WO2024018036A1 - Microalgae expressing biologically active products - Google Patents

Abstract

The present invention relates to a platform based on unicellular algae, grown in a controlled environment, which serve as a biofactory for the production of biologically active metabolites.

Description

MICRO ALGAE EXPRESSING BIOLOGICALLY ACTIVE PRODUCTS

Field of Invention

The present invention relates to a platform based on unicellular algae, grown in a controlled environment, which serve as a biofactory for the production of biologically active metabolites. In the present invention, an editing methodology has been developed in which the genomic structure is modified through a natural process attributable to a targeted genetic improvement methodology.

Background of the invention

Plant organisms are commonly used as biofactories for the production of molecules of biological or pharmaceutical interest. The term "biofactory" refers to unicellular organisms capable of producing bioactive molecules, recombinant or otherwise, for application in pharmaceutical, nutraceutical, cosmeceutical, and more generally in the industrial field. Such molecules can be of various kinds, particularly peptides and biofunctional proteins for human, animal and/or plant use such as enzymes, antigens or therapeutic proteins.

One of the advantages associated with the use of plant organisms over other microorganisms or animal cells lies in the fact that the resulting products can often be used without further purification steps. In this regard, efforts have been made over the past 20 years to develop methodologies for the production of bioactive molecules in edible plant organisms that can biosynthesize the biomolecule of interest and at the same time become the delivery medium for it, thus avoiding, in part or completely, the purification process.

Many higher plants lend themselves to this strategy leading to several advantages, but at the same time also to several disadvantages. For example, their low-cost growth and ability to synthesize bioactive molecules of interest in large quantities certainly constitute positive aspects, in contrast some post-translational modifications, which do not correspond to those performed by animal cells, could result in the production of heterologous proteins with low structural authenticity. Moreover, being mostly derived from the application of recombinant DNA methods and classifiedas GMOs, these plants must comply and respond, for their cultivation and eventual processing, to legislation regulating the use of Transgenic genotypes (Bosch & Schots, 2010; Expert Rev. Vaccines 9(8), 835-842 (2010)).

Microalgae are unicellular plant organisms belonging to both the prokaryotic and eukaryotic kingdoms and are from a biophysiological point of view full-fledged plants. Microalgae produce energy through the photosynthetic process, removing carbon dioxide from the atmosphere and releasing oxygen at the end of the process. Their special position in phylogenetic evolution makes them model organisms, as they share characteristics common to both bacteria and higher plants. The unicellular nature of microalgae, means that they must respond to environmental stresses quickly and efficiently (to ensure their survival), maintaining the genome in a highly reactive euchromatic state. Numerous studies, concerning microalgae, have highlighted how they are extremely suitable for the production of secondary metabolites that are extremely interesting in the nutraceutical and pharmaceutical fields, as these are able to perform specific functions and/or contribute specific metabolites [antioxidants, essential amino acids and polyunsaturated fatty acids (co3 and co6 and co9)]. Microalgae comprise a wide range of mainly aquatic, unicellular, and eukaryotic organisms (including green algae, diatoms, and brown algae) that engage in photosynthetic activity, and contain cytoplasmic organelles such as mitochondria and chloroplasts. The chromatinstructure of microalgae is distinct from other eukaryotic organisms; in fact, this is heavily coloredhighlighting a more compact nucleosomal structure and a close association of DNA with histone protein components. These differences, which are more present, in green microalgae, highlight a differential mechanism in the regulation of gene expression at the histone chromatin level. In addition, structural chromatin differences in microalgae may be explanatory regarding stable nuclear transgene expression that is, in fact, difficult and transient due to chromatin-mediated genesilencing itself (H. Cerutti, A.M. J., N.W. Gillham, J.E. Boynton, Epigenetic silencing of a foreigngene in nuclear transformants of Chlamydomonas, The Plant Cell 9:925-945 (1997)).

To date, several approaches for obtaining genetically modified organisms (GMOs) capable of producing biomolecules of interest are known and involve the use of biological and/or physical vectors, such as bacteria, plasmids, viruses, electroporation or biobalistic methods. These approaches can be used on unicellular algae to make genotypes for recombinant protein production (Gong et al. 2011). Several anti-apoptotic gene constructs derived from mammalian cells combined with fluorescent reporter genes were introduced within model algae to evaluate their expression. The results showed that expression was low and no fluorescence gene expression couldbe detected, confirming the difficulty in the expression of transgenes within the microalgal genome.

Scientific evidence that has emerged in recent years has highlighted, especially in bacteria, mechanisms developed in a completely natural way mechanisms that allow, in an autogenous formto modify the structure of hereditary material determining in fact a process of autogenous genomic editing that does not involve the use of vectors and allows these organisms in a completely autonomous way to modify their DNA with the intent to implement the fitness of the organism (unicellular). This new knowledge if managed in the right form by geneticists can determine in these organisms the possibility of guiding modifications to be made to the genome of these unicellular organisms, modification that follows and exploits a biophysiological potential innate in them.

Microalgae are an important source of healthy nutrients for human needs and are also further used for biomass and biofuel production. Genetic engineering and stable expression (maintained over multiple generations) of different transgenes would open up new horizons and greatly improve the value and opportunity of cultivating microalgae for multiple applications. However, as described earlier, it has been difficult to achieve a stable and sufficiently high level of gene expression, so a new methodology to help overcome this obstacle is extremely useful. Such an approach must take into account gene silencing related to the unique and resistant histone presence of microalgae, including green microalgae.

The present invention reports a totally innovative approach regarding the use of microalgae and specific growth and reproduction techniques to express and produce biologically active metabolites for use, in the form of freeze-dried biomass, as functional food for animal and humanuse.

Summary of the invention

The invention is based on the development of a novel strategy for the biosynthesis of active compounds by unicellular microalgae by implementation of the targeted horizontal gene transfer (THGT) conditions allowing introduction of specific oligos coding for peptides/proteins without using the state of the art transfection methodologies such as viral vectors, microinjection, Laserfection/Optical Transfection, Lipofection, Biolistic Methods or other kind of transfection methodologies know in the art. With this in mind, microalgae preferably belonging to the Chlorophyceae class have been selected for the production of bioactive molecules under controlled conditions that are available and usable for obtaining nutraceutical and/or pharmaceutical products. Trebouxiophyceae is a further class of green microalgae which can be used in the invention, in particular Chlorella spp.

Chlorophyceae are one of the classes of green microalgae, distinguished mainly on the basis of ultrastructural morphology. Usually, their coloration is caused by the predominance of photosynthetic pigments such as chlorophyll a and chlorophyll b. The chloroplast can have a discoidal, plate-shaped, reticulate, cup-shaped, spiral or ribbon-like conformation depending on the species to which it belongs. Most species belonging to this class, have one or more storage compartments called pyrenoids, located within the chloroplast. Pyrenoids, in turn, contend for various proteins including starch.

Some species of green microalgae can store nutrient sources in the form of oily droplets. They usually have a cell wall consisting of an inner layer of cellulose and an outer layer of pectose.

The present invention is directed toward the production of biofunctional peptides and proteins for human and animal use, preferably biologically active proteins or therapeutic proteins, within a host cell, represented by a microalgal cell such as microalgae belonging to the division Chlorophyta with particular attention to the class: Chlorophyceae and Trebouxiophyceae.

In the present invention, the host cell is used as a biofactory for protein production. In some embodiments of the invention, the recombinant Chlorophyceae host cell is Haematococcus pluvialis.

It therefore forms an object of the invention an host cell comprising an isolated nucleic acid molecule comprising or consisting of: a) a polynucleotide sequence encoding at least one peptide; and b) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21; wherein the polynucleotide linker sequence is linked to the 5’ end and/or the 3' end of the encoding polynucleotide sequence of a) and in which the encoding polynucleotide sequence is not SEQ ID No 116; and wherein said host cell is an algal cell, preferably said host cell belongs to the Chlorophyceae class.

Preferably in said host cell the isolated nucleic acid comprises or consists of: a) the polynucleotide sequence encoding at least one peptide; and b) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21, linked at the 5’ end of the polynucleotide sequence of a); and c) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21, linked at the 3’ end of the polynucleotide sequence of a).

Still preferably, in said host cell the isolated nucleic acid molecule comprises a coding polynucleotide sequence has at least 90% homology to any of SEQ ID No 22-47.

Still preferably, in said host cell the isolated nucleic acid molecule comprises a coding polynucleotide sequence has at least 90% homology to any of SEQ ID No 221-222.

In a preferred embodiments, the host cell belongs to the species Hematococcus pluvialis and/or wherein said host cell belongs to the class of Trebouxiophyceae; preferably said host 1) cell belongs to the species Haematococcus spp and/or Chlorella spp.; more preferably said host cell belongs to Haematococcus pluvialis and/or Chlorella vulgaris.

It is a further object of the invention an algal biomass comprising at least one host cell as defined above and/or a lysate and/or an extract of said host cell; preferably said algal biomass comprises at least one peptide encoded by the nucleic acid molecule as defined in any of claims 1 to 4.

It is a further object of the invention a method for obtaining the above host cell or the above algal biomass, said method comprising the following steps: a. induce thermal stress in a culture of microalgae, preferably belonging to the class of Chlorophyceae, by heating at a temperature between 35 and 50 ° C for a time between 300 and 600 seconds; b. expose the microalgae culture to UV rays (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c. inoculate the culture treated in step b in fresh liquid medium; d. suspend the microalgae culture from step c) in a solution composed of 16-50% (v / v) of 0.35 M mannitol and 0.2-0.6% of a mixture of lytic enzymes comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulose, chitinase, 0-D- glucanase, macerozyme, helicase, driselasi, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, mixtures of them; e. incubate the microalgae culture treated in step d) at a temperature between 30 - 40° C for 4-8 h; f. combine the culture incubated from step e) in 10 ml of culture medium, with 0.1 - 0.5% of a solution comprising the isolated nucleic acid previously defined, in the presence of 10-35 % polyethylene glycol (PEG-X).

It is a further object of the invention the above algal biomass according or an algal biomass obtainable from the above method for medical use, preferably for use in the treatment and/or prevention of infection caused by airborn viruses, more preferably for use in the treatment and/or prevention of a SARS COV2 infection.

Airborne viruses are most commonly transmitted through small respiratory droplets. These droplets are expelled when someone with the airborne disease sneezes, coughs, laughs, or otherwise exhales in some way. These infectious vehicles can travel along air currents, linger in the air, or cling to surfaces, where they are eventually inhaled by someone else. The list of airborn virsuses includes and not limited to SARS, MERS, Chickenpox, Respiratory Syncytial Virus (RSV), Influenza, Measles, swine-origin influenza. It is a further object of the invention a pharmaceutical composition comprising the above host cell or the above biomass or the algal biomass obtainable from the above method and at least one pharmacologically acceptable excipient.

It is a further object of the invention a supplement or food product or drinking product comprising the above host cell or the above biomass or the algal biomass obtainable from the above method.

It is a further object of the invention the non therapeutic use of the above algal or of the algal biomass obtainable from the above method or of the supplement or food product or product to drink comprising said algal biomass in the nutraceutical sector or as a basic ingredient in supplement preparations.

It is a further object of the invention the use of said algal biomass in preparations in the cosmetic sector and/or in the agricultural or vegetable sector.

It is a further object of the invention an isolated nucleic acid molecule comprising or consisting of: a) a polynucleotide sequence encoding peptides; and b) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21; wherein the polynucleotide linker sequence is linked to the 5’ end and/or the 3' end of the coding polynucleotide sequence of a) and in which the coding polynucleotide sequence is not SEQ ID No 116.

Preferably said isolated nucleic acid molecule comprises or consists of: a) a polynucleotide sequence encoding peptides and biofunctional proteins; and b) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21, linked at the 5’ end of the polynucleotide coding sequence of a); and c) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21, linked at the 3’ end of the polynucleotide coding sequence of a).

Still preferably the coding polynucleotide sequence a) has at least 90% sequence homology to any of SEQ ID No 22-47, or its functional derivatives.

The invention will be illustrated with reference to the following figures .

Figure 1. Schematic representation of the expression method according to an embodiment of the invention.

Figure 2. Cell viability reported as a function of the tested dose of algae, expressed as the absorbance value of formazan crystals that is directly proportional to cell viability. (A) 24 hours; (B) 48 hours; (C) Cell viability expressed as % relative to control (untreated cells = 0%) The results show a positive effect on cell viability at 24 and 48 hours after treatment with the GLP-2 expressing alga compared to the control alga. On HepG2, the effect is visible only at 24h.

Figure 3. PBMC treatment with microalgae for boosting immune activity

Figure 4. Monocytes after treatment with microalgae. In the graphs, 1^st bar corresponds to day 0 (DAYO), 2^nd bar corresponds to control - 48 h, 3^rd bar Haematoccus pluvialis P, 4^th bar Chlorella; A - intMo have a higher capacity to secrete cytokines in the blood and are reflective of an inflammatory environment; B - High CD 163 expression is associated to proinflammatory response; C - Pl/newMoCD14+++CD16+: Mo with anti-inflammatory potential, increased phagocytic activity and decreased antigen presentation

Figure 5. T-cells and NK-cells after microalgae treatment. D - Chlorella down-regulates CD3+ T cells; E - Chlorella upregulates mature NK cells. The effect of activity should be evaluated with respect to CTR 48h (2nd bar)

Description of the invention

The present invention is directed to a platform (biofactory) for the production of biofunctional peptides and proteins for human, animal and/or plant use, particularly peptides and therapeutic proteins of interest through targeted genome editing of unicellular algae. In the course of the present invention, a methodology has been developed that enables targeted genome editing in microalgae. The developed methodology allows for the introgression of exogenous genetic material, in a stable form, within the genome of the algae thus realizing an efficient and specializedbioreactor. The specialization lies in the fact that the methodology allows the creation of special algal lines that are characterized in relation to the gene and metabolite that they are able to biosynthesize in a specific and efficient form.

Microalgae suitable for the present invention include microalgae chosen from the group consisting Chlorophyta (green algae), Rhodophyta (red algae), Stramenopiles (heterokonts), Xanthophyceae (yellow-green algae), Glaucocystophyceae (glaucocystophytes), Chlorarachniophyceae (chlorarachniophytes), Euglenida (euglenids), Haptophyceae (coccolithophorids), Chrysophyceae (golden algae), Cryptophyta (cryptomonads), Dinophyceae (dinoflagellates), Haptophyceae (coccolithophorids), Bacillariophyta (diatoms), Eustigmatophyceae (eustigmatophytes), Raphidophyceae (raphidophytes), Scenedesmaceae, Phaeophyceae (brown algae). More particularly, said microalgae is chosen from the group consisting of Chlamydomonas, Chlorella, Dunaliella, Haematococcus, diatoms, Scenedesmaceae, Tetraselmis, Ostreococcus, Porphyridium, and Nannochloropsis.

As a non limitative example, in the present invention the algal lines belonging to the Chlorophiceae class are used, initially pretreated through the application of a genetic improvement scheme aimed at improving both biophy siological parameters and the ability to incorporate exogenous DNA fragments.

The different methodological approaches, so far applied to microalgal transformation, involve that a construct, which is nothing more than an artificially structured polynucleotide sequence capableof first being transcribed and consequently translated realizing a specific gene product, is inserted within biological vectors that are capable, by different mechanisms, of infecting microalgal cells. In this way, the biological vectors insert the construct within the microalgal cell, which is first incorporated within the genomic DNA and then transcribed and consequently generating the corresponding gene product by exploiting the transcription apparatus of the plant cell. As an alternative to the application of such vectors, it is possible to apply methodologies based on physical principles, which allow the insertion of the construct within the genome of microalgal cells without the aid of activity mediated by living organisms. These methodologies in no way involve the direct involvement of the alga for the insertion of the exogenous gene fragment. In essence, the alga does not actively participate with its specific biofunctional characteristics in the incorporation of the exogenous fragment and thus undergoes what is obtained via the two types of vectors.

The technology described here, on the other hand, constitutes an innovative and revolutionary methodology in that it involves and exploits biophysiological mechanisms inherent to the selected algal lines that enable them to incorporate gene constructs in an autonomous form. In essence, it is the selected algal lines themselves, through specific biomolecular mechanisms inherent in them, that incorporate the exogenous gene fragment. The same mechanism, in nature, would allow them to implement an evolutionary process resulting in improved fitness and survival.

In the biotechnological procedure that is the subject of the invention, microalgal species are subjected to particular changes in environmental parameters and consequent conditioning of specific biophysiological activities that make them suitable for incorporating the construct by biomolecular mechanisms innate in them. Then, in a targeted manner and with a specific procedure, normal biophysiological conditions are restored so that the algal cells resume their normal life cycle. At this point the algae having incorporated the exogenous gene fragment through their natural life functions transcribe and translate the information contained therein becoming in fact specialized biofactories (specialized in that each algal line after incorporation of a specific construct becomes a specific biofactory). In this way, the biotechnological process that is the subject of the invention reproduces in a controlled manner what would happen in nature in a spontaneous manner, effectively allowing a modified cell to be obtained. The same biomolecular mechanism exploited in the biotechnological process that is the subject of the invention allows microalgae to create new variability and consequently adapt to changes in their habitat.

In essence, this mechanism allows the microalgae to incorporate exogenous gene fragments into their genome that could bring them a possible selective advantage from a survival perspective in the short term, consequently determining an evolutionaryprocess in the medium to long term. Further advantage of this methodology lies in the fact that it allows obtaining genomic structures that are structurally and functionally stable over time since the microalga, once it incorporates the gene construct, is able to pass it on to future generations, making it an integral part of its genome. The elaborated protocol is simple to execute, inexpensive, and applicable to all microalgae belonging to the class Chlorophiceae and especially to some genera such as, by way of example only, Chlorella, Haematococcus, and Chlamidomonas for which supercompetent genotypes particularly responsive to treatment are available. It should also be pointed out that the gene construct designed for use in the expression platform of the invention requires the use of specific linker sequences at the ends of the coding sequence for the exogenous protein of interest and said specific linker sequences are derived from the algal genome of interest, and have been identified and selected within said algal genome for the development of the platform of the invention.

For this innovative process, effective but not limiting growth conditions for the host cell include (i) efficient growth media, (ii) bioreactor temperature, (iii) pH, and (iv) oxygenation. Efficient growth medium is defined as any culture medium in which a microalgal cell according to the invention, preferably a Chlorophyceae cell, is usually grown. Such medium typically includes an aqueous phase containing assimilable sources of carbon, nitrogen andphosphate, as well as mineral salts, metals and other appropriate nutrients such as, for example, vitamins. Non-limiting examples of suitable media and growth conditions are described in the"Examples" section. Cells of the present invention can be cultured in conventional fermentationphotobioreactors, shaking flasks, test tubes, microtiter plates, and Petri dishes. The culture can becarried out at temperature, pH and oxygen content appropriate for the recombinant cell. According to the present invention, the term "transformation" identifies any methodology bywhich an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can beinserted within microbial cells. Within microbial cells, the term "transformation" is used primarilyto describe a genetic, heritable change caused by the acquisition of exogenous nucleic acids by the microorganism, and is essentially synonymous with the term "transfection."

Several methodologies suitable for the introduction of exogenous nucleic acid molecules inside algal host cells are known, such as (i) shotgun with gold particles, (ii) electroporation, (iii) micro injection, (iv) lipofection, (v) adsorption, (vi) infection, and (vii) protoplastic fusion.

In the present invention, a methodology for transforming a competent algal host cell has been developed that involves two main steps: a) pre-stratifying the cell with physical stressors, b) treating the host cell with an enzyme or mixture of enzymes, and c) introducing an exogenous nucleic acid molecule into the host cell. According to the present invention, the enzyme can havecellulase, protease, P-glucoronase and various combinations of these activities.

The expression system of the invention, which allows a protein of interest to be expressed within the algal cell, includes regulatory control elements that are active in microalgal cells.

Many of these elements, including several promoters, are active in different species; therefore, the novel regulatory or linker sequences, described as aspects of the invention, can be used not only in the algal cells described here, but also in cells belonging to different species. The design and construction of the expression systems covered by the invention use standard biomolecular technologies known to persons skilled in the art. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd edition.

The expression system or expression vector comprises a polynucleotide sequence that codes for a protein of interest, for example, a SARS-COV2 virus protein, such as the spike protein or a peptide derived from it, or the GLP-2 protein hormone, or PYY protein hormone, or Xenin-25 peptide associated with any promoter sequence, or 5' linker, and possibly a terminator sequence, or 3' linker. The 5' linker, coding sequence and 3' linker are operatively linked in such a way as to be functional within the host cell. The expression system may also include additional regulatory sequences that are functional within the genome of the hostcell. Inducible or constitutively active sequences can be used. Suitable control elements, also include any regulatory element associated with the expression of the nucleic acid molecules described here.

The present invention is also directed to the algal host cell comprising the expression system described above and/or to a biomass obtained from or comprising said algal cell.

A biomass according to the present invention is a composition comprising transformed algal cells and/or their secretions and/or extracts and/or lysates or other derivatives.

The expression system of the invention preferably comprises at least one of the nucleic acid molecules isolated in the present invention and described herein. In addition, all genetic elements of the expression system are sequences associated with previously isolated nucleic acid molecules. In some embodiment protocols, the nucleic acid sequence encoding for the protein of interest, or coding sequence, is stably integrated into the genome of the host cell, while in others, said coding sequence is operatively linked to a promoter linker sequence and/or a teminator linker sequence, both of which are functional in the host cell. The linker sequences to which the coding sequence is operatively linked include, but are not limited to, the novel nucleic acid sequences described inthe present invention. In some embodiments, moreover, the coding sequence is optimized for the codon belonging specifically to the host cell of Haematococcus pluvialis so as to maximize translation efficiency.

In some embodiments, the proteins of interest produced by a recombinant host cell that are the subject of the invention include, but are not limited to, peptides and biofunctional proteins for human, animal, and/or plant use, also referred to herein as therapeutic proteins. A biofunctional peptide or protein for human, animal, and/or plant use, as used herein, includes proteins useful for the treatment or prevention of diseases, pathological conditions, and various disorders in both animals and humans and the plant kingdom. The terms "cure" and "treatment" refer to both therapeutic treatment and prophylactic or preventive measures in which the objective is to preventer slow down (reduce) an unwanted pathophysiological condition, disease, or disorder, or to achieve beneficial or desired clinical results. For the purposes of the present invention, beneficialor desired clinical results include, but are not limited to, the alleviation of symptoms or signs associated with a pathological condition or disorder of normal physiology; diminution in the magnitude of a condition, disease, or disorder; stabilization of a condition, disease, or disorder (orbetter, situations in which the condition, disease, or disorder is stable and does not worsen over time) delay in the onset or progression of the condition, disease, or disorder; improvement of the condition, disease, or disorder; remission (total or partial and detectable or undetectable) of the condition, disease, or disorder; or enhancement or improvement of a condition, disease, or disorder. Treatment includes eliciting a clinically meaningful response without excessive side effects and also prolonging survival over expected survival if treatment is not received. In some forms of realization, therapeutic proteins include, but are not limited to, biologically active proteins, such as enzymes, antibodies, or antigenic proteins, while in other forms of realization, therapeutic proteins include, but are not limited to: a viral protein such as a coronavirus Spike protein, PYY, Xenin-25, a human vaccine, an animal vaccine, and a veterinary drug.

The GLP-2 peptide is a 33 -amino acid peptide, having the following sequence: HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID No. 192).

Peptides derived from the SARS C0V2 Spike protein (or functional fragments of the Spike protein) used for the purposes of the present invention are as follows:

COV E ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK (35 aminoacids) (SEQ ID No. 193)

C0V 1.1: ATRFASVYAWNRKRISNCVADYSVLYNSASF (31 aminoacids) (SEQ ID No. 194) COV-1.2: ASVYAWNRKRISNCVADYSVLYNSASFSTFK (31 aminoacids) (SEQ ID No. 195) COV 2:

YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKL (57 aminoacids) (SEQ ID No. 196)

COV 2.1 : ADYSVLYNSASFSTFKCYGVSPTKLNDLCFT (31 aminoacids) (SEQ ID No. 197) COV 2.2: YSVLYNSASFSTFKCYGVSPTKLNDLCFTNV (31 aminoacids) (SEQ ID No. 198) COV 2.3: YNSASFSTFKCYGVSPTKLNDLCFTNVYADS (31 aminoacids) (SEQ ID No. 199) COV 2.4: SFVIRGDEVRQIAPGQTGKIADYNYKLPDDF (31 aminoacids) (SEQ ID No. 200) COV 2.5: VIRGDEVRQIAPGQTGKIADYNYKLPDDFTQ (31 aminoacids) (SEQ ID No. 201) COV 3: GYQPYRVWLSFELLHAPATVCGPKKSTNLVKNK (34 aminoacids) (SEQ ID No. 202)

COV 3.1 : PYRVWLSFELLHAPATVCGPKKSTNLVKNK (31 aminoacids) (SEQ ID No. 203) COV 3.2: WLSFELLHAPATVCGPKKSTNLVKNKCVNF (31 aminoacids) (SEQ ID No. 204) COV 3.3: SFELLHAPATVCGPKKSTNLVKNKCVNFNFN (31 aminoacids) (SEQ ID No. 205) COV 3.4: ELLHAPATVCGPKKSTNLVKNKCVNFNFNG (30 aminoacids) (SEQ ID No. 206) COV 3.5: GPKKSTNLVKNKCVNFNFNGLTGTGVLTES (30 aminoacids) (SEQ ID No. 207) COV 4: TSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKL (37 aminoacids) (SEQ ID No. 208)

COV 4.1: TSGWTFGAGAALQIPFAMQMAYRFNG (26 aminoacids) (SEQ ID No. 209)

COV 4.2: TSGWTFGAGAALQIPFAMQMAYRFNGIGVTQ (31 aminoacids) (SEQ ID No. 210) COV 4.3: FGAGAALQIPFAMQMAYRFNGIGVTQNVL (29 aminoacids) (SEQ ID No. 211) COV 5: NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI (35 aminoacids) (SEQ ID No. 212)

COV 5.1 : NTVYDPLQPELDSFKEELDKYFK (23 aminoacids) (SEQ ID No. 213)

COV 5.2: LDKYFKNHTSPDVDLGDISGI (21 aminoacids) (SEQ ID No. 214)

COV 6: ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIML (40 aminoacids) (SEQ ID No. 215)

COV 6.1: ESLIDLQELGKYEQYIKWPWYIWLGFIAG (29 aminoacids) (SEQ ID No. 216) COV 6.2: EQYIKWPWYIWLGFIAGLIAIVMVTIML (28 aminoacids) (SEQ ID No. 217) C0V_7: GSCCKFDEDDSEPVLKGVKLHYT (22 aminoacids) (SEQ ID No. 218)

Peptide YY (PYY) also known as peptide tyrosine tyrosine is a peptide that in humans is encoded by the PYY gene. [5] Peptide YY is a short (36-amino acid) peptide released from cells in the ileum and colon in response to feeding. In the blood, gut, and other elements of periphery, PYY acts to reduce appetite; similarly, when injected directly into the central nervous system, PYY is also anorexigenic, i.e., it reduces appetite.

The PYY(peptide tyrosine tyrosine) peptide is a 33 -amino acid peptide, having the following sequence:

PYY: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (36 aminoacids) (SEQ ID No. 219) Xenin-25 is a 25-amino acid peptide hormone co-secreted from the same enteroendocrine K-cell as the incretin peptide glucose-dependent insulinotropic polypeptide. Xenin-25 has been demonstrated in pancreatic islets and recently shown to possess actions in relation to the regulation of insulin and glucagon secretion, as well as promoting beta-cell survival.

The Xenin-25 peptide is a 25-amino acid peptide, having the following sequence: MLTKFETKSARVKGLSFHPKRPWIL (25 aminoacids) (SEQ ID No. 220)

In some forms of embodiment, proteins produced by a recombinant host cell included in the invention include, but are not limited to, industrial enzymes. Industrial enzymes include, but are not limited to, enzymes used in the production, preparation, storage, nutrient mobilization or processing of products, including food, medical, chemical, mechanical and other industrial products.

Industrial enzymes include, but are not limited to: alpha-amylase, alpha-galactosidase, betaamylase, cellulase, beta-glucanase, dextrin dextranase, glucoamylase, hemicellulase/pentosanase, xylanase, invertase, lactase, naringinase, pectinase, pullulase, acid proteinase, alkaline protease, bromelain, papain, pepsin, aminopeptidase, endo-peptidase (trypsin, chemotrypsin, pepsin, elastase), renin/reninachemosin, sibtilism, thermolysin, aminoacylase, glutaminase, lysozyme, penicillin acylase, triglyceridase, phospholipase, pregastric esterase, phytase, amidase, isomerase, alcohol hydrogenase, amino acid oxidase, catalase, chloroperoxidase, peroxidase, acetolactate decarboxylase, cyclodextrin glycosyltransferase, phytase and chymosin. In some forms of embodiment, the proteins produced by a recombinant host cell included in the invention include an auxotrophic marker, a dominant selection marker (such as, for example, an enzyme that degrades antibiotic resistance) or another protein involved in transformation selection, a reporter protein, an enzyme involved in protein glycosylation, and an enzyme involved in cellular metabolism.

Protein produced or expressed by the algae cell according to the invention can be produced on a commercial scale. Commercial scale includes protein production from a microorganism grown in an aerated biofermentor of size > 100 L, > 1,000 L, > 10,000 L, or > 100,000 L. In some forms of implementation, commercial-scale production is performed in an aerated biofermentor with agitation. The protein produced by the algae cell can also accumulate within the cell or can be secreted by the cell, for example, into the culture medium as a soluble protein. The protein produced can be recovered from the cell, the culture medium, or the fermentation medium in which the cell itself is grown. The same biomass expressing the GLP-2 protein or PYY protein or Xenin-25 or the SARS COV2 spike protein, or peptides derived from it, can be used directly for the purposes of the invention, which include the therapeutic and preventive uses of a pharmaceutical product, composition or kit, and non therapeutic uses, such as in the preparation of a nutraceutical product. In addition, the present invention is directed to a method for producing a recombinant protein; the methodology also includes culture conditions for the microalgal cells of the invention such that a polynucleotide sequence coding for a protein can be expressed.

Proteins produced by the method elaborated in this invention can also be purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed phase chromatography, chromatography using concanavallin A, chromatofocusing, and differential solubilization. In some embodiments, proteins produced by the method described in the present invention are isolated in "substantially pure" form. In this context, "substantially pure" refers to a purity that allows effective use of the protein as a commercial product. In some embodiments, the recombinant protein accumulates within the cell and is recovered from the cell; in some embodiments, the host cell of the method belongs to the Chlorophyceae class, while in other embodiments, the host cell is from Haematococcus pluvialis. In some embodiments, the recombinant protein is a therapeutic protein, a food enzyme or an industrial enzyme. In some embodiments, the recombinant protein is the spike protein of SARS- COV2 or a peptide derived from it. In some embodiments, the recombinant protein is the PYY protein or a peptide derived from it (a functional derivative). In some embodiments, the recombinant protein is the Xenin-25 peptide or a peptide derived from it (a functional derivative). In some realizations, the recombinant protein is a therapeutic protein that includes a secretion signal sequence. According to the invention, a “peptide derived from” or a “functional derivative” is an elongated version of the referred peptide comprising one or more aminoacids at the C and/or N- terminal and maintaining the same biological properties.

Nucleic acids Isolated nucleic acid molecules or polynucleotide sequences that constitute the expression system in the algal cell form the object of the invention. The nucleic acid sequences described here include the 5' and 3' linker sequences and coding sequences. The GLP-2 coding sequence and sequences encoding functional fragments of the SARS-COV2 spike protein are shown as examples. Other examples are the coding sequences of the PYY peptide and of the Xenin-25 peptide.

An isolated nucleic acid molecule can be a DNA molecule, RNA molecule (e.g., mRNA) or derivatives of them (e.g., cDNA). Although the phrase "nucleic acid molecule" refers primarily to the physical nucleic acid molecule, and although the phrases "nucleic acid sequence" or "polynucleotide sequence" refer primarily to the sequence of nucleotides present on the nucleic acid molecule, the phrases are used interchangeably, especially in reference to a nucleic acid molecule, polynucleotide sequence, or nucleic acid sequence encoding a protein. In some embodiments, a nucleic acid molecule isolated by the present invention is produced using recombinant DNA technology (such as, for example, cloning and amplification by polymerase in reaction (PCR)) or by chemical synthesis. Isolated nucleic acid molecules include naturally occurring nucleic acid molecules and their homologs, including, but not limited to, naturally occurring allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or reversed in such a way that these modifications provide the desired effect on the sequence, function, and/or biological activity of the encoded peptide or protein.

A double-stranded DNA present in this invention, includes a single-stranded DNA and its complementary strand, the sequence of which mirrors the sequence of the single-stranded DNA. As such, nucleic acid molecules of the present invention, may be double-stranded or single- stranded and also include those nucleic acid molecules that form stable hybrids under high "stringency" conditions with a sequence of the invention and/or with a sequence complementary to a sequence of the invention. Methods for tracing a complementary sequence are known to experts in the field. The term "protein" includes single-chain polypeptide molecules as well as multiple polypeptide complexes in which the individual constituent polypeptides are bound through covalent and noncovalent means. The term "polypeptide" includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.

The new nucleic acid molecules of the present invention can be used in any genus of microalgae in which they are found to be functional. In some embodiment the nucleic acid molecules of the invention are used in algae including Chlorophyta (green algae), Rhodophyta (red algae), Stramenopiles (heterokonts), Xanthophyceae (yellow-green algae), Glaucocystophyceae (glaucocystophytes), Chlorarachniophyceae (chlorarachniophytes), Euglenida (euglenids), Haptophyceae (coccolithophorids), Chrysophyceae (golden algae), Cryptophyta (cryptomonads), Dinophyceae (dinoflagellates), Haptophyceae (coccolithophorids), Bacillariophyta (diatoms), Eustigmatophyceae (eustigmatophytes), Raphidophyceae (raphidophytes), Scenedesmaceae, Phaeophyceae (brown algae), more particularly including Chlamydomonas, Chlorella, Dunaliella, Haematococcus, diatoms, Scenedesmaceae, Tetraselmis, Ostreococcus, Porphyridium, and Nannochloropsis. In some specific embodiments, the nucleic acid molecules are used in algae belonging to the class Chlorophyceae. In some preferred embodiment, recombinant nucleic acid molecules are used in the species Haematococccus pluvialis. As used in this invention^ recombinant microorganism has a genome that has been modified (i.e., mutated or changed) from its natural (i.e., naturally occurring or wild-type) form using recombinant technology. A recombinant microorganism according to the present invention, may include a microorganism in which nucleic acid molecules have been inserted, deleted, or modified (i.e., mutated, e.g., by nucleotide insertion, deletion, substitution, and/or inversion) such that the modifications provide the desired effect within the microorganism.

Linker promoters and terminals

The present invention is directed to the 5' and 3' linker sequences. The 5' or promoter linker is a DNA sequence that directs transcription of a coding region associated with it. The 3' or terminal linker, on the other hand, is the gene sequence that marks the end of transcription of genomic DNA. The linker of the invention (promoter or terminal) is any of the following sequences: CGGGGCAACTCAAGAAATTC (SEQ ID No 1) GTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 2) ACTGCACATCGCTGCAGTCT (SEQ ID No 3) CGCGTCGGGGCCTGCCTAAG (SEQ ID No 4) TTACCTGCCACACAAGCCTG (SEQ ID No 5) CGTGCTACTGGGGTCTGGCAG (SEQ ID No 6) CACATGCCATCCGAGTCGTC (SEQ ID No 7) CACAACCATACTGGCGAAGT (SEQ ID No 8) ATGGCCACGC (SEQ ID No 9) CTCTACCCAC (SEQ ID No 10)

CCGGACTGCCATAGCACAGCTAGACGA (SEQ ID No 11) GTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 12) ACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 13) ATTTGCTGCATGACTGGATCAATGCGACGA (SEQ ID No 14) GTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 15) ATGCCCTGATCCCAATGATGGACGA (SEQ ID No 16) GTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 17) GAGCGTGCTGAAATGCATGCGACGA (SEQ ID No 18) GTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 19) CCCGGGTATAGTAGCTGACTGCGACGA (SEQ ID No 20) GTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 21)

It therefore forms an object of the invention to have an isolated nucleic acid molecule comprising a polynucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto any one of SEQ ID NO: 1 - SEQ ID NO:21, in which the polynucleotide sequence functions as a promoter and/or terminal linker at least in algae belonging to the class Chlorophyceae.

The invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence that hybridizes with any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences or that hybridizes with a polynucleotide sequence that is at least 95 percent identical to any of the SEQ ID NO:1 - SEQ ID NO:21 sequences. The isolated nucleic acid molecule may include a polynucleotide sequence that is completely complementary to any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences or to a polynucleotide sequence that is at least 95% identical to any of the SEQ ID NO:1 - SEQ ID NO:21 sequences.

MATERIALS AND METHODS

Cell cultures

Microalgae, belonging to the genus Chlorophyceae, were grown in the culture medium shown in Table 1, under illumination intensity of 120 mmol photons m s^-2-1 in an alternating cycle of 16 h of light and 8 h of dark, at a temperature of 25 °C. The cultures were agitated by mechanical shaker(g24 environmental incubator shaker, American Laboratory Trading) at 70 rpm throughout the growth time.

Table 1 : composition of the growth medium

The percentages shown in the table may vary depending on the species, genotype and initial concentration of microalgae cultures.

Microalgal pretreatment

Several species of microalgae, belonging to the class Chlorophyceae were subjected to stress pretreatment with the aim of making the algae optimized and responsive to the treatment. The selection process, applicable to different microalgae species, involves: thermal stress induction in a temperature range of 35-50° C for a time interval of 600 to 300 seconds;

Exposure of cultures to physical mutagenic (UV) agents. The treatment involved the application of different types of UV (UV-A, UV-B and UV-C) for different time intervals(5/10/15 minutes);

Inoculation into the fresh liquid medium to restore normal biophysiological conditions;

Cultures were characterized according to the following parameters: growth capacity, cell size, adaptive capacity, and ability to incorporate exogenous fragments.

Polynucleotide design

Through bioinformatics research, some microalgae-specific genes related to the photosynthesis pathway were analyzed. The following sequences were derived from specific algal sequences:

CGGGGCAACTCAAGAAATTC (SEQ ID No 1)

GTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 2)

ACTGCACATCGCTGCAGTCT (SEQ ID No 3)

CGCGTCGGGGCCTGCCTAAG (SEQ ID No 4)

TTACCTGCCACACAAGCCTG (SEQ ID No 5)

CGTGCTACTGGGGTCTGGCAG (SEQ ID No 6)

CACATGCCATCCGAGTCGTC (SEQ ID No 7)

CACAACCATACTGGCGAAGT (SEQ ID No 8) ATGGCCACGC (SEQ ID No 9)

CTCTACCCAC (SEQ ID No 10)

CCGGACTGCCATAGCACAGCTAGACGA (SEQ ID No 11)

GTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 12)

ACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 13)

ATTTGCTGCATGACTGGATCAATGCGACGA (SEQ ID No 14)

GTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 15)

ATGCCCTGATCCCAATGATGGACGA (SEQ ID No 16)

GTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 17)

GAGCGTGCTGAAATGCATGCGACGA (SEQ ID No 18)

GTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 19)

CCCGGGTATAGTAGCTGACTGCGACGA (SEQ ID No 20) GTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 21)

The coding sequences for the Spike fragments of SARS-CoV- 2 are as follows:

COV E

GCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATT

ATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAG (SEQ. ID No 22)

COV l.l:

GCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATT

ATTCTGTCCTATATAATTCCGCATCATTT (SEQ. ID No 23)

COV 1.2:

GCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTAT

ATAATTCCGCATCATTTTCCACTTTTAAG (SEQ. ID No 24)

COV 2:

TATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCT

CTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCT

CCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTA (SEQ. ID No 25)

COV 2.1:

GCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC

TACTAAATTAAATGATCTCTGCTTTACT (SEQ. ID No 26) COV 2.2:

TATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAA

ATTAAATGATCTCTGCTTTACTAATGTC (SEQ. ID No 27)

COV 2.3:

CTATGCAGATTCATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTA

AATTAAATGATCTCTGCTTTACTAATGT (SEQ. ID No 28)

COV 2.4:

TCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCT

GATTATAATTATAAATTACCAGATGATTTT (SEQ. ID No 29)

COV 2.5:

GTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTAT

AATTATAAATTACCAGATGATTTTACAAGG (SEQ. ID No 30)

COV 3:

GGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTT

GTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAA (SEQ. ID No 31)

COV 3.1:

CCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTA

AAAAGTCTACTAATTTGGTTAAAAACAAA (SEQ. ID No 32)

COV 3.2:

GTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTA

ATTTGGTTAAAAACAAATGTGTCAATTTC (SEQ. ID No 33)

COV 3.3:

TCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTA

AAAACAAATGTGTCAATTTCAACTTCAAT (SEQ. ID No 34)

COV 3.4:

GAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACA

AATGTGTCAATTTCAACTTCAATGGT (SEQ. ID No 35) COV 3.5:

GGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAA

CAGGCACAGGTGTTCTTACTGAGTCT (SEQ. ID No 36)

COV 4:

GGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGT

TTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAATTG (SEQ. ID No 37)

COV 4.1:

ACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTT

ATAGGTTTAATGGT (SEQ. ID No 38)

COV 4.2:

ACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTT

ATAGGTTTAATGGTATTGGAGTTACACAG (SEQ. ID No 39)

COV 4.3:

TTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTA

TTGGAGTTACACAGAATGTTCTC (SEQ. ID No 40)

COV 5:

AACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATT

TTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATC (SEQ. ID No 41)

COV 5.1:

AACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATT

TTAAG (SEQ. ID No 42)

COV 5.2:

TTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATT

(SEQ. ID No 43)

COV 6:

GAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTAC

ATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTT (SEQ. ID No 44) C0V 6.1:

GAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCCATGGTAC

ATTTGGCTAGGT (SEQ. ID No 45)

COV 6.2:

GAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAG

TAATGGTGACAATTATGCTT (SEQ. ID No 46)

COV 7:

TCCTCCTCCAAATTTGATGAAGACGACTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTAC

ACA (SEQ. ID No 47)

The following coding oligonucleotides were then synthesized, comprising the above sequences (SEQ ID 22-47) and a 5' linker and/or a 3' linker of sequence between SEQ ID sequences No 1-21 (underlined in the list below):

ACTGACTGCCATAGCACAGCTAGACGAGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGG AAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTT TAAGGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 48)

CGGGGCAACTCAAGAAATTCGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAA TCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGGTC TGGCCGAGGTCTGGTTCCTGTGCC (SEO ID No 49)

GAGCGTGCTGAAATGCATGCGACGAGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAA GAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTA AG (SEQ ID No 50)

ACTGACTGCCATAGCACAGCTAGACGAGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGG AAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTGTCTGGC CGAGGTCTGGTTCCTGCCTAG (SEO ID No 51)

TTACCTGCCACACAAGCCTGGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAA TCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTT (SEQ ID No 52)

GCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATT ATTCTGTCCTATATAATTCCGCATCATTTGTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 53)

ATTTGCTGCATGACTGGATCAATGCGACGAGCCACCAGATTTGCATCTGTTTATGCTTGGAACA GGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTACTGC ACATCGCTGCAGTCT (SEQ ID No 54)

ACTGACTGCCATAGCACAGCTAGACGAGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGC AACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGGTCTGGCC GAGGTCTGGTTCCTGCCTAG (SEO ID No 55) ACTGCACATCGCTGCAGTCTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTG TTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAG (SEQ ID No 56)

GCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTAT ATAATTCCGCATCATTTTCCACTTTTAAGTTACCTGCCACACAAGCCTG (SEO ID No 57)

ACTGACTGCCATAGCACAGCTAGACGATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATG GAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATT AGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTAT

AAATTAGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEP ID No 58)

ACTGACTGCCATAGCACAGCTAGACGATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATG GAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATT AGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTAT

AAATTA (SEQ ID No 59)

CACAACCATACTGGCGAAGTTATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTC TCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTG ATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTAT

TACCTGCCACACAAGCCTG (SEQ ID No 60)

ACTGACTGCCATAGCACAGCTAGACGAGCTGATTATTCTGTCCTATATAATTCCGCATCATTTT CCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTGTCTGGCCG AGGTCTGGTTCCTGCCTAG (SEO ID No 61)

GTCTGGCCTGACGTATGATCGATGCCATAAATGCGCTGATTATTCTGTCCTATATAATTCCGCA TCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTCA CAACCATACTGGCGAAGT (SEQ ID No 62)

GCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCC TACTAAATTAAATGATCTCTGCTTTACTGTCTGGCCTGACGTATGATCGATGCCATAAATG (SEQ ID No 63)

ACTGACTGCCATAGCACAGCTAGACGATATTCTGTCCTATATAATTCCGCATCATTTTCCACTTT TAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCGTCTGGCCG AGGTCTGGTTCCTGCCTAG (SEO ID No 64)

CCCGGGTATAGTAGCTGACTGCGACGATATTCTGTCCTATATAATTCCGCATCATTTTCCACTTT TAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCATGGCCACG C_(SEQ ID No 65)

ACTGACTGCCATAGCACAGCTAGACGACTATGCAGATTCATATAATTCCGCATCATTTTCCACT TTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTGTCTGGCCG AGGTCTGGTTCCTGCCTAG (SEO ID No 66)

CTATGCAGATTCATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTA AATTAAATGATCTCTGCTTTACTAATGTGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 67)

ACTGACTGCCATAGCACAGCTAGACGATCATTTGTAATTAGAGGTGATGAAGTCAGACAAATC GCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTGTCTGGC CGAGGTCTGGTTCCTGCCTAG (SEO ID No 68) ATGGCCACGCTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGG AAAGATTGCTGATTATAATTATAAATTACCAGATGATTTT (SEQ ID No 69)

ACTGACTGCCATAGCACAGCTAGACGAGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCA GGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAAGGGTCTGG CCGAGGTCTGGTTCCTGCCTAG (SEO ID No 70)

ACTGACTGCCATAGCACAGCTAGACGAGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTG AACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAA AGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEO ID No 71)

ATTTGCTGCATGACTGGATCAATGCGACGAGGTTACCAACCATACAGAGTAGTAGTACTTTCTT TTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAA CAAAACTGCACATCGCTGCAGTCT (SEP ID No 72)

ACTGACTGCCATAGCACAGCTAGACGACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTAC ATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAAGTCTGGC CGAGGTCTGGTTCCTGCCTAG (SEO ID No 73)

CGGGGCAACTCAAGAAATTCACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCAC CAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAA (SEQ ID No 74) GTCTGGCCGAGGTCTGGTTCCTGTGCCCCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTAC ATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAA (SEQ ID No 75)

ACTGACTGCCATAGCACAGCTAGACGAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAA CTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCGTCTGGCC GAGGTCTGGTTCCTGCCTAG (SEO ID No 76)

CGCGTCGGGGCCTGCCTAAGGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTG TGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC (SEQ ID No 77)

ACTGACTGCCATAGCACAGCTAGACGATCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTG GACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGTCTGGCC GAGGTCTGGTTCCTGCCTAG (SEO ID No 78)

CACATGCCATCCGAGTCGTCTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAA AAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATCGTGCTACTGGGGTC TGGCAG (SEO ID No 79)

CTCTACCCACTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACT AATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAAT (SEQ ID No 80)

ACTGACTGCCATAGCACAGCTAGACGAGAACTTCTACATGCACCAGCAACTGTTTGTGGACCT AAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTGTCTGGCCGA GGTCTGGTTCCTGCCTAG (SEO ID No 81)

CCGGACTGCCATAGCACAGCTAGACGAGAACTTCTACATGCACCAGCAACTGTTTGTGGACCT AAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTGTCTGGCCGA GGTCTGGTTCCTGCCTAG (SEO ID No 82)

ACTGACTGCCATAGCACAGCTAGACGAGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAA TGTGTCAATTTCAACTTCAATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTGTCTGGCCGA GGTCTGGTTCCTGCCTAG (SEO ID No 83) GGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTAA CAGGCACAGGTGTTCTTACTGAGTCTGTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEO ID No 84)

ACTGACTGCCATAGCACAGCTAGACGAGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATA CCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATG AGAACCAAAAATTGGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEO ID No 85)

ATGCCCTGATCCCAATGATGGACGAGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACC ATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCTATGAG AACCAAAAATTGCTCTACCCA (SEO ID No 86)

ACTGACTGCCATAGCACAGCTAGACGAACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTAC AAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTGTCTGGCCGAGGTCTGGTTCCTG CCTAG (SEO ID No 87)

CTCTACCCAACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGC AAATGGCTTATAGGTTTAATGGTGTCTGGCCCCCGGGTATAGTAGCTGAC (SEO ID No 88)

GTCTGGCCCCCGGGTATAGTAGCTGACACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTAC AAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGT (SEQ ID No 89)

ACTGACTGCCATAGCACAGCTAGACGAACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTAC AAATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGGTCTGGCC GAGGTCTGGTTCCTGCCTAG (SEO ID No 90)

GTCTGGGAGCGTGCTGAAATGCATGACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAA ATACCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAG (SEQ ID No 91)

ACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAATGGCTT ATAGGTTTAATGGTATTGGAGTTACACAGACTGACTGCCATAGCACAGCTAGACGA (SEO ID No 92)

ACTGACTGCCATAGCACAGCTAGACGATTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTA TGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCGTCTGGCCGAGGTC TGGTTCCTGCCTAG (SEO ID No 93)

CACATGCCATCCGAGTCGTCTTTGGTGCAGGTGCTGCATTACAAATACCATTTGCTATGCAAAT GGCTTATAGGTTTAATGGTATTGGAGTTACACAGAATGTTCTCCTCTACCCA (SEQ ID No 94)

ACTGACTGCCATAGCACAGCTAGACGAAACACAGTTTATGATCCTTTGCAACCTGAATTAGAC TCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTG ACATCGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEO ID No 95)

ATTTGCTGCATGACTGGATCAATGCGACGAAACACAGTTTATGATCCTTTGCAACCTGAATTAG ACTCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAG GTGACATCACTGCACATCGCTGCAGTCT (SEQ ID No 96)

AACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATT TTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTTACCTGCCACACAAGCCTG (SEQ ID No 97) ACTGACTGCCATAGCACAGCTAGACGAAACACAGTTTATGATCCTTTGCAACCTGAATTAGAC TCATTCAAGGAGGAGTTAGATAAATATTTTAAGGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 98)

TTACCTGCCACACAAGCCTGAACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAA GGAGGAGTTAGATAAATATTTTAAGACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 99)

AACACAGTTTATGATCCTTTGCAACCTGAATTAGACTCATTCAAGGAGGAGTTAGATAAATATT TTAAGGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEO ID No 100)

ACTGACTGCCATAGCACAGCTAGACGATTAGATAAATATTTTAAGAATCATACATCACCAGAT GTTGATTTAGGTGACATCTCTGGCATTGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 101)

CACAACCATACTGGCGAAGTTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTT

AGGTGACATCTCTGGCATT (SEQ ID No 102)

TTACCTGCCACACAAGCCTGTTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTT

AGGTGACATCTCTGGCATT (SEQ ID No 103)

CCGGGTATAGTAGCTGACTGCGACGATTAGATAAATATTTTAAGAATCATACATCACCAGATG TTGATTTAGGTGACATCTCTGGCATT (SEQ ID No 104)

TTAGATAAATATTTTAAGAATCATACATCACCAGATGTTGATTTAGGTGACATCTCTGGCATTA TGGCCACGC (SEO ID No 105)

ACTGACTGCCATAGCACAGCTAGACGAGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTAT GAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAG TAATGGTGACAATTATGCTTGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 106)

CACATGCCATCCGAGTCGTCGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGT ATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGT GACAATTATGCTT (SEQ ID No 107)

CGTGCTACTGGGGTCTGGCAGGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAG TATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGG TGACAATTATGCTT (SEQ ID No 108)

ACTGACTGCCATAGCACAGCTAGACGAGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTAT GAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTGTCTGGCCGAGGTCTGGTTCCTGCC TAG (SEO ID No 109)

ATGGCCACGCGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATG GCCATGGTACATTTGGCTAGGT (SEQ ID No 110)

GTCTGGCCGAGGTCTGGTTCCTGCCTAGGAATCTCTCATCGATCTCCAAGAACTTGGAAAGTAT GAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGT (SEQ ID No 111)

GAGCAGTATATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAG TAATGGTGACAATTATGCTTCCCGGGTATAGTAGCTGACTGCGACGA (SEO ID No 112)

GTCTGGCCTGACGTATGATCGATGCCATAAATGCGAGCAGTATATAAAATGGCCATGGTACAT TTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTT (SEQ ID No 113)

ACTGACTGCCATAGCACAGCTAGACGAGAGCAGTATATAAAATGGCCATGGTACATTTGGCTA GGTTTTATAGCTGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTGTCTGGCCGAGGTCTG GTTCCTGCCTAG (SEO ID No 114)

ACTGACTGCCATAGCACAGCTAGACGATCCTCCTCCAAATTTGATGAAGACGACTCTGAGCCA GTGCTCAAAGGAGTCAAATTACATTACACAGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEO ID No 115)

In addition, oligonucleotides comprising the gene sequence of GLP2 : CATGCTGATGGTTCTTTCTCTGATGAGATGAACACCATTCTTGATAATCTTGCCGCCAGGGAC TTTATAAA CTGGTTGATTCAGACCAAAATCACTGAC (SEQ ID No. 116) and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were synthesized. Relative sequences are reported below:

CGGGGCAACTCAAGAAATTCCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGACGTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 117)

ACTGCACATCGCTGCAGTCTCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGACCGCGTCGGGGCCTGCCTAAG (SEQ ID No 118)

TTACCTGCCACACAAGCCTGCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGAC (SEQ ID No 119)

CGTGCTACTGGGGTCTGGCAGCATGCTGATGGTTCTTTCTCTGATGAGATGAACA CCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAA AATCACTGAC (SEQ ID No 120)

CACATGCCATCCGAGTCGTCCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGAC (SEQ ID No 121)

CACAACCATACTGGCGAAGTCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGAC (SEQ ID No 122)

AT CCAC CCATGCTGATGGTTCTTTCTCTGATGAGATGAACACCATTCTTGAT

AATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAAATCACTGACC

TCTACCCAC (SEQ ID No 123) CCGGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 124)

ACTGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 125)

ATTTGCTGCATGACTGGATCAATGCGACGACATGCTGATGGTTCTTTCTCTGATG AGATGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGAT TCAGACCAAAATCACTGACGTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 126)

ATGCCCTGATCCCAATGATGGACGACATGCTGATGGTTCTTTCTCTGATGAGATG AACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGA CCAAAATCACTGACGTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 127)

GAGCGTGCTGAAATGCATGCGACGACATGCTGATGGTTCTTTCTCTGATGAGATG AACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGA CCAAAATCACTGACGTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 128)

CCCGGGTATAGTAGCTGACTGCGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 129)

ACTGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACAAAAAAAAAAAAAAAAAAGTCTGGCCGAGGTCTGGTTC CTGCCTAG (SEQ ID No 130)

The coding sequence for the PYY peptide is as follows:

ATGTTAACCAAATTCGAGACCAAGAGCGCGCGGGTCAAAGGGCTCAGCTTTCACCCCA

AAAGACCTTGGATCCTG (SEQ ID 221)

The coding sequence for Xenin-25 peptide is as follows:

ATCAAACCCGAGGCTCCCGGCGAAGACGCCTCGCCGGAGGAGCTGAACCGCTACTAC

GCCTCCCTGCGCCACTACCTCAACCTGGTCACCCGGCAGCGGTAT (SEQ ID 222)

In addition oligonucleotides comprising the above coding gene sequence of PYY and Xenin-25 and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were synthesized.

Relative sequences are reported below:

ACTGACTGCCATAGCACAGCTAGACGAATGTTAACCAAATTCGAGACCAAGAGCGCG CGGGTCAAAGGGCTCAGCTTTCACCCCAAAAGACCTTGGATCCTGGTCTGGCCGAGGT CTGGTTCCTGCCTAG (SEQ ID No 223) ACTGACTGCCATAGCACAGCTAGACGAATCAAACCCGAGGCTCCCGGCGAAGACGCC TCGCCGGAGGAGCTGAACCGCTACTACGCCTCCCTGCGCCACTACCTCAACCTGGTCA CCCGGCAGCGGTATGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 224)

Treatment of microalsae

The microalgae culture was resuspended at a ratio of 1 : 5 to 1 : 25, depending on cell concentrations, in a macerating solution which constitutes a lytic mixture. The macerating solution consisted of 16- 50% (v/v) 0.35 M mannitol and 0.2-0.6% lytic enzyme mixture (Table 2). The lytic mixtures were then incubated at 30-37°C for 4-8 h. The composition of the lytic mixture, temperature and incubation time changed according to the microalgae species.

Table 2: Lytic enzymes

The microalgae solutions after treatment with the lytic mixture were combined, in a final volumeof 10 ml of culture medium, with 0.1 to 0.5 % v/v of polynucleotide diluted 2, 5, 10, 20, 25, or 50times (initial concentration 100 pM), depending on the nucleotide length and the resulting molecular weight of each polynucleotide (Table 3)

Table 3: Polynucleotide dilutions and percentages of lithium mixture used

10-35 % w/V polyethylene glycol (PEG-X) chemical melting agent was added to the culture (Table

4), calculated in consideration of the biomass obtained from the previous step.

Subsequently, the following steps were taken: i) The mixture was centrifuged at 1000 to 7000 rpm for 2 to 15 minutes; ii) The supernatant was removed and the pellet was washed with 2-15 ml of culture medium; iii) The mixture was centrifuged at 1000 to 7000 rpm for 2 to 15 minutes.

Steps i, ii and iii are repeated 2 to 5 times. When finished, the pellet is resuspended in 1 to 50 ml of standard culture medium. Centrifugation speed and duration vary according to the concentrations of PEG used (Table 4)

Table 4: PEG concentrations (% w/v) and processing parameters.

DNA extraction and amplification reaction by PCR

After several subcultures, PCR on DNA was conducted using Phire Plant Direct PCR Master Mix

(Thermo Fisher). The following primers were used in the reaction:

Pri GLP regl - Forward AGACATGCTGATGGTTCTTT (SEQ ID No 131)

Pri GLP reg2 - Forward TCTCTGATGAGATGAACACC (SEQ ID No 132) Pri GLP reg4 - Forward GATTTCCCAGAAGAGGTCG (SEQ ID No 133) Pri GLP regl - Reverse AACATTTCAAACATCCCACG (SEQ ID No 1334) Pri GLP reg2 - Reverse GCAGGTGATGTTGTGAAGAT (SEQ ID No 135)

Pri GLP reg4 - Reverse CGGCAAGATTATCAAGAATGG (SEQ ID No 136)

As evidence of successful insertion, sequencing of the amplification products was performed.

The amplification protocol is as suggested in the Phire Plant Direct PCR Master Mix brochure. (Thermo Scientific™ - Cat. Number: Fl 60S - https : //www. thermofisher, com/ order/ catalo g/ product/F 160S )

To extract metabolites contained within culture, this was sonicated for 15 min (1/2 W converter set to 100 % . Modulated pulse. UH-500B probe - Probe 600 ml - 50% pulse duty cycle). Then the sonicated culture was centrifuged at 4500 rpm for 10 min in order to separate cell debris from the supernatant. Depending on the different applications (in vivo or in vitro), the supernatant and/or biomass were subjected to lyophilization.

RESULTS

From the sequencing of the amplification products, several sequences were obtained that confirm the insertion of the chosen fragments within the algal genome. Only some of the results obtained are shown as examples, indicating in parentheses the percentages of identity returned by the Basic Local Alignment Search Tool (BLAST ®) software set to Highly similar sequences (megablast). The software performs an alignment between the sequence entered (Query) and all sequences deposited in the National Center for Biotechnology Information (NCBI) database to look for homologies and identify organisms and/or genes to which they belong. The table below shows the polynucleotide sequences related to the Spike fragments inserted in the algae, the product resulting from the sequencing analysis, and the percentage of identity that this product has, compared to the sequences deposited in the databases, indicated by their respective unique ID codes.

In vitro studies

Quantification of GLP-2 (pg/ml) in microalgae biomass by ELISA assay

To determine the amount of GLP-2 expressed by algal biomass, a human glucagon-like peptide 2 (GLP2) ELISA kit (0.156-10 ng/mL) was used. 100 mg of dry algal biomass, obtained by freeze- drying the crude product, was dissolved in 10 mL of water containing 50% acetonitrile and 0.1% trifluoroacetic acid. The extraction step performed is reported in the materials and methods, in fact, the solution was sonicated for 15 min and then, by centrifugation, the algal biomass was separated from the supernatant. The supernatant was lyophilized and 100 pl was resuspended in diluent buffer (provided by the kit).

The analysis protocol used includes the following steps:

1. Prepare all reagents, samples and standards.

2. Add 100 pl of sample to each well. Incubate 2 hours at 37°C

3. Aspirate and add 100 pl of prepared detection reagent A. Incubate 1 hour at 37°C

4. Vacuum and wash 3 times

5. Add 100 pl of prepared detection reagent B. Incubate 1 hour at 37°C

6. Vacuum and wash 5 times

7. Add 90 pl of substrate solution. Incubate 15-25 min at 37°C

8. Add 50 pl of blocking solution.

9. Read the absorbance immediately at 450 nm.

Interpolating the different measurements made, with the calibration line derived from the assay of the GLP-2 standard provided by the ELISA kit, the amount of peptide present in the sample is in the range of 150 to 2000 pg/ml.

Cytotoxicity Assays

Biocompatibility experiment conducted through MTT assay, of the extract derived from control cells of Haematococcus pluvialis and GLP-2

5x10³ IEC-6 cells were seeded and allowed to grow overnight. CTRL and GLP-2 cells of H. pluvialis were sonicated and filtered to separate the precipitated phase from the liquid phase. Different concentrations of algal extracts were tested (starting from 25% v/v. Serial 1:2 dilutions were made until the final concentration of 0.39 % v/v was reached). MTT assay was conducted after

24 and 48 hours to check cell viability.

The protocol used involves the following steps:

1. Remove the treatment medium: For adherent cells, carefully aspirate the medium.

2. Add 50 pl of serum-free medium and 50 pl of MTT reagent to each well.

3. Background control wells were prepared: 50 pL of MTT reagent + 50 pL of cell culture medium(without cells)

4. Incubate the plate at 37°C for 3 hours.

5. After incubation, add 150 pl of MTT solvent to each well.

6. Wrap the plate in aluminum foil and shake on an orbital shaker for 15 minutes.

7. Read the absorbance at OD = 590 nm.

Data analysis and statistics were performed using GraphPad Prism .

PBMCs preparations and stimulation experiments

Cell separation and Culture

PBMCs were purified using standard Ficoll-Paque gradient centrifugation according to the instructions of the manufacturer (Biochrom, Germany). Briefly, 20 ml of Ficoll-Paque gradient was pipetted into 50-ml centrifuge tubes. The heparinized blood was diluted 1 :3 in IX phosphate-buffered saline (PBS) (Euroclone s.p.a, Italy) and carefully layered over the Ficoll-Paque gradient. The tubes were centrifuged for 20 min at 2000 rpm. Mononuclear cells stratified in the Ficoll-plasma interface were extracted at the end of centrifugation, and the cells were washed twice in PBS (for 5 min at 1500 rpm), resuspended in PBS and counted in a Neubauer chamber. Dead cells were excluded using 1% trypan blue.

Once isolated, PBMCs were frozen in freezing medium (FBS/10% DMSO) and stored in liquid nitrogen.

Culture of PBMCs from healthy donors stimulated with algal coltures

PBMCs obtained from healthy donors were counted and plated at the concentration of 4xl06/ml in RPMI medium (Cell Genix, USA) with 10% FBS. PBMCs were stimulated with SARS CoV-2 antigenic peptide containing biomass The plate was incubated at 37 °C with 5% CO2 for 2 days and then analysed by FACS.

FACS analysis

Immune cells collected from Spike-stimulated PBMC cultures were stained with a viability fluorescent dye that irreversibly labels dead cells prior to fixation and/or permeabilization procedures (AQUA ThermoFisher L34966 for T, B and NK) and LIVE/DEAD ( Fixable Blue Dead Cell Thermofisher L34961 for Monocyte-MAcrophages), allowing dead cells to be excluded from analysis. Hence, in order to identify and characterize the lymphocyte populations, 10 pl of cell suspension were distributed in 4 wells of 96-well plates and stained with the mixture of primary antibodies, containing 1 pl of each antibody. Afterward, cells were incubated for 30 ' at 4°C in the dark, washed with IX PBS, centrifuged at 1200 rpm for 5'. Immunophenotypic identification and characterization of immune cell populations was based on 10-color flow cytometry staining.

Two antibody mixtures were used:

Monocyte-Macrophages identification: LIVE/DEAD (Fixable Blue Dead Cell Thermofisher L34961), CD16 (FITC eBioscience), CD80 (PE BD), CD163 (PE-CF594 BD), CD14 (BV510 Biolegend), PD-L1 (BV421 BD), CDl lc (BUV395 BD)

T, B and NK lymphocyte identification: LIVE/DEAD AQUA (ThermoFisher L34966), CDl lc (BUV395 BD), CD4 (BUV496, Biolegend), CD3 (BUV661, BD Pharmingen), IgG (BV650 BD), CD24 (BV785 Biolegend), IgD (BB515, BD Pharmingen), CD21 (PE/Dazzle 594 Biolegend), CD56 (PE BD), CD38 (PE Cy7, Biolegend), CD19 (PercPCy5.5, Biolegend), CD27 (APC, Biolegend), CD8 (PeCy5.5 Invitrogen), HLA-DR (AlexaFluor 780, Biolegend)

FACS analyses were performed on a Cytoflex flow cytometer (Beckman Coulter) and analysed with Cytexpert software (Beckman Coulter).

SARS CoV-2 Antigenic Peptide containing biomass: Immunomodulation activity

The immune system has two distinct components: mucosal and circulatory.

The mucosal immune system provides protection at the mucosal surfaces of the body. These include the mouth, eyes, middle ear, the mammary and other glands, and the gastrointestinal, respiratory and urogenital tracts. Antibodies and a variety of other anti-microbial proteins in the sticky secretions that cover these surfaces, as well as immune cells located in the lining of these surfaces, directly attack invading pathogens. Almost all infectious diseases in people and other animals are acquired through mucosal surfaces. As evident from the virus that causes COVID-19, SARS-CoV-2, enters the body via droplets or aerosols that get into nose, mouth or eyes. It can cause severe disease if it descends deep into the lungs and causes an overactive, inflammatory immune response.

This means that the virus’s first contact with the immune system is probably through the surfaces of the nose, mouth and throat. This is supported by the presence of IgA antibodies against S ARS-CoV- 2 in the secretions of infected people, including their saliva, nasal fluid and tears. These locations, especially the tonsils, have specialized areas that specifically trigger mucosal immune responses.

Sars CoV-2 is classified as airborn viurses and all the Airborn viruses have the same mechanism of infection through the mucosal compartments.

Some recent research suggests that if these IgA antibody responses form as a result of vaccination or prior infection, or occur quickly enough in response to a new infection, they could prevent serious disease by confining the virus to the upper respiratory tract until it is eliminated.

Therefore, Nasal vaccination can be effective in boositn or preventing viral infection for airborn viruses.

Microalgae expressing Sars CoV-2 peptide antigens can be in appropriate formulation spray used as boosting or preventing vaccine for airborn viruses.

In this embodiment the sars cov-2 antigen containing biomass produced according to the present invention methodology (in Chlorella spp as well as Haematococcus spp. ) was tested against PMBC (Periferal Mononucler Blood cells) of subject received vaccination against Sars Cov-2 to evaluate toxicity on blood cells, pro- or anti-inflammatory activity , ability for modulating specific macrophage families involved in the cascade events involved in immune response.

The SARS COV-2 antigenic peptide containing biomass was incubated with PBMC ad time course analysis perfomed at 24h and 48h to analyze the variation in macrophages populations.

It was detected a increase of intMo CD 14+ CD 16+ activity have a higher capacity to secrete cytokines in the blood and are reflective of an inflammatory environment for activation events. Furthermore, the SARS COV-2 antigenic peptide containing biomass increased the expression of CD 163 expression which associated to proinflammatory response ( effective for vaccine) and reduction of Pl/newMoCD14+++CD16+ activity at 48h where monocyte with anti-inflammatory potential, increased phagocytic activity and decreased antigen presentation ( effective for vaccine) . In addition, at 48h hours, no toxicity was observed and cell count was done, it was observed the down-regulation of CD3+ T cells and the up-regulation of mature NK cells compared to the control at 48h . Taken together all these results shown how this approach by expressing antigenic peptide epitopes of SARS CoV-2 can be effective in restimulating the immune system.

Claims

1) An host cell comprising a nucleic acid molecule comprising or consisting of: a) a polynucleotide sequence encoding at least one peptide; and b) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21; wherein the polynucleotide linker sequence is linked to the 5’ end and/or the 3' end of the encoding polynucleotide sequence of a) and in which the encoding polynucleotide sequence is not SEQ ID No 116; and wherein said host cell is an algal cell.

2) The host cell according to claim 1 wherein the nucleic acid comprises or consists of: a) the polynucleotide sequence encoding at least one peptide; and b) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21, linked at the 5’ end of the polynucleotide sequence of a); and c) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21, linked at the 3’ end of the polynucleotide sequence of a).

3) The host cell according to any of claims 1 and 2 wherein polynucleotide sequence a) has at least 90% homology to any of SEQ ID No 22-47.

4) The host cell according to any of claims 1 and 2 wherein the polynucleotide sequence a) has at least 90% homology to any SEQ ID No 221-222.

5) The host cell according to any of claims 1 and 2 wherein the nucleic acid is any of SEQ ID No 193-220 or any of SEQ ID No. 223-224.

6) The host cell according to any of claims 1-5 wherein said host cell belongs to the species Hematococcus pluvialis and/or wherein said host cell belongs to the class of Trebouxiophyceae.

7) The host cell according to any of previous claims wherein said host cell belongs to the species Haematococcus spp and/or Chlorella spp.

8) The host cell according to any of previous claims wherein said host cell belongs to Haematococcus pluvialis and/or Chlorella vulgaris.

9) An algal biomass comprising at least one host cell according to any one of previous claims and/or a lysate and/or an extract of said host cell. ) The algal biomass according to claim 9 comprising at least one peptide encoded by the nucleic acid molecule as defined in any of claims 1 to 5. 1) The algal biomass according to claim 10 wherein said peptide is any of SEQ ID No 193 - 220. ) A method for obtaining the host cell according to any one of claims 1-8 or the algal biomass according to any of claims 9 to 11 comprising the following steps: a) induce thermal stress in a culture of microalgae, preferably belonging to the class of Chlorophyceae, by heating at a temperature between 35 and 50° C for a time between 300 and 600 seconds; b) expose the microalgae culture to UV rays (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c) inoculate the culture treated in step b in fresh liquid medium; d) suspend the microalgae culture from step c) in a solution composed of 16-50% (v / v) of 0.35 M mannitol and 0.2-0.6% of a mixture of lytic enzymes comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulose, chitinase, 0-D- glucanase, macerozyme, helicase, driselasi, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidase, cellobiohydrolase, mixtures of them; e) incubate the microalgae culture treated in step d) at a temperature between 30 - 40° C for 4-8 h; f) combine the culture incubated from step e) in culture medium comprising 0.1 - 0.5% of a solution comprising the nucleic acid according to any one of claims 1-4, in the presence of 10-35 % polyethylene glycol (PEG-X). ) The method according to claim 12 further comprising a lysing step, preferably a sonication step.) The algal biomass according to any of claims 9 - 11 or obtainable from the method according to any of claims 11 and 12 for medical use. ) The algal biomass comprising at least one host cell according to claim 3 or comprising at least one peptide of SEQ ID No 193 - 218 for use in the treatment and/or prevention of infection caused by airborn viruses, preferably for use in the treatment and/or prevention of a SARS COV2 infection. ) A pharmaceutical composition comprising the algal biomass according to claims 9 - 11 or obtainable from the method according to claims 12-13 and at least one pharmacologically acceptable excipient. ) Supplement or food product or drinking product comprising the algal biomass according to claims 9-11 or obtainable from the method according to claims 12-13. ) Non therapeutic use of the algal biomass according to claims 9-11 or of the algal biomass obtainable from the method according to claims 12-13 or of the supplement or food product or product to drink according to claim 17 in the nutraceutical sector or as a basic ingredient in supplement preparations. ) Use of the algal biomass according to according to claims 9-11 or of the algal biomass obtainable from the method according to claims 12-13 in preparations in the cosmetic sector and/or in the agricultural or vegetable sector. ) An isolated nucleic acid molecule comprising or consisting of: a) a polynucleotide sequence encoding peptides; and b) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21; wherein the polynucleotide linker sequence is linked to the 5’ end and/or the 3' end of the polynucleotide sequence of a) and in which the polynucleotide sequence is not SEQ ID No 116.) The isolated nucleic acid molecule according to claim 20 comprising or consisting of: a) a polynucleotide sequence encoding peptides; and b) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21, linked at the 5’ end of the polynucleotide sequence of a); and c) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21, linked at the 3’ end of the polynucleotide sequence of a). ) The isolated nucleic acid molecule according to any one of claims 20 or 21 wherein the polynucleotide sequence of a) has at least 90% sequence homology to any of SEQ ID No 22-47 or has at least 90% sequence homology to any of SEQ ID No 221-222, or its functional derivatives.