Plant-based collagen-like proteins

Abstract

The present invention relates to plant-based collagen-like proteins, their uses and methods for the production thereof. The plant-based collagen-like proteins of the invention may comprise at least one G-X-X-G-X-X-G-X-X sequence wherein X is each independently any amino acid, at least 15% glycine, and 3% to 25% proline.

Description

[0001] Plant-based collagen-like proteins

[0002] Field of the invention

[0003] The present invention relates to plant-based collagen-like proteins, their uses and methods for the production thereof. The plant-based collagen-like proteins of the invention may comprise at least one G-X-X-G-X-X-G-X-X sequence wherein X is each independently any amino acid, at least 15% glycine, and 3% to 25% proline.

[0004] Background of the invention

[0005] Collagen is the most abundant protein in the human body, constituting 25 - 30% of the total protein content. It is essential for providing structural support in skin, bones, tendons, and connective tissues, contributing to their strength, elasticity, and integrity. With 29 distinct types of collagen, each serving unique biological functions, defining collagen becomes complex. As S. Blum highlighted in Cold Spring Harbor Perspectives in Biology (201 1 ), the question "What is collagen, what is not?" first posed by Fay and Miller in 1993, continues to be pertinent, with different answers emerging based on its structure and role.

[0006] A common feature of collagens is the presence of a triple helix structure, composed of three polypeptide chains known as a chains. This triple helix can constitute a significant portion of the collagen molecule, comprising up to 96% of the total structure in fibril-forming collagens (types I, II, III, V, XI, XXIV, and XXVII), or less than 10% in collagens with interrupted triple helices, such as types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. While fibril-forming collagens are the most widely recognized, there are also collagens that assemble into networks rather than fibrils, such as types IV, VIII, and X. Additionally, some collagens are involved in proteinprotein interactions and contain a von Willebrand domain (types VI, VII, and XXVIII). Another category includes membrane collagens (types XIII, XVII, XXIII, and XXV), which feature transmembrane domains.

[0007] Regardless of the type, all collagens contain at least one triple-helical domain (Blum, Cold Spring Harbor Perspectives in Biology, 2011 ; Zhang et al., J Photochem Photobio B Biol., 2020), characterized by a repeating Gly-X-Y sequence, where "Gly" stands for glycine, which appears every third residue. In this sequence, "X" is often proline, and "Y" is frequently hydroxyproline, a modified form of proline that plays a critical role in stabilizing the collagen triple helix. Interestingly, although glycine is the simplest amino acid and is considered non- essential, a recent study by Paz-Lugo et al. (Biosystems, 2023) demonstrated that a major deficiency in the procollagen cycle is due to glycine scarcity, leading to misfolding. Paz Lugo et al. suggest that increasing dietary glycine may promote cartilage regeneration by enhancing collagen synthesis and reducing waste.

[0008] With advancing age, collagen production in the human body decreases, and existing collagen in connective tissues begins to break down. To address this, collagen peptides from various sources are now widely used as dietary supplements, medicinal foods, or nutraceuticals. Clinical studies have shown that these peptides can stimulate the synthesis of collagen and other matrix proteins in fibroblasts, chondrocytes, osteoblasts, and tenocytes.

[0009] Meta-analyses and systematic reviews offer significant advantages when evaluating the benefits of collagen through clinical trials. By systematically gathering and analyzing data from multiple studies, these methods provide a comprehensive and evidence-based assessment of collagen's efficacy across various populations and conditions. Meta-analyses allow for the pooling of results from different trials, increasing the statistical power and precision of the findings, leading to more robust conclusions than those derived from individual studies. Systematic reviews ensure that all relevant research is considered, minimizing bias and providing a balanced overview of the evidence. This approach also helps identify patterns, inconsistencies, or gaps in the existing literature, guiding future research directions and informing clinical practice with greater confidence. Recent meta-analyses have highlighted the favorable effects of oral collagen supplementation: de Miranda et al. (Int J Dermatol., 2021 ) reported significant improvements in skin hydration, elasticity, and reduction of wrinkles with hydrolyzed collagen compared to a placebo; Khatri et al. (Amino Acids 53, 2021 ) found that collagen peptide supplements significantly improved joint functionality and reduced joint pain; and Pu et al. (Nutrients, 2023) demonstrated that hydrolyzed collagen supplementation notably enhanced skin hydration and elasticity compared to placebo.

[0010] The range of marketed collagen derivatives is vast, with various products available depending on their source. The most common collagen products are derived from land animals, including bovine, porcine, and poultry sources. These are widely used due to their availability and compatibility with human collagen. Marine organisms provide another significant source, with collagen extracted from sea cucumbers, mollusks, sponges, crustaceans, jellyfish, and particularly fish, which is valued for its high bioavailability and lower risk of transmitting diseases. Collagen is created by recombinant methodology as it is disclosed in several examples below.

[0011] In EP3684800, it is disclosed a recombinant yeast cell (Pichia pastoris) producing bovine collagen. The method offers advantages over traditional collagen production methods by enabling precise control over collagen hydroxylation patterns. Using recombinant yeast cells allows for scalable and efficient production of collagen with tailored properties, offering potential advancements in biotechnological collagen synthesis. In EP3687564, it is disclosed a recombinant collagen and elastin molecules and uses thereof. The invention discloses a recombinant collagen polypeptide with a truncated amino acid sequence which may be used in foods, cosmetics and many other products.

[0012] In EP4329521 , it is disclosed collagen compositions and methods of use thereof such as a recombinant collagen fragment having a molecular weight of about 50 kDa is used for treating a dermatological condition comprising administering an effective amount of the recombinant collagen fragment. The composition described herein can be a dietary composition.

[0013] Additionally, there are animal-free alternatives, such as recombinant collagen produced in yeast or through transgenic plants like tobacco. These methods allow for the production of human or animal collagen without the use of animal tissues, appealing to those seeking ethical or allergen-free options. For example, in EP2502939, it is disclosed a non-natural collagen- like proteins. As well, in W02022101804, a vegan analogue for collagen is described using plant-based free amino acid sources to replicate the amino acid composition of human collagen type I. This biomimetic free amino acid composition is created using plant-based amino acids and optional inductors to emulate the body's natural collagen building process for nutraceutical and cosmeceutical purposes. The process is extremely cumbersome and expensive, requiring the production and purification of single amino acids and carefully mixing those to obtain a mixtures with the appropriate composition. Evidently, such products lack the physicochemical and functional properties of collagen proteins and peptides.

[0014] Despite the fact that natural collagen expression is exclusive to the animal kingdom, there is a growing demand for vegetarian and vegan collagen alternatives among healthconscious consumers who follow a vegetarian or vegan diet or seek an animal-free alternative for use as a dietary supplement, medicinal food, or food ingredient. There is a need for animal- free collagen-like preparations as beneficial as animal-sourced collagen preparations for use e.g. as a dietary supplement, a medicament, or a food ingredient, with structural and functional characteristics similar to those of animal-sourced collagen.

[0015] Summary of the invention

[0016] The inventors surprisingly identified plant proteins with collagen-like properties. Therefore, in a first aspect, the invention provides a plant-based collagen-like protein comprising:

[0017] - at least one G-X-X-G-X-X-G-X-X sequence wherein X is each independently any amino acid,

[0018] - at least 15% glycine, and

[0019] - 3% to 25% proline. The plant-based collagen-like protein according to the invention may particularly comprise:

[0020] - from 30 to 40% glycine,

[0021] - at least three G-X-X repeat regions, each comprising at least 5 G-X-X repeat sequences; wherein X is at each occurrence independently any amino acid, and wherein between 9 and 15% of X in the protein is proline, and

[0022] - wherein from 20 to 70 amino acids are located between each of the three G-X-X repeat regions.

[0023] In one particular embodiment, the plant-based collagen-like protein according to the invention has a molecular weight in the range of 5 to 200 kDa.

[0024] In another particular embodiment of the invention, the plant-based collagen-like protein according to the invention comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0025] In yet another particular embodiment, the plant-based collagen-like protein according to the invention has been produced by microbial expression.

[0026] The invention furthermore provides a nucleotide sequence encoding the plant-based collagen-like protein according to the invention.

[0027] The invention also provides an expression vector comprising a nucleotide sequence according to the invention and a promotor operably linked to the nucleotide sequence to allow the expression of the encoded plant-based collagen-like protein according to the invention.

[0028] Furthermore, the invention provides a host cell comprising the nucleotide sequence according to the invention or the expression vector according to the invention.

[0029] The invention also provides a hydrolysate of the plant-based collagen-like protein according to the invention, preferably having an average molecular weight from 0.5 to 15kDa.

[0030] As well, a method is provided by the invention to produce a plant-based collagen-like protein according to the invention, comprising the following steps : providing a host cell according to the invention, culturing the host cell to express the plant-based collagen-like protein, recovering the expressed recombinant plant-based collagen-like protein, and optionally, hydrolyzing the recovered recombinant plant-based collagen-like protein.

[0031] A composition is provided by the invention such as a dietary supplement, a food ingredient, a cosmetic composition, a pharmaceutical composition, or a biomaterial comprising the recombinant plant-based collagen-like protein according to the invention, or the hydrolysate of the plant-based collagen-like protein according to the invention.

[0032] In a particular embodiment, the invention provides the plant-based collagen-like protein, the hydrolysate or the composition according to the invention for use as a medicine.

[0033] In another embodiment, the invention provides the plant-based collagen-like protein, the hydrolysate or the composition according to the invention for use in the treatment and / or prevention of a disorder selected from the group consisting of an inflammatory skin disorder, a bone or cartilage disorder, an ophthalmic disorder, and a neurodegenerative disorder.

[0034] In another particular embodiment, the invention provides with a non-therapeutic use of the plant-based collagen-like protein, the hydrolysate or the composition according to the invention, for improving skin condition, for improving the growth and / or strength nails or hair, for increasing the number of mitochondria and / or mitochondrial activity, for improving endurance performance, or for improving mental performance.

[0035] In another embodiment, the invention provides the use of the plant-based collagen-like protein, the hydrolysate or the composition according to the invention, as a gelling agent.

[0036] Brief description of the drawings

[0037] Fig. 1 : Locations of G-X-X repeat regions in the plant-based collagen-like proteins. A: SEQ ID NO :2 from Solanum lycopersicum, B: SEQ ID NO :3 from Solanum chilense, C: SEQ ID NO :4 from Solanum commersonii, D: SEQ ID NO :5 from Solanum tuberosum.

[0038] Fig. 2: SDS-PAGE analysis of tomato collagen-like protein before and after protease hydrolysis. TCLP is tomato collagen like protein. TCLPh is tomato collagen like protein after hydrolysis.

[0039] Detailed description of the invention

[0040] As described herein before, the present invention provides a plant-based collagen-like protein comprising:

[0041] - at least one G-X-X-G-X-X-G-X-X sequence wherein X is each independently any amino acid,

[0042] - at least 15% glycine, and

[0043] - 3% to 25% proline.

[0044] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclature used in connection with, and techniques of, biology and chemistry described herein are those well- known and commonly used in the art.

[0045] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), to the general background art referred to above and to the further references cited therein.

[0046] As used herein, the terms "polypeptide", "protein", “peptide”, and “amino acid sequence” are used interchangeably, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0047] As used herein, amino acid residues will be indicated either by their full name or according to the standard three-letter or one-letter amino acid code.

[0048] As used herein, the terms "nucleic acid molecule", "polynucleotide", “polynucleic acid”, “nucleic acid” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0049] As used herein, a “G-X-X repeat sequence” refers to a G-X-X sequence that is part of a repetition of such sequences. As will be evident from the descriptions herein and the examples, repeat or repetition of G-X-X refers to the repetition of such a motif, but does not require that X at the second (or third) position must always be the same amino acid across the repetitions. Just as an example, G-X-X-G-X-X-G-X-X comprises three G-X-X repeat sequences, the X at position 2, 5 and 8 (i.e. the second amino acid in each G-X-X repeat sequence) may be different amino acids. Similarly, X at position 3 can be different from X at position 6 and 9 (the third amino acids in the other G-X-X repeat sequences) as well as those at positions 2, 5 and 8 (the second amino acid in each G-X-X repeat sequence).

[0050] As used herein, a “G-X-X repeat region” refers to a region in the protein amino acid sequence comprising at least three (successive) G-X-X repeat sequences. The shortest G-X- X repeat region thus has a length of 9 amino acids and has the sequence G-X-X-G-X-X-G-X- X. Plant-based collagen-like proteins

[0051] As used herein, the term “collagen-like protein” refers to a protein which has similar physiological functions and structural properties as known collagen proteins. Collagen and collagen-like proteins are rich in glycine and proline residues and contain one or more regions with glycine-X-X (G-X-X) repeat sequences, particularly containing at least three repeats (a G- X-X repeat region). Such a three G-X-X repeat sequence can thus be presented herein as a G-X-X-G-X-X-G-X-X sequence, where "G" stands for glycine, and X designates each independently any amino acid. Such repeat sequences can interact with each other to form what is generally known as a collagen triple helix. Therefore, in a particular embodiment, a collagen-like protein comprises at least one triple-helix domain composed of three intertwined polypeptide chains, each chain comprising a G-X-X-G-X-X-G-X-X sequence.

[0052] Therefore, as described herein before, the plant-based collagen-like protein according to the invention comprises:

[0053] - at least one G-X-X-G-X-X-G-X-X sequence wherein X is each independently any amino acid,

[0054] - at least 15% glycine, and

[0055] - 3% to 25% proline.

[0056] In a further embodiment, the plant-based collagen-like protein of the invention comprises at least two sequences of G-X-X-G-X-X-G-X-X. In a particularly preferred embodiment, the plant-based collagen-like protein of the invention comprises at least three sequences of G-X-X-G-X-X-G-X-X. Thus, the plant-based collagen-like protein comprises several consecutive or non-consecutive G-X-X repeat regions. Preferably, the plant-based collagen-like protein comprises from one to eight, consecutive or non-consecutive G-X-X repeat regions. More preferably, the plant-based collagen-like protein comprises from three to six, consecutive or non-consecutive G-X-X repeat regions. If more than one G-X-X repeat regions are present in the protein, these are preferably non-consecutive, but spaced apart. Therefore, if more than one sequence of G-X-X-G-X-X-G-X-X is required to be present in the embodiments of the invention, it is preferred that there are at least 15 amino acids, more preferably at least 20 amino acids between the required sequences of G-X-X-G-X-X-G-X-X.

[0057] It is further advantageous to have longer G-X-X repeat regions, particularly containing more than three repeats of the G-X-X sequence. Therefore, in a particular embodiment, the G- X-X-G-X-X-G-X-X as disclosed herein are directly followed by one or more additional G-X-X repeats. In a particular embodiment, the plant-based collagen-like protein comprises at least one, preferably at least two, more preferably at least three G-X-X repeat regions, wherein the region comprises three to twelve consecutively repeated G-X-X sequences. Phrased otherwise, such regions comprise a G-X-X-G-X-X-G-X-X sequence followed by 0 to 9 G-X-X sequences. More preferably, the plant-based collagen-like protein comprises between three to ten consecutively repeated G-X-X sequences, preferably 4 to 8, most preferably 5 to 7 consecutively repeated G-X-X sequences. More preferably, the plant-based collagen-like protein comprises at least one G-X-X repeat region comprising between six to nine consecutively repeated G-X-X sequences. X designates each independently any amino acid within one G-X-X sequence and across repeated G-X-X sequences in the same plant-based collagen-like protein. In other words, within one G-X-X repeat region (comprising at least three repeated G-X-X sequences) each X refers each independently to any amino acid.

[0058] In a particular embodiment, the plant-based collagen-like protein according to the invention comprises:

[0059] - at least three G-X-X-G-X-X-G-X-X sequences, each optionally followed by 1 to 12 G- X-X sequences, wherein X is at each occurrence independently any amino acid, and

[0060] - wherein at least 15, preferably at least 20, amino acids are located between the G-X- X-G-X-X-G-X-X sequences.

[0061] In a further embodiment, the plant-based collagen-like protein according to the invention comprises:

[0062] - at least three G-X-X repeat regions, each comprising at least 3, such as from 4 to 15, G-X-X repeat sequences; wherein X is at each occurrence independently any amino acid, and

[0063] - wherein at least 15, preferably at least 20, amino acids are located between each of the three G-X-X repeat regions.

[0064] In an even further embodiment, the plant-based collagen-like protein according to the invention comprises:

[0065] - at least three G-X-X repeat regions, each comprising at least 5 G-X-X repeat sequences; wherein X is at each occurrence independently any amino acid, and

[0066] - wherein at least 20 amino acids are located between each of the three G-X-X repeat regions.

[0067] In yet a further embodiment, the plant-based collagen-like protein according to the invention comprises:

[0068] - at least three G-X-X repeat regions, each comprising at least 5 G-X-X repeat sequences; wherein X is at each occurrence independently any amino acid, and

[0069] - wherein from 20 to 70 amino acids are located between each of the three G-X-X repeat regions.

[0070] Glycine is the most necessary amino acid for collagen synthesis. An increase in glycine concentration results in an increased collagen production (Paz-Lugo et al., Amino Acids, 2018). Therefore, the plant-based collagen-like protein comprises at least 15% glycine, meaning that at least 15% of the amino acids in the protein sequence are glycine. In a further embodiment, the plant-based collagen-like protein comprises at least 20%, such as at least 25%, glycine. In a preferred embodiment, the plant-based collagen-like protein comprises at least 30% glycine. In another particular embodiment, the plant-based collagen-like protein comprises from 15 to 50% glycine, such as from 20 to 45% glycine, preferably from 25 to 40% glycine. In an even more preferred embodiment, the protein comprises from 30 to 40% glycine, such as from 34 to 37% glycine.

[0071] Advantageously, the plant-based collagen-like protein may comprise at least 2%, particularly at least 3%, preferably at least 4% proline. In a particular embodiment, the protein comprises from 2 to 25% proline. In a particular embodiment, the protein comprises from 3 to 20% proline, such as from 3 to 19% proline. Preferably, the protein comprises from 3 to 10% proline, such as from 4 to 8% proline.

[0072] As used herein, proline includes its hydroxylated form hydroxyproline. In one embodiment, at least 5% of the proline amino acids in the plant-based collagen-like protein is hydroxyproline In another embodiment, at least 7%, particularly at least 9%, more particularly at least 10% of the proline amino acids in the plant-based collagen-like protein is hydroxyproline.

[0073] Of particular relevance is the presence of a significant amount of proline residues in the G-X-X repeat sequences, such as in the G-X-X-G-X-X-G-X-X sequence or G-X-X repeat region(s). Therefore, in a particular embodiment, the minimal proline (and hydroxyproline) percentages described herein in relation to the plant-based collagen-like protein can be applied as particular minimal percentages of proline within the X residues of the G-X-X and G-X-X-G- X-X-G-X-X sequences or G-X-X repeat regions.

[0074] In a particular embodiment, at least 2% of X in each G-X-X repeat region is proline, particularly at least 3%, preferably at least 4 %.

[0075] Particularly advantageous are those plant-based collagen-like protein wherein X is any amino acid and wherein at least 5% of X in the protein is proline, particularly at least 7%, such as at least 8% proline. In a preferred embodiment, at least 9% of X in the protein is proline. In an alternative embodiment, between 5 and 50% of X in the protein is proline, such as between 7 and 30%, particularly between 8 and 20%, preferably between 9 and 15%.

[0076] As the skilled person will understand from the disclosures herein, the present invention also envisages a preferred plant-based collagen-like protein according to the invention comprising:

[0077] - at least three G-X-X repeat regions, each comprising at least 5 G-X-X repeat sequences; wherein X is at each occurrence independently any amino acid, and wherein 9 and 15% of X in the protein is proline,

[0078] - wherein from 20 to 70 amino acids are located between each of the three G-X-X repeat regions, and

[0079] - from 30 to 40% glycine. Preferably, said plant-based collagen-like protein comprises a collagen-like domain from the conserved domain database family with NCBI HMM accession number NF038329.2. The presence of the domain can be verified on the basis of the protein amino acid sequence using the conserved domain database (CDD database) version 3.21 (containing 62456 PSSMs) and an e-value lower or equal to 1 e-5, for example using NCBI’s CD-search in the latest version available on 17 September 2024 using default parameters.

[0080] Said collagen-like protein has similar physiological functions as animal-based collagen. Collagen is a structural protein which provides structure and integrity to most body tissues. Collagen is therefore essential to maintain the physiological role of these tissues.

[0081] The term “plant-based protein”, according to the present invention, refers to a protein having an amino acid sequence that is derived from a plant protein. The plant-based protein may be isolated from a plant or produced by synthetic or recombinant production. A plantbased protein according to the invention may have the same or substantially the same sequence as a wild-type plant protein sequence or it may have a derivative protein sequence. In a particular embodiment, a plant-based protein according to the invention has an amino acid sequence having at least 80% sequence identity to the sequence of the (wild-type) plant protein from which it is derived. Preferably, a plant-based protein according to the invention has an amino acid sequence having at least 85% sequence identity to the sequence of the (wild-type) plant protein from which it is derived, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity. In a particular embodiment, the plant is a plant from the order Solanales, particularly the Family of Solanaceae, such as the subfamily of Solanoideae. In a preferred embodiment, the plant is a Solanum species, even more preferably a plant selected from the group consisting of Solanum lycopersicum, Solanum chilense, Solanum commersonii, and Solanum tuberosum; particularly Solanum lycopersicum. As the skilled person will understand from the description herein, the percentages of sequence identity to the sequence of the wild-type plant protein apply particularly to the plants and preferred plants disclosed herein. For example, in a particular embodiment, the plant-based protein has an amino acid sequence having at least 80% sequence identity to the sequence of a wild-type protein of a Solanum species, such as the plant-based protein may have an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity to the sequence of a wild-type protein of Solanum lycopersicum. In another embodiment, the plant-based protein has an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity to the sequence of a wild-type protein of Solanum chilense. In another embodiment, the plantbased protein has an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity to the sequence of a wild-type protein of Solanum commersonii. In yet another embodiment, the plant-based protein has an amino acid sequence having at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity to the sequence of a wild-type protein of Solanum tuberosum. In a further embodiment, the plant-based protein has an amino acid sequence of a wild-type protein of a plant selected from the group consisting of Solanum lycopersicum, Solanum chilense, Solanum commersonii, and Solanum tuberosum; preferably Solanum lycopersicum.

[0082] As will be discussed in more detail elsewhere herein, the plant-based proteins may be produced through recombinant production, particularly by (recombinant) microbial expression. Preferably, plant-based collagen-like protein is a recombinantly expressed protein, wherein the protein preferably originates from a plant as described herein, such as from a Solanum plant.

[0083] In one embodiment, the plant-based collagen-like protein has a molecular weight of 5 to 200 kDa, such as a molecular weight of 5 to 100 kDa, such as a molecular weight of 5 to 50 kDa. In a further embodiment, the protein has a molecular weight of 10 to 45 kDa, preferably from 15 to 40 kDa, more preferably from 20 to 35 kDa. Such a molecular weight ranges provide an optimal protein size for the collagen-like protein to enable its physiological functions in mammals or to provide an ideal protein size for further hydrolyzation.

[0084] In one embodiment, the plant-based collagen-like protein is optimized for recombinant production and / or purification by inserting or removing protein trafficking sequences, removing / adding post translation modification sites in encoded protein (e.g. glycosylation sites), adding, removing or shuffling protein domains. Protein optimization may as well be performed to increase the nutritional value for human consumption, such as changing non- essential amino acids to essential amino acids.

[0085] As is evident from the examples, several proteins have been identified as plant-based collagen-like proteins such as the proteins with the amino acid sequences of SEQ ID NO :2 identified in Solanum lycopersicum, or SEQ ID NO :3 identified in Solanum chilense, or SEQ ID NO :4 identified in Solanum commersonii, or SEQ ID NO :5 identified in Solanum tuberosum.

[0086] In a preferred embodiment, said plant-based collagen-like protein according to the invention has an amino acid sequence that has at least 85% sequence identity to the SEQ ID NO:2, preferably, at least 90% sequence identity to the SEQ ID NO:2, more preferably, at least 95% sequence identity to the SEQ ID NO:2, even more preferably, at least 97% sequence identity to the SEQ ID NO:2. SEQ ID NO: 2 comprises an amino acid sequence identified in Solanum lycopersicum as a tomato collagen-like protein.

[0087] In another embodiment, said plant-based collagen-like protein according to the invention may comprise an amino acid sequence that has at least 85% sequence identity to the SEQ ID NO:3, preferably, at least 90% sequence identity to the SEQ ID NO:3, more preferably, at least 95% sequence identity to the SEQ ID NO:3, even more preferably, at least 97% sequence identity to the SEQ ID NO:3. SEQ ID NO: 3 comprises an amino acid sequence identified in Solanum chilense as an homolog of the tomato collagen like protein.

[0088] In a particular embodiment, said plant-based collagen-like protein according to the invention may comprise an amino acid sequence that has at least 85% sequence identity to the SEQ ID NO:4, preferably, at least 90% sequence identity to the SEQ ID NO:4, more preferably, at least 95% sequence identity to the SEQ ID NO:4, even more preferably, at least 97% sequence identity to the SEQ ID NO:4. SEQ ID NO: 4 comprises an amino acid sequence identified in Solanum commersonii as an homolog of the tomato collagen like protein.

[0089] In another embodiment, said plant-based collagen-like protein according to the invention may comprise an amino acid sequence that has at least 85% sequence identity to the SEQ ID NO:5, preferably, at least 90% sequence identity to the SEQ ID NO:5, more preferably, at least 95% sequence identity to the SEQ ID NO:5, even more preferably, at least 97% sequence identity to the SEQ ID NO:5. SEQ ID NO: 5 comprises an amino acid sequence identified in Solanum tuberosum as an homolog of the tomato collagen like protein.

[0090] As used herein, the term “sequence identity” means that two polypeptide or polynucleotide sequences are identical (i.e. on an amino acid-by-amino acid, or on a nucleotide-by-nucleotide basis, respectively) over a window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid or nucleic acid base, whichever relevant, occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0091] It is another aspect of the invention to provide a nucleotide comprising a sequence encoding a plant-based collagen-like protein as described herein. Although the amino acid sequence of the protein encoded by the nucleotide is more important than the nucleotide sequence itself, in a particular embodiment, the nucleotide comprises the same or substantially the same nucleotide sequence as a wild-type plant nucleotide sequence or it may have a derivative nucleotide sequence. In a particular embodiment, the nucleotide according to the invention has a nucleotide sequence having at least 80% sequence identity to the sequence of the (wild-type) plant nucleotide from which it is derived. Particularly, the nucleotide according to the invention may have a nucleotide sequence having at least 85% sequence identity to the sequence of the (wild-type) plant nucleotide from which it is derived, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity. In one preferred embodiment, said nucleotide according to the invention comprises a nucleotide sequence having at least 80% sequence identity to the SEQ ID NO: 1 , preferably, at least 85% sequence identity to the SEQ ID NO: 1 , more preferably, at least 90% sequence identity to the SEQ ID NO: 1 , even more preferably, at least 95% sequence identity to the SEQ ID NO: 1

[0092] The nucleotide sequence according to the invention may be optimized in order to enhance protein expression in an heterologous host and / or to provide an optimal folding of said plant-based collagen-like protein to ensure its functions. Optimization includes any modifications of said nucleotide sequence which aims to provide an improved expression of said plant-based collagen-like protein encoded by said nucleotide sequence. Codon optimization is for example a codon modification to match codon frequencies in target and host organisms. Many other types of modifications can be considered to participate in optimizing said nucleotide sequence such as changing bias GC content to increase mRNA stability or reducing secondary structures, minimizing tandem repeat codons or base runs that may impair gene construction or expression, customizing transcriptional and translational control regions, , inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or reducing / eliminating problematic secondary structures within the mRNA. Optimization of the nucleotide sequence may include modifications such that the encoded protein has the protein optimizations discussed herein before.

[0093] In one preferred embodiment, the nucleotide comprises a nucleotide sequence according to SEQ ID NO: 6 or a sequence having at least 85% sequence identity thereto. SEQ ID NO: 6 is an example of a nucleotide sequence originating from Solanum lycopersicum w ic has been optimized for heterologous expression in Pichia Pastoris.

[0094] In a particular embodiment, said nucleotide sequence encodes a fusion protein comprising the plant-based collagen-like protein fused to a secretion signal; for example to enhance the plant-based collagen-like protein secretion from the host cell. For example, the secretion signal may be a yeast secretion signal. In one embodiment, the secretion signal is a pre-pro-a-factor from Saccharomyces cerevisiae.

[0095] Advantageously, the invention provides an expression cassette or expression vector comprising a nucleotide sequence as described herein and a promotor operably linked to said nucleotide sequence to allow the expression of the encoded plant-based collagen-like protein.

[0096] The invention therefore provides an expression cassette comprising the nucleotide sequence encoding the protein of the invention. The expression cassette typically includes a promoter operably linked to the nucleotide sequence, optionally along with other regulatory elements, such as an enhancer, polyadenylation signal, and selectable marker. These are the minimum elements ensure efficient transcription and translation of the protein in a host cell, however a person skilled in the art could also incorporate purification or solubility tags (e.g histidine tag or SUMO fusion) to ensure protein folding, improving solubility and yield of the heterologous protein. The expression cassette may be introduced into a variety of host cells, including bacterial, yeast, insect, plant, and mammalian cells, to achieve high-level expression of the recombinant protein. Preferred host cells are microbial host cells, such as bacterial or yeast host cells. Such host cells are preferably genetically modified for improved protein expression and / or reduced protein degradation. The choice of promoter and regulatory elements can be tailored to the specific host cell, allowing for controlled and inducible expression of the protein. In one particular embodiment, the expression cassette is integrated into the host cell genome for stable, long-term production of the protein. Alternatively, it can be maintained as an nonintegrated element, such as a plasmid, for example to ensure high-copy number replication and transient expression of the protein.

[0097] By the term “expression vector”, it is understood a vector designed for protein expression in cells. According to the invention, said expression vector comprises said nucleotide sequence encoding said plant-based collagen-like protein. Moreover, said nucleotide sequence preferably preceded by a promotor in order to initiate expression of the nucleotide sequence. The promotor and nucleotide sequence are linked operably to allow the initiation of expression of said plant-based collagen-like protein.

[0098] Thus present invention also provides an expression vector comprising the nucleotide sequence encoding the protein of the invention, in particular comprising the expression cassette of the invention. The expression vector can be a plasmid, viral vector, or other suitable vector used for introducing the nucleotide or expression cassette into a host cell. In preferred embodiments, the expression vector is designed for recombinant protein production in prokaryotic or eukaryotic host cells, preferably prokaryotic, more preferably microbial host cell.

[0099] Typically, the expression vector includes selectable markers, an origin of replication, and regulatory sequences to ensure efficient propagation, selection, and expression of the protein in the host cell. For instance, vectors designed for bacterial host cells may contain antibiotic resistance genes, while vectors for mammalian host cells may include mammalian selectable markers such as hygromycin or neomycin resistance genes. The expression vector can be introduced into the host cell via various methods, including transformation, transfection, electroporation, or viral transduction, depending on the host cell type.

[0100] In a preferred embodiment, the expression vector is used for large-scale recombinant protein production in a suitable expression system, ensuring high yield and quality of the protein. The vector may also be adapted for use in cell-free protein expression systems, providing flexibility in protein production across different platforms.

[0101] For the present invention, a variety of promoters can be used in the expression cassette or expression vector, each selected to drive optimal expression of the nucleotide sequence encoding the protein of interest. A promoter is a regulatory element that controls the initiation of transcription, and the choice of promoter can significantly affect the level, timing, and location of protein expression within a host cell.

[0102] In one embodiment, the promoter is a constitutive promoter, which allows for continuous, high-level expression of the nucleotide sequence in the host cell. Alternatively, inducible promoters can be used to regulate the expression of the nucleotide sequence in response to specific environmental or chemical signals. Inducible promoters offer the advantage of controlled expression, enabling protein production to be switched on or off as needed.

[0103] The selection of a promoter for use in the expression vector depends on the specific host cell system chosen for recombinant protein production. For bacterial expression systems, strong promoters such as the T7 or araBAD promoter are preferred, as they can drive high levels of transcription in bacteria like E. coli. In yeast expression systems, promoters such as the GAL1 promoter (induced by galactose) or the AOX1 promoter (induced by methanol in Pichia pastoris) are preferred, as these can regulate protein production. For expression in mammalian cells, promoters like the CMV promoter are preferred, as the have the ability to achieve high-level, constitutive expression, while inducible systems, like the Tet-On / Tet-Off promoters, are alternatively used when controlled or time-specific expression is necessary. Plant expression systems may utilize the CaMV 35S promoter for constitutive expression or other plant-specific promoters depending on the species and tissue targeted.

[0104] For the present invention, the use of a yeast expression system is particularly preferred, therefore, in a particular embodiment, the host cell is a yeast, such as a Saccharomyces or Pichia species. Therefore, the present invention also relates to the use of a yeast promoter for driving the expression of the nucleotide sequence encoding the protein of interest in a yeast host cell. Yeast is a regularly used expression system for recombinant protein production due to its eukaryotic machinery, which enables proper folding, glycosylation, and post-translational modifications of the expressed proteins. Moreover, yeast combines the ease of genetic manipulation and high growth rates, typical of simpler organisms, with the ability to produce complex proteins, making it a preferred system for industrial-scale protein production.

[0105] A variety of yeast promoters can be used to regulate the transcription of the nucleotide sequence in different yeast species, such as Saccharomyces cerevisiae and Pichia pastoris. In a particular embodiment, the promoter is an inducible yeast promoter. In a particular embodiment, the inducible yeast promoter is the GAL1 promoter. It is derived from the S. cerevisiae gene involved in galactose metabolism and is induced by the presence of galactose and repressed in the presence of glucose, allowing for tight control over gene expression.

[0106] In one embodiment, said yeast promoter is the Alcohol Oxidase promoter (AOX1 ). In Pichia pastoris, the AOX1 (alcohol oxidase 1 ) promoter is a highly inducible promoter used for driving high-level expression of foreign genes. The AOX1 promoter is tightly regulated and induced by methanol, making it ideal for achieving high yields of recombinant protein in fermentation processes. In some embodiments, variants of the AOX1 promoter with modified regulatory elements may be used to further optimize expression levels or to fine-tune the induction profile.

[0107] In another embodiment, said expression vector comprises the expression cassette comprising the the formate dehydrogenase promoter (PDF).

[0108] The expression vector is the key element for driving the expression of the heterologous gene in the host cell and for providing selection markers for generating the recombinant cell line. Expression vectors may include a constitutive or inducible promoter capable of robust transcriptional activity; optimized mRNA processing and translational signals, a translation termination codon, mRNA cleavage and polyadenylation signals, as well as mRNA splicing signals; a transcription terminator; selection markers for the preparation of stable cell lines and for gene amplification.

[0109] A selection marker allows to specifically select host cells comprising said expression vector. For example, the selection marker confers a resistance to the host cell against an antibiotics. When the selection marker is expressed in the host cell, this cell can survive antibiotic treatments when the others which do not express the selection marker will be eliminated. Therefore the remaining cells after antibiotic treatments contain said expression vector.

[0110] In one particular embodiment, said expression vector comprise a selection marker gene which confers Zeocin resistance. In another particular embodiment, said expression vector comprise a selection marker gene which confers Nourseothricin resistance.

[0111] In another aspect, the invention provides a host cell comprising said plant-based collagen-like protein or said expression vector. The host cell is typically a cell-based expression system. Preferably the host cell is selected amongst microbial host cells which may be bacteria or fungi, preferably fungi.

[0112] In some embodiments, host cells / organisms can be selected from a yeast species from the group consisting of Saccharomyces, Pichia, Hansenula, Yarrowia, Arxula, Kluyveromyces, and Schizosaccharomyces. More particurly from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe. In certain further, preferred, embodiments, the host cell is Pichia pastoris.

[0113] Hydrolysates

[0114] Plant-based collagen-like proteins may be hydrolyzed for their use to break down the large, complex proteins into smaller, more easily absorbable peptides. This involves cleaving the protein into shorter chains of amino acids, which my offer several benefits, including at least one of the following:

[0115] - Improved Digestibility and Absorption: Native plant-based collagen-like proteins may have a relatively low digestibility and / or absorbability by the human body. Hydrolysis reduces its molecular weight, allowing the peptides to be easily digested and absorbed through the gut and into the bloodstream.

[0116] - Enhanced Bioavailability: Hydrolyzed Plant-based collagen-like peptides are more bioavailable, meaning they can be quickly used by the body to support e.g. the improvement of skin, joints, and connective tissues.

[0117] - Solubility: Hydrolyzed Hydrolysis of plant-based collagen-like proteins may increase solubility and may make it easier to incorporate it into supplements, food products, and beverages.

[0118] - Reduced Allergenicity: Hydrolyzing plant-based collagen-like proteins reduces its potential to cause allergic reactions or immune responses, making it more suitable for consumption in a broader population.

[0119] - Functional Properties: In industrial applications, hydrolyzed plant-based collagen-like proteins may provide a range of functional benefits, such as improved texture, emulsification, and stability when used in products like cosmetics and pharmaceuticals.

[0120] Therefore, the invention also provides plant-based collagen-like protein hydrolysates. These may be produced through hydrolytic processes, in particular enzymatic hydrolysis, of the plant-based collagen-like protein. These plant-based collagen-like protein hydrolysates represent a mixture of peptides, the molecular weights of which are distributed over certain size ranges. Sequence analysis of the peptides in a mixture allow to determine whether or not these peptides constitute a hydrolysate of the plant-based collagen-like protein, as these peptides have sequences that are parts of the original proteins and as typically the overall amino acid composition remains substantially the same throughout the hydrolysis process.

[0121] In a particular embodiment, the hydrolysate of the plant-based collagen-like protein according to the invention comprises peptides having an average molecular weight from 0.5 to 15 kDa, more preferably from 2 to 12 kDa, even more preferably from 5 to 10 kDa. Such a range in the molecular weight of the hydrolysate of plant-based collagen-like protein allows for an optimal hydrolysate size in order to induce collagen physiological role once it has been administered through a composition.

[0122] Plant-based collagen-like protein production

[0123] The present invention also provides methods for the production of the proteins of the invention. Proteins may be produced synthetically, may be purified from plants that naturally contain them, or may be produced recombinantly. Recombinant production is particularly preferred.

[0124] In a particular embodiment, the invention provides a method to produce a plant-based collagen-like protein according to the invention, comprising the following steps :

[0125] - providing a host cell according to the invention,

[0126] - culturing the host cell to express the plant-based collagen-like protein,

[0127] - recovering the expressed plant-based collagen-like protein, and

[0128] - optionally, hydrolyzing the recovered recombinant plant-based collagen-like protein. Application of said method, allows to produce a plant-based collagen-like protein on a commercial scale. Moreover such a method produce a plant-based collagen-like protein fulfilling expectations of animal-free products required by vegetarian or vegan consumers.

[0129] The method comprise a step of providing a host cell according to the invention. Advantageously, host cells comprising the expression vector according to the invention are provided. Host cells may be selected through the expression of a selection marker. Preferably, the selection marker confers a cell resistance to a host cell elimination agent, such as an antibiotic or antifungal compound.

[0130] In one embodiment, the expression vector comprises an antibiotic resistance gene such that when expressed in the host cells, it confers a resistance to antibiotic treatments applied to the host cells. The antibiotic resistance gene may be a zeocin resistance gene or a nourseothricin resistance gene.

[0131] By the terms ‘culturing the host cell”, it is understood according to the invention, proceeding to a cell culture where the host cells are grown under controlled conditions, generally outside of their natural environment. The controlled conditions may include specific temperature parameters, specific media compositions, or other specific parameters allowing to cultivate, propagate and grow the host cell for the purpose of producing a protein in a gene expression system, such as a host cell. For systems using inducible promoters, protein expression is triggered by adding specific inducers to the culture medium at the appropriate time.

[0132] In one particular embodiment, the expression of the plant-based collagen-like protein takes place in the context of controlled temperature such as the temperature is maintained in between 20 to 50°C depending on the host cell. Preferably, the temperature is maintained between 25 and 40°C. The specific conditions which enable an expression of plant-based collagen-like protein are known to the person skilled in the art and depend on the expression system used and the expression cassette used, in particular, on the promoter contained therein. In another embodiment, the culturing media of the host cell may comprise glucose, amino acids, hormones, growth factors, salts or organic solvent such as alcohol, for example, methanol.

[0133] In another aspect, the method comprises a step recovering the expressed recombinant plant-based collagen-like protein. The recovering step allows to obtain a purified plant-based collagen-like protein ready to use in a composition.

[0134] Once the host cells have reached the desired density and protein expression has been achieved, the cells are typically separated from the culture medium by centrifugation or filtration. The recombinant plant-based collagen-like protein is then extracted from the cells or culture medium, depending on whether it is intracellular or secreted. This step may involve host cell lysis, followed by purification processes such as affinity chromatography, ionexchange chromatography, or size-exclusion chromatography to isolate the protein from other cellular components. Thus, in one embodiment, the recovering step comprises a purification step in order to increase the purity of the plant-based collagen-like protein, isolating it from other components. The purification step may be operated through chromatography techniques, ultrafiltration, precipitation techniques, centrifugation, or filtration.

[0135] In another aspect, according to the invention, the method may comprise an additional step as hydrolyzing the recovered recombinant plant-based collagen-like protein.

[0136] By the term ‘hydrolysis”, it has to be understood a process to obtain smaller peptides or amino acids from an original protein. Protein hydrolysis may be conducted e.g. through enzymatic hydrolysis, acid hydrolysis or alkaline hydrolysis, or any techniques allowing to obtain smaller peptides or amino acids originating from an initial protein.

[0137] Compositions

[0138] In another aspect, the invention provides a composition comprising the plant-based collagen-like protein, or the hydrolysate of the plant-based collagen-like protein. Such a composition may, for example, be a dietary supplement, a food ingredient, a cosmetic composition, a pharmaceutical composition, or a biomaterial.

[0139] Preferably, said composition further comprise a suitable carrier. According to the present invention, by the term “a suitable carrier”, it is understood a compound which allows to carry, distribute, or preserve the plant-based collagen-like protein or hydrolysate thereof. It may keep the proteins or peptides in their optimal form.

[0140] In one embodiment, according to the present invention, said collagen composition comprises at least one plant-based collagen-like protein and / or at least one hydrolysate of the invention.

[0141] Preferably, the composition may be liquid or solid. The composition may be any types of product encapsulated or not, selected in the group of solid tablets, powder, solution, gel, lotion, cream, ointment, foam, emulsion, microemulsion, milk, serum, aerosol, spray, aerosol, spray, dispersion or soap.

[0142] Advantageously, the composition may comprise additional compounds enhancing, supporting or completing the physiological role of the plant-based collagen-like protein and hydrolysate thereof, such as for example vitamins (vitamin A, B, C, D, E), hyaluronic acids and salts thereof, amino acids, or proteins.

[0143] Preferably, the composition comprises an acceptable excipient. By the term “excipient”, it is designated any compound or ingredient having the purpose to guarantee the stability, and bioavailability of the active ingredient of the composition, or providing a texture or taste to the composition.

[0144] Advantageously, the composition may comprise one or several additional active ingredients or compounds which will not impair the effectiveness of the plant-based collagen- like protein or hydrolysate thereof in their physiological function.

[0145] In one embodiment, the excipient may be a solubilizing agent with the purpose to increase bioavailability of active ingredients present in the composition.

[0146] In another embodiment, the excipient may be an antimicrobial agent with the purpose to avoid bacterial or fungal contamination of the composition.

[0147] In another embodiment, the composition may comprise a thickener agent such as glycerin.

[0148] Dietary supplement or food ingredient

[0149] By the term “dietary supplement”, it is understood, an ingestible composition that may be added to the diet to provide beneficial properties. Preferably, a dietary supplement is an oral composition in the form of pills, capsules, tablets, powders or liquids. The plant-based collagen-like proteins or hydrolysates of the invention can be incorporated into various dietary supplements designed to e.g. improve the condition of the skin, improve the growth and / or strength of nails or hair or improve endurance or mental performance. Hydrolyzed plant-based collagen-like proteins are particularly advantageous for dietary supplements due to its enhanced digestibility and bioavailability. These peptides are broken down into smaller, easily absorbed fragments, facilitating their uptake in the gastrointestinal tract and subsequent utilization by the body.

[0150] In dietary supplements, the plant-based collagen-like proteins or hydrolysates of the invention can be formulated alone or combined with other complementary nutrients such as vitamins, minerals, and herbal extracts. As one example, the dietary supplement may further include vitamin C, which is important for collagen synthesis. The formulation may also include flavoring agents and stabilizers to improve palatability and shelf life. The collagen protein of the invention can also be utilized as a food ingredient in various food products. Its inclusion in foods can enhance the nutritional profile by providing a source of protein and offering benefits associated with collagen. Food applications include its incorporation into protein bars, beverages, dairy products, baked goods, and snacks.

[0151] In food formulations, the plant-based collagen-like proteins or hydrolysates of the invention can be used as a texturizing agent, improving the mouthfeel and consistency of products. For example, it can enhance the texture of yogurt or provide a gelatinous consistency in confectionery items. Additionally, the plant-based collagen-like proteins or hydrolysates of the invention can be added to protein-enriched beverages, smoothies, and meal replacement shakes to boost protein content and deliver the benefits normally associated with animal-based collagen.

[0152] The integration of the plant-based collagen-like proteins or hydrolysates of the invention into food products requires consideration of factors such as solubility, taste, and stability. The hydrolysates of the invention are preferred for these applications due to its solubility in cold and hot liquids and its minimal impact on flavor. Furthermore, the use of the plant-based collagen-like proteins or hydrolysates of the invention in food products can be tailored to meet dietary preferences and requirements, including options for gluten-free, dairy- free, vegetarian and vegan formulations where suitable substitutes are used.

[0153] Pharmaceutical composition

[0154] By the terms “pharmaceutical composition”, it is understood a composition comprising the plant-based collagen-like proteins or hydrolysates of the invention and one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity. Pharmaceutically acceptable carriers enhance or stabilize the composition or can be used to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, as is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329; Remington: The Science and Practice of Pharmacy, 21 st Ed. Pharmaceutical Press 2011 ; and subsequent versions thereof). Nonlimiting examples of said pharmaceutically acceptable carrier comprise any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. These compositions include, for example, liquid, semi-solid and solid dosage formulations, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, or liposomes. In some embodiments, a preferred form may depend on the intended mode of administration and / or therapeutic application. Pharmaceutical compositions containing the proteins or hydrolysates of the invention can be administered by any appropriate method known in the art, including, without limitation, oral, mucosal, byinhalation, topical, buccal, nasal, rectal, or parenteral (e.g. intravenous, infusion, , subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, intrajoint or other kinds of administration involving physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue). Such a formulation may, for example, be in a form of an injectable or infusible solution that is suitable for intradermal or subcutaneous administration, for intravenous infusion or for injection in or at the joint or cartilage. The administration may involve intermittent dosing. Alternatively, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time, simultaneously or between the administration of other compounds.

[0155] Formulations of the invention generally comprise therapeutically effective amounts of the plant-based collagen-like proteins or hydrolysates of the invention. “Therapeutic levels”, “therapeutically effective amount” or “therapeutic amount” means an amount or a concentration of an active agent that has been administered that is appropriate to safely treat the condition to reduce or prevent a symptom of the condition, in particular a disorder selected from the group consisting of an inflammatory skin disorder, a bone or cartilage disorder, an ophthalmic disorder, and a neurodegenerative disorder.

[0156] In a preferred embodiments- of the present invention, the subject for which the proteins, hydrolysates and compositions of the invention are intended is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, hamster, mouse, rat, rabbit, or guinea pig, but most preferably the subject is a human.

[0157] In some embodiments, the plant-based collagen-like proteins or hydrolysates can be prepared with carriers that protect it against rapid release and / or degradation, such as a controlled release formulation, such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used.

[0158] In another embodiment, the pharmaceutical composition comprises an optimal concentration of the pharmaceutically active compound eliciting an expected biological response according to the common knowledge of the skilled person, and comprises the composition comprising the plant-based collagen-like protein and hydrolysate thereof in order to support the delivery of the pharmaceutically active compound.

[0159] Pharmaceutical compositions comprising the plant-based collagen-like proteins or hydrolysates of the invention are particularly valuable for the treatment of inflammatory skin disorders, such as eczema, psoriasis, and dermatitis. These compositions can be formulated as topical applications, including creams, gels, or ointments, which deliver collagen directly to the affected skin areas. The collagen, often hydrolyzed to enhance absorption, helps to repair and rejuvenate the skin by promoting collagen synthesis and reducing inflammation. These topical formulations can also be combined with other anti-inflammatory agents and moisturizers to enhance their therapeutic efficacy. In some embodiments, collagen-based compositions may be used in conjunction with systemic therapies to provide comprehensive management of chronic inflammatory skin conditions.

[0160] For bone and cartilage disorders, such as osteoarthritis and osteoporosis, pharmaceutical compositions comprising the plant-based collagen-like proteins or hydrolysates of the invention also offer therapeutic benefits. These compositions are typically formulated as oral supplements, including tablets, capsules, or powders, which provide bioactive the plant-based collagen-like proteins or hydrolysates that support joint health and bone regeneration. Hydrolyzed plant-based collagen-like proteins in these formulations allow to be easily absorbed and utilized by the body to enhance cartilage repair and bone density. Additionally, injectable formulations, such as solutions or suspensions, can be used for targeted delivery into joints or bone tissues to provide localized relief and support tissue regeneration. Such compositions may be used as part of a comprehensive treatment regimen that includes physical therapy and other medications.

[0161] Pharmaceutical compositions comprising the plant-based collagen-like proteins or hydrolysates of the invention also allow for treating ophthalmic and neurodegenerative disorders. In ophthalmic applications, the formulations can be developed as eye drops, gels, or inserts that deliver the plant-based collagen-like proteins or hydrolysates of the invention directly to the cornea or other eye tissues, supporting healing in conditions such as dry eye syndrome or corneal ulcers. These formulations help to maintain the integrity of the ocular surface and promote tissue regeneration. For neurodegenerative disorders, such as Alzheimer's disease or Parkinson's disease, the plant-based collagen-like proteins or hydrolysates of the invention can be formulated as oral supplements or injectable solutions aimed at supporting neuroprotection and brain tissue repair. By providing essential collagen- like proteins or peptides that contribute to extracellular matrix stability and neuronal support, these compositions offer therapeutic benefits, which may be provided alongside conventional treatments.

[0162] Cosmetic composition

[0163] In one embodiment, the invention provides a cosmetic composition comprising the plant-based collagen-like proteins or hydrolysates of the invention. Preferably, the cosmetic composition is a topical composition which may comprise a topical carrier and / or a preservative. Topical compositions include but are not limited to creams, serums, and masks. These are designed to deliver he plant-based collagen-like proteins or hydrolysates directly to the skin. In a preferred embodiment, the plant-based collagen-like proteins are hydrolyzed to facilitate skin absorption and efficacy. The incorporation of the plant-based collagen-like proteins or hydrolysates into these formulations support the restoration of skin elasticity, firmness, and hydration by reinforcing the natural collagen matrix within the dermal layers. Additionally, these compositions may include synergistic components such as hyaluronic acid, vitamins, and antioxidants to further amplify the anti-aging effects, diminish fine lines and wrinkles, and improve overall skin texture. Regular application of cosmetic compositions results in a visibly healthier, more resilient skin.

[0164] Another aspect of the invention pertains to cosmetic compositions for the improvement of nail growth and strength. Such formulations are designed as topical treatments including nail creams, conditioners, and treatment oils, which are applied directly to the nails and cuticles. The plant-based collagen-like proteins or hydrolysates in these compositions are incorporated to strengthen the nail structure and enhance hydration, thus reducing brittleness and promoting healthier nail growth. The invention further includes cosmetic compositions comprising plant-based collagen-like proteins or hydrolysates for the promotion of hair health. These formulations, which can be provided as shampoos, conditioners, and hair masks, are designed to deliver plant-based collagen-like proteins or peptides to the hair and scalp. The inclusion of plant-based collagen-like proteins or hydrolysates in these compositions enhances the structural integrity of the hair by reinforcing the protein matrix and improving moisture retention. This results in stronger, more resilient hair with reduced breakage and split ends.

[0165] Preferably, the topical carrier is selected from the group consisting of liposome, biodegradable microcapsule, lotion, spray, aerosol, dusting powder, biodegradable polymer, mineral oil, triglyceride oil, silicone oil, glycerin, glycerin monostearate, alcohols, emulsifying agents, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and water.

[0166] Advantageously, the preservative is selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1 ,3-diol, cis isomer 1 -(3-chloroallyl)- 3,5,7-triaza-1 -azoniaadamantane chloride, glutaraldehyde, 4,4-dimethyl oxazolidine, 7- ethylbicyclooxazolidine, methyl paraben, sorbic acid, rosemary extract, and ethylenediaminetetraacetic acid (EDTA).

[0167] Preferably, according to the invention, the cosmetic composition comprises a concentration of the plant-based collagen-like protein or hydrolysate that is dermatologically effective, such a concentration range of the plant-based collagen-like protein or hydrolysate thereof, in the composition, elicits a dermatological response on the subject receiving the composition as expected from healthcare professional (for example improving skin condition).

[0168] Biomaterial composition

[0169] In one embodiment, the present invention provides a biomaterial comprising the plantbased collagen-like protein or hydrolysate of the invention. Such a biomaterial may be advantageous in order to repair tissue, particularly joint or cartilage tissue.

[0170] By the term “biomaterial”, it is meant according to the invention, a material engineered to interact with biological systems. For example, a bandage on a wound containing an active compound which aims to accelerate wound recovery.

[0171] Preferably, the biomaterial may be in the form of a coating material, a monolith, microspheres, a foam or a gel.

[0172] Advantageously, the biomaterial comprise a mean to stabilize the composition such as for example crosslinking collagen proteins together or integrating the collagen protein inside a synthetic hydrogel network.

[0173] Collagen application

[0174] Collagen is one of the main components of the extracellular matrix of the dermis and articular cartilage and influences the body's mechanical, organizational, and tissue formation properties. Literature data have shown that collagen and hydrolyzed collagen supplementation promotes skin changes, such as decreased wrinkle formation; increased skin elasticity; increased hydration; increased collagen content, density, and synthesis, which are factors closely associated with aging-related skin damage. Regarding orthopedic changes, collagen supplementation increases bone strength, density, and mass; improves joint stiffness / mobility, and functionality; and reduces pain.

[0175] Collagen depletion occurs in aging process. The natural aging process and various pathologies correlate with alterations in the composition and the structural and mechanical integrity of the connective tissue. Collagens represent the most abundant matrix proteins and provide for the overall stiffness and resilience of tissues. The structural changes of collagens and their susceptibility to degradation are associated with skin wrinkling, bone and cartilage deterioration, as well as cardiovascular and respiratory malfunctions. Therefore collagen supplementation may prevent physiological alterations or improve physiological functions altered by collagen depletion. Furthermore, during hemostasis, collagen induces platelet activation and aggregation, thereby depositing fiber clots at the injured site. In the inflammatory stage, the activation of immune cells promotes the secretion of inflammatory cytokines, thus affecting the migration of fibroblasts, epithelial cells, and endothelial cells, and fibroblasts contribute to the deposition of collagen. Collagen degradation releases fragments that promote fibroblast proliferation and growth factor synthesis, leading to angiogenesis and reepithelialization (Zhou et al., Pharmaceutics 2023 15(5): 1443). Various studies confirmed that the administration of collagen or collagen hydrolysates reduced inflammation, at least partly through the inhibition of inflammatory cytokines.

[0176] In one aspect, according to the invention, it is provided a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a (pharmaceutical) composition of the invention for use in the treatment and / or prevention of a disorder selected from the group consisting of an inflammatory skin disorder, a bone or cartilage disorder, an ophthalmic disorder, neurodegenerative disorder, and neuromuscular disorder.

[0177] Advantageously, the composition for use in the treatment and / or prevention of a disorder selected from the group consisting of an inflammatory skin disorder, a bone or cartilage disorder, an ophthalmic disorder, and a neurodegenerative disorder, is the pharmaceutical composition described herein.

[0178] In a particular embodiment, the inflammatory skin disorder is a disorder selected from the group consisting of atopic dermatitis (eczema), psoriasis, contact dermatitis, seborrheic dermatitis, rosacea, lichen planus, lupus erythematosus, dermatitis herpetiformis, pityriasis rosea, urticaria, bullous pemphigoid, cutaneous vasculitis, perioral dermatitis, lichen sclerosus, and alopecia areata. In a particular embodiment, the inflammatory skin disorder is dermatitis, in particular atopic dermatitis (eczema), contact dermatitis, seborrheic dermatitis, dermatitis herpetiformis, and perioral dermatitis. In a preferred embodiment, the inflammatory skin disorder is atopic dermatitis (also known as eczema).

[0179] Collagen and collagen hydrolysates are beneficial in treating bone and cartilage disorders by supporting tissue repair, reducing joint pain, and promoting overall joint health. In conditions like osteoarthritis, where cartilage deteriorates, collagen supplements stimulate the production of new cartilage by enhancing the activity of chondrocytes (cartilage cells). This helps repair damaged cartilage, reduce pain, and improve joint function. Collagen also strengthens tendons and ligaments, aiding in recovery from injuries and improving flexibility. Additionally, it supports the production of synovial fluid, which lubricates joints, further reducing friction and wear.

[0180] For bone health, collagen plays a key role in maintaining the bone matrix and promoting mineral deposition, crucial for preventing bone loss in conditions like osteoporosis. Collagen hydrolysates can enhance bone density and reduce fracture risk by stimulating the production of bone-forming cells (osteoblasts). The supplementation also speeds up recovery from fractures and improves the quality of bone and cartilage tissue regeneration. While not a cure, collagen supplements provide valuable support in managing bone and cartilage disorders by improving tissue structure and function. Therefore, in a particular embodiment, the plant-based collagen-like protein of the invention, the hydrolysate of the invention, or the (pharmaceutical) composition of the invention is for use in the treatment and / or prevention of a bone or cartilage disorder. Bone disorders for prevention and / or treatment include in particular:

[0181] - Osteoporosis: Collagen can enhance bone mineral density and reduce fracture risk by supporting the bone matrix.

[0182] - Osteopenia: Collagen can improve bone density and prevent the progression to osteoporosis.

[0183] Bone Fractures: Collagen can speed up healing by promoting bone regeneration and improving structural integrity.

[0184] - Paget’s Disease of Bone: Collagen may aid in normalizing bone remodeling and strengthening the bone matrix.

[0185] - Osteogenesis Imperfecta: Collagen supplementation may help strengthen bones, though it won’t correct the underlying genetic defect.

[0186] Cartilage disorders for prevention and / or treatment include in particular:

[0187] - Osteoarthritis: Collagen supports cartilage repair and regeneration, reduces pain, and improves joint function.

[0188] - Rheumatoid Arthritis: Collagen can help reduce inflammation, support cartilage repair, and improve joint mobility.

[0189] - Chondromalacia Patellae: Collagen may aid in repairing damaged cartilage and reduce knee pain.

[0190] - Cartilage Injuries (e.g., Sports Injuries): Collagen promotes cartilage regeneration and speeds up recovery from injury.

[0191] - Achondroplasia: Collagen may help support cartilage health, although it does not address the genetic cause of the condition.

[0192] Ehlers-Danlos Syndrome (Cartilage-Related Forms): Collagen supplementation may improve connective tissue strength and joint function.

[0193] Therefore, in a particular embodiment, the bone or cartilage disorder is selected from the group of osteoporosis, osteopenia, bone fractures, Paget’s disease of bone, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, chondromalacia patellae, cartilage injuries, achondroplasia, and Ehlers-Danlos syndrome. In particular from the group consisting of osteoporosis, osteopenia, osteoarthritis and rheumatoid arthritis. A preferred embodiment concerns the treatment and / or prevention of a bone disorder, particularly osteopenia or osteoporosis. Another preferred embodiment concerns the treatment and / or prevention of a cartilage disorder, particularly osteoarthritis or rheumatoid arthritis.

[0194] Collagen plays a crucial role in maintaining eye health and can benefit various eye diseases due to its structural and functional properties. It is a primary component of the cornea, providing strength, shape, and transparency, and collagen-based eye drops or implants may enhance corneal healing after injuries or surgeries, promoting tissue regeneration. In cases of dry eye syndrome, collagen's hydrophilic properties allow it to attract and retain moisture, and topical collagen eye drops can provide lubrication and improve tear stability. Collagen is also involved in the extracellular matrix of the retina, supporting retinal cell integrity and potentially benefiting conditions like diabetic retinopathy and age-related macular degeneration through neuroprotective effects. Additionally, collagen supports the eye lens’s structure, and its supplementation may help prevent cataract formation by maintaining lens transparency. In glaucoma, collagen helps maintain optic nerve structure, and its neuroprotective properties may safeguard optic nerve cells from damage. Furthermore, collagen's significance in the sclera and vitreous body is essential for overall eye health, with potential treatments aiding conditions like posterior vitreous detachment. Lastly, collagen is vital in the wound healing process, promoting recovery from ocular surface injuries or surgeries by providing a scaffold for new cell growth. Overall, collagen’s unique properties make it a valuable component in treating and managing various eye diseases, enhancing eye health and promoting tissue repair.

[0195] Therefore, in a particular embodiment, the plant-based collagen-like protein of the invention, the hydrolysate of the invention, or the (pharmaceutical) composition of the invention is for use in the treatment and / or prevention of an ophthalmic disorder. Ophthalmic disorders include in particular:

[0196] - Corneal disorder: Injuries to the cornea, such as abrasions or chemical burns, can be treated with collagen-based eye drops or patches that promote healing and regeneration of corneal tissue.

[0197] Dry Eye Syndrome: This condition, characterized by insufficient tear production or rapid tear evaporation, benefits from collagen administration to provide lubrication and improve tear stability.

[0198] - Ocular Surface Disorders: Conditions affecting the conjunctiva and cornea that lead to inflammation or discomfort can be treated with collagen-based therapies that help repair and regenerate damaged tissue.

[0199] - Osteogenesis Imperfecta (Ocular Symptoms): This genetic disorder, which can lead to various eye issues, benefits from collagen supplementation to improve overall connective tissue health and ocular integrity.

[0200] - Cataracts: Clouding of the eye lens, leading to vision impairment, is supported by collagen supplementation to slow cataract development.

[0201] - Glaucoma: This condition, characterized by increased intraocular pressure damaging the optic nerve, may be treated with collagen that provides neuroprotective effects to safeguard optic nerve cells. Retinal Diseases (e.g., Diabetic Retinopathy, Age-Related Macular Degeneration): Conditions affecting the retina that can lead to vision loss may benefit from collagen-derived peptides that support retinal cell survival and function.

[0202] - Chondromalacia Patellae (Ocular Symptoms): While primarily a knee condition, collagen may improve overall joint health, which can have secondary benefits for ocular mobility and function.

[0203] - Scleral Disorders: Conditions affecting the sclera, the white outer coating of the eye, can be treated with collagen-based treatments that help maintain scleral integrity and support overall eye health.

[0204] - Vitreous Degeneration: Changes in the vitreous body that can lead to issues like floaters or retinal detachment may be addressed with collagen that supports the structural integrity of the vitreous humor.

[0205] Postoperative injuries (e.g., after LASIK or Cataract Surgery): Recovery from eye surgeries can be enhanced with collagen-based eye drops or implants that promote healing and reduce inflammation.

[0206] Therefore, in a particular embodiment, the ophthalmic disorder is selected from the group consisting of corneal disorder, dry eye syndrome, ocular surface disorder, osteogenesis imperfecta, cataract, glaucoma, retinal diseases (particularly diabetic retinopathy or age- related macular degeneration), chondromalacia patellae, scleral disorder, vitreous degeneration, and postoperative injuries. In a preferred embodiment, the ophthalmic disorder is selected from glaucoma, cataract, corneal disorder, diabetic retinopathy, and age-related macular degeneration. More preferably glaucoma or cataract.

[0207] In another aspect, the invention provides the (non-therapeutic) use of a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a (cosmetic) composition of the invention. Such use may comprise the use for improving a skin condition, in particular, to promote densification and / or homogenization of the epidermis, reduce wrinkles, reduce stretch marks, plump and / or firm the skin, to reduce for accelerating and strengthening the nails and hair, for improving endurance performance and / or for improving mental performance.

[0208] The present invention furthermore provides a method for the treatment and / or prevention of a disorder, comprising administering a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a composition of the invention to a subject in need thereof. In a particular embodiment, the present invention provides a method for the treatment and / or prevention of an inflammatory skin disorder, comprising administering a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a (pharmaceutical) composition of the invention to a subject in need thereof. In embodiments related to the treatment and / or prevention of an inflammatory skin disorder, the plant-based collagen-like protein, the hydrolysate of the invention, or the composition of the invention is preferably administered topically, more preferably wherein the composition is a cream, gel, or ointment.

[0209] In a particular embodiment, the present invention provides a method for the treatment and / or prevention of a bone or cartilage disorder, comprising administering a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a (pharmaceutical) composition of the invention to a subject in need thereof. In embodiments related to the treatment and / or prevention of an inflammatory bone or cartilage disorder, the plant-based collagen-like protein, the hydrolysate of the invention, or the composition of the invention is preferably administered orally, more preferably wherein the composition is a pill, tablet, capsule, powder or liquid. The provision of a protein hydrolysate of the invention is particularly preferred.

[0210] In another particular embodiment, the present invention provides a method for the treatment and / or prevention of an ophthalmic disorder, comprising administering a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a composition of the invention to a subject in need thereof. In embodiments related to the treatment and / or prevention of an ophthalmic disorder, the plant-based collagen-like protein, the hydrolysate of the invention, or the composition of the invention is preferably administered to the eye, more preferably the composition is formulated as an eye drop, gel, or insert.

[0211] In another particular embodiment, the present invention provides a method for improving skin condition, for improving the growth and / or strength of nails or hair, for improving endurance performance, or for improving mental performance; the method comprising administering a plant-based collagen-like protein of the invention, a hydrolysate of the invention, or a composition of the invention to a subject in need thereof. In embodiments related to improvements for skin, hair, or nails, the plant-based collagen-like protein, the hydrolysate of the invention, or the composition of the invention is preferably administered topically. Preferred embodiments relate to improvements for skin by administering a cosmetic composition to the skin, in particular a cream, gel, or ointment.

[0212] As will be understood from the disclosures herein, the present invention also provides the use of the proteins, hydrolysates, or compositions of the invention for the manufacture of a medicament, in particular a medicament for the treatment and / or prevention of the disorders as specified herein.

[0213] Collagen and collagen hydrolysates are used as gelling agents in a variety of industrial applications due to their unique structural and functional properties. Collagen forms a triplehelix structure that can create gels with varying degrees of firmness, depending on the concentration and temperature. When subjected to hydrolysis, collagen is broken down into smaller peptides, resulting in collagen hydrolysates that exhibit enhanced solubility and bioavailability while retaining gelling capabilities. These materials can be utilized as effective gelling agents in food products, pharmaceuticals, cosmetics, and biomedical applications, where they can form biocompatible gels. Additionally, collagen hydrolysates possess the advantage of being able to gel at lower temperatures, offering versatility in formulations where heat-sensitive components are involved. The adjustable rheological properties of collagen and its hydrolysates make them highly adaptable for use in novel gel-based systems, contributing to the creation of tailored textures, controlled release mechanisms, and structural integrity in a variety of end products. Therefore, in another aspect, the invention provides the use of the plant-based collagen-like protein , hydrolysates thereof, or the composition as a gelling agent. Such a gelling agent provides a three-dimensional structural network having a high degree of physical / chemical cross-linking to produce gel or semisolid systems when dissolved or dispersed in an appropriate media. The plant-based collagen-like protein used as a gelling agent, in appropriate concentrations, may enhance thickness or viscosity of a composition.

[0214] Examples

[0215] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not construed as limiting the scope thereof.

[0216] Example 1. Identification of plant-based collagen-like proteins

[0217] The inventors surprisingly identified a particular hypothetical protein from Solanum lycopersicum as having collagen-like properties. Although collagen and collagen-like molecules were considered to be generally exclusive to animals, the inventors thus surprisingly identified an alternative in plants, which is assumed to be the first identified plant-based collagen-like protein. The protein is available in the UniProt database as a predicted, uncharacterized and unreviewed protein with low annotation score. The protein sequence is available under UniProt reference K4CAX9 (sequence entry version 1 ). The protein has the amino acid sequence of: MYGGLMAGNGGSGGVGKPGTRGGDDGLVLGNGGSDGIGKPGIRGEDGELGDPGNEGTIG IGNPGTRGGDDGLVLGNGGSDGIGKPGISGEDGELGEPGNEGTNGIGKPGTRGGDVGLVL GNGGIDGIGKPGIRGEDREVCDPGIRGGDDGLALGNGGGDGIGKPGIRGEHGDPGNEGTN GIGKPGIRGGDGELGDPGNEGTNGIGKPGIRGEDGELGDPGNEGTNGIGKPGIRGEDGDP GNEGTNGIGKPEIRGEDGEAGDPGNGIGKPGTEGRDDGLVLGNGGSDGIGKPGIRGKDGE LDEPGTEGGLAPGNGGISDGAKRDGSRGLIKGGIVPDKGEGDTEIGVDAKHT (SEQ ID NO: 2).

[0218] Remarkably, this tomato collagen-like protein exhibits a high glycine content, contains six G- X-X repeat regions. Bioinformatical analysis indicates the presence of the domain from the conserved domain database family with NCBI HMM accession number NF038329.2. Analysis with AHRD v1 also suggests the presence of a collagen alpha-1 (IV) chain in the protein (Hallab, A., Kelle, K., Boecker, F., & Schoof, H. (2022). Automated Assignment of Human Readable Descriptions (AHRD)). After identification of the plant-based collagen-like protein in Solanum lycopersicum, the inventors were able to retrieve homologs of the tomato collagen- like protein in the genomes of other plants, including proteins with the amino acid sequence of SEQ ID NO: 3 (GenBank accession number TMW84546.1 ) from Solanum chilense, SEQ ID NO: 4 (GenBank accession number KAG5593631 .1 ) from Solanum commersonii, and SEQ ID NO: 5 (GenBank accession number XP 015162519.1 ) from Solanum tuberosum:

[0219] EDGELGDPGNAGSGGVGKPGTRGGDDGLVLGNGGSDGIGKPGIRGEDGELGDPGNEGTI GTGNPGTRGGDDGLVLGNGGSDGIGKPGISGENGELGEPGNEGTNGIGKPGTRGGDDGL VLGNGGIDGIGKPGIRGEDRELCHPGIRGGDDGLALGNGGSDGIGKPGIRGEDGDPGNEGT

[0220] TGIGKPGIRGEDDELGDSGTEGTNGIGKPGIRGEDGEDGDPGTEGTNGIGKPGIRGEDGDP GNEGTNGIGKPEIRGEDGEVGDPGNGIGKPGTKGGDDGLVLGNGGSDGISKPGIRGKDGE LDEPGTEGGLAPGNGGISDGAKRDGSRGLIEGGIVPDKGEGDTEIGVDEKHT (SEQ ID NO:

[0221] 3)

[0222] MYGGIMAGNGGIDGTRKIGLKGGDGGLVAGSGGMDCTGNIGMEGGDDGLVLGNGGSEGI GKPGIRGEEGELGEPGNGGTSGIGKPGTIGRDDGLVLGNGGIDGIGKLGIRGEDGELGDPG NEGTIGIGKNPGTRGGLVLGNGGCDGIGKPRIRGEDGELGDPGNEGTNGIGKPGNRGEDG

[0223] ELGDPGSDGIGKPGIRGEDGDPGTRGEDDGLVLGNGGSDGIGKPGIRSKDGEPGTKGADR GLAPGNGGTDGIGKVGTDGGLVPGNGGISDGAKRDGIKGGIVPENGEGDTEIGVDA (SEQ ID NO: 4)

[0224] MYGGIMAGNGGIDGTRKIGLKGGDGGLVAGSGGMDGTGNIGIEGGDDGLVLGNGGSDGIG KPGIRGEDGELGDPGNAGTNGIGKRGARGGDDGLVLGNGGSEGIGNPGVRGEDGELGEP GNGGTSGIGKPGTIGRDDGLVLGNGGIDGIGKPGIRGEEGELGDPGNEGTIGIGNPGTRGG

[0225] LVLGNGGSDGIGMPGIRGKDGEPGTKGADRGLAPGNGGTDGIGKVGTDGGLVPGNGGISD GAKRDGSRGGIVPENGEGDTEIGLDAKHT (SEQ ID NO: 5).

[0226] Table 1. Percent identity matrix for the four plant-based proteins based on a multiple sequence alignment of their amino acid sequences.

[0227] Table 1 presents a percent identity matrix for the four plant-based proteins based on a multiple sequence alignment of their amino acid sequences. Identified plant-based collagen-like proteins identified as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, share together at least 75% sequence identity.

[0228] Remarkably, the tomato collagen-like protein also share some sequence homology with mammalian collagens, in particular with several human collagens: 47.8% sequence homology with collagen type VII (NP 000085.1 ), 46.2% sequence homology with collagen type XXIV (AAP80185.1), 46.0% sequence homology with collagen type XXVIII (XP 054214070.1), 45.7% sequence homology with collagen type III (NP 000081 .2), 45.2% sequence homology with XXV (NP_115907.2), 45.2% sequence homology with collagen type I (CAA98968.1) and 44.7% sequence homology with collagen type XI (NP 001411040.1).

[0229] The alignment of Tomato collagen like protein and the sequences of 29 known human collagens was done using Geneious aligner inbuild in Geneious Prime software version 2024.0.7 (Dotmatics, USA). Geneious aligner is a progressive pairwise aligner, similar to ClustalW. The following software settings were used: Alignment type- Global alignment with free end gaps;

[0230] Cost matrix-Blosum62;

[0231] Gap open penalty-12;

[0232] Gap extension penalty-3;

[0233] Refinement iterations-2.

[0234] The protein sequences were also aligned using ClastalW algorithm inbuild in MegAlign software, version 7.1 (Lasergene, USA) with following alignment parameters:

[0235] Gap penalty-10;

[0236] Gap length penalty-0.1 ;

[0237] Protein Weight Matrix-Gonnet 250.

[0238] Glycine is the primary amino acid in collagen, appearing at every third position in the polypeptide chain, and it plays a crucial role in maintaining the stability and structure of collagen fibers. Computational prediction using theProtParam tool from the ExPASy server (Gasteiger et al., 2015) revealed a high abundance of glycine in the plant-based collagen-like proteins. This is illustrated in Table 2, which shows the amino acid frequency of three plantbased collagen-like proteins: K4CAX9 (from Solanum lycopersicum), TMW84546.1 (from Solanum chilense), and KAG5593631.1 (Solanum commersonii), and XP 015162519.1 (Solanum tuberosum). For comparison, the table also includes five human collagen proteins: type I (uniprot reference P02452, NCBI reference: NP 000079.2), type V (uniprot reference P20908, NCBI reference: KAI4009058.1), type VII (uniprot reference Q02388, NCBI reference: NP 000085.1), type XIX (uniprot reference Q14993, NCBI reference; NP 001849.2), and type XXV (uniprot reference Q9BXS0, NCBI reference: NP 942014.1). Table 2. Amino acid composition of plant-based collagen like proteins and human collagen Another notable characteristic of collagen's amino acid composition is the rare presence of certain important amino acids, including lysine (Lys), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), and arginine (Arg) (Owczarzy et al., 2017), which are also present in the plant-based collagen-like protein as shown in Table 2.

[0239] All human collagens share a characteristic triple-helical structure formed by a repeating (Gly- X-X) sequence in each of the three a-chains. The key difference between fibril-forming collagens and non-f ibrillar collagens, such as type IV, lies in the interruptions of these triplehelical sequences. This repeating pattern is a distinctive feature crucial for the elasticity and structural integrity of collagen. Fig. 1 shows the G-X-X repeat regions (grey boxes) that are present in the proteins of the invention.

[0240] Example 2. Production of a tomato collagen-like protein in Plchla pastorls

[0241] The nucleotide sequence of the gene encoding the tomato collagen-like protein is presented in SEQ ID NO: 1 . A codon optimized version was synthetically created with selective substitutions to improve the translational efficiency of the target gene in Pichia pastoris (SEQ ID NO:6). The synthetic gene was cloned into the expression vector pJUGaMF (Biogramatics, USA) using Gibson Assembly. Two different expression cassettes were constructed using different promoters and selection marker cassettes. In version 1 , the plasmid pSNY-023-10 was assembled by cloning the tomato collagen-like gene under the control of the Alcohol Oxidase promoter (AOX1 ), with Zeocin resistance as the selection marker gene. In version 2, the plasmid pSNY-023-11 was assembled by cloning the tomato collagen-like gene under the control of the Formate Dehydrogenase promoter (PDF), with Nourseothricin resistance as the selection marker.

[0242] The plasmids resulting from the Gibson assembly were transformed into Escherichia co / / NEB 10 and subjected to standard selection and screening for recombinants. E. co / / clones carrying the expression vectors were selected, and their plasmids were isolated to be used as templates to amplify the expression cassette by PCR, which was then linearized before being transformed into Pichia pastoris by electroporation.

[0243] Genetic transformation of Pichia strains was performed using the individual expression cassettes as well as the combined integration where the two expression cassettes were simultaneously integrated in the genome of Pichia pastoris. For this purpose, both AOX1 - and PDF-driven cassettes were amplified. The PCR products were separated on a 0.8% agarose gel and purified using the QIAquick Gel Extraction Kit (QIAGEN). Two hundred nanograms of each cassette were added to 50 pl of electrocompetent Pichia pastoris Bg10 cells and electroporated simultaneously at 1150V using a MicroPulser Electroporator (BioRad). After a 4-hour recovery in 1 ml of liquid YPD supplemented with sorbitol to a final concentration of 500 mM (28°C, 100 RPM), 200 pl of the yeast suspension was inoculated onto YPD agar plates supplemented with Zeocin (100 mg / L) and Nourseothricin (80 mg / L). Plates were then incubated at 28°C for 2xON (two overnights). The resulting single colonies were screened for target protein production in a small-scale fermentation with a final volume of 2.5 ml, with methanol feeding. The protein expression capacity of the selected clone, named SNY-023-12, was confirmed in a 20L-scale pilot fermenter (see Example 3).

[0244] SDS-PAGE gel analysis confirmed the expression of the tomato collagen-like protein in Pichia pastoris BG10, under the control of the AOX1 or PDF promoters. The experiment furthermore demonstrated enhanced expression of the recombinant tomato collagen-like protein after 96 hours in the clones containing both expression cassettes simultaneously, as determined by more intense protein bands between 37 and 50 kDa, having the expected protein size.

[0245] Example 3. Pilot-Scale Fermentation and Yield Characteristics

[0246] To evaluate scalability, the recombinant Pichia pastoris strain SNY-023-12 described in Example 2 was cultivated in a pilot-scale 20 litter bioreactor under fed-batch conditions. The working volume was 12 L, with dissolved oxygen maintained through automated agitation and aeration control. A glycerol-based feed was supplied to sustain high biomass accumulation for 13-18 hours, followed by a methanol-fed induction phase lasting 18-135 hours. The culture achieved very high cell densities, with optical density values at 600 nm exceeding 400, corresponding to a dry cell weigh approximately 135 g / L. Recombinant protein of tomato collagen like protein yields exceeded 20 grams per litter.

[0247] Following fermentation, cells were harvested by centrifugation and the supernatant clarified through crossflow microfiltration (0.2 pm pore diameter). The clarified supernatant was subjected to desalination and concentration by ultrafiltration (10 kDa cutoff), and the resulting protein concentrate was spray-dried using a spray granulator.

[0248] Example 4. Preparation of Hydrolyzed Recombinant Protein for Cosmetic Applications

[0249] In the cosmetic industry, hydrolyzed collagen is valued as a moisturizer, film-former, and skin / hair conditioner, contributing to hydration, improved texture, and enhanced elasticity. Conventionally, such materials are obtained from animal sources using proteolytic enzymes such as alcalase, papain, or pepsin.

[0250] In this example, the recombinant protein obtained in Example 3 was digested with alcalase, a widely used nonspecific protease in cosmetic and food applications. Hydrolysis was performed under mild conditions at pH 7.0 and 37 °C for one hour. SDS-PAGE analysis confirmed hydrolysis of the recombinant protein (Fig. 2). Most of the protein in the intact tomato collagen-like protein (TCLP) sample was present in a range of about 30-50 kDa. Following enzymatic digestion with alcalase, the hydrolyzed protein (TCLPh) displayed a clear shift toward lower molecular weight fragments, consistent with proteolytic cleavage. The resulting hydrolysate comprised peptides in the range of 3-10 kDA, demonstrating generation of collagen-like hydrolysate suitable for cosmetic and food applications.

[0251] The hydrolysate exhibited amino acid profiles enriched in glycine, alanine, serine, and aspartate, consistent with natural moisturizing factors known to support skin hydration. In addition, the recombinant hydrolysate contained functional groups such as hydroxyl (-OH), amide (-CONH2), carboxyl (-COOH), amine (-NH2), and sulfhydryl (-SH), which are capable of forming hydrogen bonds with water molecules and thereby enhancing moisture retention. These properties demonstrate the suitability of the recombinant hydrolysed protein as a vegan alternative to animal-derived collagen for use in cosmetic formulations.

Claims

39Claims1 . A plant-based collagen-like protein comprising- at least one G-X-X-G-X-X-G-X-X sequence wherein X is each independently any amino acid,- at least 15% glycine, and- 3% to 25% proline.

2. The plant-based collagen-like protein according to claim 1 comprising:- from 30 to 40% glycine,- at least three G-X-X repeat regions, each comprising at least 5 G-X-X repeat sequences; wherein X is at each occurrence independently any amino acid, and wherein between 9 and 15% of X in the protein is proline, and- wherein from 20 to 70 amino acids are located between each of the three G-X-X repeat regions.

3. The plant-based collagen-like protein according to claim 1 or 2, wherein the molecular weight is in the range of 5 to 50 kDa.

4. The plant-based collagen-like protein according to any of the previous claims, comprising an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

5. The plant-based collagen-like protein according to any of the previous claims, which has been produced by microbial expression.

6. A nucleotide sequence encoding the plant-based collagen-like protein according to any of the previous claims.

7. An expression vector comprising a nucleotide sequence according to claim 6, and a promotor operably linked to the nucleotide sequence to allow the expression of the encoded plant-based collagen-like protein according to claim 1 to 5.

8. A host cell comprising the nucleotide sequence according to claim 6, or the expression vector according to claim 7.

9. A hydrolysate of the plant-based collagen-like protein according to any of claims 1 to 5, preferably having an average molecular weight from 0.5 to 15 kDa.4010. A method to produce a plant-based collagen-like protein according to any of claims 1 to 5, comprising the following steps :- providing a host cell according to claim 8,- culturing the host cell to express the plant-based collagen-like protein,- recovering the expressed recombinant plant-based collagen-like protein, and- optionally, hydrolyzing the recovered recombinant plant-based collagen-like protein.

11. A composition, such as a dietary supplement, a food ingredient, a cosmetic composition, a pharmaceutical composition, or a biomaterial, comprising the recombinant plant-based collagen-like protein according to any of claims 1 to 5, or the hydrolysate of the plant-based collagen-like protein according to claim 9.

12. The plant-based collagen-like protein according to any of claims 1 to 5, the hydrolysate according to claim 9, or the composition according to claim 11 , for use as a medicine.

13. The plant-based collagen-like protein according to any of claims 1 to 5, the hydrolysate according to claim 9, or the composition according to claim 11 , for use in the treatment and / or prevention of a disorder selected from the group consisting of an inflammatory skin disorder, a bone or cartilage disorder, an ophthalmic disorder, and a neurodegenerative disorder.

14. Non-therapeutic use of the plant-based collagen-like protein according to any of claims 1 to 5, the hydrolysate according to claim 9, or the composition according to claim 1 1 , for improving skin condition, for improving the growth and / or strength of nails or hair, for improving endurance performance, or for improving mental performance.

15. Use of the plant-based collagen-like protein according to any of claims 1 to 5, the hydrolysate according to claim 9, or the composition according to claim 1 1 , as a gelling agent.

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