB (Origin of replication) - promoter - signal peptide - transcription terminator - MCS. In a preferred embodiment, the MCS comprises or consists of any of the following sequences SEQ ID NO 11 to SEQ ID NO 13 [SEQ ID NO 11] GTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAG [SEQ ID NO 12] GTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAG [SEQ ID NO 13] GGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGC or any other nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any of the sequences SEQ ID NO: 10 to SEQ ID NO: 13. Preferably, the MCS sequence comprises or consists of SEQ ID NO: 14 (comprising a chloramphenicol resistance marker) [SEQ ID NO: 14] GTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAG tcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttgtcgagattttcagga It is noted that any element of interest such as a selectable marker, for example a resistance marker, or an expression cassette can be inserted into the MCS. In yet another preferred embodiment, the expression vector or plasmid of the first aspect of the invention additionally comprises at least one histidine tag, preferably as shown below: MRB (Origin of replication) - oriT (origin of transfer) - promoter - signal peptide - HisTag - transcription terminator - MCS Where the histidine tail is preferably encoded by SEQ ID NO 16: CATCATCATCATCATCATTG. The histidine tail is inserted for the detection and purification of said gene of interest. It is noted that any length of the histidine tail (for example between 4 and 10 or more) is encompassed in the present invention as long as it serves for the detection and purification of said gene of interest. In another preferred embodiment, the expression vector or plasmid of the first aspect of the invention comprises or consists of the nucleotide sequence of vector 1: pVibexp vector (SEQ ID NO 17): [SEQ ID NO 17] CTTTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTTTTTCGGTATATCCATCCTTTTTC caatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatc GCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGGCTAGCggtcaggatctttggatgatcatcggcttgag ttatttcaagttggagagtagtatctttgcccagatgtgaaaaaaccagccttgcagggccggttttttcggagttacttaaaacg attttcgagctcgttaagttcttttcatgatagccatatcacgatggcacttcaagtattcctgcactctatttggatcaagatgttt tggatagagcgatgcgcatcaccaccgaccaagggaaattccataggtggaggtaaaggtcttcgggaaaatgacgtaatgt gtcgcccaactaaagcgttcaggttggggtcgcgacttaatgataataacctcgaagattgtgtgactggaatggggggcgaa agcctttacacacagcgcaggaacgcccgaaagggtaagagacaaatcccgatctgatgcgaccaaaaagatactcgatttc gctaaaattcagctgaaattgggtaggggtcggcgcagccgaaaggcaatcaagttgagcctttcgcgacttcctttttgctcgg cttttctatgtcaaaattaaatacacatcttattgagcgctgagcgtacctttctgcgatcttattctaattaggcgattgtgtgtaa agtcgaggtcaacttgttgtcctgacttttccacatgagacgttgtcaccttttcgtactgattcttctcgttcagaataactcgatt aatgtcctggtgaagttgttcctggataacatgactaggcaaactgctttgatttaataccaggtatgcttgaatcagttttttacc tctcttgtagagctctctgaggggttttggagtgcattcggattctgactcgaatttctcgcttaggattgaattagaggcttttgaaacts Recovery Gateways expression vector or plasmid of the first aspect of the invention comprises or consists of the nucleotide sequence of vector 2: pA-162- Vibexp vector (SEQ ID NO 18): [SEQ ID NO 18] TATTGTGTAATGGTAATAGTATTTATCATAGAAAATTAATAACTGATTTTTGGTTGTTAATCCTT t ttt t tt GGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCG In another preferred embodiment of the present invention, the expression vector or plasmid of the first aspect of the invention comprises an origin of replication and / or an insertion site flanked by restriction enzyme recognition sequences such as Nhel, Ndel or BamHI. A second aspect of the invention relates to a process for bacterial conjugation between a donor bacterial strain and a recipient bacterial strain, characterized in that the transfer of genetic information from the donor or donor cell to the recipient cell is carried out by means of a plasmid or vector as defined in the first aspect of the invention. Preferably, the recipient bacterial strain belongs to the family Vibrionaceae, preferably to any of the genera Photobacterium or Vibrio, more preferably to Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida or V. natriegens. Preferably, the donor bacterial strain is E.coli, more preferably E. coli β-3914. With respect to the donor bacterial strain, it is noted that the Escherichia coli strains used as donors in the RP4 / RK2 type plasmid conjugation processes (strains S-17-1-^-pir and β-3914) are in the public domain and widely known by the international scientific community. They are widely distributed in laboratories of public and private institutions and, in fact, the publications in which these strains are described have been cited by countless studies to date. Although in the present invention we propose the use of the β-3914 strain, it is true that the use of any other Escherichia coli strain containing the RK2 / RP4 type conjugation machinery would be perfectly valid to obtain transconjugating clones (in species of Photobacterium, Vibrio and other genera of marine bacteria) with the vectors described in the present invention.A third aspect of the invention relates to a recipient bacterial strain belonging to the Vibrionaceae family, preferably to any of the genera Photobacterium or Vibrio , more preferably to Photobacterium damselae subsp. damselae , P. damselae subsp. piscicida or V. natriegens ( Vibrio natriegens ), which comprises the plasmid or vector as defined in the first aspect of the invention. A fourth aspect of the invention relates to a method for producing a recombinant protein comprising introducing the expression vector of the first aspect of the invention into a host cell, culturing the host cell under conditions suitable for expressing the protein and recovering the protein. Preferably, the host cell is selected from any of the Vibrionaceae family, preferably to any of the genera Photobacterium or Vibrio , more preferably to Photobacterium damselae subsp. damselae , P. damselae subsp. piscicida or V. natriegens.The present invention provides the following examples for merely illustrative purposes. Examples Materials and methods. Design and construction of the plasmid (pVIBEXP). Much of the knowledge about the physiology and virulence of Pdd has been obtained through the use of genetic engineering techniques. For the performance of these methodologies, plasmid vectors constitute essential molecular tools, being widely used for the genetic manipulation of marine bacteria. In the specific case of Pdd, observations obtained over the years in the laboratory suggest that the vectors currently used for the genetic engineering of Pdd are, in many cases, inefficient, unstable and require the addition of antibiotics that, in some cases, lead to the selection of spontaneous mutants and / or interfere with the performance of certain tests.The observed limitations motivated us, in the present invention, to construct a modular vector as a new tool for the genetic engineering of Pdd and whose use could be extended to other marine bacteria belonging to the Vibrionaceae family. Regarding the construction of the pVibexp vector, the sequences corresponding to the modules: (1) origin of transfer (oriT) of the RK2 / RP4 type (obtained from the pSEVA plasmids), (2) promoter and peptide are from . damselae subsp. damselae, (3) the histidine tail (6-HisTag) and the T1 and T2 terminators of the rrnB gene (obtained from plasmid pTrc-His2A) and finally (4) the sequence corresponding to the multiple cloning site (MCS) (from plasmid pWKS30), were designed in silico using the SnapGene v4.1.9 program. The NheI target sequence was added to this 1895 bp DNA sequence at its ends, and the resulting complete molecule was chemically synthesized and cloned into the EcoRV position of the pUC57-Simple (AmpR) vector (GenScript).The obtained construct was transformed by heat shock into competent E. coli DH5α cells, which were selected in TSA-Amp (resistance conferred by the pUC57-Simple vector) giving rise to clone pAVZ185 (pUC57-Vibexp). Next, the DNA sequence corresponding to the pVibexp vector was marked by cloning the chloramphenicol resistance gene (cat) into the BamHI site of the MCS. This gene was amplified from plasmid pKD3 with the primers indicated in Table 1 (PC3 and PC4). The ligation was transformed into competent E. coli DH5α cells that were selected in TSA-Cm20. From the positive transforming clones, it was decided to continue the procedure with clone pAVZ190. The platform sequence, tagged with the chloramphenicol resistance gene, was released from plasmid pAVZ190 (pUC57-Vibexp-CmR) by cutting with NheI and ligated with the oriV of plasmid pPHDP10 amplified with primers PC1 and PC2 (OriV_NheI_F and R, respectively) from strain DI-21.Selection of E. coli DH5α transformants was performed on TSA-Cm20 plates and the grown colonies were streaked on TSA-Amp to verify the loss of the pUC57-Simple sequence. Those clones that grew on TSA-Cm20, but not on TSA-Amp, were confirmed by PCR, giving rise to clone pAVZ192. This plasmid was then transformed by heat shock into the DAP-auxotrophic E. coli β-3914 strain, giving rise to clone AVZ198. The illustrative scheme of the construction process of the pVibexp vector is shown in Figure 11. Example 1. Description of the modules that make up the pVibexp plasmid In order for a plasmid to be transformed and selected in the host organism, it must meet a series of essential characteristics: the vector must be replicative and selectable and, furthermore, it can be introduced into the host cell by conjugation.To meet these requirements, the constructed vector, named Vibrioexpress (pVibexp), is composed of 5 functional modules, of which the origin of replication (oriV) and the antibiotic marker (AbR) are also interchangeable modules, flanked by NheI and BamHI restriction sites, respectively (Figure 1). The first essential component of any vector is the origin of replication (oriV), which must be recognized by the cellular replication machinery and determines the copy number of the plasmid in the bacterial host cells. For the construction of the pVibexp vector, the origin of replication of plasmid pPHDP10 from the P. damselae subsp. piscicida (Pdp) strain DI-21 was selected. This plasmid, of almost 10 kB, encodes the main virulence factor of Pdp, the AIP56 toxin, being a key determinant of the pathogenicity of this subspecies.The minimal region of oriV required for replication of this plasmid, approximately 1080 bp, was previously identified and constitutes the core element of our platform (it corresponds to nucleotide positions 20-1100 of the sequence deposited in GenBank under accession number NZ_AKYG01000457). It should be noted that when the functionality of this minimal oriV was first demonstrated, the mechanisms controlling its replication were still unknown. However, research published almost simultaneously years later (Le Roux et al., 2011; Pan et al., 2010), allowed pPHDP10 to be classified as a member of a new family of plasmids, whose presence was restricted to the Vibrionaceae family. One of the main characteristics of this new family of plasmids is that its replication mechanism depends on two RNA molecules, which is why they were named MRB (Marine RNA-based) plasmids (Le Roux et al., 2011).However, given the similarity of this RNA-dependent system to the Col-E1 replication system of E. coli, other authors designated these replicons as CLV (ColE1-like Vibrionaceae) plasmids (Pan et al., 2010). The particularities of MRB-type replicons mean that the origin of replication of the Pdp plasmid pPHDP10 meets a series of characteristics that make it ideal for vector design. The specific properties that motivated the use of this oriV as the scaffold for the designed vector were the following: -High copy number. Despite the extensive knowledge about the heterogeneity of Pdd, little is known about the systems that govern the replication of its plasmids or their copy number. However, visualization of plasmid pPHDP10 in an agarose gel suggests that it is a high copy number plasmid.Multicopy plasmids are widely used as protein expression vectors since the higher the plasmid copy number, the higher the expression of the target gene (Rouches et al., 2022). This property of pPHDP10 motivated us to use its oriV sequence as a base element for the design of an expression vector. -Stability. The plasmid pPHDP10 of P. damselae subsp. piscicida encodes the AIP56 exotoxin, one of the main virulence factors of this subspecies (DoVale et al., 2005). Recent research shows that, unlike other unstable plasmids, such as plasmid pPHDPT3, which encodes a type 3 secretion system (T3SS), plasmid pPHDP10 constitutes a highly stable and prevalent mobile element, almost 100%, in isolates of the subsp. piscicide (Abushattal et al., 2020, 2022).Furthermore, supporting this enormous stability, the genetic organization of plasmid pPHDP10 is highly conserved in Pdp isolates regardless of their geographical origin (Baseggio et al., 2021). -Temperature sensitive. Previous studies have shown that the replication of this plasmid is temperature sensitive, with its copy number being significantly higher at temperatures of 30°C compared to 37°C (Le Roux et al., 2011). Although the genetic basis of this thermoregulation is unknown, this characteristic offers the possibility of using this vector as a conditional replicon in vibrios (Le Roux et al., 2011). -Possibility of generating compatible vectors with minimal modifications in the oriV. LeRoux et al., (2011) demonstrated that plasmids from the same family with minimal modifications in their origins of replication became compatible.This unusual feature would provide additional utility to the vector since, by using plasmids with minimally modified oriV regions, proteins of interest could be co-expressed, including egu as well as ep esion. In order to generate versions of Vibexp modified in its oriV, the minimal sequence used was flanked by an NheI restriction site. In addition to the origin of replication (oriV), the absence of a transformation protocol in Pdd requires the incorporation into pVibexp of an origin of transfer (oriT) that allows the mobilization of the plasmid by conjugation. The sequence for the oriT, of 246 bp, from the conjugative plasmid RK2 / RP4 of broad host range, was selected from the pSEVA plasmids (Silva-Rocha et al., 2013).To obtain a region in which to clone the selectable marker, as well as any other genes of interest, we included the multiple cloning site (MCS) of plasmid pWKS30, which provides a large number of single-cut restriction sites in our vector (Figure 1) (Wang & Kushner, 1991). The universal M13 primers, which were used for amplification and sequencing of the cloned genes, hybridize to the ends of this 248-bp cassette. The second interchangeable cassette of our platform, the antibiotic resistance marker (AbR), was cloned into the BamHI site of the MCS. As shown in Figure 1, the DNA sequences encoding the selectable markers include the structural gene for antibiotic resistance (AbR) and its native promoter.In this case, markers commonly used in the selection of Gram-negative bacteria were included, such as the chloramphenicol resistance gene (Cm, the cat gene, from plasmid pKD3; Datsenko & Wanner, 2000) and, as an alternative, the akanamycin resistance gene (Km, kanR gene from plasmid pKD4; Datsenko & Wanner, 2000). In this way, the MCS sequence constitutes a "cargo module" that, beyond serving as a cloning point for the resistance marker (AbR), attributes to this vector countless utilities, such as for genetic complementation through the cloning of genes with a native promoter, or for the cloning of additional "gadgets", that is, dispensable DNA sequences that confer added utility to our platform.These functional modules constitute the initial version of the pVibexp vector and, as will be seen later, serve as the skeleton for the design of a preliminary platform for the production and secretion of recombinant proteins and peptides in P. damselae. Example 1.1. The pVibexp vector is replicative in E. coli and can be mobilized by conjugation to different vibrios. As mentioned in the previous example, the functionality of the oriV sequence selected for the pVibexp vector had already been previously demonstrated in E. coli. Curiously, when this minimal replicon, tagged with a chloramphenicol resistance gene, was transformed into E. coli, the transformant colonies grown at 37°C were negative for the plasmid, suggesting that this replicon could be thermosensitive. In agreement with these observations, subsequent studies by Le Roux et al., (2011), empirically demonstrated that the copy number for the minimal oriV of a V.cholerae was much higher when cells were grown at 30°C compared to 37°C. For this reason, during the course of this work, a temperature of 25°C was established for the selection and growth of transformant E. coli clones with the pVibexp platform. Transformation of the Vibexp plasmid (CmR) in E. coli resulted in clones that were PCR-positive for the vector (Figure 2), demonstrating that pVibexp is a selectable and replicative vector in E. coli. This initial transformation step was performed in the E. coli strain DH5alpha, commonly used as a plasmid storage strain. The next step was to demonstrate the functionality of the origin of transfer (oriT) of pVibexp. It is worth mentioning that the absence of a transformation protocol in Pdd makes conjugation transfer, for the moment, an essential step to test the functionality of our construct in P. damselae.To do this, it was necessary to first transform the pVibexp platform into two of the most commonly used conjugative strains in the laboratory, the E. coli S-17-1-^-pir strain (Herrero, M., De Lorenzo, V., & Timmis, KN ​​(1990). Transposon vector containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. Journal of bacteriology, 172(11), 6557-6567) and the β-3914 strain, the latter being auxotrophic for DAP. PCR analysis of the transformant clones of E. coli S-17-1^^- pir for pVibexp revealed that a proportion of the selected colonies had amplicons of a larger size than expected for the P1+P2 PCR (Figure 2B). Sequencing of some of these amplicons revealed the presence of an IS1 insertion sequence in the region corresponding to the MCS.The insertion of this transposable element into a non-functional sequence such as the polylinker suggests that this mutation does not confer any advantage for vector replication. In contrast, when pVibexp was transformed into the E. coli strain β-3914, no insertion event was detected in the vector sequence. These results seem to indicate that the E. coli strain S-17-1-^-pir seems to be more prone to skipping insertion elements in the plasmid sequence. In line with these findings, skipping of IS1 elements in plasmids harbored in ^-pir strains has previously been reported in other vectors designed for the genetic manipulation of Gram-negative bacteria (pCVD442) (Philippe et al., 2004). Among the E. coli insertion elements, the IS1 element appears to be the most active, with a rate of 2.79 × 10. -5transpositions / generation (Sousa et al., 2013). The possibility of these IS sequences being introduced into the pVibexp platform should be taken into account, as this may lead to non-specific recombination events that limit the usefulness of our vector (Philippe et al., 2004). Since the use of the E. coli strain β-3914 did not appear to lead to any insertion events in the pVibexp sequence, it was used as the platform donor strain for subsequent conjugations. Various strains of Pdd (strains RM-71 and LD-07), P. damselae subsp. piscicida (strains PP3, AQV27.1 and AQP18.1) and the V. natriegens strain CCUG16374 were selected as recipient strains. PCR analysis of the transconjugant clones grown on TSA-1-Cm5 revealed that all clones were positive for the platform (Figure 2). Furthermore, the integrity and absence of transposable elements in the transconjugant clones was verified by PCR and sequencing.The results obtained demonstrate that the plasmid pVibexp is a mobilizable vector, easy to introduce by conjugation into various strains of vibrios. Example 1.2. The pVibexp vector allows the efficient expression and secretion of phospholipase Dly in P. damselae subsp. damselae and in subsp. piscicidaOnce it was demonstrated that the modular plasmid pVibexp is replicative, selectable and mobilizable to various vibrios, its functionality as a recombinant protein expression vector was tested. With this objective in mind, the sequences corresponding to a strong promoter and a native P. damselae signal peptide dependent on the type II secretion system (T2SS) were included in the platform design.For this purpose, the promoter and signal peptide of the dly gene encoding the phospholipase Damselysine were selected because it is a high-expression promoter (Matanza and Osorio, 2018) and because of the evidence that this toxin is secreted very efficiently by the T2SS. In this way, the cloning of a gene of interest (GOI, “Gene of Interest”) under the control of both components will direct the secretion of the target protein into the culture supernatant, greatly simplifying subsequent protein recovery procedures. Furthermore, to facilitate the detection of the protein using His-Tag antibodies as well as purification with chelating resins, a polyhistidine tail (6xHis) was added to the C-terminus of the recombinant protein in the in silico design. In order to prevent the transcription of sequences adjacent to the GOI and to stabilize the mRNA produced, the transcriptional terminators T1 and T2 of the E.coli, frequently used in expression vectors. The sequence corresponding to the polyhistidine tag (6xHis) and the transcriptional terminator were selected from the sequence of the expression plasmid pTrcHis2A (Invitrogen). One of the main limitations of the production of recombinant proteins in microbial factories, such as E. coli or yeast, is that the post-translational modifications that the proteins undergo may be different from those that occur in the original host. For this reason, the use of this modifiable platform, designed to order and with native elements, would offer us the possibility of producing recombinant proteins in P. damselae as a factory organism, which would provide numerous advantages.First, recombinant proteins and peptides would be produced in the same native cellular context, thereby ensuring the introduction of post-translational modifications that ensure the protein's antigenicity and functionality. Second, the use of a strong promoter and a T2SS signal peptide allows the produced recombinant protein / peptide to be efficiently secreted into the extracellular medium. As previously mentioned, this constitutes a considerable technical advantage since it minimizes the need to disrupt cells and facilitates subsequent purification processes. Third, we have extensive knowledge about the nutritional requirements of Pdd and Pdp strains, allowing them to be produced in a minimal medium with a defined composition and establish optimal conditions for the production of the proteins of interest.To test the functionality of the pVibexp platform as a vector for the expression of recombinant proteins, and to demonstrate that the insertion of a PCR-amplified gene of interest (GOI) at the NdeI site maintains the reading frame intact, allowing expression of the cloned gene, we performed the first test with an amplicon of the Pdd RM-71 cloning gene itself, without promoter or signal peptide. One of the advantages of cloning a phospholipase is that its phenotype is easily testable and monitored on plates supplemented with egg yolk. As shown in Table 1, the primers for cloning any gene of interest (GOI) have a universal sequence that includes the target sequence for the NdeI / NsiI restriction enzyme in the forward primer, and the sequence for the NdeI target in the reverse primer. The low cloning efficiency obtained in the first attempts at the NdeI site led to certain modifications in the primer design.These improvements consisted of the introduction of an 8-nucleotide staple at the 5' end of the NdeI recognition sequence. The incorporation of this non-palindromic sequence, whose in silico analysis did not reveal the formation of primer dimers, considerably improved the cutting and cloning efficiency of the Damselysin gene. For this reason, this modification was carried over to the primers designed for any other gene cloned in the NdeI position. Taking these universal sequences into account, specific primers were designed for the cloning of gendly on the pVibexp platform (Table 1). The initial screening for the selection of E. coli DH5^ transformants positive for the pVibexp+dly platform allowed us to visualize a high percentage of clones with a phospholipase halo on TSA-1-Cm20 plates supplemented with egg yolk (Figure 3B). It is worth noting that this halo of phospholipase activity began to be seen in E.coli from 72 h and in some clones even after 5 days of incubation. These results highlight one of the main disadvantages of using E. coli as a microbial factory for recombinant proteins: the limited capacity of its transport apparatus to secrete proteins into the extracellular medium. The same occurred when the platform was transformed into the conjugative E. coli strain β-3914, where the number of clones with phospholipase activity was even lower. The pVibexp+dly platform was then mobilized by conjugation to V. natriegens and various P. damselae strains. In this case, recipient strains selected were those into which the empty version of the pVibexp vector had been previously transferred and which were also negative or had low phospholipase activity.As shown in Figure 4A, the Pdd and Pdp transconjugants obtained for pVibexp+dly presented a high phospholipase activity compared to the transconjugant clones for the empty vector (pVibexp). PCR analysis with primers specific for the platform and for the dly gene confirmed the presence of the construct in all the clones analyzed (Figure 4). This result demonstrates that the pVibexp vector is a functional vector for the production and secretion of the Dly toxin in several P. damselae strains. Regarding the P. damselae subsp. piscicida transconjugants, a previous study has shown that the recipient strains AQV27.1 and AQP18.1 are PCR positive for the pPHDP10 plasmid markers (Abushattal et al., 2020).Considering that the origin of replication of the pVibexp plasmid comes from the pPHDP10 plasmid and considering the principle of plasmid incompatibility, it would be expected that by applying selective pressure with antibiotics the pVibexp platform would be maintained in the transconjugants, causing the cure of the pPHDP10 plasmid. However, all the transconjugants analyzed from both Pdp strains were positive for the aip56 gene, indicating the presence of the pPHDP10 plasmid (Figure 5A). It would then be expected that these clones would be negative for pVibexp, however, PCR analysis revealed that some clones were positive for the platform modules (Figure 5B and C), suggesting the possibility that both plasmids were coexisting in some clones. Considering that, MRB type replicons with minimal modifications in the oriV RNAI sequence could coexist (Le Roux et al., 2011), it would be possible that, by forcing antibiotic selection, and at the cost of not losing the plasmid pPHDP10, the oriV sequence of pVibexp was suffering from some point mutation, which allows the coexistence of both plasmids. However, apart from those clones in which the plasmid pVibexp and the plasmid pPHDP10 seemed to coexist, other transconjugant clones were negative for the amplification of the region corresponding to the oriV of pVibexp (Figure 5B), suggesting the presence of rearrangements in the plasmid structure or a possible integration. Curiously, despite these possible rearrangements, those Pdp clones positive for the pVibexp+dly construct showed a strong phospholipase activity, suggesting that the determining modules for the expression and secretion of Damselysin were intact, a result that was also confirmed by PCR. Example 1.3.Construction of the pA-162-Vibexp vector, a cloning vector based on a native MRB-like plasmid of Photobacterium damselae subsp. damselae. Simultaneous studies by Pan et al., (2010) and Le Roux et al., (2011), demonstrated the presence of MRB (Marine RNA-based) replicons in various members of the Vibrionaceae family. However, since these works, only one additional MRB plasmid has been recently described in a V. cholerae isolate (Ceccarelli et al., 2017) and to date, the presence of plasmids of this family has not been reported in any Pdd strain. To investigate the presence of MRB-type replicons in the Pdd genomes deposited in the NCBI database, a BlastN search was performed by entering the minimal sequence corresponding to the origin of replication of plasmid pPHDP10 of P. damselae subsp. piscicida as a query. This search revealed the presence of regions homologous to this oriV in two strains of P. damselae subsp.damselae: strain A-162 and strain TW250 / 03. The sequence identified in the genome of strain A-162 (2863 bp) (Contig_53;LZFN01000155.1) presented a region homologous to the minimal oriV of pPHDP10, as well as two ORFs for hypothetical proteins, which we called ORF1 (636 bp) and ORF2 (405 bp). To close this sequence and verify its circular nature, primers were designed at the ends of the contig (PC7 and PC8, Table 1) and the resulting PCR amplicon was analyzed by Sanger sequencing. The result obtained confirmed that the sequence identified in the genome of Pdd strain A-162 constitutes an MRB type plasmid. The sequence of this native plasmid, which was named pA-162, was used to design an expression vector.To do this, the complete sequence of pA-162 was amplified by inverse PCR with two pairs of primers designed in different intergenic positions of the plasmid: PC9 and PC10 (designed between ORF2 and the oriV region); PC11 and PC12 (between oriV and. la ORF1).The product resulting from the amplification with the second pair of primers was cloned into the Vibexp-CmR construct, previously released with NheI from the AVZ190 molecule (pUC57-Vibexp-CmR). This cloning gave rise to a new vector, which was called pA-162- Vibexp (pAVZ368), composed of several modules: an MRB type origin of replication, the two ORFs of unknown function encoded in the sequence of the native plasmid of P. damselae and the rest of the modules from the sequence corresponding to plasmid pVibexp (oriT, promoter and signal peptide dependent on the T2SS, terminator, polylinker and chloramphenicol resistance marker). Transformation of this vector into competent E. coli DH5α cells gave rise to numerous transforming clones that were confirmed by PCR (Figure 7). To avoid insertion sequence (IS) skipping, the pA-162-Vibexp platform was transformed into the conjugative E. coli strain β-3914.The results obtained from PCR amplification with specific primers demonstrate that the pA-162-Vibexp platform, whose origin of replication comes from the native Pdd plasmid pA-162, is replicative and selectable in E. coli. The pA-162-Vibexp vector was then mobilized by conjugation to different vibrios. Various Pdd strains (strains RM-71 and LD-07), a P. damselae subsp. piscicida strain (strain DS11, negative for pPHDP10; Abushatta et al., 2020) and the V. natriegens strain CCUG16374 were selected as recipient bacteria. The transconjugants selected in TSA-1-Cm5 were positive by PCR for the pA-162-Vibexp vector, giving amplicons of the expected size (Figure 7). The results obtained demonstrate that the oriV sequence of the native MRB plasmid of the Pdd A-162 strain is functional in Pdd and other vibrios.Although the minimal region of oriV that ensures replication is still unknown, the structure of the plasmid has allowed the design of a new vector that can be mobilized in P. damselae and V. natriegens, opening the way to its use as a new tool for gene expression in marine bacteria. Example 1.4. The vector pA-162-Vibexp allows the expression of native and heterologous genes in P. damselae subsp. damselae. To test the applicability of pA-162-Vibexp as a gene expression vector in Pdd, a native gene and a heterologous gene, both with their own promoter, were cloned into the MCS of the vector. The TbpD gene, mutated by deletion in the PddRM-71 strain, was selected as the native gene. The E. coli β-3914 transformant clones analyzed were positive by PCR for the pA-162-Vibexp+tbpD construct (Figure 8A and C). The pA-162-Vibexp+tbpD vector was then mobilized by conjugation to the tbpD mutant strain RM-71 clone (AVZ159).ΔtbpD transconjugants complemented with the pA-162-Vibexp+tbpD vector were confirmed by PCR with primers specific for the internal region of the gene (Mut_tbpD primers specific for the 5' and 3' construct, Figure 8B). Considering that TbpD is an iron-regulated outer membrane protein, the genetic complementation of the mutant was evaluated by analyzing the outer membrane proteins, under iron-limiting conditions, of one of the complemented clones obtained (AVZ410). This analysis showed that the band corresponding to the TbpD protein, absent in the tbpD mutant profile, was recovered in the complemented clone AVZ410, demonstrating that the pA-162-Vibexp vector is a functional expression vector in Pdd. This result demonstrates that the pA-162-Vibexp vector constructed in the present invention is a useful tool for genetic complementation studies in Pdd. Furthermore, since this plasmid vector is also functional in the subsp.piscicida, as well as in V. natriegens, the benefits offered by this platform could also be extended to other members of the Vibrionaceae family. The phospholipase gene from the marine bacterium Pseudoalteromonas citrea, homologous to the Dly phospholipase of Pdd and which we named Dly-like, was cloned as a heterologous gene. As with Damselysine, the system for tracing the production of this heterologous phospholipase is easily detectable on plates supplemented with egg yolk. Cloning the gene for the Dly-like phospholipase of P. citrea with its own promoter, in the MCS of the vector pA-162-Vibexp (Figure 4.40A), gave rise to E. coli clones with phospholipase activity, showing a quite satisfactory cloning efficiency. These clones were positive by PCR for the construct pA-162-V be pdy e. cot uat ón, the construct was mobilized by conjugation to the Pdd strain LD-07, negative for the plasmid pPHDD1 and which presents a low level of phospholipase activity.As shown in the figures of the present invention, the transconjugant LD-07 clones for the pA-162+dly-like platform showed high phospholipase activity, while the strain carrying the empty vector showed a narrow halo of phospholipid degradation. This result demonstrates that the heterologous phospholipase Dly-like cloned in the pA-162-Vibexp vector is expressed and secreted in Pdd. Interestingly, the Dly-like phospholipase-positive strain of P. citrea showed lower growth with respect to the strain carrying the empty vector. This observation suggests that the expression of this phospholipase could entail a metabolic cost capable of reducing the fitness of the LD-07 strain.Consistent with these observations, numerous studies have reported that the expression of certain plasmid genes can divert resources initially intended for bacterial replication, causing cell stress and reducing their ability to survive in different environments (Baltrus, 2013). Example 1.5. The pA-162-Vibexp vector: a genetic tool with potential for recombinant protein production in P. damselae subsp. damselaeBeyond their use as genetic tools in basic molecular research, expression vectors are essential in the biotechnology industry, and both their design and optimization constitute a fundamental challenge for the efficient production of recombinant proteins. As previously mentioned, the design of a plasmid vector that allows the production of recombinant proteins directly in P. damselae as a factory organism would offer several very interesting advantages.To this end, the native sequence corresponding to a strong promoter and a Pdd-dependent Sec signal peptide was included in the design of the pA-162-Vibexp vector. As a proof of concept, the heterologous Dly-like phospholipase gene from P. citrea was cloned (Figure 10A). For primer design, the cleavage site of the native protein signal peptide was predicted using the SignalP server (v. 6.0). The primer with the appropriate universal sequence (Table 1) was designed from the cleavage residue predicted by the server. Cloning of the dly-like gene under the control of the promoter and signal sequence of the pA-162-Vibexp vector resulted in 5 colonies that were PCR positive for the platform. However, E. coli colonies positive for the construct did not show phospholipase activity, even after 4 days of incubation. This absence of phospholipase activity could be related to the limited capacity of E.coli to secrete proteins into the extracellular space, so that the recombinant protein produced could be retained in the cytoplasm. Considering that Pdd has a high capacity to secrete proteins into the extracellular medium, the construct was mobilized into the Pdd strain, LD-07. The transfer of pA-162-Vibexp-rDly-like by conjugation gave rise to LD-07 clones with a phospholipase halo slightly larger than the control strain, which carried the empty plasmid. As observed in Figure 10B, the phospholipid degradation halo was much smaller than when this heterologous phospholipase was cloned with its own promoter in the MCS of the platform (Figure 9C). The reasons underlying this result could be diverse and are mostly supported by the difficulties involved in the production of recombinant proteins in microbial hosts (Overton, 2014).The main problems include those related to DNA instability, such as the loss of the expression plasmid or mRNA degradation; those related to protein folding, such as improper folding, the formation of inclusion bodies or proteolysis; and / or the difficulty in translocating the produced protein to the periplasm. While it has not been experimentally determined whether the absence of phospholipase activity is due to an instability of the expression plasmid or mRNA degradation, the most common problems reported in the literature refer to improper protein folding, the formation of inclusion bodies, and the difficulty in translocation to the external medium (Overton, 2013). To address these limitations, there are several strategies that are commonly used to improve protein production.These include signal peptide engineering, i.e., the de novo modification or synthesis of signal sequences specifically designed for the protein to be secreted, and strategies aimed at modifying culture conditions to optimize the production of recombinant proteins. These are just a few examples of the many strategies available, although the choice will always depend on the characteristics of the protein of interest and the host organism used for production (Freudl, 2018; Kleiner-Grote et al., 2018). Example 2. The pA-162-Vibexp vector is stable in the Pdd LD-07 strain and unstable in the RM-71 strain. One of the main disadvantages of plasmid vectors is that most of them require the addition of antibiotics to ensure their maintenance in bacterial cell lines.Although selectable markers are very powerful, their use is not always practical, as is the case in in vivo infection models or in vitro genetic complementation assays. To determine whether the pA-162-Vibexp vector was stable in the different host strains, passages (1:100) of the strains were grown in TSB-1 medium without antibiotic. To ensure that 100% of the initial cell population was positive for the vector, the initial inoculum was made from a single isolated colony grown in TSA-1-Cm5. At each passage, cells were diluted and plated on TSA-1 plates, and 100 colonies from each passage were spotted onto antibiotic-supplemented plates (TSA-1-Cm5). The results obtained show that while 100% of the colonies of the LD-07 strain retained the pA-162-Vibexp vector at first passage, only 4% of the colonies of the RM-71 strain retained it. Regarding the E.coli DH5α, as well as the DS11 strain, the plasmid remained stable in 100% of the analyzed colonies from the first passage. Considering that, of the host strains, only the RM-71 strain naturally carries another plasmid (plasmid pPHDD1), it could be hypothesized that some underlying molecular mechanism could be preventing the coexistence of both plasmids in this strain. This result supports the enormous stability of the pPHDD1 plasmid, whose curation attempts in the laboratory have always been unsuccessful. The instability of the pA-162-Vibexp vector in the Pdd strain RM-71, already in the first passage, must be taken into account and although it limits its practicality, it points to the need to always supplement the culture medium when performing experiments with this vector in this strain. In short, our results have led to the design of a vector that is stable in Pdd and Pdp, easily selectable, and mobilizable from routinely used E. coli strains.coli laboratory tests. As a first test of its functionality, the pA-162-Vibexp vector has successfully complemented the ΔtbpD mutant of Pdd. Furthermore, we have demonstrated that this vector is functional in P. damselae subsp. piscicida and in Vibrionatriegens. Of particular interest has also been the finding that the expression and secretion platform, based on the promoter and signal peptide of the damselysin gene, allows the effective secretion of a heterologous phospholipase from Pseudoalteromonas citrea in P. damselae strains. This finding would pave the way for the use of this platform for the production of complete proteins or small peptides in P. damselae and with the potential for its successful use in other genera of marine bacteria. With this study, we have contributed to the design of a new category of vectors for use in biotechnology, based on native MRB-type plasmids.

[0002] Table 1. Primers used to construct and sequence the expression vector platforms of the present invention. Example 3 For the efficient expression and secretion of a recombinant protein using the Vibrioexpress platform, object of the present invention, the use of the promoter and signal peptide of the Dly protein and the promoter and signal peptide of the PirA protein (also native to Pdd) is proposed. The scientific evidence that supports the use of the promoter and signal peptide of the Dly protein and the PirA protein for our Vibrioexpress proposal is presented below: 1- High level of expression For a recombinant protein to be produced efficiently and in large quantities, it is essential that the gene that encodes it is under the control of a strong promoter, which controls its expression. Both the promoter of the dly gene and the promoter of the pirA gene have a very high level of expression in Pdd under standard culture conditions (25 ºC and 1% NaCl).In a first study (Matanza & Osorio, 2018), which investigated the expression levels of Pdd genes at two incubation temperatures (15 ºC and 25 ºC, at 1% NaCl), it was shown that the dly gene is among the most expressed genes in the entire transcriptome, both at 15 ºC and 25 ºC, even at levels comparable to those of ribosomal protein genes (Figure 12A). Likewise, evidence derived from a subsequent study (Barca et al., 2023) shows that the pirA gene is among the 5 most expressed genes at 1% NaCl, showing an expression value even higher than that of the genes encoding ribosomal proteins (Figure 12B). This evidence supports the use of both sequences as strong promoters for the expression of recombinant proteins on the Vibrioexpress platform.2- Efficient secretion through the type 2 secretion system (T2SS)Several works carried out in our laboratory show that both the Dly protein (Rivas et al., 2015; Terceti et al., 2019) and the PirA protein (Terceti et al., 2019; Barca et al., 2023) are secreted very efficiently into the extracellular medium by a type 2 secretion system (T2SS). As shown in Figure 13, analysis of Pdd supernatants on SDS-PAGE gels shows that the Dly and PirA proteins, which correspond to bands 4 and 12, respectively, are the most abundant proteins in the entire Pdd secretome. The mutation in the epsL gene, which encodes a protein essential for translocation across the T2SS, results in the absence of most of the secreted proteins from Pdd, including the proteins Dly and PirA. This result confirms that both proteins are secreted into the extracellular medium via the T2SS.The data presented in the previous axes of this patent demonstrate that the use of the promoter and the signal peptide of the Dly protein allow the efficient expression and secretion of a heterologous phospholipase into the extracellular medium using the Vibrioexpress platform. However, the high levels of expression of the PirA protein in the Pdd secretome, at levels comparable to or even higher than the expression level of Dly (Figures 13 and 14B), also support its use as an additional option in our Vibrioexpress platform. 3- Modular expression level in response to changes in salinity One of the most practical ways to modulate the expression levels of a recombinant protein without resorting to the use of genetic engineering tools is to modify the expression conditions of its promoter. As shown in Figure 14A, both the dly gene and the pirA gene are highly upregulated in Pdd at 1% NaCl compared to 3% NaCl.More specifically, the dly and pirA genes are 11 and 84 times more induced at low salinity. In agreement with this result, the SDS-PAGE analysis of the Pdd supernatants obtained at 1 and 3% NaCl shows that the Dly and PirA proteins are much more abundant at low NaCl (Figure 13B, lanes 4 and 12, respectively). Taking into account this evidence, the use of the promoter and signal peptide of the dly gene on the one hand (already described in the initial proposal), and of pirA as an additional option, in the Vibroexpress platform would allow us to: (1) a strong expression of the genes of interest under its control, (2) an efficient secretion through the T2SS and finally (3) modulate the expression levels of the recombinant proteins by changes in the NaCl concentration in the culture medium. Sequences >PirA promoter (11Kda) GAGTCCTTGACGATTGAGCTTGTGATATCAGAAGTGGATTACTATAGCGATTTTATCATGAGAAATTAAGA. Referencias Abushattal, S., Vences, A., & Osorio, C. R. (2020). A virulence gene typing scheme for Photobacterium damselae subsp. piscicida, the causative agent of fish photobacteriosis, reveals a high prevalence of plasmid-encoded virulence factors and of type III secretion system genes. Aquaculture, 521. Abushattal, S., Vences, A., & Osorio, C. R. (2022). A highly unstable and elusive plasmid that encodes the Type III Secretion System is necessary for full virulence in the marine fish pathogen Photobacterium damselae subsp. piscicida. International Journal of MolecularSciences, 23(9). Abushattal, S., Vences, A., Dos Santos, N. M. S., do Vale, A., & Osorio, C. R. (2019). Draft genome sequences of Photobacterium damselae subsp. piscicida SNW-8.1 and PP3, twofish-isolated strains containing a Type III secretion system. Microbiology resourceannouncements, 8(21), e00426-19. Baltrus, D. A. (2013). Exploring the costs of horizontal gene transfer.Trends in Ecology and Evolution, 28(8), 489–495. Baseggio, L., Silayeva, O., Buller, N., Landos, M., Englestädter, J., & Barnes, A. C. (2021). Complete, closed and curated genome sequences of Photobacterium damselae subsp. piscicida isolates from Australia indicate mobilome-driven localized evolution and novel pathogenicity determinants. Microbial Genomics, 7(4), 1– 14.Ceccarelli, D., Garriss, G., Choi, S. Y., Hasan, N. A., Stepanauskas, R., Pop, M., Huq, A., & Colwell, R. R. (2017). Characterization of two cryptic plasmids isolated in Haiti from clinical Vibrio cholerae non-O1 / non-O139. Frontiers in Microbiology, 8, 1–9.Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6640–6645.Do Vale, A., Silva, M. T., Dos Santos, N. M. S., Nascimento, D. S., Reis-Rodrigues, P., Costa- Ramos, C., Ellis, A. E., & Azevedo, J. E.(2005). AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils. Molecular Microbiology, 58(4), 1025–1038.Freudl, R. (2018). Signal peptides for recombinant protein secretion in bacterial expression systems. Microbial Cell Factories, 17(1), 1– 10.Herrero, M., De Lorenzo, V., & Timmis, K. N. (1990). Transposon vector containing non- antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. Journal of bacteriology, 172(11), 6557-6567. Kleiner-Grote, G. R. M., Risse, J. M., & Friehs, K. (2018). Secretion of recombinant proteins from E. coli. Engineering in Life Sciences, 18(8), 532–550.Le Roux, F., Binesse, J., Saulnier, D., & Mazel, D. (2007). Construction of a Vibriosplendidus mutant lacking the metalloprotease gene vsm by use of a novelcounterselectable suicide vector.Applied and Environmental Microbiology, 73(3), 777–784. Le Roux, F., Davis, B. M., & Waldor, M. K. (2011). Conserved small RNAs govern replication and incompatibility of a diverse new plasmid family from marine bacteria. Nucleic Acids Research, 39(3), 1004–1013. Overton T. W. (2014). Recombinant protein production in bacterial hosts. Drug discovery today, 19(5), 590–601. Pan, L., Leung, P. C., & Gu, J. D. (2010). A new ColE1-like plasmid group revealed by comparative analysis of the replication proficient fragments of Vibrionaceae plasmids. Journal of Microbiology and Biotechnology, 20(8), 1163–1178.Rouches, M. V., Xu, Y., Cortes, L. B. G., & Lambert, G. (2022). A plasmid system with tunable copy number. Nature Communications, 13(1), 1–12.Silva-Rocha, R., Martínez-García, E., Calles, B., Chavarría, M., Arce- Rodríguez, A., De Las Heras,A., Páez-Espino, A. D., Durante- Rodríguez, G., Kim, J., Nikel, P. I., Platero, R., & DeLorenzo, V. (2013).The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Research, 41 (Database issue), D666. Sousa, A., Bourgard, C., Wahl, L. M., & Gordo, I. (2013). Rates of transposition in Escherichia coli. Biology Letters, 9(6), 2–5.Wang, R. F., & Kushner, S. R. (1991). Construction of versatile lowcopy- number vectors forcloning, sequencing and gene expression in Escherichia coli. Gene, 100, 195–199.Barca, A. V., Vences, A., Terceti, M. S., do Vale, A., & Osorio, C. R. (2023). Low salinity activates a virulence program in the generalist marine pathogen Photobacterium damselae subsp. damselae. mSystems, 8(3), e0125322. https: / / doi.org / 10.1128 / msystems.01253-22. Matanza, X. M., & Osorio, C. R. (2018). Transcriptome changes in response to temperature in the fish pathogen Photobacterium damselae subsp. damselae: Clues to understand the emergence of disease outbreaks at increased seawater temperatures.PloS one, 13(12), e0210118. https: / / doi.org / 10.1371 / journal.pone.0210118. Rivas, A. J., Vences, A., Husmann, M., Lemos, M. L., & Osorio, C. R. (2015). Photobacterium damselae subsp. damselae major virulence factors Dly, plasmid-encoded HlyA, and chromosome-encoded HlyA are secreted via the type II secretion system. Infection and immunity, 83(4), 1246–1256. https: / / doi.org / 10.1128 / IAI.02608-14. Terceti, M. S., Vences, A., Matanza, X. M., Barca, A. V., Noia, M., Lisboa, J., Dos Santos, N. M. S., do Vale, A., & Osorio, C. R. (2019). The RstAB System Impacts Virulence, Motility, Cell Morphology, Penicillin Tolerance and Production of Type II Secretion System-Dependent Factors in the Fish and Human Pathogen Photobacterium damselae subsp. damselae. Frontiers in microbiology, 10, 897. https: / / doi.org / 10.3389 / fmicb.2019.00897.

Claims

CLAIMS 1 . Vector de expresión o plásmido caracterizado porque comprende: a. Un origen de replicación OriV del tipo MRB de P. damselae subsp. piscicida (Pdp) y / o P. damselae subsp. damselae (Pdd); b. un origen de transferencia (oriT), preferiblemente del tipo RK2 / RP4; c. un sitio de inserción para insertar un gen de interés; d. un promotor unido funcionalmente a una secuencia, preferiblemente de ADN, that encodes a signal peptide and a sequence, preferably DNA, that encodes the gene of interest, where i) the sequence that encodes the signal peptide is that of the signal peptide of the damselisin (Dly) gene of Photobacterium damselae subsp. damselae, and the promoter is that of the damselisin (Dly) gene of P hotobacterium damselae subsp. Damselae, o donde ii) la secuencia que The signal peptide encodes the signal peptide of the PirA gene of Photobacterium damselae subsp. damselae, and the promoter is that of the PirA gene of Photobacterium d amselae subsp. damselae; y e. una región de terminación de la transcripción; and because said expression vector or plasmid is characterized by the absence of transposases genes and their inactivated versions (pseudogenes). 2 . Vector de expresión o plásmido caracterizado porque consiste en: a. Un origen de replicación OriV del tipo MRB de P. damselae subsp. piscicida (Pdp) y / o P. damselae subsp. damselae (Pdd); b. un origen de transferencia (oriT), preferiblemente del tipo RK2 / RP4; c. un sitio de inserción para insertar un gen de interés; d. un promotor unido funcionalmente a una secuencia, preferiblemente de ADN,that encodes a signal peptide and a sequence, preferably DNA, that encodes the gene of interest, where i) the sequence that encodes the signal peptide is that of the signal peptide of the damselisin (Dly) gene of Photobacterium damselae subsp. damselae, and the promoter is that of the damselisin (Dly) gene of P hotobacterium damselae subsp. Damselae, o donde ii) la secuencia que encodes the signal peptide of the PirA gene of Photobacterium d amselae subsp. damselae, y el promotor es el del gen PirA de Photobacterium damselae subsp. damselae; e. una región de terminación de la transcripción, y f. opcionalmente uno o más de cualquiera de los siguientes elementos selected from the list consisting of: positive selection genes such as antibiotic resistance genes and other molecules with antimicrobial activity; genes that solve auxotrophies; or metal resistance genes, negative selection genes (sacB, rpsL, ccdB), reporter genes (lacZ, luxCDABE, galK), toxin-antitoxin systems, restriction-modification systems, fluorescent protein genes (GFP, CFP, YFP, dsRed2, m Cherry) y genes de rutas catabólicas.

3. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 o 2, dondeThe sequence encoding the signal peptide is that of the signal peptide of the damselysin (Dly) gene of Photobacterium damselae subsp. damselae, and the promoter is that of the D gene. amselisina (Dly) de Photobacterium damselae subsp. Damselae.

4. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 o 2, donde The sequence encoding the signal peptide is that of the signal peptide of the PirA gene of P hotobacterium damselae subsp. damselae, y el promotor es el del gen PirA de Photobacterium damselae subsp. damselae.

5. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 4, donde the origin of replication of the MRB type consists of SEQ ID No 1 or a nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 8 5%, 90%, 95%, 98% o 99% de identidad de secuencia con la SEQ ID Nº 1 y que sea capable of acting as an origin of replication (initial sequence with that the plasmic DNA will be replicated in the recipient cell).

6. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 4, donde the MRB type origin of replication consists of SEQ ID No 3 or a nucleotide sequence having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID No. 3 and that is capable of acting as an origin of replication (initial sequence with which the plasmic DNA will be replicated in the recipient cell).

7. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 4, donde The origin of replication of the MRB type consists of any one of the sequences: SEQ ID No 1, 2, 3 or 6.

8. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 7, donde el origen de transferencia consiste en la SEQ ID NO 7 o en una secuencia nucleotídica having at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO: 7 and being capable of functioning as a conjugative transfer initiation point .

9. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 8, donde the promoter consists of SEQ ID NO 8 or a nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 8 and that is capable of promoting the transcription of the gene of interest to be expressed.

10. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 8, dondethe signal peptide consists of SEQ ID NO 9 or a nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO 9 and that codes for a protein that maintains the function of the protein encoded by SEQ ID NO 9.

11. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 10, donde la región de terminación de la transcripción consiste en la SEQ ID NO 10 o en any other nucleotide sequence that has at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the sequence SEQ ID NO: 10 and maintains the transcription termination function.

12. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 11, where the signal peptide consists of SEQ ID NO 9, where the promoter consists of SEQ ID NO 8 and where the transfer origin consists of SEQ ID NO 7.

13. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 12, where the expression vector or plasmid additionally comprises a multiple cloning site (MCS).

14. El vector de expresión o plásmido según cualquiera de las reivindicaciones 1 a 13,where the expression vector or plasmid additionally comprises a coding sequence of a histidine tail in the N or C terminal position to the gene of interest.

15. El vector de expresión o plásmido según la reivindicación 1 o 2, donde el vector de expression or plasmid consists of the nucleotide sequence SEQ ID NO 17 or any other nucleotide sequence that has at least 99% sequence identity with the sequence SEQ ID NO 17.

16. El vector de expresión o plásmido según la reivindicación 1 o 2, donde el vector de expresión o plásmido consiste en la secuencia nucleotídica SEQ ID NO 18 o cualquier another nucleotide sequence that has at least 99% sequence identity with the sequence SEQ ID NO.

18.

17. Cepa bacteriana perteneciente a Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida o V. natriegens (Vibrio natriegens) que comprende el plasmid or vector as defined in any of claims 1 to 16.

18. Método para producir una proteína recombinante que comprende introducir el vector expression according to any of claims 1 to 16 in a host cell, culturing the host cell under conditions suitable for expressing the protein and recovering the protein.

19. El método según la reivindicación 18, donde la célula huésped se selecciona de la lista que consiste en Photobacterium damselae subsp. damselae, P. damselae subsp. piscicida o V. natriegens.

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