Methods of producing collagen

Abstract

This invention relates to methods of producing exogenous collagen in photosynthetic organelles and photosynthetic organisms, in particular methods of producing exogenous collagen in the plastid of an alga or plant, particularly in chloroplasts. Genetically altered algae and genetically altered plants, plant parts thereof or plant cells which express collagen in their chloroplasts are also included.

Description

[0001] Methods of producing collagen

[0002] FIELD OF INVENTION

[0003] This invention relates to methods of producing exogenous collagen in photosynthetic organelles and photosynthetic organisms, in particular methods of producing exogenous collagen in the plastid of an alga or plant, particularly in chloroplasts. Genetically altered algae and genetically altered plants, plant parts thereof or plant cells that express collagen in plastids, and specifically in chloroplasts are also included.

[0004] BACKGROUND

[0005] Collagen is the most abundant protein in animals, making up a significant portion of the skin, hair, nails, muscles, and tendons. Collagen's main role is to provide structure, strength and support throughout the body. Sufficient levels of collagen are important for all animals to stay healthy, including animals commonly kept as pets and humans. Adequate levels of collagen are also important for maintaining a youthful appearance.

[0006] Collagen production decreases when animals age, leading to both health problems and cosmetic concerns. Specifically, the loss of collagen with age is associated with wrinkles, sagging skin, and weakened joints. These detrimental effects lead many individuals to resort to supplements to boost or maintain their collagen levels.

[0007] Collagen for supplements is primarily sourced from animal or cadaver sources. In addition to being unsuitable for individuals living a vegan or vegetarian lifestyle, collagen sourced from animals or cadavers involves several risks including allergenicity and cross-contamination of pathogens. The current methods of collagen production also face significant qualitative and environmental challenges. Current methods for synthetic collagen production contribute substantially to deforestation, high carbon emissions (159.57 kg CO2 eq / Kg) and often achieves only low-availability of ultra-pure collagen.

[0008] These issues have led to an interest in synthetic collagen. Although synthetic routes resolve the ethical challenges of animal-sourced collagen, they are still associated with high carbon emissions and low availability of ultra-pure collagen. As a result of low

[0009] 15252506-1availability and high costs, synthetic collagen remains an expensive, inadequate alternative.

[0010] The global demand for collagen is increasing and the existing issues with production systems mean that global demand is unlikely to be met.

[0011] The anticipated global demand for collagen is estimated to reach 194,819 metric tons by 2035 and poses a significant environmental concern, with CO₂e emissions projected to reach 3.10 million metric tons CO₂e

[0012] There is therefore a need for new methods of producing and sourcing collagen, which are more ethical and sustainable. Such new methods would ideally also increase the availability of ultra-pure collagen. The present invention addresses this problem.

[0013] SUMMARY OF INVENTION

[0014] In a first aspect of the invention, there is a method of producing collagen in a plastid of a photosynthetic organism, the method comprising introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0015] at least one collagen a chain suitable for producing collagen;

[0016] at least one plastid regulatory sequence, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0017] a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0018] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0019] optionally a selectable marker.

[0020] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the photosynthetic organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0021] at least one collagen a chain suitable for producing collagen;

[0022] 15252506-1at least one plastid regulatory sequence selected from a group consisting of the promoter of rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR of rrn, psaA, psbA, rps16, rbcL and atpA;

[0023] a plastid terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA;

[0024] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene suitable for integrating the sequence encoding at least one collagen a chain into a plastid genome and

[0025] optionally a selectable marker, selected from neo, aadA, nptII, aphA6, badh and npII-aphA6.

[0026] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0027] a sequence encoding at least one collagen a chain suitable for producing collagen;

[0028] at least one plastid regulatory sequence comprising the promoter of psbA, psaA, or atpA and / or the 5’UTR of psbA, psaA, or atpA;

[0029] a plastid terminator comprising TrbcL,

[0030] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, suitable for integrating the sequence encoding at least one collagen a chain into a plastid genome, and optionally a selectable marker, comprising aadA.

[0031] In one embodiment, the photosynthetic organism comprising the nucleic acid construct is grown at a first temperature and subsequently is grown at a second temperature;

[0032] wherein the second temperature is higher than the first temperature, preferably wherein the first temperature is at or around 15 °C to 25 °C, and the second temperature is at or around 28 °C to 40 °C,

[0033] and wherein the change from the first temperature to the second temperature is associated with an increase in collagen production per unit of time.

[0034] 15252506-1In a further aspect of the invention, there is provided a method of producing collagen in a plastid of a photosynthetic organism, the method comprising introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding at least one collagen a chain suitable for producing collagen and at least one plastid regulatory sequence comprising the promoter of psaA from Chlamydomonas reinhardtii and / or the 5’UTR of psbA from Chlamydomonas reinhardtii.

[0035] In one embodiment, the at least one plastid regulatory sequence comprises the 5’UTR of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

[0036] In one embodiment, the at least one plastid regulatory sequence comprises the promoter of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 12 or 83 or functional variant or homolog thereof.

[0037] In a further aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding:

[0038] at least one collagen a chain suitable for producing collagen;

[0039] at least one plastid regulatory sequence, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0040] a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0041] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0042] optionally a selectable marker.

[0043] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0044] at least one collagen a chain suitable for producing collagen;

[0045] at least one plastid regulatory sequence selected from a group consisting of the promoter of rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR of rrn, psaA, psbA, rps16, rbcL and atpA;

[0046] 15252506-1a plastid terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA,

[0047] a pair of flanking sequences comprising comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene for integrating the sequence encoding at least one collagen α chain into a plastid genome; and optionally a selectable marker, selected from neo, aadA, nptll, aphA6, badh and npll-aphA6.

[0048] In a further aspect of the invention, there is provided a genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, wherein the genetically modified organism is a plant, plant part thereof or a plant cell and / or an algae or alga.

[0049] In one embodiment, the genetically modified organism is further characterised by the expression of at least one nucleic acid construct of this invention in the plastid.

[0050] In a further aspect of the invention, there is provided a method of producing a genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, the method comprising introducing and expressing at least one nucleic acid as described herein in the plastid of the genetically modified organism.

[0051] The following embodiments apply to all aspects of the invention.

[0052] In one embodiment, the plastid regulatory sequence and plastid terminator comprises or consists of sequences obtained from Chlamydomonas reinhardtii.

[0053] In one embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two plastid-encoded genes comprise a sequence from two chloroplast-encoded genes endogenous to the organism.

[0054] In one embodiment, the plastid regulatory sequence is a promoter selected from rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR selected from rrn, psaA, psbA, rps16, rbcL and atpA, wherein the promoter of rrn, psaA, psbA, rps16, rbcL and atpA comprises or consists of SEQ ID NO: 14 or 15, 12 or 83, 16, 17, 18 and 81 or functional variant of

[0055] 15252506-1homolog thereof, respectively, and wherein the 5’UTR of rrn, psaA, and atpA comprises or consists of SEQ ID NO: 13, any one of SEQ ID NO: 84 to 86, and SEQ ID NO: 82 or functional variant of homolog thereof, respectively.

[0056] In one embodiment, plastid regulatory sequence is or comprises the promoter of psaA and / or 5’ UTR of psaA derived from Chlamydomonas reinhardtii, wherein preferably the sequence of the promoter of psaA comprises or consists of SEQ ID NO: 12 or 83 or functional variant or homolog thereof and the 5’ UTR of psaA comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

[0057] In one embodiment, the plastid regulatory sequence comprises the psaA 5’UTR from Chlamydomonas reinhardtii and comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

[0058] In one embodiment, the plastid regulatory sequence is selected from psaA 5’UTR from Chlamydomonas, prfA 5’UTR from Listeria monocytogenes, agsA 5'UTR from Salmonella, ibpA 5 ‘UTR from Vibrio cholerae, PIF75’ UTR from Arabidopsis thaliana, and the hsp175’UTR from Synechocystis.

[0059] In one embodiment, the plastid terminator comprises TpsbA, TrbcL or Trsp16, wherein TpsbA, TrbcL or Trsp16 comprises or consists of SEQ ID NO: 19, 21 or 92, 20 or 91, and 92 or functional variant or homolog thereof, respectively.

[0060] In one embodiment, the plastid terminator comprises TrbcL and comprises or consists of SEQ ID NO: 19 or 21 or functional variant or homolog thereof.

[0061] In one embodiment, the nucleic acid sequence comprises the selection marker aadA, wherein aadA comprises or consists of a sequence defined in SEQ ID NO: 22 or functional variant or homolog thereof.

[0062] In one embodiment, the nucleic acid construct encodes a nucleic acid sequence encoding at least one collagen a chain, wherein the at least one collagen a chain comprises or consists of a sequence selected from SEQ ID NO: 1 or 24 to 52 or 88 to 90 or functional variant or homolog thereof.

[0063] 15252506-1In one embodiment, the photosynthetic organism is selected from a plant, plant part thereof or plant cell, and / or an alga or algae.

[0064] In one embodiment, the photosynthetic organism is a plant, part thereof or plant cell, and wherein the plant is selected from the group consisting of tobacco (Nicotiana), lettuce, maize (Zea mays), alfalfa (Medicago sativa), rice (Oryza sativa), potato (Solanum tuberosum), soybean (Glycine max), tomato (Solanum lycopersicum), wheat (Triticum), barley (Hordeum vulgare), canola (Brassica napus) and cotton (Gossypium), spinach (Spinacia oleracea) and sugar beet (Beta vulgaris), preferably tobacco (Nicotiana) or lettuce.

[0065] In one embodiment, the photosynthetic organism is a plant organism is an algae or alga, preferably Chlamydomonas reinhardtii.

[0066] In one embodiment, the plastid is a chloroplast.

[0067] DESCRIPTION OF FIGURES

[0068] Figure 1 shows the selection process of potential collagen transformants demonstrated in tobacco RMOP+S: RMOP medium is supplemented with spectinomycin (S) to select for transformed plants or tissues that are resistant to this antibiotic. RMOP+SS: RMOP medium supplemented with spectinomycin and streptomycin.

[0069] Figure 2 shows PCR confirmation of transgene integration and collagen gene detection in transgenic tobacco plant samples using specific primers. PCR amplification confirms the transgene integration and collagen gene detection in plant samples using primers F1 and R1. Transgenic samples (LC009#1A, LC009#6B, and LC009#6A) display both a 6872 bp band (collagen transgene) and a 208 bp band (endogenous chloroplast DNA) showing heterozygous plants, while LC009#1B shows only the 6872 bp band, indicating successful integration without detectable wild-type DNA and confirms homozygous plants. The wild type displays only the 208 bp band. The empty vector control (EV) shows an 1826 bp band, and the plasmid control confirms the transgene size (6872 bp). The blank control shows no amplification, confirming the absence of contamination. A 1kb plus DNA ladder provides size references. Lane 1: 1kb plus DNA Ladder; Lane 2: Wild

[0070] 15252506-1Type = 208 bp; Lane 3-6: Mutant band = 6872 bp; Lane 7: Empty Vector LC008 = 1826 bp. Gene specific primers F1 and R1.

[0071] Figure 3 shows the expression and detection of collagen protein in transgenic plants via SDS-PAGE and western blot analysis (A) SDS-PAGE gel analysis of collagen (human type l / Bovine type I) protein extracted from plants (LC009#1B, LC009#1A, LC009#6B) and controls (2.5 pg and 5 pg collagen protein (Sigma Aldrich 234138; Human skin collagen type I)). " Wild type" represents no detectable collagen. Protein molecular weight markers (kDa) are shown on the left. (B) Western blot analysis of the same samples probed with a collagen-specific antibody. Collagen bands are observed in plant samples and controls, with no signal in the wildtype lane.

[0072] Figure 4 shows a method for transforming the plastid genome of Chlamydomonas reinhardtii with an expression cassette containing the collagen gene (COL1A1). a) shows the transformation plasmid used in the process, comprising: A trnE2 region for homologous recombination into the plastid genome; a collagen gene (COL1A1 CDS) driven by the psaA 5' UTR as the promoter and untranslated region; the rbcL 3' UTR, functioning as a transcription terminator for stable gene expression; the psbH gene, included as part of the homologous arms for restoration of its function in the recipient plastome; and the flanking sequences also include psbN, which serves as an additional structural element of the plastome. b) shows the initial state of the plastome in the HT72 strain, characterized by: disruption of the psbH gene, replaced with an aadA cassette conferring spectinomycin resistance (SpecR); aadA cassette flanked by the atpA 5' UTR and rbcL 3' UTR, ensuring its expression in the chloroplast genome; and presence of trnE2 and psbN as adjacent plastid loci, providing target sites for homologous recombination, c) shows the plastome after successful transformation and homologous recombination:

[0073] Figure 5 shows a method for transforming the plastid genome of Nicotiana tabacum with an expression cassette containing the collagen gene (COL1A1) under the control of Chlamydomonas reinhardtii-derived regulatory elements, a) shows the transformation plasmid used in the process, comprising: Gene of Interest (GOI), COL1A1 CDS, encoding the desired collagen protein; regulatory elements Cr_psaA 5' UTR (Chlamydomonas reinhardtii) Promoter and untranslated region for driving transcription of the collagen gene in Nicotiana tabacum plastids and Cr_rbcL 3' UTR

[0074] 15252506-1(Chlamydomonas reinhardtii) ensures proper termination and transcript stability of the COL1A1 gene in tobacco plastids; the selectable marker aadA, providing resistance to spectinomycin for selecting transformed plastids (flanked by loxP sites, facilitating marker excision using Cre recombinase); additional regulatory sequences Nt_psbA 5' UTR, a tobacco-derived untranslated region included for supporting downstream expression of the selectable marker and Nt_Prrn 5' UTR, a Tobacco-derived promoter driving marker gene transcription; and homologous arms including trnG and trnfM, facilitating precise integration of the transformation cassette into the target region of the tobacco plastid genome, b) shows the wild-type plastome of tobacco prior to transformation, featuring: the trnG and trnfM loci flanking the targeted insertion site, an insertion site, indicated as the region for homologous recombination and Primers (F1 and R1) included for molecular validation of successful integration and orientation of the expression cassette, c) illustrates the plastome after successful transformation characterised by the integrated cassette comprising the collagen gene (COL1A1 CDS) driven by the Cr_psaA 5' UTR promoter and terminated by the Cr_rbcL 3' UTR in the tobacco plastid genome and the aadA selectable marker in the plastome. The selectable marker is flanked by loxP sites, enabling its removal through recombinase-mediated excision.

[0075] Figure 6 shows a method for transforming the plastid genome of Nicotiana tabacum with an expression cassette containing multiple collagen genes under the control of Chlamydomonas reinhardtii-derived regulatory elements, a) shows the transformation plasmid used in the process, comprising: collagen peptides / genes COL1A1, COL2A1, COL3A1; Regulatory elements Nt_Prrn (promoter that drives transcription of the collagen genes), Nt_Trps16 and Nt_TrbcL (5' UTRs that enhance translational efficiency of the collagen constructs), cr_TrbcL (terminator derived from Chlamydomonas reinhardtii, ensuring transcript stability and proper termination of transcription), Nt_TpsbA (3' UTR for additional transcriptional regulation), IEE: an intercistronic expression element (IEE), that mediates the efficient intercistronic cleavage of polycistronic mRNAs into stable monocistronic transcripts; selectable marker aadA, flanked by loxP sites for subsequent removal of marker; direct repeats (DR) to support marker removal; homologous arms comprising flanking sequences corresponding to trnG and trnfM regions of the tobacco plastid genome to enable precise integration via homologous recombination, b) shows the wild-type plastome of tobacco prior to transformation, featuring: the trnG and trnfM loci flanking the targeted insertion site, an insertion site, indicated as the region for homologous recombination and Primers (F1 and R1) included for molecular validation of

[0076] 15252506-1successful integration and orientation of the expression cassette, c) illustrates the plastome after successful transformation characterised by the integrated cassette comprising the collagen genes driven by the Cr_psaA 5' UTR promoter and terminated by the Cr_rbcL 3' UTR in the tobacco plastid genome and the aadA selectable marker in the plastome. The selectable marker is flanked by loxP sites, enabling its removal through recombinase-mediated excision.

[0077] Figure 7 shows a method for transforming the plastid genome of Lactuca sativa with an expression cassette containing multiple collagen genes under the control of Chlamydomonas reinhardtii (Cr)-derived or Nicotiana tabacum (Nt) regulatory elements, a) shows the transformation plasmid used in the process, comprising: collagen genes COL1A1, COL2A1, COL3A1; Regulatory elements Nt_Prrn (promoter that drives transcription of the collagen genes), Nt_Trps16 and Nt_TrbcL (5' UTRs that enhance translational efficiency of the collagen constructs), cr_TrbcL (terminator derived from Chlamydomonas reinhardtii, ensuring transcript stability and proper termination of transcription), Nt_TpsbA (3' UTR for additional transcriptional regulation), IEE: an intercistronic expression element (IEE), that mediates the efficient intercistronic cleavage of polycistronic mRNAs into stable monocistronic transcripts; selectable marker aadA, flanked by loxP sites for subsequent removal of marker; direct repeats (DR) to support marker removal; homologous arms comprising flanking sequences corresponding to trnl and trnA regions of the lettuce plastid genome to enable precise integration via homologous recombination, b) shows the wild-type plastome of lettuce prior to transformation, featuring: the trnG and trnfM loci flanking the targeted insertion site, an insertion site, indicated as the region for homologous recombination and primers (F1 and R1) included for molecular validation of successful integration and orientation of the expression cassette, c) illustrates the plastome after successful transformation characterised by the integrated cassette comprising the collagen genes driven by the Cr_psaA 5' UTR promoter and terminated by the Cr_rbcL 3' UTR in the lettuce plastid genome and the aadA selectable marker in the plastome. The selectable marker is flanked by loxP sites, enabling its removal through recombinase-mediated excision.

[0078] Figure 8 shows the expression of collagen protein in transgenic alga, a) Genotyping of Chlamydomonas reinhardtii HT72 mutants, b) Algal liquid culture of mutant, Wild type CC1690 and mutant strains of HT72 Chlamydomonas reinhardtii mutants, c) Purification of alga-Derived Collagen: SDS-PAGE analysis illustrates the purification of collagen from transgenic alga d) Growth of Chlamydomonas reinhardtii strains on TAP, HSM, and

[0079] 15252506-1TAP supplemented with spectinomycin (TAP+spec) media. 1. CC1690: Wild-type strain showing growth on TAP and HSM medium and no growth on TAP+spec. 2. HT72: Recipient strain showing no growth on HSM medium as non-photosynthetic in nature however shown growth on TAP+spec confirms presence of aadA. 3. Transformants for Collagen (TAP): Growth of marker-free transformants COL-1, COL-2 and COL-4 on TAP and HSM medium confirming the photosynthetic activity and no growth on TAP+spec confirming marker -free. 4. Transformants empty vector (EV): Growth of marker-free transformants Control (EV)TAP and on TAP and HSM medium confirming the photosynthetic activity and no growth on TAP+spec confirming marker free.

[0080] Figure 9 shows two constructs used to compare constitutive versus temperature-inducible plastid translation of collagen mediated by distinct 5' untranslated regions. A) shows a nucleic acid construct comprising a native tobacco plastid ribosomal RNA operon promoter (NtPrrn), a native tobacco plastid 5' untranslated region (Nt 5'UTR), the COL1A1 coding sequence, and a tobacco rbcL 3' untranslated region (Nt rbcL 3'UTR). B) shows a nucleic acid construct comprising a Chlamydomonas reinhardtii psaA promoter (CrPpsaA), the psaA 5' untranslated region (CrpsaA 5'UTR) containing a temperature-responsive RNA structure, the COL1A1 coding sequence, and the psaA 3' untranslated region (Cr psaA 3'UTR). Both constructs are suitable for expression in algae and higher plants - specifically, Chlamydomonas reinhardtii psaA promoter / UTRs function heterologously in a higher plant system. Both constructs in Figure 9 were also experimentally expressed in tobacco plastids.

[0081] Figure 10 shows the immunoblot analysis of collagen accumulation in transplastomic tobacco lines harbouring plastid-integrated COL1A1 expression cassettes controlled by either (i) native tobacco plastid regulatory elements (TPrrn-Nt 5'UTR / TrbcL-Nt 3'UTR) or (ii) Chlamydomonas reinhardtii regulatory elements (CrPpsaA-Cr psaA 5'UTR / CrpsaA 3'UTR). Total soluble protein was extracted from leaves maintained at 25 °C or following temperature elevation to 40 °C. For the Chlamydomonas regulatory construct (CrPpsaA-Cr psaA 5'UTR / CrpsaA 3'UTR), protein extracts from two independent transplastomic plants (P1 and P2) are shown at 25 °C and at 40 °C (middle lanes). Purified human collagen type I (skin) is included as a reference control (right lane; expected migration 140 kDa). Collagen was detected using an anti-collagen I antibody (upper panel). Coomassie Brilliant Blue staining of the corresponding gel is shown as a loading control (lower panel).

[0082] 15252506-1DETAILED DESCRIPTION OF INVENTION

[0083] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0084] We have demonstrated for the first time that exogenous collagen can be expressed and accumulated in the chloroplast of photosynthetic organisms. In particular, we have shown that human collagen can be expressed and accumulated in the chloroplast of the model plant, tobacco. Our studies in a model organism and archetypal plastid is proof of principle that collagen can be expressed and synthesised in planta and algal plastids.

[0085] Although chloroplasts have been used to express exogenous proteins in plants, neither chloroplasts nor any other plastid has been successfully used to synthesise collagen. Indeed, attempts to express collagen in plastids have, to date, failed due to low yields and unstable collagen protein production.

[0086] In some embodiments described herein, the invention may preferably use Chlamydomonas reinhardtii-derived regulatory elements to drive or induce gene expression in a plastid genome, enabling efficient (and optionally temperature-controlled) collagen expression in chloroplasts. This has been demonstrated, as nonlimiting examples, for tobacco (Nicotina tabacum) and lettuce (Lactuca sativa).

[0087] In particular, we have also developed a novel temperature-responsive transgene expression system using the Chlamydomonas reinhardtii-derived psaA 5' UTR regulatory element to achieve high and stable yields of collagen in plants. This system enables efficient and economical collagen production by simply adjusting the temperature during cultivation, eliminating the need for costly chemical inducers or growth regulators.

[0088] 15252506-1This is the first demonstration of a plant-based collagen production system that leverages temperature control for scalable and sustainable protein expression. The technology presents a significant advancement in molecular farming, providing a cost-effective solution for industrial production of high-value proteins such as collagen for use in cosmetics, healthcare, and food applications.

[0089] Thus, the present invention addresses the need for new methods of producing and sourcing collagen, which are more ethical and sustainable to the predominant sources of collagen (animals and cadavers). Indeed, as we explain below, the present invention can significantly mitigate CO2emissions from the global collagen production:

[0090] Net CO2e Reduction

[0091] Net Reduction = Total CO2e (Animal-derived) - CO2e Footprint according to the present invention = 159.57 - 0.5 = 159.07 kg CO2e / kg Collagen

[0092] Percentage of Mitigation:

[0093] Mitigation percentage=159.57÷ 159.07×100=99.69%.

[0094] The Total Addressable Carbon (TAC) assessment demonstrates a significant potential for mitigating CO2emissions associated with the global collagen market. By replacing animal-derived collagen, which has a CO2e value of 159.57 kg per kilogram of bovine collagen, with Applicant’s plant-based collagen, which emits only 0.5 kg CO2e per kilogram, a net reduction of 159.07 kg CO2e per kilogram of collagen can be achieved.

[0095] The global collagen production is expected to grow from 118,983 metric tons in 2025 to 194,819 metric tons in 2035, with a CAGR of 5.09%, assuming a steady increase in annual production. Using this growth trajectory, applicant’s annual total addressable carbon (TAC) is calculated by multiplying the global production for each year by the net CO2e reduction per kilogram.

[0096] By 2035, global bovine collagen production will reach 194,819 metric tons, with a cumulative TAC of approximately 3.10 million metric tons CO2e over the period 2025-2035. This represents a nearly complete elimination (99.69%) of the CO2emissions currently generated by the production of animal-derived collagen. Addressable CO2e

[0097] 15252506-1mitigation is calculated by multiplying annual collagen production by the net CO2e reduction per kilogram of collagen.

[0098] A further key feature of the invention is the provision of a system capable of expressing collagen sequences, facilitated by the use of Intercistronic Expression Elements (lEEs) which generate stable monocistronic transcripts from polycistronic mRNA.

[0099] Our use of plastid expression for collagen provides significant advantages over other synthetic methods. For example, plastids show an impressive ability to accumulate large amounts of foreign protein (up to 46% of total leaf protein) when the transgene is stably integrated. Additionally, because chloroplasts lack transgene silencing, transcripts accumulate at a significantly higher level in chloroplasts than in nuclear transgenic plants. This results in a large quantity of high-quality collagen produced by the invention described herein.

[0100] Another benefit afforded by the use of chloroplasts to express and synthesise exogenous collagen is that the site-specific integration into the chloroplast genome by homologous recombination of flanking chloroplast DNA sequences present in the chloroplast vector eliminates the concerns of position effect, frequently observed in nuclear transgenic lines.

[0101] Indeed, it has been found that the integration of the expression cassettes described herein yields:

[0102] • stable, maternally inherited transformants,

[0103] • high plastome copy number, leading to high dosage of the collagen cassette, • robust, homogeneous expression across leaf tissues,

[0104] • capacity for polycistronic expression of collagen, modifying enzymes, and folding factors,

[0105] • compatibility with temperature-responsive translation control via the thermoresponsive regulatory elements.

[0106] The expression of collagen in chloroplasts also offers significant biosafety advantages over nuclear transgene expression. Chloroplast genomes are maternally inherited in most plants, which significantly reduces the risk of gene flow into wild populations through pollen (Ruf et al., 2001; Daniell et al., 2002). This containment feature is

[0107] 15252506-1particularly important in ensuring that genetically modified traits remain restricted to cultivated crops, preventing the unintended spread of transgenes in the environment.

[0108] Furthermore, chloroplast-based expression systems can achieve high levels of transgene expression due to the polyploidy of chloroplast genomes, resulting in stable and efficient production of recombinant proteins (Maliga, 2004). This makes chloroplasts an ideal platform for the large-scale, environmentally safe production of plant-made collagen, minimizing the risks associated with traditional nuclear transformation methods.

[0109] We have determined that plastids are a successful expression system for exogenous collagens through the expression of a nucleic acid comprising a novel and innovative chloroplast-specific expression cassette, comprising a selection of chloroplast-specific recombination regions, selection markers and flanking regions. These components contribute individually and in combination to target and drive exogenous collagen expression and accumulation in the chloroplast. The identified methods advantageously produce a high yield of collagen.

[0110] Genetically altered organisms

[0111] Accordingly, in a first aspect of the invention, there is provided a genetically modified organism characterised by the expression of exogenous collagen in at least one plastid.

[0112] In one embodiment, the genetically modified organism is a photosynthetic organism.

[0113] In one embodiment, the genetically modified organism is an alga or a plant, plant part thereof or a plant cell.

[0114] Accordingly, in one aspect of the invention, there is provided a genetically modified plant, plant part thereof or plant cell characterised by the expression of exogenous collagen in at least one plastid.

[0115] In another aspect of the invention, there is provided a genetically modified alga or algae characterised by the expression of exogenous collagen in at least one plastid.

[0116] 15252506-1In one embodiment, the at least one plastid is selected from a group consisting of: at least one chloroplast, chromoplast, leucoplast, etioplasts, gerontoplasts or an apicoplast. Preferably, the at least one plastid is a chloroplast.

[0117] In a preferred embodiment, the genetically modified organism is characterised by the expression of exogenous collagen in a plurality of plastids. For example, a plurality of chloroplasts, a plurality of chromoplasts, a plurality of leucoplasts or a plurality of apicoplasts. As a second example, a plurality of plastids may consist of at least one of a first plastid and at least one of a second, different plastid. For example, at least one chloroplast and at least one plastid selected from a chromoplast, leucoplast or an apicoplast. As a third example, a plurality of plastids may consist of a plurality of first plastids and plurality of a second, different plastids.

[0118] Preferably, the genetically modified organism is characterised by the expression of exogenous collagen in a plurality of chloroplasts (belonging to said genetically modified organism).

[0119] Plants

[0120] A plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a monocot or a dicot plant.

[0121] The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned comprise the nucleic acid construct as described herein. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the nucleic acid construct as described herein.

[0122] As used herein, a “genetically modified plant” or “mutant plant” or “transgenic plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, a mutant plant is a plant that has been modified compared to the naturally occurring wild type (WT) plant using any of the transformative methods described herein. In one embodiment, the method is polyethylene glycol method for protoplasts or a biolistic particle delivery system, most preferably a biolistic particle

[0123] 15252506-1delivery system. In one embodiment, the plant genome has been altered compared to wild type sequences using a method of transformation. Such plants have a modified phenotype as described herein, namely the expression of collagen chains. Therefore, in this example, the transformation of a plant to express a collagen chain is conferred by the presence of an altered plant genome, for example, the expression of a selection marker described herein.

[0124] In one embodiment, the plant is a flowering plant, such as one belonging to the Asteraceae family.

[0125] In one embodiment, the plant is selected from asparagus, grasses, palms, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, coriander, sunflower, peanuts, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe vera, yews (taxus) aubergine, pineapple, sumac, chickpea, rosemary, lychee, liquorice (Glycyrrhiza glabra), spinach, soybean, Chinese cabbage, brassicas, carrot, corn, rice, Thuja, Juniper, Pine, Garlic, cucumber, chillies, peppers, lettuce, peaches, peas, watermelons, grapes, apples, onions, mandarins, bananas, peas, mangos, oranges, tea, sugarcane, cotton, barley, sorghum, wheat, rice, quassia, Magnolia, the Quillija spp., and Cupressaceae family. The present invention can also be used with other plants, of course.

[0126] In a preferred embodiment, the plant is selected from the group consisting of tobacco (Nicotiana), lettuce (Lactuca sativa), maize (Zea mays), alfalfa (Medicago sativa), rice (Oryza sativa), potato (Solanum tuberosum), soybean (Glycine max), tomato (Solanum lycopersicum), wheat (Triticum), barley (Hordeum vulgare), canola (Brassica napus) and cotton (Gossypium), quinoa (Chenopodium quinoa), spinach (Spinacia oleracea) and sugar beet (Beta vulgaris).

[0127] Algae

[0128] By ‘alga’ is meant a single cellular or multi-cellular photosynthetic eukaryote, belonging to the kingdom Protista. Algae is the plural of alga.

[0129] Any photosynthetic algae is intended to be within the scope of this invention. In one embodiment, the alga may be selected from one of the algae classes Chlorophyceae (Green Algae), Xanthophyceae (Yellow green algae), Chrysophyceae, Bacillariophyceae (Diatoms), Cryptophyceae, Dinophyceae, Chloromonodineae, Euglinineae,

[0130] 15252506-1Phaeophyceae (Brown algae), Rhodophyceae (Red algae) and Myxophyceae (Cyanophyceae or Blue green algae).

[0131] In a preferred embodiment, the algae is selected from the group consisting of: Chlamydomonas reinhardtii, Dunaliella salina, Spirulina (Arthrospira platensis), Phaeodactylum tricornutum, Nannochloropsis spp., Haematococcus pluvialis, Porphyridium cruentum and Euglena gracilis. In a preferred embodiment, the alga or algae is Chlamydomonas reinhardtii. More preferably, the alga or algae is Chlamydomonas reinhardtii HT72 strain.

[0132] Plastids

[0133] By ‘plastid’ is meant a membrane-bound organelle found in the cells of plants and alga, typically involved in the manufacture and store of energy sources.

[0134] Examples of plastids include chloroplasts, chromoplasts and leucoplasts.

[0135] By ‘chloroplast’ is meant the biconvex shaped, semi-porous, double membraned, cell organelle found within the mesophyll of photosynthetic cells. They are the sites for synthesizing food by the process of photosynthesis. Chloroplasts convert into chromoplasts, which store carotenoid pigments. Leucoplasts are non-pigmented organelles involved in the production and storage of nutrients. Leucoplasts can be subdivided into amyloplast, proteinoplasts and elaioplasts, which store starch, proteins and fats / oils respectively.

[0136] In one embodiment, the plastid is selected from a group comprising a chloroplast, chromoplast, and leucoplast. Preferably, the plastid is a chloroplast.

[0137] The transgenic expression of an exogenous protein into a plastid, such as a chloroplast, is known in the art. However, it has not previously been shown that collagen can be expressed in a plastid.

[0138] Collagen

[0139] 15252506-1By ‘collagen’ is meant a protein or precursor thereof belonging to the collagen family, which is characterised as containing at least one triple-helix domain. In particular, collagen refers to an exogenous collagen, defined as a collagen molecule not expressed naturally in a wild type plant or algae. In this way, a plant or alga can be used as a source of human or other animal collagen. Thus, also included in the scope of the invention is engineered collagen-like domains or recombinant collagens, truncated procollagen fragments or stabilised triple-helix motifs, or synthetic collagen-mimetic peptides designed for enhanced solubility or function.

[0140] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

[0141] The synthesis and modification of collagen Alpha (a) chains into the final collagen fibre structure is well described in the art, for example in Amirrah et al. (2022). The application intends for all precursors and intermediates of collagen fibres to be included within the scope of the present application.

[0142] Proteins in the collagen family are formed of three or more single collagen Alpha (a) chains (also known in the art as pre-procollagen chains). Depending on the collagen type, the a chain(s) undergo post-translational enzymatic hydroxylation and glycosylation to form pro- a chains which adopt a left-handed triple helix conformation. Specifically, pre-peptide sequences are removed, the hydroxylation of lysine and proline residues occurs, the pre-peptides become glycosylated, and disulphide bonding occurs.

[0143] The association of three post-translationally modified pro-a chains forms a right-handed coil triple helix structure known as procollagen. Procollagen is cleaved by procollagen N- and C-proteinases to form tropocollagen. Tropocollagen is the monomer of collagen and is defined as a right-handed bundle of three parallel, left-handed polyproline Il-type helices. The tight packing of polyproline Il-type within the triple helix mandates that every third residue be Gly, resulting in a repeating XaaYaaGly sequence, where Xaa and Yaa can be any amino acid. Multiple tropocollagen triplex helixes self-assemble into collagen microfibrils. Cross-linking and self-assembly between triple helices of microfibrils forms a collagen fibre.

[0144] 15252506-1Accordingly, as used herein, ‘collagen’ may refer to any molecule selected from a group consisting of: a collagen a chain, procollagen, tropocollagen, a collagen microfibril, and a collagen fibre.

[0145] As such, in one embodiment, the genetically altered organism of the invention is characterised by the expression of at least one collagen a chain.

[0146] In one embodiment, the genetically altered organism is characterised by the expression of at least one procollagen.

[0147] In one embodiment, the genetically altered organism is characterised by the expression of at least one tropocollagen.

[0148] In one embodiment, the genetically altered organism is characterised by the expression of at least one collagen microfibril.

[0149] In one embodiment, the genetically altered organism is characterised by the expression of at least one collagen fibre.

[0150] The application may refer to any of a collagen a chain (also referred to as a pro-a chain), procollagen and tropocollagen as a ‘collagen precursor’. As explained above, collagen a chain (also referred to as a pro-a chain), procollagen and tropocollagen are the precursors from which mature collagen microfibrils and fibres are produced.

[0151] In one embodiment, collagen may refer to a collagen peptide. By collagen peptide is meant a peptide fragment of a collagen molecule.

[0152] In one embodiment, one or more collagen peptide(s) may be produced from the collagen produced by the methods disclosed herein, i.e., one or more collagen peptide(s) are produced from a collagen a chain procollagen, tropocollagen, a collagen microfibril or collagen fibre obtained in a plastid by the methods described herein.

[0153] In one embodiment, the collagen produced by the present invention may be denatured to produce individual a chains (e.g., by heat treatment) and hydrolysed into small peptides with a smaller molecular weight.

[0154] 15252506-1The hydrolysation of collagen into collagen peptides can be achieved for example by using proteolytic enzymes (such as alcalase, papain, pepsin etc.,) or by using chemical products in acidic (acetic acid, hydrochloric acid, and phosphoric acid) or alkaline media, as described in Leon-Lopez et al (2019). Collagen peptides produced by hydrolysation are appropriately called hydrolysed collagen (peptides) and typically have a molecular weight of 3-6 KDa.

[0155] Alternatively, at least one collagen peptide may be produced directly by the methods disclosed herein. That is, in one embodiment, at least one collagen peptide is synthesised and extracted from a plastid, preferably a chloroplast, according to the methods described herein.

[0156] Accordingly, in one embodiment, there is provided a genetically altered plant, part thereof or plant cell characterised by the expression at least one of: an collagen a chain, procollagen, tropocollagen, a collagen microfibril and / or a collagen fibre.

[0157] Collagen types

[0158] Members of the collagen family are classified as a collagen ‘type’ according to the a chains forming the molecule (as shown in table 1).

[0159] The unique domain structures of the various chains contribute to the differentiation of the collagen types. As such, any type of collagen alpha chain can be expressed in a plastid according to the present invention by expressing the appropriate a chains which constitute each type of collagen. The constituent alpha chains of each collagen type is summarised in Table 1. The alpha chain also includes engineered collagen-like domains or recombinant collagens, truncated procollagen fragments or stabilised triple-helix motifs, or synthetic collagen-mimetic peptides designed for enhanced solubility or function.

[0160] Examples of collagen types include fibril-forming collagens (types I, II, III, V, and XI), networks forming collagens (types IV, VIII, and X), collagens associated with fibril surfaces (types IX, XII, and XIV), collagens which occur as transmembrane proteins (types XIII and XVII), or form 11-nm periodic beaded filaments (type VI).

[0161] 15252506-1Accordingly, in one example, collagen refers to a collagen type selected from type I, II, III, V, IX, VI, IX, XII, XIV, XIX or XX. Also, collagen may refer to an engineered collagen-like domain or recombinant collagen, truncated procollagen fragment or stabilised triplehelix motif, or synthetic collagen-mimetic based on collagen type type I, II, III, V, IX, VI, IX, XII, XIV, XIX or XX.

[0162] In a preferred embodiment, collagen refers to a fibril-forming collagen selected from Types I, II, III, V, and IX.

[0163] Type I collagen constitutes the predominant collagen in bone and tendon, and is found in significant quantities in skin, aorta, and lung. Type I collagen is a heterotrimer is composed of two alpha 1 and one alpha 2 chains, constructed from repeating Gly-X-Y triplets, where X and Y can represent any amino acid but are typically proline and hydroxyproline. Type II collagen is a fibrillar collagen, and the main component of cartilage and plays an important role in joint support. Type III collagen is found as a major structural component in hollow organs such as large blood vessels, uterus and bowel, tissues that must withstand stretching. Type III collagen is also found in many other tissues in association with type I collagen. Type V collagen is a low-abundance fibrillar collagen that is co-expressed with collagen I in many tissues and forms with it heterotypic fibrils. Collagen V plays a crucial role in the assembly of these heterotypic fibres and in regulating their diameter. Type IX is found primarily in interstitial cartilage.

[0164] Table 1. Structure of the members of the collagen family.

[0165] Collagen type a Chains Molecular species Collagen I a1(l), a2(l) [a1(l)]2, a2(l)

[0166] [a1(l)]3

[0167] Collagen II a1(ll) [a1(ll)]3

[0168] Collagen III a1(lll) [a1(lll)]3

[0169] Collagen IV a1(IV), a2(IV), a3(IV), a4(IV), [a1(IV)]2, a2(IV)

[0170] a5(IV), a6(IV) a3(IV), a4(IV), a5(IV)

[0171] [a5(l V)]2, a6(IV) Collagen V a1(V), a2(V), a3(V), a4(V)a[a1(V)]2, a2(V)

[0172] [a1(V)]3

[0173] [a1(V)]2a4(V) a1(XI)a1(V)a3(XI) Collagen VI a1(VI), a2(VI), a3(VI), a4(VI)b,

[0174] a5(VI)c, a6(V)

[0175]

[0176] Collagen VII a1(VII) [a1(VII)]3

[0177] 15252506-1M& C PC933728W0

[0178] 23

[0179] Collagen VIII α1(VIII) [α1(VIII)]2, α2(VIII)α1(VIII), [α2(VIII)]2[α1(VIII)]3[α2(VIII)]3Collagen IXeα1(IX), α2(IX), α3(IX) [α1(IX), α2(IX), α3(IX)] Collagen X α1(X) [α1(X)]3Collagen XI α1(XI), α2(XI), α3(XI)dα1(XI)α2(XI)α3(XI)α1(XI)α1(V)α3(XI) Collagen XIIeα1(XII) [α1(XII)]3Collagen XIII α1(XIII) [α1(XIII)]3Collagen XIVeα1(XIV) [α1(XIV)]3Collagen XV α1(XV) [α1(XV)]3Collagen XVIeα1(XVI) [α1(XVI)]3Collagen XVII α1(XVII) [α1(XVII)]3Collagen XVIII α1(XVIII) [α1(XVIII)]3Collagen XIXeα1(XIX) [α1(XIX)]3Collagen XXeα1(XX) [α1(XX)]3Collagen XXIeα1(XXI) [α1(XXI)]3Collagen XXIIeα1(XXII) [α1(XXII)]3Collagen XXIII α1(XXIII) [α1(XXIII)]3Collagen XXIV α1(XXIV) [α1(XXIV)]3Collagen XXV α1(XXV) [α1(XXV)]3Collagen XXVI α1(XXVI) [α1(XXVI)]3Collagen XXVII α1(XXVII) [α1(XXVII)]3Collagen α1(XXVIII) [α1(XXVIII)]3

[0180]

[0181] XXVIII

[0182] As examples of the nomenclature used in Table 1: a1(IX), a2(IX), a3(IX) refers to Type 9 alpha chains 1, 2 and 3 respectively; α1(XXVIII) refers to Type 28 collagen alpha 1 chain;

[0183] aThe a4(V) chain is solely synthesized by Schwann cells;bThe a4(VI) chain does not exist in humans;cThe a5(VI) has been designated as a 1 (XXIX);dThe a3(XI) chain has the same sequence as the a 1(11) chain but differs in its posttranslational processing and cross-linking.

[0184] Accordingly, in one embodiment, the collagen α chain(s) for producing a collagen type are selected for a collagen type listed in Table 1. As an example, the a chains for producing collagen type I are cd and a2. As a further example, the a chain for producing collagen type III is cd.

[0185] That is, in one embodiment, there is provided a genetically altered organism characterised by the expression of at least one collagen α chain(s) for producing at least one collagen type listed in Table 1.

[0186] 15252506-1In one embodiment, there is provided a genetically altered organism characterised by the expression of at least one of a collagen type listed in Table 1.

[0187] In one embodiment, collagen may refer to a collagen molecule, selected from a group consisting of: a procollagen, tropocollagen, a collagen microfibril or collagen fibre, wherein the collagen type is selected from at least one collagen type in Table 1. As a non-limiting example, collagen may refer to at least one Type I tropocollagen collagen molecule; or as another non-limiting example, collagen may refer to at least one Type III collagen fibre.

[0188] Included within the scope of the invention is native collagens and also engineered collagens. By ‘native collagens’ is meant the collagen sequence or a portion thereof found naturally within an organism. By ‘engineered collagen’ is meant a collagen sequence or portion thereof that has been genetically modified, such as to improve a characteristic of the collagen. For example, engineered collagen may show improved assembly or helix stability.

[0189] In one embodiment, the nucleic acid sequence encodes at least one collagen α chain(s) comprising or consisting of any one of SEQ ID NO: 1 or 24 to 52 or functional variant or homolog thereof.

[0190] Preferably, the nucleic acid sequence encodes at least one collagen α chain(s) comprising or consisting of any one of SEQ ID NO: 1 or 24 or functional variant or homolog thereof. SEQ ID NO: 1 and / or SEQ ID NO: 24 may be referred to as human type I collagen.

[0191] In one embodiment, the genetically altered photosynthetic organism expresses of at least one collagen α chain(s), wherein the collagen α chain(s) comprises or consists of any one of SEQ ID NO: 1 or 24 to 52 or functional variant or homolog thereof.

[0192] In a preferred embodiment, the genetically altered photosynthetic organism expresses a collagen α chain(s) comprising or consisting of any one of SEQ ID NO: 1 or 24 or functional variant or homolog thereof. SEQ ID NO: 1 and / or SEQ ID NO: 24 may be referred to as human type I collagen.

[0193] 15252506-1M& C PC933728W0

[0194] 25

[0195] For any embodiments described herein, the nucleic acid sequence encodes at least one collagen α chain(s) comprising or consisting of a sequence in Table 2. Also, the genetically altered photosynthetic organism of the invention may expresse a collagen α chain(s) comprising or consisting of a sequence in Table 2.

[0196] For example, in one embodiment, type I collagen may be defined as sequence comprising or consisting of SEQ ID NO: 1 (UniProt ID: P08123). For example, in one embodiment, Type I collagen a1 chain may comprise or consist of a sequence defined in SEQ ID NO: 2.

[0197] Table 2. Amino acid sequences of human collagen genes

[0198] SEQ ID Collagen Protein Name UniProt ID Similarity to NO: Type P02452

[0199] (CO1A1_HUMAN) 1 Type I Collagen alpha-1 (I) P02452 100%

[0200] chain

[0201] 24 Type I Collagen alpha-2(l) P08123

[0202] chain

[0203] 25 Type II Collagen alpha-l(ll) P02458 -72%

[0204] chain

[0205] 26 Type III Collagen alpha-l(lll) P02461 -62%

[0206] chain

[0207] 27 Type IV Collagen alpha-1 (IV) P02462 -45%

[0208] chain

[0209] 28 Type V Collagen alpha-1 (V) P20908 -45%

[0210] chain

[0211] 29 Type VI Collagen alpha-1 (VI) P12109 -30%

[0212] chain

[0213] 30 Type VII Collagen alpha-1 (VII) Q02388 -41%

[0214] chain

[0215] 31 Type VIII Collagen alpha-1 (VI II) P27658 -40%

[0216] chain

[0217] 32 Type IX Collagen alpha-1 (IX) Q14050 -47%

[0218] chain

[0219]

[0220] 15252506-133 Type X Collagen alpha-1 (X) Q03692 -38% chain

[0221] 34 Type XI Collagen alpha-1 (XI) P12107 -46% chain

[0222] 35 Type XII Collagen alpha-1 (XII) Q99715 -37% chain

[0223] 36 Type XIII Collagen alpha-1 (XI II) Q5TAT6 -44% chain

[0224] 37 Type XIV Collagen alpha-1 (XIV) Q05707 -36% chain

[0225] 38 Type XV Collagen alpha-1 (XV) Q13425 -19% chain

[0226] 39 Type XVI Collagen alpha-1 (XVI) Q07092 -43% chain

[0227] 40 Type Collagen alpha-1 (XVII) Q9UMD9 -40%

[0228] XVII chain

[0229] 41 Type Collagen alpha-1 (XVIII) P39059 -39%

[0230] XVIII chain

[0231] 42 Type XIX Collagen alpha-1 (XIX) Q14993 -41% chain

[0232] 43 Type XX Collagen alpha-1 (XX) Q8NFW1 -45% chain

[0233] 44 Type XXI Collagen alpha-1 (XXI) Q96P44 -42% chain

[0234] 45 Type Collagen alpha-1 (XXII) Q8NBS9 -23%

[0235] XXII chain

[0236] 46 Type Collagen alpha-1 (XXIII) Q5TAT7 -22%

[0237] XXIII chain

[0238] 47 Type Collagen alpha-1 (XXIV) Q8IZC6 -41%

[0239] XXIV chain

[0240] 48 Type Collagen alpha-1 (XXV) Q96PD7 -17%

[0241] XXV chain

[0242] 49 Type Collagen alpha-1 (XXVI) Q8N119 -20%

[0243] XXVI chain

[0244]

[0245] 15252506-150 Type Collagen alpha- Q8IZC6 -41% XXVII 1 (XXVII) chain

[0246] 51 Type Collagen alpha- Q8N2U9 -21%

[0247] XXVIII 1 (XXVIII) chain

[0248] 52 Type Collagen alpha-1 (XXIX) Not well- N / A

[0249] XXIX chain characterized

[0250]

[0251] In one embodiment, the collagen-encoding sequence comprises or consists of SEQ ID NO: 89. We have found that adding a portion of the collagen alpha-1 (III) chain (COL3A1) sequence (i.e., 589–510 bp of the COL3A1 sequence sequence) to other collagen sequences produces beneficial properties.

[0252] In one embodiment, the collagen-encoding sequence comprises or consists of SEQ ID NO: 88. This includes two portions of the collagen alpha-1 (111) chain, namely 908-1136 bp and 589–510 bp (SEQ ID NO: 26).

[0253] In one embodiment, the collagen-encoding sequence may be codon-optimised for plastid translation in a plastid-containing organism, such as Chlamydomonas, lettuce or tobacco. Codon-optimisation is a well-known process in the art, referring to a molecular biology technique wherein a polynucleotide sequence is modified, without changing the amino acid sequence of the resulting protein, so that the genetic sequence aligns with the target organism’s codon usage bias. For example, a human collagen sequence can be codon-optimised for expression in Chlamydomonas, so that the genetic sequence of the modified sequence aligns with Chlamydomonas codon usage bias without changing the amino acid sequence of the human collagen sequence.

[0254] Functional variants and homologs are also included within the scope of the present invention.

[0255] The term “functional variant” (or “variant”) as used herein with reference to any of the sequences described herein refers to a variant sequence or part of the sequence which retains the biological function of the full non-variant sequence. For example, this may include engineered variants, synthetic variants, truncated portions (i.e., fragments), A variant of a plastid regulatory sequence refers to any sequence capable of initiating or regulating transcription or translation, for example a promoter or 5’ UTR. For example, a

[0256] 15252506-1variant of a promoter will retain the wild-type function of being able to participate in the binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. A variant of a 5’ UTR will retain the wild-type function of recruiting ribosome complexes and modulating (and initiating) translation of the transcript, for example through ribosome-binding elements, leader sequences, secondary structural features (such as such as hairpin loops and G-quadruplexes), or translation-enhancing elements that modulate interactions with RNA-binding proteins, microRNA and influence mRNA stability. A variantof a plastid terminator sequence refers to any sequence capable of bringing transcription to cessation. A variant of a pair of flanking sequences comprising at least one portion of a sequence of two plastid-encoded genes for refers to any sequence capable of integrating the nucleic acid sequence (i.e., comprising the collagen-encoded sequence) into a plastid genome. A functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0257] The term, “homolog” as used herein with reference to any of the sequences described herein refers to a variant sequence or part of the sequence which shows a high homolog, i.e., sequence similarity. Due to the high sequence similarity, it can be deduced that the sequences are equivalents and are also in the scope of the application. For example, both the alpha chain 1 and alpha chain 2 of type I collagen from rats, sheep, pig, bovine

[0258] 15252506-1and equine all show over 90% protein sequence homology with human collagen. Such homologs are included within the scope of the invention.

[0259] A homolog may have, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to any sequence disclosed herein.

[0260] The term, “ortholog” as used herein with reference to any of the sequences described herein refers to the corresponding gene in different species that evolved from a common ancestral gene by speciation.

[0261] In one embodiment, the ortholog, functional variant or homolog may be defined by a sequence encoding an amino acid sequence comprising or consisting of any sequence in Table 3.

[0262] Table 3. Orthologs of the human type I collagen gene (CO1A1_HUMAN) in various species.

[0263] SEQ Accession Gene ID Species Similarity to ID No. P02452

[0264] NO: (CO1A1_HUMAN) 53 P02452 CO1A1_HUMAN Homo sapiens (Human) Ident. 100% 54 Q9XSJ7 CO1A1_CANLF Canis lupus familiaris (Dog) Ident. 97.81% 55 P02453 CO1A1_BOVIN Bos taurus (Bovine) Ident. 97.54% 56 P02457 CO1A1_CHICK Gallus gallus (Chicken) Ident. 90% 57 P02458 CO1A1_SUSSC Sus scrofa (Pig) Ident. 72.18% 58 P02459 CO1A1_ORCU Oryctolagus cuniculus (Rabbit) Ident. 72.12% 59 P02460 CO1A1_OVIA Ovis aries (Sheep) Ident. 72.93% 60 P02461 CO1A1_CAPHI Capra hi reus (Goat) Ident. 61.51% 61 P02465 CO1A1_EQUCA Equus caballus (Horse) Ident. 94% 62 P02463 CO1A1_EQUAS Equus asinus (Donkey) Ident. 43.72% 63 P02464 CO1A1_CAMEL Camelus dromedarius (Camel) Ident. 93% 64 Q6P4Z2 CO1A1_XENLA Xenopus laevis (African clawed Ident. 70.91% frog)

[0265] 65 P02466 CO1A1_ICTPU Ictalurus punctatus (Catfish) Ident. 64.49%

[0266]

[0267] 66 P02467 CO1A1_SALSA Salmo salar (Atlantic salmon) Ident. 65.15%

[0268] 15252506-167 093484 CO1A1_ONCMY Oncorhynchus mykiss (Rainbow Ident: 62.41% trout)

[0269] 68 P02470 CO1A1_PROAN Protopterus annectens Ident: 16.30%

[0270] (Lungfish)

[0271] 69 C0HJN5 CO1A1_HIPAM Hippocampus abdominalis Ident.: 96.13%

[0272] (Seahorse)

[0273] 70 P02468 CO1A1_AURAU Aurelia aurita (Jellyfish) Ident.: 12.92% 71 C0HM85 CO1A1_SEAB Dicentrarchus labrax (Seabass) Ident.: 63.81% 72 C7DZK3 CO1A1_DANRE Danio rerio (Zebrafish) Ident.: 42.50% 73 C0HJN8 CO1A1_SEACU Holothuria scabra (Sea Ident.: 65.60% cucumber)

[0274] 74 Q28668 CO1A1_TILNI Oreochromis niloticus (Tilapia) Ident.: 61.76% 75 C0HM84 CO1A1_ACHFU Achatina fulica (Snail) Ident.: 79.40% 76 C0HM86 CO1A1_MYTED Mytilus edulis (Mussel) Ident.: 75.60% 77 C0HJN6 CO1A1_CRAGI Crassostrea gigas (Oyster) Ident.: 66.40% 78 P02479 CO1A1_GADMO Gadus morhua (Codfish) Ident.: 16.30% 79 P11087 CO1A1_MOUSE Mus musculus (Mouse) Ident.: 92.43%

[0275]

[0276] 80 P02454 CO1A1_RAT Rattus norvegicus (Rat) Ident.: 92.70%

[0277] Nucleic acid constructs and genetically modified organisms expressing said constructs.

[0278] We have identified an expression cassette, taking the form of a nucleic acid construct, which enables algae, plants and plant cells to be genetically modified to express exogenous collagen in their plastids.

[0279] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences.

[0280] 15252506-1It will be understood by the skilled person that the constructs described herein may comprise a single nucleic acid or multiple nucleic acids. For example, a construct may comprise a nucleic acid comprising feature A and feature B. Alternatively, a construct may describe a nucleic acid comprising feature A and a (separate) nucleic acid comprising feature B. Such arrangements of nucleic acids within or between nucleic acid constructs is immaterial to the present invention. Instead, the below embodiments are intended to describe the innovative features of the invention, which may be arranged within or between nucleic acid constructs in any routine manner.

[0281] Accordingly, in one aspect of the invention, there is provided at least one nucleic acid construct comprising a nucleic acid sequence encoding at least one collagen a chain for producing collagen, at least one plastid gene promoter, at least one plastid gene terminator, at least one pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, and at least one selectable marker gene.

[0282] In another aspect of the invention (wherein the plastid is a chloroplast), there is provided a nucleic acid construct comprising a nucleic acid sequence encoding at least one collagen a chain for producing collagen, a chloroplast gene promoter, a chloroplast gene terminator, a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, and a selectable marker gene.

[0283] In a preferred embodiment, the chloroplast gene promoter and the chloroplast gene terminator are from Chlamydomonas reinhardtii.

[0284] In one embodiment, the chloroplast gene promoter and the chloroplast gene terminator are Nicotiana sequences.

[0285] The phrase ‘at least one collagen a chain for producing collagen’ has been used above. For completeness, it refers to at least one collagen a chain forming part of at least one collagen type. In one embodiment, ‘at least one’ means one, two, three, four or five types of collagen a chain for producing collagen, as well as one, two, three, four or five copies (or subtypes) of the same type of collagen. For example, as shown in Figures 6 and 7, the invention can be used to express multiple, different collagen a chains in plastids.

[0286] 15252506-1Accordingly, the invention also provides for methods of producing at “least two collagen a chains for producing collagen” as described herein.

[0287] By ‘plastid regulatory sequence’ is meant a promoter and / or a 5’ untranslated region of a plastid-encoded gene.

[0288] The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in the binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and / or external stimuli, or in a tissue- specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and / or -10 box transcriptional regulatory sequences.

[0289] The promoters upstream of the nucleotide sequences useful in the nucleic acid constructs described herein can also be modified by one or more nucleotide substitution(s), insertion(s) and / or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'-regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoter is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the GQY1 nucleic acid sequence is, as described above, preferably linked operably to or comprises a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.

[0290] The term "5’ untranslated region (5’IITR)" typically refers to a nucleic acid sequence that, when linked to a transcript, is capable of recruiting ribosome complexes and modulating (and initiating) translation of the transcript. Typically, a 5' UTR is positioned directly upstream of the initiation codon of a transcript; specifically, between the cap site and the initiation codon. The 5' UTR may comprise native, synthetic, or modified nucleotide

[0291] 15252506-1sequences and often includes regulatory motifs, such as ribosome-binding elements, leader sequences, secondary structural features (such as such as hairpin loops and G-quadruplexes), or translation-enhancing elements that modulate interactions with RNA-binding proteins, microRNA, as well as influence mRNA stability.

[0292] The term "terminator" typically refers to a nucleic acid control sequence located downstream from the transcriptional start of a gene and is involved in initiating the release of the newly synthesized RNA from the transcription machinery. Thus, by ‘plastid terminator’ is meant a sequence that inhibits the expression of genes encoded within the plastid genome, for example by signalling the end of the gene to the polymerase.

[0293] The term, ‘plastid terminator’ and ‘plastid promoter’ may be collectively referred to as ‘regulatory elements’.

[0294] Examples of plastid regulatory sequence and plastid terminators and, in particular, chloroplast promoters and / or 5’ UTR, and chloroplast terminators are well known, and generally are the regulatory elements adjacent to genes involved in photosynthesis.

[0295] We have identified that in order for collagen to be expressed within the chloroplast or other plastids, the sequences encoding collagen a chains must be placed under the transcriptional control of elements that regulate plastid genes.

[0296] In one embodiment, the plastid regulatory sequence(s) and / or plastid terminator(s) are endogenous to the organism expressing the nucleic acid construct. For example, if the organism expressing the nucleic acid construct is Nicotiana, then the plastid regulatory sequence and / or plastid terminators will be or comprise Nicotiana sequences. For example, as shown in Figure 4, the organism expressing the nucleic acid construct is Chlamydomonas reinhardtii and the plastid regulatory sequence and plastid terminators are Chlamydomonas reinhardtii sequences.

[0297] In a preferred embodiment, the plastid regulatory sequence(s) and / or plastid terminator(s) are exogenous to the organism expressing the nucleic acid construct. For example, if the organism expressing the nucleic acid construct is Nicotiana, then the plastid regulatory sequence and / or plastid terminator will not comprise Nicotiana sequences.

[0298] 15252506-1In a preferred embodiment, the plastid regulatory sequence(s) and / or plastid terminator(s) are exogenous to the organism expressing the nucleic acid construct, wherein the plastid promoter and / or plastid terminator is derived from Chlamydomonas reinhardtii. For example, as shown in Figure 5 and 9 and 10, if the organism expressing the nucleic acid construct is Nicotiana, then the plastid regulatory sequence and / or plastid terminator are derived from Chlamydomonas reinhardtii sequences. As shown in Figure 10, the expression of Chlamydomonas reinhardtii regulatory elements is associated with increased collagen synthesis in a plastid.

[0299] As a further example, as shown in Figure 7, 9 and 10 if the organism expressing the nucleic acid construct is lettuce (Lactuca sativa), then the plastid regulatory sequence and / or plastid terminator is from Nicotiana and / or Chlamydomonas reinhardtii sequences.

[0300] It will also be apparent to the skilled person that the nucleic acid construct may comprise more than one plastid regulatory sequence and plastid terminator. For example, as shown in Figures 6 and 7, multiple plastid promoters and terminators have been used to facilitate the expression of more than one collagen chain.

[0301] In one embodiment, the plastid regulatory sequence (s) comprises at least one promoter and / or 5’ UTR selected from a group consisting of: rrn, psaA, psbA, rps16, rbcL or atpA. rrn refers to the plastid ribosomal operon, rrn. psaA encodes the P700 chlorophyll a apoprotein A1. psbA encodes the D1 protein that plays an important role in protecting photosystem II from oxidative damage in higher plants. rps16 encodes the ribosomal protein S16. PrbcL encodes ribulose bisphosphate carboxylase. PatpA encodes the alpha subunit of the ATP synthase enzyme.

[0302] In one embodiment, the promoter of any of rrn, psaA, psbA, rps16, rbcL or atpA refers to the sequence found ~ -200 bp upstream of the transcriptional start position of rrn, psaA, psbA, rps16, rbcL or atpA, repsectively.

[0303] In some embodiments, the plastid regulatory sequence(s) are derived from Chlamydomonas, and in particularly Chlamydomonas reinhardtii. We have found that promoters and 5’ UTR derived from Chlamydomonas reinhardtii strongly activate the

[0304] 15252506-1transcription of chloroplast genes, and that these activities can be leveraged to increase collagen synthesis in chloroplasts and other plastids.

[0305] In particular, we have found that the promoter and / or 5’ UTR of psaA from Chlamydomonas reinhardtii is effective in regulating the expression of exogenous collagen in plastids (Figures 4 and 5). More specifically, we have found that the psaA promoter supports plastid transcription, while the psaA 5'UTR confers temperature-dependent translational control of collagen expression, particularly in higher plants. Of note, this latter property is likely associated with specific Chlamydomonas regulatory elements (e.g. the psaA 5'UTR) rather than Chlamydomonas regulatory elements in general.

[0306] Accordingly, in a preferred embodiment, the plastid regulatory sequence(s) comprises or consists of a sequence from a promoter and / or 5’ UTR sequence obtained from Chlamydomonas reinhardtii. We have found that regulatory elements originating from the plastid genome of Chlamydomonas reinhardtii or from related photosynthetic organisms can provide translational regulation of collagen genes in algae and also higher plants, and in some embodiments in a temperature-responsive manner (Figure 5, Figure 10).

[0307] Our preliminary data provides direct evidence that engineered plastid regulatory elements can retain function across divergent taxa. Specifically, we expressed the Chlamydomonas reinhardtii psaA promoter and 5'UTR in tobacco chloroplasts and observed robust translation of recombinant collagen together with the same temperature-responsive ‘RNA-thermometer’ behaviour reported in the native algal context. This cross-kingdom functionality indicates that the core regulatory mechanism is encoded in cis within the promoter / 5'UTR architecture (e.g., RNA structure-mediated control of translation initiation) and can therefore be repurposed as a modular, chimeric plastid regulatory element for predictable control of transgene expression in heterologous plastid hosts.

[0308] Accordingly, in one embodiment, the promoter and / or 5’ UTR sequence is or comprises a thermoresponsive regulatory element. That is, the regulation of translation of a downstream polynucleotide sequence is dependent on temperature.

[0309] 15252506-1In a preferred embodiment, the sequence of the promoter and / or 5’ UTR sequence is thermoresponsive and obtained from Chlamydomonas reinhardtii.

[0310] In a more preferred embodiment, the plastid regulatory sequence comprises or consists of the psaA 5’ UTR sequence and / or psaA promoter obtained from Chlamydomonas reinhardtii. The psaA 5'UTR contains a cis-acting structured RNA hairpin that acts as an ‘RNA thermometer’. The RNA hairpin represses translation at a first, lower temperature by occluding a ribosome-binding region. Upon shifting to a second, higher temperature, the hairpin melts, exposing the ribosome-binding region and enabling efficient plastid translation. This mechanism operates in both algal and plant plastids, providing a universal, portable temperature-inducible expression system.

[0311] Thus, at a first, ‘non-inducing’ temperature (e.g., 15-25 °C), the 5' UTR adopts a stable secondary structure that: partially or fully occludes a ribosome-binding region (e.g., Shine-Dalgarno-like sequence), reducing the accessibility of the translation initiation machinery which thereby represses translation of the downstream collagen coding sequence.

[0312] Upon exposure to a second, higher ‘inducing’ temperature (e.g., at least 25, 28, 30, 35 or 40 °C), the hairpin undergoes partial or complete melting, resulting in: increased single-strandedness of the ribosome-binding region, enhanced ribosome recruitment, a sharp increase in plastid translation initiation rates, and corresponding elevation of collagen accumulation.

[0313] This mechanistic behaviour is supported by Figure 10 which shows that collagen synthesis under the psaA 5'UTR regulatory element (SEQ ID NO: 84) is associated with minimal change in collagen abundance at 25 °C but is associated with a substantial increase in collagen accumulation at elevated temperatures. This demonstrates that the regulation is cis-acting and post-transcriptional and translational, not transcriptional.

[0314] Thus, in one embodiment, the plastid regulatory sequence (from Chlamydomonas or otherwise) comprises a temperature-responsive regulatory element, such as an RNA secondary structure. Preferably, the thermoresponsive regulatory element is a secondary hairpin loop structure, e.g., that modulates translation of a downstream collagen coding sequence in response to temperature.

[0315] 15252506-1In one embodiment, the thermoresponsive regulatory element selectively occludes a ribosome binding region at a first temperature and becomes destabilised at a second, higher temperature, resulting in increased plastid translation of collagen.

[0316] In a preferred embodiment, the plastid regulatory sequence comprises PpsaA i e., the promoter) and / or 5’ UTR of the gene psaA (which encodes the P700 chlorophyll apoprotein A1)).

[0317] In a preferred embodiment, the plastid regulatory sequence comprises the 5’ UTR of the gene psaA (i.e., psaA 5’UTR). Preferably, the sequence of psaA 5’UTR is from Chlamydomonas reinhardtii.

[0318] In one embodiment, the plastid regulatory sequence comprises the 5’ UTR of the gene psaA from Chlamydomonas reinhardtii and promoter of gene psaA from Chlamydomonas reinhardtii, wherein the 5’UTR comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof and the promoter comprises or consists of SEQ ID NO: 12 or 83 or functional variant or homolog.

[0319] In one embodiment, the plastid promoter comprises or consists of SEQ ID NO: 12 or a variant thereof.

[0320] In one embodiment, the plastid promoter comprises or consists of SEQ ID NO: 83 or a variant thereof. SEQ ID NO: 83 encodes for the promoter region of the psaA promoter from Chlamydomonas reinhardtii.

[0321] In one embodiment, the plastid regulatory sequence comprises or consists of SEQ ID NO: 84 or a variant thereof. SEQ ID NO: 84 encodes the 5’ UTR region of the psaA promoter from Chlamydomonas reinhardtii. SEQ ID NO: 84 is the wild-type sequence and comprises a hairpin loop structure that is sensitive to temperature changes. At temperatures at or around 25 °C the 5’UTR mRNA sequence forms a stable hairpin that prevents or reduces protein expression. At higher temperatures, for example at or around 40 °C, the hairpin melts or unwinds, leading to translation or substantially increased rates of translation. Positions 124 to 134 correspond to the hairpin loop structure. By introducing at least one mutation in the hairpin structure, it is possible to alter the

[0322] 15252506-1thermoresponsive properties of the region as desired. Exemplar variants with modified hairpins are shown in SEQ ID NO: 85 and 86.

[0323] Thus, in one embodiment, the variant of SEQ ID NO: 84 comprises at least one mutation at positions 124 to 134 of SEQ ID NO: 84. In one embodiment, the variant of SEQ ID NO: 84 is SEQ ID NO: 85 or 86.

[0324] In one embodiment, the plastid regulatory sequence comprises or consists of SEQ ID NO: 85 or a variant thereof. SEQ ID NO: 85 encodes a 5’UTR region of the psaA gene from Chlamydomonas reinhardtii that has been modified in the hairpin loop structure, such that the thermoresponsive profile of the promoter is altered, so that protein expression is increased (i.e. translational activity) downstream of the 5’ UTR region at or around 25 °C.

[0325] In one embodiment, the plastid promoter comprises or consists of SEQ ID NO: 86 or a variant thereof. SEQ ID NO: 86 encodes a 5’IITR region of the psaA gene from Chlamydomonas reinhardtii that has been modified in the hairpin loop structure, such that the thermoresponsive profile of the promoter are altered, so that protein expression is increased (i.e. translational activity) downstream of the 5’ UTR region at or around 28 °C. to 30 °C.

[0326] Known examples of other mechanistically analogous thermoresponsive elements-i.e., comprising a temperature-responsive, cis-acting RNA regulatory element - are also included in the scope of this invention. For example, the prfA 5'UTR thermoregulatory element from Listeria monocytogenes. The 5' UTR of prfA forms a stable hairpin structure at environmental temperatures (<30oQ is less than 30 raised to the composed with power <30oQ), which masks the Shine-Dalgarno sequence, preventing ribosome binding and translation of the prfA mRNA. As temperature rises to 37 °C, this hairpin structure "melts" (unfolds), unmasking the Shine-Dalgarno sequence and allowing for translation of the PrfA protein.

[0327] A further example is a ‘FourU-type RNA thermometer’, a compact cis-regulatory element found in 5-UTR of certain bacterial mRNAs that controls gene expression in response to temperature changes. Examples include agsA 5'UTR from Salmonella (Johansson et al.

[0328] 2002). At low temperatures (e.g., 30°C), the FourU structure forms a stable hairpin that

[0329] 15252506-1occludes the Shine-Dalgarno sequence, preventing ribosome binding and translation. As temperatures rise (e.g., to 37°C or 42°C), the non-canonical base pairs between the uridines and the Shine-Dalgarno sequence melt, releasing the ribosome binding site and allowing translation to proceed.

[0330] A further example is the ROSE or ROSE-like 5'UTR elements regulating small heat-shock genes, for example ibpA 5 ‘UTR from Vibrio cholerae (Weber et al. 2014).

[0331] A further example is the Phytochrome-Interacting Factor 7 (PIF7) 5’IITR that acts as an RNA-based thermometer in Arabidopsis thaliana (thale cress). At lower temperatures (e.g., 17°C), the hairpin structure is compact, which restricts the translation of PIF7 mRNA. At higher temperatures (e.g., 27°C), the hairpin structure relaxes and partially unfolds, allowing for increased translation and accumulation of the PIF7 protein.

[0332] A further example is the hsp17 chaperone ribonucleic acid thermometer, formed of the hsp17 5’IITR from Synechocystis (Wagner et al. 2015). At low temperatures (e.g., 28°C), the hairpin structure sequesters the ribosome binding site inhibiting translation of the Hsp17 protein. Upon a rise in temperature (e.g., 42°C), the hairpin melts, releasing the ribosome binding site and allowing translation to proceed.

[0333] Accordingly, in one embodiment, the thermoresponsive regulatory element is selected from the psaA 5’UTR from Chlamydomonas, prfA 5’UTR from Listeria monocytogenes, agsA 5'UTR from Salmonella, ibpA 5 ‘UTR from Vibrio cholerae, PIF7 5’ UTR from Arabidopsis thaliana, and the hsp175’UTR from Synechocystis.

[0334] In the present invention, the regulatory principle of temperature-responsive, cis-acting RNA regulatory elements are adapted to plastid gene expression. Indeed, this application represents the first demonstration of a 5'UTR-encoded thermoresponsive regulatory element being used to control to recombinant collagen production, and the first demonstration that an algal plastid 5'UTR-based temperature-responsive regulatory module can be functionally deployed in higher-plant plastids.

[0335] The thermoresponsive regulatory element may comprise at least one mutation, wherein the mutation alters the element’s response to temperature. For example, the mutation may alter (increase or decrease) the stability of a hairpin structure that contributes to the response to temperature. Also included within the scope of the invention are targeted

[0336] 15252506-1nucleotide substitutions within the 5'UTR hairpin region that stabilise or destabilise the RNA secondary structure enable tuning of the temperature threshold at which translation is induced. This allows precise control of collagen translation / collagen protein accumulation in plastids of both microalgae and higher plants.

[0337] This invention represents the first application of a plastid RNA thermoregulatory element to recombinant collagen production and the first demonstration of a temperature-responsive translational switch being used to control plastid gene expression at the level of translation in higher plants and microalgae.

[0338] In one embodiment, the plastid regulatory sequence(s) comprises at least one promoter selected from a group consisting of: rrn, psaA, psbA, rps16, rbcL or atpA. Preferably, the promoter is obtained from Chlamydomonas reinhardtii.

[0339] In a preferred embodiment, the plastid regulatory sequence(s) comprises the promoter of PpsbA. The promoter of the PpsbA sequence is highly conserved, making it an ideal choice for this invention. PpsbA also contains many conserved regions including a stemloop region that is an important determinant of mRNA stability and translation efficiency.

[0340] In one embodiment, the plastid regulatory sequence(s) comprises at least one promoter selected from the promoter sequences of rrn, psaA, psbA, rps16, rbcL or atpA and at least one 5’ UTR selected from rrn, psaA, psbA, rps16, rbcL or atpA.

[0341] In on embodiment, the plastid regulatory sequence(s) comprises at least one promoter selected from the promoter sequences of rrn, psaA, and atpA and at least one 5’ UTR selected from rrn, psaA, and atpA.

[0342] In one embodiment, the promoter of rrn comprises or consists of a sequence defined in SEQ ID NO: 14 or 15 or functional variant or homology thereof. In one embodiment, the 5’ UTR of rrn comprises or consists of SEQ ID NO: 13.

[0343] In one embodiment, the promoter of psaA comprises or consists of a sequence defined in SEQ ID NO: 12 or SEQ ID NO: 83 or a functional variant or homology thereof. In one embodiment, the 5’UTR of psaA comprises or consists of a sequence defined in SEQ ID NO: 84 to 86.

[0344] 15252506-1In one embodiment, the promoter of psbA comprises or consists of a sequence defined in SEQ ID NO: 16 or a functional variant or homolog thereof.

[0345] In one embodiment, the promoter of rps16 comprises or consists of a sequence defined in SEQ ID NO: 17 or a functional variant or homolog thereof.

[0346] In one embodiment, the promoter of rbcL comprises or consists of a sequence defined in SEQ ID NO: 18 or a functional variant or homolog thereof.

[0347] In one embodiment, the promoter of atpA comprises or consists of SEQ ID NO: 81 or a functional variant or homology thereof. In one embodiment, the 5’UTR of atpA comprises or consists of SEQ ID NO: 82 or a functional variant or homolog thereof.

[0348] Thus, in one embodiment, the plastid regulatory sequence is a promoter selected from rm, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR selected from rrn, psaA, psbA, rps16, rbcL and atpA, wherein the promoter of rrn, psaA, psbA, rps16, rbcL and atpA comprises or consists of SEQ ID NO: 14 or 15, 12 or 83, 16, 17, 18 and 81 respectively, and wherein the 5’UTR of rrn, psaA, and atpA comprises or consists of SEQ ID NO: 13, any one of SEQ ID NO: 84 to 86, and SEQ ID NO: 82 or functional variant of homolog thereof, respectively.

[0349] In one embodiment, the chloroplast terminator(s) is selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA. TpsbA refers to the 3’ UTR or terminator region of the psbA gene. Trps16 refers to the 3’ UTR or terminator region for the chloroplast rps16 gene encoding the ribosomal protein S16. TrbcL refers to the 3’ UTR or terminator region sequence of the rbcL gene. TatpA refers to the 3’ UTR or terminator region sequence of the atpA gene, encoding for the a-subunit of the chloroplast ATP synthase. Preferably, the chloroplast terminator is TpsbA or TrbcL.

[0350] In a preferred embodiment, the chloroplast terminator(s) comprises or consists of a sequence from a terminator sequence obtained from Chlamydomonas reinhardtii or Nicotiana.

[0351] 15252506-1In one embodiment, the chloroplast terminator(s) is selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA obtained from Chlamydomonas reinhardtii. In a preferred embodiment, the chloroplast terminator is TrbcL obtained from Chlamydomonas reinhardtii and comprises of consists of SEQ ID NO: 19 or 21 or a functional variant or homolog thereof.

[0352] In one embodiment, the sequence of TpsbA comprises or consists of a sequence defined in SEQ ID NO: 20 or 91 or a functional variant or homolog thereof.

[0353] In one embodiment, the sequence of TrbcL comprises or consists of a sequence defined in SEQ ID NO: 19 or 21 or a functional variant or homolog thereof.

[0354] In one embodiment, the sequence of Trps16 comprises or consists of a sequence defined in SEQ ID NO: 92 or a functional variant or homolog thereof.

[0355] In one embodiment, the nucleic acid construct comprises a psaA 5' untranslated region (UTR) from Chlamydomonas reinhardtii as a plastid regulatory sequence and a rbcL 3' UTR from Chlamydomonas reinhardtii as a terminator. As described herein, and shown in the Figures, such a construct can be used to drive expression of collagen in plastids, preferably chloroplasts of plants. Preferably, psaA 5' UTR enhances translation efficiency through ribosome binding optimization, and rbcL 3' UTR increases mRNA stability, preventing degradation in plastids.

[0356] For the first time, we have shown that using a promoter and 5' UTR from Chlamydomonas chloroplasts (as shown in Figure 5) can confer temperature inducibility to transgene (collagen) expression in seed plants. Since the yield of recombinant protein is key to the cost effectiveness of any production system in biotechnology, the possibility to boost protein expression levels by applying a temperature shift before harvest of the algal culture provides a simple and inexpensive method to improve product yields in molecular farming. We have also used and tested the psaA thermometer from Chlamydomonas in higher plant plastids (tobacco, lettuce) to confer heat inducibility to gene and transgene expression in seed plants.

[0357] By ‘pair of flanking sequences comprising at least one portion of a sequence of two plastid-encoded genes’ is meant at least one portion from a first plastid-encoding gene

[0358] 15252506-1and a second portion from a second plastid-encoding gene that enables site-specific recombination of the nucleic acid into the plastid genome. By directing homologous recombination, the pair of flanking sequences ensures that the nucleic acid construct is inserted into the plastid genome at an appropriate position, thereby eliminating the concerns of position effect that is observed in nuclear transgenic lines. Suitable flanking sequences for plastid and, in particular, chloroplast nucleic acid constructs are well known in the art.

[0359] The skilled person will understand that the flanking sequences do not need to comprise or consist of the entire plastid gene in order to direct homologous recombination of the plastid nucleic acid construct into the plastid genome, but rather only a suitable portion may be necessary. The size of the portion required will vary depending on the plastid-encoded genes and this can be determined by routine experimentation by the skilled person. Accordingly, the pair of flanking sequences comprising at least one portion of a sequence of two plastid-encoded genes is for directing transgene integration.

[0360] Preferably, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes endogenous to the organism expressing the construct. As explained in Examples 4 to 6, the use of endogenously encoded chloroplast genes ensures targeted integration of the gene of interest into the plasmid.

[0361] In one embodiment, the pair of flanking sequences comprising at least a portion of a sequence of two chloroplast-encoded genes is selected from a group consisting of:

[0362] • trnI and trnA

[0363] • rbcL and accD

[0364] • (3’)rpl32 and trn

[0365] • pet and psbJ

[0366] • tm\l and (3’)rps7 / 12

[0367] • rrn16, trn\l and 3’rps7 / 12

[0368] • trnfM and trnG

[0369] • trnN and trnR

[0370] • ycf3 and trnS

[0371] • prs14 and trnG

[0372] • trnG and trnF

[0373] • trnE2 and / or psbN

[0374] 15252506-1These loci allow stable and predictable insertion without disrupting essential plastid functions. In certain embodiments, homologous high-expression loci in Chlamydomonas chloroplasts may be used.

[0375] In a preferred embodiment, the pair of flanking sequences comprises at least a portion of a sequence of the two chloroplast-encoded genes trnI and trnA. In one embodiment, the sequences of trnI and trnA comprises or consists of a sequence defined in SEQ ID NO: 10 and 11 or functional variants or homologs thereof, respectively. Accordingly, in one embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes, comprises or consists of a sequence defined in SEQ ID NO: 10 and 11 or functional variants or homologs thereof respectively.

[0376] In another preferred embodiment, the pair of flanking sequences comprises at least a portion of a sequence of the two chloroplast-encoded genes trnFM and trnG. In one embodiment, the sequences of trnFM and trnG comprises or consists of a sequence defined in SEQ ID NO: 8 and 9 or functional variants or homologs thereof respectively. Accordingly, in one embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes, comprises or consists of a sequence defined in SEQ ID NO: 8 and 9 or functional variants or homologs thereof respectively.

[0377] In one embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes comprise a sequence from two chloroplast-encoded Nicotiana genes.

[0378] In one embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes comprise a sequence from two chloroplast-encoded Lactuca genes.

[0379] By ‘selectable marker’ is meant a gene or nucleic acid that enables the selection of transformed cells. The concept of a selectable markers is well known to the skilled person. A suitable marker can be bar-phosphinothricin or PPT. Alternatively, the transformed organisms are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS (β-glucuronidase).

[0380] 15252506-1The selectable marker must be suitable for selecting organisms with transformed plastids. Such selectable markers are known in the art. The neo gene confers kanamycin resistance and has been shown to be an effective selectable marker for plastid transformation. aphA6 is another kanamycin resistance gene with good transformation efficiency. Another selection strategy utilizes a “double barrel” vector; the use of two genes encoding two different enzymes, namely aphA6 and nptll, is known to be efficient in selecting for chloroplast-directed nucleic acid constructs. The bar gene, encoding phosphinothricinacetyltransferase (PAT) and conferring herbicide resistance, has been shown to be an effective plastid-selectable marker. The spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (badh) gene was developed as a plant-derived selectable marker gene to transform chloroplast genomes. The aadA gene, which encodes the enzyme aminoglycoside 3’ adenylyltransferase, and confers resistance to streptomycin and spectinomycin, is a highly effective selectable marker for chloroplast-targeted nucleic acid constructs.

[0381] Accordingly, in one embodiment, the selectable marker is selected from a group consisting of neo, aadA, nptII, aphA6, badh and npII-aphA6.

[0382] Preferably, the selectable marker is the aadA gene. Even more preferably, the aadA gene is the Chlamydomonas reinhardtii aadA gene. In one embodiment, aadA comprises or consists of a sequence defined in SEQ ID NO: 22 or a functional variant or homolog thereof respectively.

[0383] It will be understood to the skilled person that the selectable marker will be under the transcriptional control of regulatory elements, as part of a selection cassette. As shown in Figure 4, in one embodiment, the selectable marker is under the transcriptional control of regulatory elements that are endogenous or exogenous to the organism that cassette is being expressed in. Examples 5 and 6 describe the use of direct repeats and flanking loxP sites to enable the removal of the aadA gene using direct repeat homologous recombination or by using Cre recombinanse respectively.

[0384] Accordingly, in one embodiment, the nucleic acid construct may further comprise either a pair of LoxP sites or at least one direct repeat for facilitating the removal of the selectable marker.

[0385] 15252506-1The elements of the nucleic acid constructs of this invention may be arranged within a single polycistronic plastid cassette or distributed across multiple plastome integration sites. Intercistronic expression elements may optionally be included to ensure coordinated expression of all required proteins.

[0386] In one embodiment, the nucleic acid further comprises at least one intercistronic expression element (IEE). In one embodiment, the IEE is defined as a sequence comprising or consisting of SEQ ID NO: 23 or variant or homologs thereof.

[0387] Accordingly, in one embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0388] at least one collagen a chain for producing collagen,

[0389] at least one plastid regulatory sequence, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0390] a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0391] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0392] optionally a selectable marker.

[0393] In a preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0394] a sequence encoding at least one collagen a chain for producing collagen; at least one plastid regulatory sequence, for regulating the expression of the sequence encoding at least one collagen α chain in a chloroplast;

[0395] a plastid terminator, for regulating the expression of the sequence encoding at least one collagen α chain in a chloroplast;

[0396] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0397] optional a selectable marker.

[0398] 15252506-1In a more preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0399] a sequence encoding at least one collagen a chain for producing collagen; at least one plastid regulatory sequence, selected from a group consisting of: the promoter of rrn, psaA, psbA, rps16 or rbcL and / or the 5’UTR of rrn, psaA, psbA, rps16 or rbcL;

[0400] at least one chloroplast terminator, selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA;

[0401] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome; and

[0402] optionally, a selectable marker, selected from neo, aadA, nptII, aphA6, badh and npII-aphA6.

[0403] In a preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0404] a sequence encoding at least one collagen a chain for producing collagen; at least one plastid regulatory sequence comprising the promoter of psaA and / or the 5’ UTR of psaA;

[0405] a plastid terminator comprising TrbcL,

[0406] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome, wherein the chloroplast-encoded genes are trnE2 and psbN; and

[0407] optionally a selectable marker, comprising aadA.

[0408] In a preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0409] a sequence encoding at least one collagen a chain for producing collagen; at least one plastid regulatory sequence comprising the promoter of psaA and / or the 5’ UTR of psaA;

[0410] a plastid terminator comprising TrbcL,

[0411] 15252506-1a pair of flanking sequences comprising a first sequence from a or a portion of a first cholroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome, selected trnG and trnFM; and

[0412] optionally a selectable marker, comprising aadA.

[0413] In another preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0414] a sequence encoding at least one collagen a chain for producing collagen, preferably at least two collagen a chains for producing collagen;

[0415] at least one plastid regulatory sequence comprising the promoter sequence of the promoter of rm, psaA, psbA, rps16 or rbcL and / or the 5’UTR of rm, psaA, psbA, rps16 or rbcL;

[0416] chloroplast terminators comprising Trps16 and / or TpsbA

[0417] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome, selected trnG and trnFM; and

[0418] optionally a selectable marker, comprising aadA.

[0419] In another preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0420] a sequence encoding at least one collagen a chain for producing collagen, preferably at least two collagen a chains for producing collagen;

[0421] at least one plastid regulatory sequence comprising the promoter of rm, psaA, psbA, rps16 or rbcL and / or the 5’UTR of rm, psaA, psbA, rps16 or rbcL;

[0422] chloroplast terminators comprising Trps16 and / or TpsbA

[0423] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome, selected trnI and trnA; and

[0424] optionally a selectable marker, comprising aadA.

[0425] In another preferred embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding:

[0426] 15252506-1(a) a codon-optimised sequence encoding at least one collagen a chain for producing collagen, preferably at least two collagen a chains for producing collagen, preferably wherein the sequence is selected from vertebrate collagens (i.e., COL1A1-COL29A), collagen-like domains or recombinant collagens, truncated procollagen fragments or stabilised triple-helix motifs, or synthetic collagen-mimetic peptides;

[0427] (b) a plastid promoter derived from Chlamydomonas reinhardtii, preferably PpsaA, PpsbA or PatpA or variant thereof, more preferably PpsaA;

[0428] (c) a psaA 5' untranslated region (5'UTR) or variant thereof;

[0429] (d) a plastid terminator, preferably comprising a psbA terminator, rbcL terminator, or variant thereof; and

[0430] (e) a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen a chain into a plastid genome, preferably wherein the plastid-encoded genes are from Lactuca sativa, Nicotiana tabacum, or Chlamydomonas reinhardtii.

[0431] A similar construct is exemplified in SEQ ID NO: 87, comprising a combination of higher plant (tobacco) promoters and the psaA 5’UTR from Chlamydomonas reinhardtii, to regulate the expression of collagen.

[0432] According to any embodiment, preferably, the sequences of the chloroplast promoter and / or chloroplast terminator are obtained from Chlamydomonas reinhardtii.

[0433] According to any embodiment, preferably the sequence encoding at least one collagen a chain for producing collagen is selected from a collagen listed in Table 1 and defined by a sequence encoding a nucleic acid which encodes an amino acid sequence listed within Table 2.

[0434] The nucleic acid construct may further comprise a nucleic acid sequence comprising a leader sequence. A leader sequence is a polynucleotide region located between the promoter and the coding region that may regulate gene expression at the level of transcription or translation due to their propensity to form mutually exclusive secondary structures (stem-loops) by base-pairing of complementary sequences.

[0435] 15252506-1In one embodiment, the nucleic acid construct may further comprise a nucleic acid sequence encoding at least one enzyme for the post-translational modification of the expressed collagen α chain(s). Such a leader sequence may be encoded on the same nucleic acid encoding at least one collagen a chain, or alternatively, on a separate nucleic acid. In both embodiments, the at least one enzyme is under the transcriptional control of a promoter functional in chloroplasts.

[0436] The expression of at least one enzyme assists with the modification of collagen precursors into high-order structures, as described above.

[0437] In one embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding at least one enzyme for the post-translational modification of at least one collagen a chain.

[0438] Typically, a collagen chains expressed in plants may or may not include their terminal propeptides (i.e. propeptide C and propeptide N). The processing of procollagen by plant proteolytic activity is different to the processing in mammals and propeptide C is removed by plant proteolytic activity although the cleavage site is unknown. In a case where N and / or C propeptides are included in the expressed collagen chain, the nucleic acid may also comprise a sequence encoding for the respective protease (i.e. C or N or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NOs: 3 (protease C) and 4 (Protease N) or functional variants or homologs thereof. Such proteases can be expressed such that they are accumulated in the same subcellular compartment as the collagen chain.

[0439] Accordingly, in one embodiment, the at least one enzyme is a C-protease and / or a N-protease. In one embodiment, the sequence of the C-protease and / or N-protease comprises or consists of a sequence defined in SEQ ID NO: 3 and 4 respectively and variants thereof.

[0440] The expression of the C-protease and / or N-protease will assist in modifying expressed collagen α chain(s) to enable their self-assembly into a tropocollagen molecule.

[0441] 15252506-1In another embodiment, the at least one enzyme is prolyl-4-hydroxylase (P4H), wherein the P4H hydroxylates the collagen α chain(s) [i.e. hydroxylates the proline (Y) position of the Gly-X-Y triplets], P4H is an enzyme composed of two subunits, alpha and beta. Both are needed to form an active enzyme while the Beta subunit also possesses a chaperone function. The 4-hydroxyproline residues that result from the activity of P4H stabilizes the triple helices.

[0442] Accordingly, in one embodiment, the nucleic acid construct further comprises a sequence encoding a prolyl-4-hydroxylase (P4H), preferably a mammalian P4H. In one embodiment, P4H comprises or consists of a sequence encoding SEQ ID NO: 5 and 6, or a homolog or functional variant thereof. Human P4H is preferably encoded by, for example, by SEQ IDs NO: 5 and 6. A suitable P4H homologue is the Arabidopsis oxidoreductase identified by NCBI accession NP_179363. Pairwise alignment of the protein sequence of the Arabidopsis oxidoreductase identified by NCBI accession NP_179363 and a human P4H alpha subunit revealed the highest homology between functional domains of any known plant P4H homolog, thereby confirming it as an appropriate choice..

[0443] In another embodiment, the method may further comprise expressing a nucleic acid sequence encoding a Lysyl hydroxylase, galactosyltransferase and / or a glucosyltransferase. These three enzymes are involved in post-translational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues.

[0444] In one embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding a single human enzyme, Lysyl hydroxylase 3 (LH3). LH3 can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation.

[0445] In one embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding LH3, wherein LH3 is defined as a sequence comprising or consisting of a sequence encoding SEQ ID NO: 7 or a functional variant or homolog thereof.

[0446] In another aspect of the invention, there is provided a cell comprising the nucleic acid construct of the invention.

[0447] 15252506-1In another aspect of the invention, there is provided a genetically modified organism (stably or transiently) expressing any of the nucleic acids or nucleic acid constructs described herein. In an alternative aspect of the present invention, there is provided an isolated cell (transiently or stably) transfected with at least one nucleic acid or nucleic acid construct as described herein. Preferably, the genetically modified organism is a plant or an alga.

[0448] In some embodiments, the genetically modified organism is Chlamydomonas reinhardtii. Through a genetically modified organism of the invention, i.e., an organism characterised by the plastid genome comprising an expression cassette encoding collagen polypeptides under the control of a strong promoter, it is possible to control collagen expression in microalgae for applications in cosmetics, nutraceuticals, or biomaterials, filling an unmet need in algal biotechnology.

[0449] Indeed, the methods described herein, including the use of thermoresponsive regulatory elements, are compatible with:

[0450] • greenhouse cultivation,

[0451] • open field

[0452] • vertical farming,

[0453] • controlled-environment agriculture,

[0454] • photobioreactors (for Chlamydomonas).

[0455] In one embodiment, the collagen is produced in Chlamydomonas reinhardtii plastids and produced in photobioreactors, algal fermenters, or outdoor cultivation systems.

[0456] In one aspect of the invention, there is provided a genetically modified plant, part thereof or plant cell characterised by the expression of exogenous collagen in at least one plastid, further characterised by the expression of any of the nucleic acids or nucleic acid constructs described herein.

[0457] In one aspect of the invention, there is provided a genetically modified alga characterised by the expression of exogenous collagen in at least one plastid, further characterised by the expression of any of the nucleic acids or nucleic acid constructs described herein.

[0458] 15252506-1In one embodiment, the genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, wherein preferably the organism comprises and expresses a nucleic acid construct of the invention, and wherein the organism is further characterised by the expression of at least one nucleic acid encoding at least one enzyme selected from a group consisting of:

[0459] • C-protease, wherein C-protease is defined by a nucleic acid sequence comprising or consisting of SEQ ID NO: 3 or a homolog or functional variant thereof;

[0460] • N-protease, wherein N-protease is defined by a nucleic acid sequence comprising or consisting of SEQ ID NO: 4 or a homolog or functional variant thereof;

[0461] • prolyl-4-hydroxylase (P4H), preferably human PH4, wherein the human PH4 is defined by a nucleic acid sequence comprising or consisting of SEQ ID NO: 6 or 6, or a homolog or functional variant thereof; and

[0462] • human Lysyl hydroxylase 3, wherein LH3 is defined by a nucleic acid sequence comprising or consisting of SEQ ID NO: 7 or a homolog or functional variant thereof.

[0463] •

[0464] In another aspect of the invention, there is provided a method of producing a genetically modified organism or a genetically modified cell characterised by the expression of exogenous collagen in at least one plastid, the method comprising transforming said organism or cell with a nucleic acid or nucleic acid construct of the present invention.

[0465] In another aspect of the invention, there is provided a method of producing a genetically modified plant, plant part thereof or plant cell characterised by the expression of exogenous collagen in at least one plastid, the method comprising transforming said plant, plant part thereof or plant cell with a nucleic acid or nucleic acid construct of the invention as described herein.

[0466] The transfer of foreign genes or nucleic acid constructs into the nuclear or plastid genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Any of several transformation methods known to the skilled person may be used to introduce the nucleic acid construct or sgRNA molecule of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation.

[0467] 15252506-1The methods and principles for transformation are largely discussed in the context of plants. However, the methods and principles disclosed herein also apply to the transformation of other photosynthetic organisms, such as alga.

[0468] There are two principal methods of effecting stable genomic integration of exogenous sequences such as those included within the nucleic acid constructs of the present invention into plastid genomes:

[0469] (i) Agrobacterium-mediated gene transfer: The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. The details of this method are well known to a skilled person. In short, plant transformation by Agrobacterium results in the integration into the nuclear genome of a sequence called T-DNA, which is carried on a bacterial plasmid. The use of T-DNA transformation leads to stable single insertions. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. Another common approach is the floral dip / Agrobacterium vacuum infiltration method as described in Clough & Bent (1998) and incorporated herein by reference.

[0470] (ii) direct DNA uptake: for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant (microinjection), gene guns (or biolistic particle delivery systems (biolistics)) as described in the examples, lipofection, transformation using viruses or pollen and microprojection.

[0471] The essential steps to producing a genetically modified plant or alga expressing an exogenous nucleic acid or protein in at least one plastid, such as a chloroplast, are described below. First, plant or alga cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Plastid transformation is typically based on DNA delivery by the biolistic process (Daniell et al., 1990) or occasionally by polyethylene glycol (PEG) treatment of protoplasts (O'Neill et al., 1993). This is followed by transgene integration into the chloroplast genome via homologous recombination facilitated by a RecA-type system between the plastid-targeting sequences of the transformation vector (i.e., the pair of flanking sequences) and the targeted region of the plastid genome. Chloroplast transformation vectors are thus designed with homologous flanking

[0472] 15252506-1sequences on either side of the transgene cassette to facilitate double recombination. Transformation is accomplished by integration of the transgene into a few genome copies, followed by 25 to 30 cell divisions under selection pressure to eliminate untransformed plastids, thereby achieving a homogeneous population of plastid genomes. If the transgene is targeted into the inverted repeat (IR) region, integration in one IR is followed by the phenomenon of copy correction that duplicates the introduced transgene into the other IR as well. In addition, the exogenous nucleic acid may include a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous nucleic acid.

[0473] Accordingly, in one embodiment a plastid of the present invention is transformed by a method selected from the calcium / polyethylene glycol method for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibres, electroporation of protoplasts, microinjection into plant material, infection with (non-integrative) viruses and the like.

[0474] Preferably, the method is selected from the calcium / polyethylene glycol method for protoplasts or a biolistic particle delivery system, most preferably a biolistic particle delivery system.

[0475] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be self-crossedand homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. Appropriate propagation methods will be discernible to those in the art, and are reviewed in detail in Belanger et al. (2024).

[0476] Transient expression methods which can be utilized for transiently expressing the isolated nucleic acid included within the nucleic acid construct of the present invention include, but are not limited to, microinjection and bombardment as described above but under conditions which favour transient expression, and viral mediated expression wherein a packaged or unpackaged recombinant virus vector including the nucleic acid construct is utilized to infect plant tissues or cells such that a propagating recombinant virus established therein expresses the non-viral nucleic acid sequence.

[0477] 15252506-1Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV and BV. Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87 / 06261.

[0478] Accordingly, in one embodiment, at least one nucleic acid construct as described herein can be introduced to at least one plant cell or alga using any of the above described methods. In an alternative embodiment, any of the nucleic acid constructs described herein may be first transcribed to form a preassembled Cas9-sgRNA ribonucleoprotein and then delivered to at least one plant cell or alga using any of the above described methods, such as lipofection, electroporation or microinjection.

[0479] Optionally, to select transformed organisms, the organic material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed organisms can be distinguished from untransformed organisms. For example, for plants, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. As described in the examples, a suitable marker can be the aad / X gene which confers resistance to streptomycin and spectinomycin. Alternatively, the transformed plants or alga are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS (β-glucuronidase).

[0480] Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using PCR to detect the presence of the gene of interest, copy number and / or genomic organisation. Alternatively or additionally, integration and expression levels of the newly introduced DNA may be monitored using Southern, Northern and / or Western analysis, all techniques being well known to persons having ordinary skill in the art.

[0481] Methods of producing collagen

[0482] In one aspect of the invention there is provided a method of producing collagen in a plastid of a photosynthetic organism, the method comprising introducing and expressing at least one nucleic acid or nucleic acid construct of the invention in the plastid of the photosynthetic organism.

[0483] 15252506-1In one embodiment, the method further comprises introducing and expressing the at least one nucleic acid or nucleic acid construct by transformation. Appropriate methods and embodiments relating to transformation are described elsewhere.

[0484] In a preferred embodiment, the plastid is a chloroplast. Accordingly, in one embodiment, there is provided a method of producing collagen in a chloroplast of a photosynthetic organism, the method comprising introducing and expressing a nucleic acid construct of the invention in the chloroplast of the photosynthetic organism.

[0485] In a preferred embodiment, the plastid is a chloroplast and the photosynthetic organism is an alga or algae. Accordingly, in one embodiment, there is a method of producing collagen in a chloroplast of an alga or algae, the method comprising introducing and expressing a nucleic acid construct of the invention in the chloroplast of the alga or algae.

[0486] In a preferred embodiment, the plastid is a chloroplast and the photosynthetic organism is a plant, plant part thereof or plant cell. Accordingly, in one embodiment, there is a method of producing collagen in a chloroplast of a plant, part thereof or plant cell, the method comprising introducing and expressing a nucleic acid construct of the invention in the chloroplast of plant, part thereof or plant cell.

[0487] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises at least one nucleic acid encoding at least one collagen α chain for producing collagen, a plastid gene promoter, a plastid gene terminator, a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, and optionally a selectable marker gene.

[0488] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding at least one collagen a chain suitable for producing collagen and at least one plastid regulatory sequence comprising the promoter of psaA from Chlamydomonas reinhardtii and / or the 5’UTR of psbA from Chlamydomonas reinhardtii.

[0489] 15252506-1In one embodiment, the at least one plastid regulatory sequence comprises the promoter of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 12 or 83 or functional variant or homolog thereof.

[0490] In one embodiment, the at least one plastid regulatory sequence comprises the 5’UTR of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

[0491] In one embodiment, the at least one plastid regulatory sequence comprises the promoter of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 12 or 83 or functional variant or homolog thereof and the 5’UTR of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

[0492] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0493] at least one collagen a chain suitable for producing collagen;

[0494] a plastid promoter, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0495] a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0496] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0497] a selectable marker.

[0498] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0499] at least one collagen a chain suitable for producing collagen;

[0500] at least one plastid regulatory sequence for regulating the expression of the sequence encoding at least one collagen α chain in a chloroplast;

[0501] 15252506-1a chloroplast terminator for regulating the expression of the sequence encoding at least one collagen α chain in a chloroplast;

[0502] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0503] a selectable marker.

[0504] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a acid sequence encoding:

[0505] at least one collagen a chain suitable for producing collagen;

[0506] at least one plastid regulatory sequence selected the promoter of rrn, psaA, psbA, rps16 orrbcL and / or the 5’UTR of rrn, psaA, psbA, rps16 orrbcL;

[0507] a plastid terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA;

[0508] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0509] optionally a selectable marker, selected from neo, aadA, nptII, aphA6, badh and npII-aphA6.

[0510] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the chloroplast of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0511] at least one collagen a chain suitable for producing collagen;

[0512] at least one plastid regulatory sequence comprising the promoter of psaA and / or the 5’ UTR of psaA;

[0513] a plastid terminator comprising TrbcL,

[0514] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0515] optionally a selectable marker, comprising aadA.

[0516] 15252506-1In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0517] a sequence encoding at least one collagen a chain for producing collagen, preferably at least two collagen a chains for producing collagen;

[0518] at least one plastid regulatory sequence selected the promoter of rrn, psaA, psbA, rps16 orrbcL and / or the 5’UTR of rrn, psaA, psbA, rps16 orrbcL;

[0519] a plastid terminator comprising Trps16 and / or TpsbA;

[0520] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0521] optionally a selectable marker, comprising aadA.

[0522] In one embodiment, the method of producing collagen in a plastid of a photosynthetic organism is thermoregulated i.e. the yield of collagen is modulated by altering plant or algal growth temperatures. Accordingly, in an embodiment, there is provided a method for producing recombinant collagen in a plastid of a photosynthetic organism, wherein the yield of collagen is modulated by altering plant growth temperatures. In one embodiment, this is achieved by using a themoresponsive promoter or 5' UTR and / or a thermoresponsive terminator or 3' UTR regulatory elements.

[0523] In a preferred embodiment, the themoresponsive promoter or 5' UTR and / or a thermoresponsive terminator or 3' UTR regulatory element is obtained from Chlamydomonas reinhardtii. In a more preferred embodiment, this is achieved by leveraging the psaA 5' UTR and optionally rbcL 3' UTR regulatory elements from Chlamydomonas reinhardtii, as described above to enhance expression of collagen at elevated growth temperatures. In an embodiment, the photosynthetic organism is grown at a temperature above 20ºC, preferably above 25ºC, more preferably above 30ºC. In an embodiment, the photosynthetic organism is grown at a temperature at or around 40 ºC. Such a temperature can be used to induce or increase the expression of collagen in the plastid.

[0524] 15252506-1In one embodiment, the photosynthetic organism comprising a nucleic acid construct of the invention is grown (i.e., cultivated) at a first temperature, for example at or around 15 °C-25 °C and subsequently, the photosynthetic organism is grown at a second temperature, such as above 25 °C, more preferably above 30 °C, even more preferably at or around 40 °C wherein the second temperature is higher than the first temperature.

[0525] In one embodiment, the change from the first temperature to the second temperature occurs over a number of hours, preferably 2 to 8 hours.

[0526] In one embodiment, the change from the first temperature to the second temperature is associated with an increase in collagen production per unit of time.

[0527] In a preferred embodiment, the first temperature is at or around 15 °C to 25 °C, preferably at or around 15 °C, 20 °C or 25 °C, or less than 28 °C.

[0528] In a preferred embodiment, the second temperature is greater than 25 °C, such as at or around 28 °C to 40 °C, preferably 30 °C to 40 °C, more preferably 35 °C to 40 °C, more preferably again at or around 40 °C, most preferably at or around 45 °C.

[0529] The change in temperature may be transient, sustained, or pulsed.

[0530] The temperature at which expression is increased (i.e., a hairpin conformation is altered) may be species-specific and adjustable based on engineered variants of the psaA 5'UTR with altered stem stability.

[0531] In some embodiments, multiple cycles of altering the temperature between a first and a second temperature may be used to maximise yield while minimising metabolic burden.

[0532] Thus in a preferred embodiment, the method comprises:

[0533] a) introducing and expressing a nucleic acid construct of the invention in the plastid of the organism, wherein the at least one nucleic acid sequence encodes at least one plastid regulatory sequence from Chlamydomonas, preferably Chlamydomonas reinhardtii, more preferably the psaA 5’ UTR sequence and / or promoter of psaA obtained from Chlamydomonas reinhardtii;

[0534] 15252506-1b) growing or cultivating the organism at a first temperature, preferably at or around 15 °C-25 °C;

[0535] c) growing or cultivating the organism at a second temperature, preferably above 25 °C, more preferably above 30 °C, even more preferably at or around 40 °C, most preferably at or around 45 °C; and

[0536] d) wherein the second temperature is higher than the first temperature.

[0537] In another preferred embodiment, the method comprises:

[0538] a) introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:

[0539] i) a sequence encoding at least one collagen a chain for producing collagen, preferably at least two collagen a chains for producing collagen;

[0540] ii) at least one plastid regulatory sequence from Chlamydomonas, preferably Chlamydomonas reinhardtii, more preferably the psaA 5’ UTR sequence and / or promoter of psaA obtained from Chlamydomonas reinhardtii

[0541] iii) at least one chloroplast terminator;

[0542] iv) a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome; and

[0543] v) optionally a selectable marker;

[0544] b) growing or cultivating the organism at a first temperature, preferably at or around 15 °C -25 °C

[0545] c) growing or cultivating the organism at a second temperature, preferably above 25 °C, more preferably above 30 °C, even more preferably at or around 40 °C wherein the second temperature is higher than the first temperature, and preferably wherein the temperature is raised for between 2 to 8 hours.

[0546] In one embodiment, the step further comprises harvesting (i.e., extracting) collagen from the organism.

[0547] 15252506-1Preferably, the organism is an algae or higher plant, more preferably selected from tobacco, lettuce or Chlamydomonas spp.

[0548] In another aspect, there is provided a method of producing collagen in a plastid of a photosynthetic organism comprising expressing psaA 5'UTR obtained from a Chlamydomonas spp, preferably Chlamydomonas reinhardtii, in a photosynthetic organism, wherein the photosynthetic organism is not Chlamydomonas spp. Preferably, the photosynthetic organism is a higher plant, more preferably lettuce or tobacco.

[0549] In one embodiment, the method further comprises extracting the collagen from the plastid.

[0550] In one embodiment, the method further comprises extracting the collagen from the plastid and processing the extracted collagen into collagen peptides. In one embodiment, processing the extracted collagen comprises denaturing and hydrolysing the extracted collagen. In one embodiment, hydrolysis may comprise the use of proteolytic enzymes or acidic and / or basic compounds.

[0551] In another aspect, there is provided a method for producing at least one collagen peptide in a plastid of a photosynthetic organism, the method comprising introducing and expressing at least one nucleic acid construct of the invention in the plastid of the photosynthetic organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding a protease suitable for hydrolysing collagen.

[0552] According to any methods described herein, in one embodiment, the amount of collagen produced is at least 5 mg / g fresh weight of extracted protein, preferably 10 mg / g fresh weight. In an additional or alternative embodiment, the amount of collagen produced by a method described herein is at least 5 g / kg biomass. This is shown in Figure 10, wherein the collagen produced under expression psaA 5UTR from Chlamydomonas yields 5mg / g FW (weight data not shown). The recombinant collagen is produced at yields measurable as mg per g fresh weight biomass, where fresh weight refers to the mass of harvested biological material prior to drying. The collagen comprises a short-repeat (~3 kDa) type III collagen architecture that retains biologically relevant collagen motifs, exhibits ECM-and cell-interaction bioactivity, and offers improved solubility and formulation compatibility. The 589–510 bp collagen repeat encodes a functional collagen domain,

[0553] 15252506-1and inclusion of three tandem repeats provides an optimal balance between biological functionality, molecular size, and recombinant manufacturability.

[0554] In a final aspect of the invention, there is provided the collagen produced by the methods described herein. The methods disclosed herein allow for collagen with high purity to be synthesised in plastids.

[0555] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0556] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, " A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0557] Example I - Plant Chloroplast Transformation Protocol

[0558] Plant Material and Growth Conditions:

[0559] Seeds of plants were surface sterilized and germinated on Murashige and Skoog (MS) medium supplemented with 30 g / L sucrose. Plants were grown under controlled conditions: 16-hour light / 8-hour dark cycle with a light intensity of 50 pmol photons m-2s-1at 23±1°C and 75% relative humidity. These conditions ensured optimal growth and development of the plants for transformation.

[0560] Chloroplast Transformation via Biolistic Bombardment:

[0561] Chloroplast genomes were transformed using the biolistic bombardment method. Plasmid DNA constructs were prepared at a concentration of 1 pg / pL. To prepare DNA-loaded gold particles, 10 pL of plasmid DNA was thoroughly mixed with 1 mg of washed gold particles (0.6 pm diameter, Bio-Rad), along with 2.5 M CaCI2and 0.1 M spermidine. The mixture was washed and resuspended in 50 pL of 100% ethanol.

[0562] 15252506-1The plasmid DNA-loaded gold particles were delivered into young tobacco leaf cells using a helium-driven particle gun (PDS-1000He, Bio-Rad) set to 1100 psi. The leaves were placed on regeneration RMOP medium during the bombardment. This method ensures high transformation efficiency and minimal damage to the leaf tissue.

[0563] Selection and Regeneration of Transgenic Lines:

[0564] Following bombardment, leaf tissues were incubated for 24 hours and then cut into smaller pieces for selection. The tissues were cultured on RMOP medium supplemented with 500 mg / L spectinomycin to select for recombinant chloroplasts. Within 6 weeks, mutant lines were regenerated, with visible shoots arising from the transformed tissue.

[0565] Validation of Transgenic Lines:

[0566] Regenerated plants were subsequently analyzed to confirm transgene integration into the chloroplast genome. PCR amplification using specific primers binding on native chloroplast genome verified the presence of the transgene, and further analysis, including western blot and protein expression assays, was conducted to confirm successful transformation and expression of the target gene

[0567] Example 2 - Method for constructing transformation plasmids and introducing them into chloroplasts for the stable expression of the collagen gene (COLA1) in Chlamydomonas reinhardtii.

[0568] The method involves the following steps:

[0569] 1. Construction of Transformation Plasmids and Vector configuration:

[0570] The collagen gene (COLA1) is inserted into the chloroplast expression vector pRSAPI, comprising regulatory elements including the psaA promoter and 5' untranslated region (5' UTR), and the rbcL transcription terminator (3' UTR). The insertion of the collagen gene is facilitated by the use of Sapl and Sphl restriction sites.

[0571] The pRSAPI transformation vector is flanked by homologous arms, including left and right flanking regions, corresponding to plastome sequences. These flanking regions include the mutated version of psbH locus and an aadA, which serves as a selectable marker for successful integration.

[0572] Figure 4a represents an exemplar transformation plasmid used in the process. The plasmid comprises:

[0573] 15252506-1• A trnE2 region for homologous recombination into the plastid genome.

[0574] • A collagen gene (COLA1 CDS) driven by the psaA 5' UTR as the promoter and untranslated region.

[0575] • The rbcL 3' UTR, functioning as a transcription terminator for stable gene expression.

[0576] • The psbH gene, included as part of the homologous arms for restoration of its function in the recipient plastome.

[0577] • The flanking sequences also include psbN, which serves as an additional structural element of the plastome.

[0578] 2. Chloroplast Transformation:

[0579] The pRSAPI vector is introduced into the chloroplast genome of the HT72 recipient strain, which is a psbH knockout mutant. In this strain, the psbH gene and downstream regions have been replaced with an aadA gene expression cassette. As shown in Figure 4, the use of the psbH knockout HT72 strain provides an additional level of selection; restoration of the psbH gene function in transformants can be used to select for successfully transformed algae.

[0580] Figure 4b depicts the initial state of the plastome in the HT72 recipient strain, characterized by:

[0581] • A disruption of the psbH gene, replaced with an aadA cassette conferring spectinomycin resistance (SpecR).

[0582] • The aadA cassette is flanked by the atpA 5' UTR and rbcL 3' UTR, ensuring its expression in the chloroplast genome.

[0583] • The presence of trnE2 and psbN as adjacent plastid loci, providing target sites for homologous recombination.

[0584] 3. Genomic Outcomes:

[0585] Upon transformation, the following genomic modifications are achieved:

[0586] • Restoration of psbH gene function.

[0587] • Stable integration of the collagen gene (COL1A1) into the chloroplast genome.

[0588] • The aadA cassette is replaced by the transformation cassette from the plasmid, resulting in: the restoration of the psbH gene function; stable integration of the COLA1 CDS, driven by the psaA 5' UTR promoter and terminated by the rbcL 3'

[0589] 15252506-1UTR; and the loss of the spectinomycin resistance conferred by the aadA cassette.

[0590] 4. Selection of Transformants:

[0591] Transformants are grown on acetate-free medium (Hsm) to enable phototrophic selection. Transformants exhibit sensitivity to spectinomycin and streptomycin, confirming the loss of the aadA gene. Positive transformants carrying the collagen gene (COLA1) are identified based on these phenotypic markers.

[0592] The described method enables efficient expression of the collagen gene (COLA1) in chloroplasts with stable genomic integration. Transformants are phototrophic, eliminating reliance on external acetate, and are devoid of antibiotic resistance markers, enhancing safety for downstream applications.

[0593] The success of such a strategy is verified in Figure 8, which shows the expression of collagen protein in transgenic alga.

[0594] Example 4: Transformation and Integration of Collagen Expression Cassette using Chlamydomonas-derived regulatory elements for expression in Tobacco Plastids.

[0595] Figure 5A represents the transformation plasmid, which comprises the following components:

[0596] • Gene of Interest (GOI): COL1A1 CDS: Encodes the desired collagen protein, optimized for expression in the Tobacco chloroplast.

[0597] • Regulatory Elements:

[0598] o Cr_psaA 5' UTR (Chlamydomonas reinhardtii): Promoter and untranslated region driving transcription of the collagen gene in Nicotiana tabacum plastids.

[0599] o Cr_rbcL 3' UTR (Chlamydomonas reinhardtii): Ensures proper termination and transcript stability of the COL1A1 gene in tobacco plastids.

[0600] • Selectable Marker:

[0601] o aadA: Provides resistance to spectinomycin for selecting transformed plastids. This gene is flanked by loxP sites, facilitating marker excision using Cre recombinase.

[0602] • Additional Regulatory Sequences:

[0603] 15252506-1o Nt_psbA 5' UTR: A tobacco-derived untranslated region included for supporting downstream expression of the selectable marker.

[0604] o Nt_Prrn 5' UTR: Tobacco-derived promoter driving marker gene transcription.

[0605] • Homologous Arms:

[0606] o Flanking regions include trnG and trnfM, facilitating precise integration of the transformation cassette into the target region of the tobacco plastid genome.

[0607] Figure 5b, represents the wild-type (WT) plastome of tobacco prior to transformation, featuring:

[0608] • The trnG and trnfM loci flanking the targeted insertion site.

[0609] • An insertion site, indicated as the region for homologous recombination.

[0610] • Primers (F1 and R1):

[0611] o Primer regions (red arrows) are included for molecular validation of successful integration and orientation of the expression cassette.

[0612] Figure 5c illustrates the plastome after successful transformation:

[0613] • Integrated Cassette:

[0614] o The collagen gene (COL1A1 CDS) is driven by the Cr_psaA 5' UTR promoter and terminated by the Cr_rbcL 3' UTR in the tobacco plastid genome.

[0615] o The aadA selectable marker remains in the plastome, flanked by loxP sites, enabling its removal through recombinase-mediated excision. • Molecular Validation:

[0616] o Primer sites (F1 and R1) are utilized to confirm successful recombination and proper integration of the expression cassette.

[0617] Together, these components achieve the following outcomes:

[0618] 1. Collagen Expression:

[0619] o The Chlamydomonas reinhardtii-derived regulatory elements (Cr_psaA 5' UTR and Cr_rbcL 3' UTR) are effectively expressed in the tobacco plastid genome, ensuring high levels of COL1A1 protein synthesis. 2. Selection and Validation:

[0620] 15252506-1o Transformants are resistant to spectinomycin due to the aadA gene, enabling selection of successful plastid transformants.

[0621] o Primer regions confirm correct orientation and integration of the cassette via PCR diagnostics.

[0622] 3. Marker Removal:

[0623] o The loxP sites allow excision of the aadA gene, resulting in marker-free transformants suitable for downstream applications.

[0624] Example 5. Strategy for introducing a multi-gene collagen gene (human) construct (COL1A1, COL2A1, and COL3A1) into the plastid genome of Nicotiana tabacum (Nt).

[0625] Figure 6a shows the transformation plasmid constructed for the tobacco plastid genome. The plasmid contains the following key components:

[0626] • Collagen Genes:

[0627] o COL1A1: An 87-nucleotide sequence encoding a 29-amino-acid peptide, o COL2A1: A 51 -nucleotide sequence encoding a 17-amino-acid peptide, o COL3A1: A 96-nucleotide sequence encoding a 32-amino-acid peptide.

[0628] • Regulatory Elements:

[0629] o Nt_Prrn: Promoter that drives transcription of the collagen genes.

[0630] o Nt_Trps16 and Nt_TrbcL: 5' untranslated regions (UTRs) that enhance translational efficiency of the collagen constructs.

[0631] o cr_TrbcL: A terminator derived from Chlamydomonas reinhardtii, ensuring transcript stability and proper termination of transcription. o Nt_TpsbA: A 3' UTR for additional transcriptional regulation. o IEE: an intercistronic expression element (IEE), that mediates the efficient intercistronic cleavage of polycistronic mRNAs into stable monocistronic transcripts.

[0632] • Selectable Marker:

[0633] o aadA: Confers resistance to spectinomycin, enabling selection of transformed plastids. The aadA gene is flanked by loxP sites, which allow for its removal using Cre recombinase after successful transformation.

[0634] • Direct Repeats (DR): included to support marker removal via homologous recombination or recombinase activity.

[0635] 15252506-1• Homologous Arms: The plasmid includes flanking sequences corresponding to trnG and trnfM regions of the tobacco plastid genome to enable precise integration via homologous recombination.

[0636] Figure 6b, represents the wild-type (WT) plastome of tobacco prior to transformation, featuring: trnG and trnfM loci, which flank the targeted insertion site; an insertion site located between these loci, identified as the target for homologous recombination, and primer sites (F1 and R1, indicated as red arrows), to verify successful transformation and confirm proper cassette integration.

[0637] Figure 6c illustrates the plastome after successful transformation, characterised by:

[0638] o Collagen constructs (COL1A1, COL2A1, and COL3A1) integrated into the plastid genome under the control of associated regulatory elements (Nt_Prrn promoter, Nt_Trps16 UTR, Nt_TrbcL UTR, and cr_TrbcL terminator).

[0639] o The aadA selectable marker is retained initially for selection purposes and flanked by loxP sites for eventual excision using Cre recombinase.

[0640] o The plastid genome is flanked by trnG and trnfM regions, confirming precise integration.

[0641] o Primer Sites (F1 and R1): Red arrows indicate primers used to confirm the presence of the transformed cassette via conventional molecular diagnostic methods

[0642] This strategy achieves the following outcomes:

[0643] 1. Collagen Gene Expression: The regulatory elements ensure efficient transcription, translation, and stability of the collagen genes, allowing for the synthesis of functional collagen peptides in the chloroplast.

[0644] 2. Selectable Marker Removal: The flanking loxP sites enable removal of the aadA gene using Cre recombinase, ensuring a clean plastid genome for downstream applications.

[0645] 3. Stable Integration: Homologous recombination ensures precise integration of the transformation cassette into the targeted region of the plastid genome.

[0646] The present transformation strategy is associated with numerous advantages that include: a high-yield production of collagen peptides using tobacco chloroplasts as a bioreactor; incorporation of regulatory elements to optimize transcription, translation, and

[0647] 15252506-1stability of the recombinant collagen genes; and a selectable marker system with marker removal capability for safety and regulatory compliance in downstream applications.

[0648] Example 6. Strategy for introducing a multi-gene collagen gene (human) construct (COL1A1, COL2A1, and COL3A1) using combination of tobacco and Chlamydomonas-derived regulatory elements for expression in tobacco / lettuce chloroplasts.

[0649] Figure 7a shows the transformation plasmid constructed for the lettuce plastid genome. The plasmid contains the following key components:

[0650] • Collagen Genes:

[0651] o COL1A1: An 87-nucleotide sequence encoding a 29-amino-acid peptide, o COL2A1: A 51 -nucleotide sequence encoding a 17-amino-acid peptide, o COL3A1: A 96-nucleotide sequence encoding a 32-amino-acid peptide.

[0652] • Regulatory Elements:

[0653] o Nt_Prrn: Promoter that initiates and drives polycistronic transcription of the collagen genes.

[0654] o Nt_Trps16 and Nt_TrbcL: 5' untranslated regions (UTRs) that enhance translational efficiency of the collagen constructs.

[0655] o cr_TrbcL: A terminator derived from Chlamydomonas reinhardtii, ensuring transcript stability and proper termination of transcription. o Nt_TpsbA: A 3' UTR for additional transcriptional regulation. o IEE: an intercistronic expression element (IEE), that mediates the efficient intercistronic cleavage of polycistronic mRNAs into stable monocistronic transcripts.

[0656] • Selectable Marker:

[0657] o aadA: Confers resistance to spectinomycin, enabling selection of transformed plastids. The aadA gene is flanked by loxP sites, which allow for its removal using Cre recombinase after successful transformation.

[0658] • Direct Repeats (DR): included to support marker removal via homologous recombination or recombinase activity.

[0659] • Homologous Arms: The plasmid includes flanking sequences corresponding to trnA and trnl regions of the lettuce plastid genome to enable precise integration via homologous recombination.

[0660] • PCR primers (F1 and R1) confirm correct integration and marker removal.

[0661] 15252506-1Figure 7b, represents the wild-type (WT) plastome of lettuce prior to transformation, featuring: trnA and trnl loci, which flank the targeted insertion site; an insertion site located between these loci, identified as the target for homologous recombination, and primer sites (F1 and R1, indicated as red arrows), to verify successful transformation and confirm proper cassette integration.

[0662] Figure 7c illustrates the plastome after successful transformation of lettuce chloroplasts, characterised by:

[0663] o Collagen constructs (COL1A1, COL2A1, and COL3A1) integrated into the plastid genome under the control of associated regulatory elements (Nt_Prrn promoter, Nt_Trps16 UTR, Nt_TrbcL UTR, and cr_TrbcL terminator). The multiple collagnen genes are transcribed as polycistronic mRNA, cleaved into stable monocistronic transcripts by I EEs, and translated efficiently.

[0664] o The aadA selectable marker is retained initially for selection purposes and flanked by loxP sites for eventual excision using Cre recombinase.

[0665] o The plastid genome is flanked by trnl and trnA regions, confirming precise integration in the lettuce plastid genome.

[0666] o Primer Sites (F1 and R1): Red arrows indicate primers used to confirm the presence of the transformed cassette via conventional molecular diagnostic methods

[0667] This strategy achieves the following outcomes:

[0668] 1. Efficient collagen gene expression: The regulatory elements and I EEs ensure stable and high-yield production of collagen peptides in lettuce plastids.

[0669] 2. Marker Removal: Dual strategies (LoxP-mediated or DR-mediated) allow for marker-free plastids, ensuring regulatory compliance.

[0670] 3. Stable Integration: Homology-based recombination at trnl and trnA loci ensures precise and reliable integration of the expression cassette.

[0671] The present transformation strategy is associated with numerous advantages that include: a scalable, high-yield production of collagen peptides using lettuce chloroplasts as a bioreactor; incorporation of regulatory elements that optimize transcription, translation, and stability of the recombinant collagen genes; and a selectable marker system with marker removal capability for safety and regulatory compliance in downstream applications.

[0672] 15252506-1Example 7.

[0673] Strategy for temperature-inducible plastid translation of collagen in algae and higher plants.

[0674] Figure 9 shows two constructs used to compare constitutive versus temperature-inducible plastid translation of collagen mediated by distinct 5' untranslated regions. Specifically, the purpose of the two constructs was to compare native tobacco plastid regulatory elements with algal psaA regulatory elements under elevated temperature conditions, with the aim of increasing collagen yield.

[0675] Figure 9A shows a nucleic acid construct a native tobacco plastid ribosomal RNA operon promoter (NtPrrn), a native tobacco plastid 5' untranslated region (Nt 5'UTR), the COL1A1 coding sequence, and a tobacco rbcL 3' untranslated region (Nt rbcL 3'UTR). This is suitable for expression in algae and higher plants.

[0676] Figure 9B shows a nucleic acid construct comprising a Chlamydomonas reinhardtii psaA promoter (CrPpsaA), the psaA 5' untranslated region (Cr psaA 5'UTR) containing a temperature-responsive RNA structure, the COL1A1 coding sequence, and the psaA 3' untranslated region (CrpsaA 3'UTR). This is suitable for expression in algae and higher plants.

[0677] Although not depicted in Figure 9, it is understood that the plasmid may further comprise optional features such as selectable marker, flanking regions, primer sites etc. as described herein.

[0678] Figure 10 shows that induction of plastid-expressed human collagen using Chlamydomonas regulatory elements, and specifically the thermoresponsive Chlamydomonas psaA 5'UTR, produces increased collagen production compared to constitutive regulatory elements.

[0679] Specifically, the expression of a nucleic acid construct comprising a Chlamydomonas regulatory element, specifically the thermoresponsive psaA 5'UTR (shown in Figure 8B) is associated with equal or greater production of collagen compared to constructs comprising regulatory elements native to the host organism, tobacco, at 25 °C.

[0680] 15252506-1Surprisingly, the expression of constructs comprising Chlamydomonas psaA 5'UTR is associated with an impressive increase in the production of collagen compared to constructs comprising regulatory elements native to the host organism, tobacco, at 40 °C, as shown by band size.

[0681] In particular, Figure 10 shows immunoblot analysis of collagen accumulation in transplastomic tobacco lines harbouring plastid-integrated COL1A1 expression cassettes controlled by either (i) native tobacco plastid regulatory elements (TPrrn-Nt 5'UTR I TrbcL-Nt 3'UTR) or (ii) Chlamydomonas reinhardtii regulatory elements (CrPpsaA-Cr psaA 5'UTR / CrpsaA 3'UTR). Total soluble protein was extracted from leaves maintained at 25 °C or following temperature elevation to 40 °C. For the tobacco regulatory construct (TPrrn-Nt 5'UTR / TrbcL-Nt 3'UTR), samples from plants grown at 25 °C and 40 °C are shown (left lanes). For the Chlamydomonas regulatory construct (CrPpsaA-Cr psaA 5'UTR / CrpsaA 3'UTR), protein extracts from two independent transplastomic plants (P1 and P2) are shown at 25 °C and after induction at 40 °C (middle lanes). Purified human collagen type I (skin) is included as a reference control (right lane; expected migration 140 kDa). Collagen was detected using an anti-collagen I antibody (upper panel). Coomassie Brilliant Blue staining of the corresponding gel is shown as a loading control (lower panel).

[0682] The elevated yields of collagen observed in tobacco lines expressing Chlamydomonas reinhardtii regulatory elements is understood to arise from the synergistic combination of:

[0683] • Strong plastid promoters derived from Chlamydomonas (e.g., PpsaA, PpsbA and PatpA);

[0684] • Efficient ribosome recruitment mediated by the psaA 5'UTR;

[0685] • Temperature-inducible translational activation via the RNA hairpin structure; • High plastome copy number, creating thousands of template copies per cell; and • Potential polycistronic co-expression of collagen-modifying enzymes.

[0686] The invention may be summarised by the following clauses:

[0687] In one aspect of the invention, there is provided a method of producing collagen in a or at least one plastid of a photosynthetic organism, the method comprising introducing and

[0688] 15252506-1expressing a nucleic acid construct in the plastid of the photosynthetic organism, wherein the nucleic acid construct comprises at least one nucleic acid sequence encoding at least one collagen a chain suitable for producing collagen; and at least one regulatory element selected from:

[0689] a plastid promoter, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0690] a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0691] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and

[0692] a selectable marker.

[0693] In one embodiment, the photosynthetic organism is selected from: a plant, plant part thereof or plant cell, and / or an alga or algae.

[0694] In one embodiment, the plastid is a chloroplast.

[0695] In one embodiment, the method comprises introducing and expressing a nucleic acid construct in the chloroplast of the organism, wherein the nucleic acid construct comprises at least one nucleic acid sequence encoding:

[0696] at least one collagen a chain suitable for producing collagen;

[0697] a chloroplast promoter selected from a group consisting of: Prrn, PpsaA, PpsbA, Prps16 orPrbcL;

[0698] a chloroplast terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA;

[0699] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, suitable for integrating the sequence encoding at least one collagen a chain into a chloroplast genome and a selectable marker, selected from neo, aadA, nptII, aphA6, badh and npII-aphA6.

[0700] 15252506-1In an embodiment, the chloroplast promoter and chloroplast terminator comprises or consists of sequences obtained from Chlamydomonas reinhardtii.

[0701] In an embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes comprise a sequence from two chloroplast-encoded genes endogenous to the organism.

[0702] In an embodiment, the chloroplast promoter comprises Prrn, PpsaA, PpsbA, Prps16 or PrbcL. Preferably, Prrn, PpsaA, PpsbA, Prps16 or PrbcL comprises or consists of a sequence defined in SEQ ID NO: 12 to 18 or a functional variant or homolog thereof.

[0703] In an embodiment, the chloroplast terminator comprises TpsbA or TrbcL,. Preferably TpsbA or TrbcL comprises or consists of a sequence defined in SEQ ID NO: 19 to 21 or a functional variant or homolog thereof.

[0704] In an embodiment, the selection marker is aadA, wherein preferably aadA comprises or consists of a sequence defined in SEQ ID NO: 22 or a functional variant or homolog thereof.

[0705] In an embodiment, the nucleic acid construct encodes a nucleic acid sequence encoding at least one collagen a chain, wherein the at least one collagen a chain comprises or consists of a sequence defined in any one of SEQ ID NO: 1 or 25 to 51.

[0706] In another aspect of the invention, there is provided a method of producing collagen in a or at least one chloroplast of a plant cell, plant part or plant cell thereof, the method comprising introducing and expressing a nucleic acid construct in a chloroplast of the organism, wherein the nucleic acid construct comprises at least one nucleic acid sequence encoding:

[0707] a sequence encoding at least one collagen a chain suitable for producing collagen;

[0708] a chloroplast promoter comprising PpsbA;

[0709] a chloroplast terminator comprising TrbcL,

[0710] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion

[0711] 15252506-1of a second chloroplast-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome, and

[0712] a selectable marker, comprising aadA.

[0713] In another aspect of the invention, there is provided a nucleic acid construct comprising at least one nucleic acid sequence encoding:

[0714] at least one collagen a chain suitable for producing collagen;

[0715] a chloroplast promoter, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0716] a chloroplast terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;

[0717] a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen a chain into a chloroplast genome; and

[0718] a selectable marker.

[0719] In another aspect of the invention, there is provided a nucleic acid construct comprising at least one nucleic acid sequence encoding:

[0720] at least one collagen a chain suitable for producing collagen;

[0721] a chloroplast promoter selected from a group consisting of: Prrn, PpsaA, PpsbA, Prps16 and PrbcL;

[0722] a chloroplast terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA’,

[0723] a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene, suitable for integrating the sequence encoding at least one collagen a chain into a chloroplast genome; and a selectable marker, selected from neo, aadA, nptll, aphA6, badh and npll-aphA6.

[0724] In an embodiment, the chloroplast promoter and chloroplast terminator comprises or consists of sequences obtained from Chlamydomonas reinhardtii.

[0725] 15252506-1In an embodiment, the pair of flanking sequences comprising at least one portion of a sequence of two chloroplast-encoded genes comprise a sequence from two chloroplast-encoded genes.

[0726] In an embodiment, the chloroplast promoter comprises at least one of Prrn, PpsaA, PpsbA, Prps16 or PrbcL, wherein preferably Prrn, PpsaA, PpsbA, Prps16 or PrbcL comprises or consists of a sequence defined in SEQ ID NO: 12 to 18 or a functional variant or homolog thereof.

[0727] In an embodiment, the chloroplast terminator comprises at least one of TpsbA or TrbcL, and wherein preferably TpsbA or TrbcL comprises or consists of a sequence defined in SEQ ID NO: 19 to 21 or a functional variant or homolog thereof.

[0728] In an embodiment, the selection marker is aadA, wherein preferably aadA comprises or consists of a sequence defined in SEQ ID NO: 22 or a functional variant or homolog thereof.

[0729] In an embodiment, the nucleic acid construct encodes a nucleic acid sequence encoding at least one collagen a chain, wherein preferably the at least one collagen a chain comprises or consists of a sequence defined in any one of SEQ ID NO: 1 or 25 to 51.

[0730] In another aspect of the invention, there is provided a genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, wherein the genetically modified organism is a plant, plant part thereof or a plant cell and / or an algae or alga.

[0731] In an embodiment, the genetically modified organism is further characterised by the expression of at least one nucleic acid of the invention in the plastid.

[0732] In an embodiment, the at least one plastid is a chloroplast.

[0733] In an embodiment, the organism is a plant, part thereof or plant cell, and wherein the plant is selected from the group consisting of tobacco (Nicotiana), lettuce, maize (Zea mays), alfalfa (Medicago sativa), rice (Oryza sativa), potato (Solanum tuberosum), soybean (Glycine max), tomato (Solanum lycopersicum), wheat (Triticum), barley

[0734] 15252506-1(Hordeum vulgare), canola (Brassica napus) and cotton (Gossypium), spinach (Spinacia oleracea) and sugar beet (Beta vulgaris).

[0735] In another aspect of the invention, there is provided a method of producing a genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, the method comprising introducing and expressing at least one nucleic acid of any of the invention, in the plastid of the genetically modified organism.

[0736] In an embodiment, the genetically modified organism is a plant, plant part thereof or cell and / or an alga or algae.

[0737] Sequence Listing

[0738] SEQ ID NO: 1. COL1A1_HUMAN Collagen alpha-1 (I) chain OS=Homo sapiens OX=9606 GN=COL1A1 PE=1 SV=6. SwissProt accession: P02452 MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPEPCRI CVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEGPKGDTGPR GPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYDEKSTGGISVPGP MGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPG RPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPG QMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQ GPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGIAGAPGFPGARGPS GPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGP TGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGA KGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAG ERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEA GKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGA PGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGP AGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAG AKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAG PPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQG IAGQRGWGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESG REGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAG PAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSG ASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSA GFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPAR TCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPK DKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQT GNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPII DVAPLDVGAPDQEFGFDVGPVCFL

[0739] SEQ ID NO: 2. COL1A1 collagen type I alpha 1 chain [Homo sapiens (human)] ATGTTCTCGTTCGTGGATCTTCGACTTCTTCTTCTTTTGGCCGCTACCGCCTTGCTTACCCA TGGACAAGAAGAAGGACAAGTCGAGGGGCAAGATGAGGATATTCCTCCTATCACCTGTGT GCAGAACGGGCTTCGATATCACGATCGAGATGTATGGAAGCCCGAACCTTGTCGAATCTG CGTATGCGATAACGGGAAAGTGCTTTGTGATGATGTGATCTGTGATGAGACTAAGAACTGC CCTGGGGCTGAAGTACCTGAGGGAGAATGTTGTCCTGTATGTCCCGATGGGTCCGAATCT CCTACTGATCAAGAAACCACCGGTGTAGAAGGGCCCAAAGGTGATACTGGACCTAGAGGT CCTAGAGGACCTGCTGGACCTCCTGGAAGAGATGGAATTCCTGGACAACCTGGATTACCT

[0740] 15252506-1GGTCCTCCTGGGCCTCCAGGACCACCTGGACCACCAGGTTTAGGTGGAAATTTCGCTCCC CAGCTTTCCTATGGGTATGATGAGAAATCCACTGGTGGGATCTCTGTGCCTGGACCTATGG GACCTTCTGGTCCAAGAGGTTTACCAGGACCTCCAGGTGCTCCAGGTCCTCAAGGTTTCC AAGGTCCACCTGGTGAGCCTGGTGAACCAGGTGCTAGTGGACCAATGGGTCCCCGAGGT CCGCCAGGTCCACCAGGGAAAAATGGTGATGATGGTGAAGCTGGAAAACCTGGGCGACCT GGTGAAAGAGGGCCTCCTGGTCCACAAGGTGCTAGAGGATTGCCTGGAACTGCTGGTTTA CCTGGAATGAAGGGACATCGAGGATTCTCCGGACTTGATGGTGCTAAAGGTGATGCAGGT CCTGCTGGTCCTAAGGGTGAACCTGGATCTCCCGGTGAAAATGGTGCTCCTGGTCAAATG GGACCTCGTGGATTACCAGGTGAACGAGGTAGACCTGGTGCTCCCGGACCTGCCGGTGC AAGAGGTAATGATGGTGCAACTGGTGCAGCTGGGCCACCTGGGCCTACTGGACCCGCCG GTCCTCCAGGATTTCCTGGTGCTGTTGGAGCAAAAGGTGAAGCCGGACCTCAAGGACCAA GAGGATCTGAAGGGCCTCAAGGTGTAAGAGGTGAACCCGGGCCACCAGGTCCAGCCGGT GCTGCCGGTCCGGCTGGAAATCCAGGTGCAGATGGACAACCGGGTGCCAAAGGTGCTAA TGGTGCACCCGGAATTGCTGGTGCCCCTGGTTTTCCCGGTGCTCGTGGTCCTTCTGGGCC ACAAGGTCCCGGTGGACCGCCTGGTCCGAAAGGTAATAGTGGTGAACCCGGTGCACCTG GGAGTAAAGGTGATACCGGTGCAAAAGGTGAGCCTGGGCCTGTTGGAGTACAAGGACCG CCAGGGCCAGCTGGTGAAGAGGGAAAACGAGGTGCAAGGGGAGAACCTGGTCCAACTGG TTTGCCTGGGCCTCCTGGTGAACGAGGTGGTCCCGGATCTAGAGGTTTCCCGGGTGCTGA TGGTGTTGCTGGACCAAAAGGACCCGCTGGTGAGCGAGGATCTCCTGGACCTGCAGGGC CTAAAGGTTCTCCAGGTGAGGCAGGACGACCCGGTGAAGCAGGATTGCCAGGTGCAAAG GGATTAACTGGATCCCCTGGTAGTCCTGGTCCTGATGGAAAAACTGGACCACCGGGTCCT GCCGGACAAGATGGTAGACCTGGACCGCCAGGTCCTCCTGGTGCTAGAGGTCAAGCTGG TGTAATGGGATTCCCAGGACCGAAAGGTGCCGCCGGTGAGCCAGGTAAAGCCGGTGAAC GTGGTGTACCTGGACCTCCGGGTGCCGTTGGACCTGCTGGTAAAGATGGTGAGGCTGGT GCTCAAGGACCTCCTGGTCCGGCTGGTCCCGCCGGTGAAAGGGGTGAACAAGGACCAGC TGGAAGTCCTGGGTTTCAAGGATTACCCGGGCCTGCTGGTCCGCCTGGTGAAGCCGGGA AGCCCGGTGAACAAGGTGTACCAGGTGATCTTGGTGCCCCAGGACCAAGTGGTGCCAGA GGTGAGAGGGGTTTCCCAGGTGAGAGAGGTGTACAAGGGCCACCTGGACCGGCTGGGCC AAGAGGTGCAAACGGTGCTCCGGGAAACGATGGTGCAAAGGGTGATGCTGGTGCACCCG GTGCTCCCGGATCTCAAGGTGCTCCTGGATTACAAGGTATGCCCGGTGAAAGAGGTGCTG CAGGATTACCTGGACCTAAAGGTGATAGAGGTGATGCCGGACCAAAGGGTGCAGATGGTT CTCCTGGAAAGGATGGTGTACGAGGACTTACCGGTCCTATTGGACCACCTGGACCTGCTG GTGCTCCTGGTGATAAGGGTGAAAGTGGACCATCTGGTCCCGCTGGACCAACAGGTGCAC GAGGTGCCCCGGGTGATCGAGGTGAGCCCGGTCCTCCTGGACCAGCTGGGTTTGCTGGT CCTCCAGGTGCCGATGGTCAGCCTGGTGCTAAGGGTGAGCCAGGTGATGCTGGTGCCAA AGGTGATGCTGGACCTCCTGGGCCTGCTGGTCCTGCTGGACCGCCTGGACCTATTGGAAA TGTAGGTGCTCCCGGTGCCAAGGGTGCACGAGGATCTGCCGGACCACCTGGTGCTACTG GGTTTCCAGGTGCTGCTGGAAGAGTTGGACCTCCAGGACCTTCTGGAAATGCAGGGCCGC CTGGTCCACCTGGTCCTGCAGGTAAAGAAGGTGGAAAAGGACCTAGGGGAGAAACAGGA CCCGCAGGGCGTCCAGGTGAAGTAGGTCCGCCTGGACCTCCAGGGCCTGCCGGTGAAAA AGGATCCCCAGGTGCTGATGGTCCCGCTGGTGCTCCAGGGACTCCTGGACCTCAAGGTAT TGCTGGACAACGAGGTGTAGTAGGACTACCTGGTCAGAGGGGAGAGAGAGGTTTTCCTGG ATTGCCCGGACCTTCAGGTGAACCGGGGAAACAAGGTCCTAGTGGTGCATCAGGTGAGCG TGGACCACCTGGTCCAATGGGTCCTCCTGGTTTAGCTGGACCACCTGGTGAATCTGGAAG AGAAGGTGCCCCAGGTGCCGAAGGATCTCCAGGTAGAGATGGGAGTCCAGGTGCCAAGG GTGATCGTGGTGAAACTGGACCCGCTGGACCGCCGGGTGCTCCAGGTGCTCCCGGTGCT CCTGGACCTGTAGGACCTGCTGGGAAATCTGGTGATAGGGGAGAGACTGGTCCTGCCGG GCCAGCTGGGCCTGTAGGTCCTGTTGGTGCTCGTGGGCCAGCTGGACCCCAAGGACCTA GAGGTGATAAGGGTGAGACTGGTGAACAAGGTGATCGAGGGATCAAAGGACATCGTGGGT TCTCTGGATTGCAAGGGCCTCCTGGACCTCCTGGTAGTCCGGGTGAACAAGGGCCTTCTG GTGCTAGTGGTCCTGCCGGTCCTCGTGGTCCACCAGGATCCGCTGGTGCCCCAGGTAAA GATGGACTTAATGGTTTGCCAGGACCGATTGGGCCACCAGGACCAAGAGGTAGAACTGGT GATGCTGGGCCCGTTGGTCCGCCAGGGCCTCCAGGTCCGCCGGGTCCGCCTGGGCCAC CTAGTGCTGGATTTGATTTTTCCTTCTTGCCCCAACCGCCTCAAGAGAAGGCTCATGATGG TGGGAGATATTATCGAGCCGATGATGCCAATGTGGTGCGAGATCGAGATCTTGAAGTGGA TACCACCTTGAAGTCCTTGTCGCAGCAGATCGAGAACATTCGATCGCCTGAGGGATCCCG AAAGAATCCTGCTAGAACCTGCCGAGATTTGAAGATGTGCCACTCCGATTGGAAGTCCGG GGAGTATTGGATTGATCCCAATCAGGGGTGCAACCTTGATGCCATCAAAGTGTTCTGCAAC

[0741] 15252506-1ATGGAAACCGGGGAGACTTGTGTGTATCCCACTCAACCATCCGTGGCTCAGAAGAACTGG TATATCTCGAAGAATCCCAAGGATAAGCGACACGTATGGTTCGGGGAATCCATGACCGATG GGTTCCAATTCGAGTATGGTGGACAAGGGTCTGATCCCGCTGATGTAGCTATCCAGCTTAC CTTCCTTCGACTTATGTCCACCGAGGCCTCTCAGAACATCACCTATCACTGCAAGAACTCC GTGGCCTATATGGATCAACAGACCGGGAATCTTAAGAAGGCCTTGCTTCTTCAGGGGTCCA ACGAAATCGAAATCCGAGCCGAAGGGAACTCCCGATTCACCTATTCCGTAACCGTAGATG GGTGCACCTCTCATACTGGTGCTTGGGGAAAAACCGTGATCGAGTATAAGACCACCAAGA CCTCTCGATTGCCGATCATTGATGTGGCTCCTCTTGATGTAGGGGCACCCGATCAAGAATT CGGATTCGATGTAGGGCCCGTGTGCTTCCTTTAA

[0742] SEQ ID NO: 3. Synthetic sequence containing the coding regions of the human Procollagen C - proteinase and flanking regions. SwissProt accession: p13497 SEQ ID NO: 4. Synthetic sequence containing the coding regions of the human Procollagen I N - proteinase and flanking regions. SwissProt accession: o95450 SEQ ID NO: 5. SwissProt: P07237.3. Prolyl 4-hydroxylase subunit beta

[0743] SEQ ID NO: 6. SwissProt: p13674. Prolyl 4-hydroxylase subunit alpha

[0744] SEQ ID NO: 7 LH3 amino acid sequence. UniProtKB / Swiss-Prot: 060568.1

[0745] Flanking regions

[0746] SEQ ID NO: 8. trnfM sequence from Nicotiana CCTTGCTCTAGCTTCTTTAGGGGTTATTACTTCTTTGGTAGCTCAACACATGTACTC TTTACCTGCTTATGCATTCATAGCACAAGACTTTACTACTCAAGCTGCATTATATAC CCACCACCAATATATCGCAGGATTCATCATGACAGGAGCTTTTGCTCATGGAGCTA TATTTTTCATTAGAGATTACAATCCGGAGCAAAATGAAGATAATGTATTGGCAAGAA TGTTAGAGCATAAAGAAGCTATCATATCTCATTTAAGTTGGGCCAGCCTCTTTCTG GGATTCCATACCCTGGGACTTTATGTTCATAATGATGTCATGCTTGCCTTTGGCAC TCCGGAGAAGCAAATCTTGATTGAACCTATATTTGCTCAATGGATACAATCCGCTC ATGGTAAAACTTCATATGGGTTCGATGTACTTTTATCTTCAACGAGTGGTCCAGCA TTCAATGCGGGTCGAAGCATCTGGTTGCCGGGTTGGTTAAATGCTGTTAATGAAA ATAGTAATTCATTATTTTTAACAATAGGTCCTGGAGACTTTTTGGTTCATCATGCTA TTGCTCTTGGTTTACATACAACTACATTGATCTTAGTAAAAGGTGCTTTAGATGCAC GTGGTTCCAAGTTAATGCCAGATAAAAAGGATTTCGGTTATAGTTTTCCGTGCGAT GGCCCAGGACGAGGCGGTACTTGTGATATTTCGGCATGGGACGCGTTTTATTTGG CAGTTTTTTGGATGTTAAATACTATTGGATGGGTTACTTTTTATTGGCATTGGAAGC ACATCACATTATGGCAGGGTAACGTTTCACAGTTTAATGAATCTTCCACTTATTTGA TGGGCTGGTTAAGGGATTATTTATGGTTAAACTCTTCACAACTTATCAACGGATAT AATCCTTTTGGTATGAATAGTTTATCGGTTTGGGCATGGATGTTCTTATTTGGACAT CTTGTTTGGGCTACTGGATTTATGTTCTTAATTTCTTGGCGTGGATATTGGCAGGA ATTGATTGAAACTTTAGCATGGGCTCATGAACGCACACCTTTGGCCAATTTGATTC GCTGGAGAGATAAACCAGTGGCCCTTTCCATTGTACAAGCAAGATTGGTTGGATT AGCTCACTTTTCTGTAGGTTATATATTCACTTATGCGGCTTTCTTGATTGCCTCTAC GTCGGGCAAATTTGGTTAATATTTTTAATGTGTGTATCTGCGATAATCTCATTTCTT TCGACGGAGAGGGGGTCCACCTTCTTCTATTTCTACATCTAGGATTCGACTTGTAT

[0747] 15252506-1CATGGATACTAATAGGAATTCAACCATTATGGCAAGGAAAAGTTTGATTCAGAGGG AGAAGAAGAGGCAAAAATTGGAACAGAAATATCATTCGATTCGTCGATCCTCAAAG AAAGAAATAAGCAAGGTTCCGTCGTTGAGTGACAAATGGGAAATTTATGGAAAGTT ACAATCCCCACCACGGAATAGTGCACCTACACGCCTTCATCGACGTTGTTTTTTGA CCGGAAGGCCGAGAGCTAACTATCGAGACTTTGGACTATCCGGACACATACTTCG TGAAATGGTTCATGCATGTTTGTTGCCAGGAGCAACAAGATCAAGTTGGTAAGGAT TAACGCTTCATTTCTATTTCTATGGTCGATGATCATAGAAGCCCCTTTACCATTCTG TATAAATGGGCTATTCTATTTGTACAGATAGGGTGGAGGGGCGCATTTAATCCTTG TTTATCTATTAGTTTTCAGTTCTTATCTTCGGCGCGGGGTAGAGCAGTTTGGTAGC TCGCAAGGCTCATAACCTTGAGGTCACGGGTTCAAATCCTGTCTCCGCAACATCT TGTTTTGCCAAACTATTTTAGGGTTGGAAACAGCTATGACCATGATTACGCCAAGC GCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTC

[0748] SEQ ID NO: 9. trnG flanking sequence from Nicotiana ACCTAGCTAGTAATTAATTCCCGCCTTTCGCTTTTTGGGGGTGGAAGGAAAAAGAA AACGTAGGGGAGGGATAGAATCACTACACTATCACGGCCAACTATACCAAATCCT TAATTTAAGGATATATTTAATGCTATTTATGAAATTAAATAATAAATAAATAGTAATA AAATTACTTTATCTTGGATCTTGGGCGGATAGCGGGAATCGAACCCGCATCTTCTC CTTGGCAAAGAGAAATTTTACCATTCGACCATATCCGCATTTTTTTGTTCTTGATAC ACAATATGTACCCACATATATGATATATAACCGGATCTTTTTTGTGCAGTGCCGGG ACACATATTCTCTTCGGAACGATTCCAATTATTTTTTTAATTGTATTCTTTTTATTCA AGAAGTTTGACCCCCCTCTAATTTTTTTGTTTTCTTTATTTGATTTGCGTTTTCTTTG GGGACTTAGATTCCAATTTAATGTGTCTCACAACCGAGAAAAATTAGGGGGGTCAT TTTGGTTTTGGGTCTGCGACGAATAGGTTCAAGAGATGAGAGAATTAAGGATACC CACCAGAAAGACTAATCCAATCCATAAGGATGTACCAGAAAATACAACATTTTTGT TACTTGACCAGCCATCAGGAGAAGCAAATACAACGGGTACGCTAATCA

[0749] SEQ ID NO: 10. trnA flanking region from lettuce (Ls_trnA) GGGGATATAGCTCAGTTGGTAGAGCTCCGCTCTTGCAATTGGGTCGTTGCGATTA CGGGTTGGATGTCTAATTGTCCAGGCGGTAATGATAGTATCTTGTACCTGAACCG GTGGCTCACTTTTTCTAAGTAATGGGGAAGAGGACCGAAACATGCCACTGAAAGA CTCTACTGAGACAAAGATGGGCTGTCAAGAACGTCAAGAACGTAGAGGAGGTAG GATGGGCAGTTGGTCAGATCTAGTATGGATCGTACATGGACGGTAGTTGGAGTCG GCGGCTCTCCTAGGGTTCCCTTATCGGGGATCCCTGGGGAAGAGGATCAAGTTG GCCCTTGCGAACAGCTTGATGCACTATCTCCCTTCAACCCTTTGAGCGAAATGCG GCAAAAGGAAGGAAAATCCATGGACCGACCCCATCATCTCCACCCCGTAGGAACT ACGAGATTACCCCAAGGACGCCTTCGGCATCCAGGGGTCACGGACCGACCATAG AACCCTGTTCAATAAGTGGAACGCATTAGCTGTCCGCTCTCAGGTTGGGCAGTAA GGGTCGGAGAAGGGCAATCACTCATTCTTAAAACCAGCGTTCTTAAGGCCAAAGA GTCGGCGGAAAAGGGGGGAAAGCTCTCCGTTCCTGGTTTCCTGTAGCTGGATCC TCCGGAACCACAAGAATCCTTAGTTAGAATGGGATTCCAACTCAGCACCTTTTGAG TGAGATTTTGAGAAGAGTTGCTCTTTGGAGAGCACAGTACGATGAAAGTTGTAAG CTGTGTTCGGGGGGGAGTTATTGTCTATCGTTGGCCTCTATGGTAGAATCAGTCG GGGGACCTGAGAGGCGGTGGTTTACCCTGCGGCGGATGTCAGCGGTTCGAGTC CGCTTATCTCCAACTCGTGAACTTAGCCGATACAAAGCTATATGACAGCACCCAAT TTTTCCGATTTGGCGGTTCGATCTATGATTTATCATTCAT

[0750] SEQ ID NO: 11. trnl flanking region from lettuce (Ls_trnA) TCGACAGTGAAGTAAGACCAAGCTCATGAGCTTATTATCTCAGGTCGGAACAAGTT GATAGGATCCCCCTTTTTACGTCCCCATGCCCCCTGTGTGGCGACATGGGGGCG

[0751] 15252506-1AAAAAAGGAAAGAGAGAGATGGGGTTTCTCTCGCTTTTGGCATAGTGGGCCCCCA GTGGGGGGCTCGCACGACGGGCTATTAGCTCAGTGGGTAGAGCGCGCCCCTGA TAATTGCGTCGTTGTGCCTGGGCTGTGAGGGCTCTCAGCCACATGGATAGTTCAA TGTGCTCATCGGCGCCTGACCCTGAGATGTGGATCATCCAAGGCACATTAGCATG GCGTACTCCTCCTGTTCGAACCGGGGTTTGAAACCAAACTTCTCCTCAGGAGGAT AGATGGGGCGATTCAGGTGAGATCCAATGTAGATCCAACTTTCGATTCACTCGTG GGATCCGGGCGGTCCGGGGGGGACCACCATGGCTCCTCTCTTCTCGAGAATCCA TACATCCCTTATCAGTGTATGGACAGCTATCTCTCGAGCACAGGTTTAGGTTCGGC CTCAATGGGAAAATAAAATGGAGCACCTAACAACGCATCTTCACAGACCAAGAACT ACGAGATCACCCCTTTCATTCTGGGGTGACGGAGGGATCATACCATTCGAGCCTT TTTTTTTCATGCTTTTCCCCGAGGTCTGGAGAAAGCTGAAATCAATAGGATTTCCC TAATCCTCCCTTACCGAAAGGAAGAGCGTGAAATTCTTTTTCCTTTCCGCAGGGAC CAGGAGATTGGATCTAGCCGTAAGAAGAATGCTTGGTATAAATAACTCACTTCTTG GTCTTCGACCCCCGCAGTCACTACGAACGCCCCCGATCAGTGCAATGGGATGTG TCTATTTATCTATCTCTTGACTCGAAATGGGAGCAGGTTTGAAAAAGGATCTTAGA GTGTCTAGGGTTGGGCCAGGAGGGTCTCTTAACGCCTTCTTTTTTCTTCTCATCG GAGTTATTTCACAAAGACTTGCCATGGTAAGGAAGAAGGGGGGAACAGGCACACT TGGAGAGCGCAGTACAACGGAGAGTTGTATGCTGCGTTCGGGAAGGATGAATCG CTCCCGAAAAGGAATCTATTGATTCTCTCCCAATTGGTTGGACCGTAGGTGCGAT GATTTACTTCACGGGCGAGGTCTCTGGTTCAAGTCCAGGATGGCCCAGCTGCGC CAGGGAAAAGAATAGAAGAAGCGTCTGACTCCTTCAA

[0752] Plastid regulatory sequences

[0753] SEQ ID NO: 12. PpsaA sequence from Chlamydomonas reinhardtii (Promoter 5’ UTR Cr_psaA) TTCTTAATTCAACATTTTTAAGTAAATACTGTTTAATGTTATACTTTTACGAATACAC ATATGGTAAAAAATAAAACAATATCTTTAAAATAAGTAAAAATAATTTGTAAACCAAT AAAAAATATATTTATGGTATAATATAACATATGATGTAAAAAAAACTATTTGTCTAAT TTAATAACCATGCATTTTTTATGAACACATAATAATTAAAAGCGTTGCTAATGGTGT AAATAATGTATTTATTAAATTAAATAATTGTTATTATAAGGAGAAATCC SEQ ID NO: 13. Prrn sequence from Nicotiana (5’ UTR Nt_prrn) TAATTAATCGACAGATCCCGAATTGGGATCGGTACCCAAAGCTCCCCCGCCGTCG TTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGGCAGGGATGG CTATATTTCTGGGAGCGAACTCCGGGCGAATACGAAGCGCTTGGATACAGTTGTA GGGAGGGATTT

[0754] SEQ ID NO: 14 portion of Prrn sequence from Nicotiana (promoter of rrn, Prrn) AAATCCCTCCCTACAACTGTATCCAAGCGCTTCGTATTCGCCCGGAGTTCGCTCC CAGAAATATAGCCATCCCTGCCCCCTCACGTCAATCCCACGAGCCTCTTATCCATT CTCATTGAACGACGGCGGGGGAGCTTTGGGTACCGATCCCAATTCGGGATCTGT CGATTAATTA

[0755] SEQ ID NO: 15. Portion of rRNA operon promoter from tobacco (Nt-Prrn) fused to the 5' UTR from genelO of phage T7 GGTACCCCAAAGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTG GGATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCG

[0756] 15252506-1AATACGAAGCGCTTGGATACGGATCCAAATACTGCAGTTTAACTTTAAGAAGGAGA TATACCC

[0757] SEQ ID NO: 16. Promoter of psbA from tobacco GCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTG AGGGGGCAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCGAATACGAAGCG CTTGGATACA

[0758] SEQ ID NO: 17. Promoter of rps16 sequence from tobacco ATTATGGAATCATGAATAGTCATTGGTTGGGCTGATGTATAAACACCATAATCTATA CTTTGTTCTATATCTA

[0759] SEQ ID NO: 18. Promoter of rbcL sequence from tobacco AAATCCCTCCCTACAACTGTATCCAAGCGCTTCGTATTCGCCCGGAGTTCGCTCC CAGAAATATAGCCATCCCTGCCCCCTCACGTCAATCCCACGAGCCTCTTATCCATT CTCATTGAACGACGGCGGGGGAGC SEQ ID NO: 81. atpA promoter sequence from Chlamydomonas reinhardtii (Cr_PatpA Promoter).

[0760] ACGCGTCTCCAATATAGTAGACTTTATTAGAGGCAGTGTTTATATACCATAAACGT CAAAAGTCATTTTTATAACTGGATCTCAAAATACCTATAAACCCATTGTTCTTCTCTT TTAGCTCTAAGAACAATCAATTTATAAATATATTTATTATTATGCTATAATATAAATA CTATATAAATACATTTACCTTTTTATAAAT

[0761] SEQ ID NO: 82. atpA 5’ UTR sequence from Chlamydomonas reinhardtii (Cr_atpA_5UTR) ACATTTACCTTTTTTTTAATTTGCATGATTTTAATGCTTATGCTATCTTTTTTATTTAG TCCATAAAACCTTTAAAGGACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAG CAATCGGCGTCATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCCCCCTTC CCCTTACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCTCTTC GGGCAAATGAATTTTAGTATTTAAATATGACAAGGGTGAACCATTACTTTTGTTAAC AAGTGATCTTACCACTCACTATTTTTGTTGAATTTTAAACTTATTTAAAATTCTCGAG AAAGATTTTAAAAATAAACTTTT TTAATCTTTTATTTATTTTTTCTTTTTT

[0762] SEQ ID NO: 83. psaA promoter sequence from Chlamydomonas reinhardtii (Cr_PpsaA_Promoter) TCTTAATTCAACATTTTTAAGTAAATACTGTTTAATGTTATACTTTTACGAATACACA

[0763] 15252506-1TATGGTAAAAAATAAAACAATATCTTTAAAATAAGTAAAAATAATTTGTAAACCAATA AAAAATATATTTATGGTATAATATAACAT

[0764] SEQ ID NO: 84. Wildtype psaA 5’UTR sequence from Chlamydomonas reinhardtii (Cr_PpsaA_5UTR). This 5' untranslated region (5'UTR) confers temperature-responsive translational control, with translation (and downstream protein accumulation) induced at or around 40 °C. (Underlined bases are hairpin loop sequence) ATGATGTAAAAAAAACTATTTGTCTAATTTAATAACCATGCATTTTTTATGAACACAT AATAATTAAAAGCGTTGCTAATGGTGTAAATAATGTATTTATTAAATTAAATAATTGT TATTATAAGGAGAAATCC

[0765] SEQ ID NO: 85. psaA 5'UTR sequence from Chlamydomonas reinhardtii (Cr_psaA 5'UTR, AHPLC1), containing targeted modifications within the hairpin loop region (AHPLC1). This variant confers temperature-responsive translational control, with translation (and downstream protein accumulation) induced at or around 25 °C.

[0766] (Underlined bases indicate the hairpin loop sequence.) ATGATGTAAAAAAAACTATTTGTCTAATTTAATAACCATGCATTTTTTATGAACACAT AATAATTAAAAGCGTTGCTAATGGTGTAAATAATGTATTTATTAAATTAAATAATTGT TATTATAAGGAGAAAAAAA

[0767] SEQ ID NO: 86. psaA 5’UTR sequence from Chlamydomonas reinhardtii (Cr_PpsaA_5UTR_ AHPLC2), modified in the hairpin loop sequence. Indicated as AHPLC2. This variant confers temperature-responsive translational control, with translation (and downstream protein accumulation) induced at or around 28-30 °C. (Underlined bases are hairpin loop sequence) ATGATGTAAAAAAAACTATTTGTCTAATTTAATAACCATGCATTTTTTATGAACACAT AATAATTAAAAGCGTTGCTAATGGTGTAAATAATGTATTTATTAAATTAAATAATTGT TATTATAAGGAGAAAAAACC

[0768] SEQ ID NO: 87. Tested, exemplar nucleic acid construct (portion) comprising a combination of higher plant (tobacco) promoters and the Cr_psaA’5UTR to express collagen. Specifically, SEQ ID NO: 87 comprises the following: Nt_prrn-Cr_psaA 5'UTR-Collagen-Nt rbcL 3'UTR.

[0769] 15252506-1This exemplary nucleic acid construct is designed for plastid expression in higher plants and comprises tobacco-derived regulatory elements flanking the coding sequence, with the sole heterologous component being the Chlamydomonas reinhardtii psaA 5'UTR. Specifically, the construct includes the tobacco plastid prrn promoter (Nt_prrn), the C. reinhardtii psaA 5' untranslated region (Cr psaA 5'UTR), a collagen coding sequence (e.g., COL1A1 CDS), and the tobacco rbcL 3' untranslated region (Nt rbcL 3'UTR) as the 3' regulatory / terminator sequence.

[0770] Nt_prrn_ portion = italic

[0771] Cr psaA’5UTR portion = underlined

[0772] Collagen coding sequence - SEQ ID NO: 88 COL3A1-1281-collagen alpha-1(III) chain 908-1136 bp + 589–510 bp

[0773] Nt_PrbcL’3UTR= bold

[0774] GGTA CCCCAAAGCTCCCCCGCCG TOG TTCAA TGA GAA TGGA TAA GA GGCTCG TG GGATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCG AA TA CGAA GCGCTTGGA TA CGATGATGTAAAAAAAACTATTTGTCTAATTTAATAAC CATGCATTTTTTATGAACACATAATAATTAAAAGCGTTGCTAATGGTGTAAATAATG TATTTATTAAATTAAATAATTGTTATTATAAGGAGAAATCC ATGGCGGGTAACACTGGTGCTCCTGGCAGCCCTGGAGTGTCTGGACCAAAAGGT GATGCTGGCCAACCAGGAGAGAAGGGATCGCCTGGTGCCC GGGCCCACCAG AGCTCCAGGCCCACTTGGGATTGCTGGGATCACTGGAGCACGGGGTCTTGCAGG ACCACCAGGCATGCCAGGTCCTAGGGGAAGCCCTGGCCCTCAGGGTGTCAAGG GTGAAAGTGGGAAACCAGGAGCTAACGGTCTCAGTGGAGAACGTGGTCCCCCTG GACCCCAGGGTCTTCCTGGTCTGGCTGGTACAGCTGGTGAACCTGGAAGAGATG GAAACCCTGGATCAGATGGTCTTCCAGGCCGAGATGGATCTCCTGGTGGCAAGG GTGATCGTGGTGAAAATGGCTCTCCTGGTGCCCCTGGCGCTCCTGGTCATCCAG GCCCACCTGGTCCTGTCGGTCCAGCTGGAAAGAGTGGTGACAGAGGAGAAAGTG GCCCTGCTGGCCCTGCTGGTGCTCCCGGTCCTGCTGGTTCCCGAGGTGCTCCTG GTCCTCAAGGCCCACGTGGTGACAAAGGTGAAACAGGTGAACGTGGAGCTGCTG GCATCAAAGGACATCGAGGATTCCCTGGTAATCCAGGTGCCCCAGGTTCTCCAGG CCCTGCTGGTCAGCAGGGTGCAATCGGCAGTCCAGGACCTGCAGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTCTAGAGTAGACATTAGCAGATAAATTAGCAGGAAATAAAGAAGGATAAGGAGA AAGAACTCAAGTAATTATCCTTCGTTCTCTTAATTGAATTGCAATTAAACTCGGC

[0775] 15252506-1CCAATCTTTTACTAAAAGGATTGAGCCGAATACAACAAAGATTCTATTGCATATA TTTTGACTAAGTATATACTTACCTAGATATACAAGATTTGAAATACAAAATCTAG CAAGCTT

[0776] SEQ ID NO: 19. TrbcL sequence from Chlamydomonas reinhardtii (3’ UTR of Cr_TrbcL) GTACTCAAGCTCGTAACGAAGGTCGTGACCTTGCTCGTGAAGGTGGCGACGTAAT TCGTTCAGCTTGTAAATGGTCTCCAGAACTTGCTGCTGCATGTGAAGTTTGGAAAG AAATTAAATTCGAATTTGATACTATTGACAAACTTTAATTTTTATTTTTCATGATGTTT ATGTGAATAGCATAAACATCGTTTTTATTTTTATGGTGTTTAGGTTAAATACCTAAA CATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTGTTTAAATTTGCCTGT CTTTATAAATTACGATGTGCCAGAAAAATAAAATCTTAGCTTTTTATTATAGAATTTA TCTTTATGTATTATATTTTATAAGTTATAATAAAAGAAATAGTAACATACTAAAGCGG ATGTA

[0777] SEQ ID NO: 20. TpsbA sequence from Tobacco (3' UTR of Nt_TpsbA) AGGTCGAATATAGCTCTTCTTTCTTATTTCAATGATATTATTATTTCAAAGATAAGAG ATATTCAAAGATAAGAGATAAGAAGAAGTCAAAATTTGATTTTTTTTTTGGAAAAAA AAAATCAAAAAGATATAGTAACATTAGCAAGAAGAGAAACAAGTTCTATTTCTACAA TTTTAAACAAATACAAAATCAAAATAGAATACTCAATCATGAATAAATGCAAGAAAA TAACCTCTCCTTCTTTTTCTATAATGTAAACAAAAAAGTCTATGTAAGTAAAATACTA GTAAATAAATAAAAAGAAAAAAAGAAAGGAGCAATAGCACCCTCTTGATAGAACAA GAAAATGATTATTGCTCCTTTCTTTTCAAAACCTCCTATAGACTAGGCCAGGATCG CTCTAGACA

[0778] SEQ ID NO: 21. TrbcL sequence from Chlamydomonas reinhardtii (terminator of Cr_TrbcL) TTTTTCATGATGTTTATGTGAATAGCATAAACATCGTTTTTATTTTTATGGTGTTTAG GTTAAATACCTAAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTG TTTAAATTTGCCTGTCTTTATAAATTACGATGTGCCAGAAAAATAAAATCTTAGCTTT TTATTATAGAATTTATCTTTATGTATTATATTTTATAAGTAATAAAAG

[0779] SEQ ID NO: 91. TpsbA sequence from Tobacco (terminator 3’ UTR Nt_TpsbA) TGTCTAGAGCGATCCTGGCCTAGTCTATAGGAGGTTTTGAAAAGAAAGGAGCAAT AATCATTTTCTTGTTCTATCAAGAGGGTGCTATTGCTCCTTTCTTTTTTTCTTTTTAT TTATTTACTAGTATTTTACTTACATAGACTTTTTTGTTTACATTATAGAAAAAGAAGG AGAGGTTATTTTCTTGCATTTATTCATGATTGAGTATTCTATTTTGATTTTGTATTTG TTTAAAATTGTAGAAATAGAACTTGTTTCTCTTCTTGCTAATGTTACTATATCTTTTT GATTTTTTTTTTCCAAAAAAAAAATCAAATTTTGACTTCTTCTTATCTCTTATCTTTGA ATATCTCTTATCTTTGAAATAATAATATCATTGAAATAAGAAAGAAGAGCTATATTC GAC

[0780] SEQ ID NO: 92. Terminator 3’UTR of rps16 sequence from Tobacco (terminator 3’ UTR of Nt_rps16)

[0781] 15252506-1AGAAATTCAATTAAGGAAATAAATTAAGGAAATACAAAAAGGGGGGTAGTCATTTG TATATAACTTTGTATGACTTTTCTCTTCTATTTTTTTGTATTTCCTCCCTTTCCTTTTC TATTTGTATTTTTTTATCATTGCTTCCATTGAATT

[0782] SEQ ID NO: 93. Terminator 3’ UTR of rbcL sequence from Tobacco (Terminator 3’ UTR of Nt_rbcL) CTAGAGTAGACATTAGCAGATAAATTAGCAGGAAATAAAGAAGGATAAGGAGAAA GAACTCAAGTAATTATCCTTCGTTCTCTTAATTGAATTGCAATTAAACTCGGCCCAA TCTTTTACTAAAAGGATTGAGCCGAATACAACAAAGATTCTATTGCATATATTTTGA CTAAGTATATACTTACCTAGATATACAAGATTTGAAATACAAAATCTAGCAAGCTT

[0783] Selectable marker

[0784] SEQ ID NO: 22, aadA CDS TTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCA ACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCT AGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTC GGCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCG GGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTT CCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAAGAACCGGAT CAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTT TGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCA AGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAAC GCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAAT CTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGC CGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATCAATATCACTGTG TGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGT CGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACT TCGGCGATCACCGCTTCTGCCAT

[0785] Other components

[0786] SEQ ID NO: 23. Identical intercistronic expression elements (I EE) TAGGATCGTTTATTTACAACGGAATGGTATACAAAGTCAACAGATCTCAA

[0787] Collagen sequence

[0788] SEQ ID NO: 88 COL3A1 -1281 -collagen alpha-1 (III) chain 908-1136 bp + 589–510 bp ATGGCGGGTAACACTGGTGCTCCTGGCAGCCCTGGAGTGTCTGGACCAAAAGGT GATGCTGGCCAACCAGGAGAGAAGGGATCGCCTGGTGCCCAGGGCCCACCAGG AGCTCCAGGCCCACTTGGGATTGCTGGGATCACTGGAGCACGGGGTCTTGCAGG ACCACCAGGCATGCCAGGTCCTAGGGGAAGCCCTGGCCCTCAGGGTGTCAAGG GTGAAAGTGGGAAACCAGGAGCTAACGGTCTCAGTGGAGAACGTGGTCCCCCTG GACCCCAGGGTCTTCCTGGTCTGGCTGGTACAGCTGGTGAACCTGGAAGAGATG GAAACCCTGGATCAGATGGTCTTCCAGGCCGAGATGGATCTCCTGGTGGCAAGG GTGATCGTGGTGAAAATGGCTCTCCTGGTGCCCCTGGCGCTCCTGGTCATCCAG

[0789] 15252506-1GCCCACCTGGTCCTGTCGGTCCAGCTGGAAAGAGTGGTGACAGAGGAGAAAGTG GCCCTGCTGGCCCTGCTGGTGCTCCCGGTCCTGCTGGTTCCCGAGGTGCTCCTG GTCCTCAAGGCCCACGTGGTGACAAAGGTGAAACAGGTGAACGTGGAGCTGCTG GCATCAAAGGACATCGAGGATTCCCTGGTAATCCAGGTGCCCCAGGTTCTCCAGG CCCTGCTGGTCAGCAGGGTGCAATCGGCAGTCCAGGACCTGCAGGGTTCCGAGG ACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGG T

[0790] SEQ ID NO: 89 COL3 A1 589–510 bp GGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCT GGAGAGCGTGGT

[0791] SEQ ID NO: 90 COL3 A1 3X 589–510 bp ATGGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCT GCTGGAGAGCGTGGTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGA GAAAAGGGTCCTGCTGGAGAGCGTGGTGGGTTCCGAGGACCTGCTGGACCAAAT GGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTTGA

[0792] References

[0793] Amirrah, I. N., Lokanathan, Y., Zulkiflee, I., Wee, M. F. M. R., Motta, A., & Fauzi, M. B. (2022). A Comprehensive Review on Collagen Type I Development of Biomaterials for Tissue Engineering: From Biosynthesis to Bioscaffold. Biomedicines, 10(9), 2307. Belanger, J. G., Copley, T. R., Hoyos-Villegas, V. et al. A comprehensive review of in planta stable transformation strategies. Plant Methods 20, 79 (2024).

[0794] Daniell H, Vivekananda J, Nielsen BL, Ye GN, Tewari KK (1990) Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors. Proc Natl Acad Sci USA 87: 88-92.

[0795] Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9

[0796] Leon-Lopez A, Morales-Penaloza A, Martinez-Juarez VM, Vargas-Torres A, Zeugolis DI, Aguirre-Alvarez G. Hydrolyzed Collagen-Sources and Applications. Molecules. 2019 Nov 7;24(22):4031. doi: 10.3390 / molecules24224031.

[0797] 15252506-1O'Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P. Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J. 1993 May;3(5):729-38. PMID: 8397038.

[0798] Ruf, S., et al. (2001). Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nature Biotechnology, 19(9), 870-875.

[0799] Daniell, H., et al. (2002). Molecular farming of pharmaceuticals and vaccines in plants. Nature Reviews Genetics, 3(1), 22-29.

[0800] Maliga, P. (2004). Plastid transformation in higher plants. Annual Review of Plant Biology, 55, 289-313.

[0801] Wagner D, Rinnenthal J, Narberhaus F, Schwalbe H. Mechanistic insights into temperature-dependent regulation of the simple cyanobacterial hsp17 RNA thermometer at base-pair resolution. Nucleic Acids Res. 2015 Jun 23;43(11):5572-85. doi: 10.1093 / nar / gkv414. Epub 2015 May 4. PMID: 25940621; PMCID: PMC4477652.

[0802] Johansson J, Mandin P, Renzoni A, Chiaruttini C, Springer M, Cossart P. 2002. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110:551-561.

[0803] G. G. Weber, J. Kortmann. F. Narberhaus, & K. E. Klose, RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae, Proc. Natl. Acad. Sci. U. S. A. 111 (39) 14241-14246, (2014).

[0804] 15252506-1

Claims

CLAIMS:

1. A method of producing collagen in a plastid of a photosynthetic organism, the method comprising introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:at least one collagen a chain suitable for producing collagen;at least one plastid regulatory sequence, for regulating the sequence encoding at least one collagen a chain in a plastid;a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; andoptionally a selectable marker.

2. The method of claim 1, wherein the photosynthetic organism is selected from: a plant, plant part thereof or plant cell, and / or an alga or algae.

3. The method of claims 1 or 2, wherein the plastid is a chloroplast.

4. The method of any one of claims 1 to 3, wherein the method comprises introducing and expressing a nucleic acid construct in the plastid of the photosynthetic organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:at least one collagen a chain suitable for producing collagen;at least one plastid regulatory sequence selected from a group consisting of the promoter of rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR of rrn, psaA, psbA, rps16, rbcL and atpA;a plastid terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA;a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a15252506-1portion of a second plastid-encoded gene suitable for integrating the sequence encoding at least one collagen a chain into a plastid genome andoptionally a selectable marker, selected from neo, aadA, nptII, aphA6, badh and npII-aphA6.

5. The method of any preceding claim, wherein the plastid regulatory sequence and plastid terminator comprises or consists of sequences obtained from Chlamydomonas reinhardtii.

6. The method of any preceding claim, wherein the pair of flanking sequences are endogenous to the organism.

7. The method of any one of claims 1 to 4, wherein the plastid regulatory sequence is a promoter selected from rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR selected from rrn, psaA, psbA, rps16, rbcL and atpA, wherein the promoter of rrn, psaA, psbA, rps16, rbcL and atpA comprises or consists of SEQ ID NO: 14 or 15, 12 or 83, 16, 17, 18 and 81 or functional variant of homolog thereof, respectively, and wherein the 5’UTR of rrn, psaA, and atpA comprises or consists of SEQ ID NO: 13, any one of SEQ ID NO: 84 to 86, and SEQ ID NO: 82 or functional variant of homolog thereof, respectively.

8. The method of any one of claims 1 to 6, wherein the plastid regulatory sequence is or comprises the promoter of psaA and / or 5’ UTR of psaA derived from Chlamydomonas reinhardtii, wherein preferably the sequence of the promoter of psaA comprises or consists of SEQ ID NO: 12 or 83 or functional variant or homolog thereof and the 5’ UTR of psaA comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

9. The method of claim 8, wherein the plastid regulatory sequence comprises the 5’UTR of psaA from Chlamydomonas reinhardtii and comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

10. The method of any one of claims 1 to 6, wherein the plastid regulatory sequence is selected from psaA 5’UTR from Chlamydomonas, prfA 5’UTR from Listeria monocytogenes, agsA 5'UTR from Salmonella, ibpA 5 ‘UTR from Vibrio cholerae,15252506-1PIF7 5’ UTR from Arabidopsis thaliana, and the hsp17 5’IITR from Synechocystis.

11. The method of any one of claims 1 to 4, wherein the plastid terminator comprises Tpsb / X, TrbcL or Trsp16, wherein Tpsb / X, TrbcL or Trsp16 comprises or consists of SEQ ID NO: 19, 21 or 92, 20 or 91, and 92 or functional variant or homolog thereof, respectively.

12. The method of claim 11, wherein the plastid terminator comprises TrbcL and comprises or consists of SEQ ID NO: 19 or 21 or functional variant or homolog thereof.

13. The method of any preceding claim, wherein the nucleic acid sequence comprises the selection marker aadA, wherein aadA comprises or consists of a sequence defined in SEQ ID NO: 22 or functional variant or homolog thereof.

14. The method of any preceding claim, wherein the nucleic acid construct encodes a nucleic acid sequence encoding at least one collagen a chain, wherein the at least one collagen a chain comprises or consists of a sequence selected from SEQ ID NO: 1 or 24 to 52 or 88 to 90 or functional variant or homolog thereof.

15. The method of any one of claims 1 to 6, comprising introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding:a sequence encoding at least one collagen a chain suitable for producing collagen;at least one plastid regulatory sequence comprising the promoter of psbA, psaA, oratpA and / or the 5’UTR of psbA, psaA, oratpA;a plastid terminator comprising TrbcL,a pair of flanking sequences comprising a first sequence from a or a portion of a first chloroplast-encoded gene, and a second sequence from a or a portion of a second chloroplast-encoded gene suitable for integrating the sequence encoding at least one collagen a chain into a plastid genome, and optionally a selectable marker, comprising aadA.15252506-116. The method of claim 9 or 10, wherein the photosynthetic organism comprising the nucleic acid construct is grown at a first temperature and subsequently is grown at a second temperature;wherein the second temperature is higher than the first temperature, preferably wherein the first temperature is at or around 15 °C to 25 °C, and the second temperature is at or around 28 °C to 40 °C,and wherein the change from the first temperature to the second temperature is associated with an increase in collagen production per unit of time.

17. A method of producing collagen in a plastid of a photosynthetic organism, the method comprising introducing and expressing a nucleic acid construct in the plastid of the organism, wherein the nucleic acid construct comprises a nucleic acid sequence encoding at least one collagen a chain suitable for producing collagen and at least one plastid regulatory sequence comprising the promoter of psaA from Chlamydomonas reinhardtii and / or the 5’UTR of psbA from Chlamydomonas reinhardtii.

18. The method of claim 17, wherein the at least one plastid regulatory sequence comprises the 5’UTR of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

19. The method of claim 17, wherein the at least one plastid regulatory sequence comprises the promoter of psaA from Chlamydomonas reinhardtii comprising or consisting of SEQ ID NO: 12 or 83 or functional variant or homolog thereof.

20. A nucleic acid construct comprising a nucleic acid sequence encoding:at least one collagen a chain suitable for producing collagen; at least one plastid regulatory sequence, for regulating the sequence encoding at least one collagen a chain in a plastid;a plastid terminator, for regulating the sequence encoding at least one collagen a chain in a plastid;a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; and15252506-1optionally a selectable marker.

21. The nucleic acid construct of claim 20, comprising a nucleic acid sequence encoding:at least one collagen a chain suitable for producing collagen; at least one plastid regulatory sequence selected from a group consisting of the promoter of rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR of rrn, psaA, psbA, rps16, rbcL and atpA;a plastid terminator selected from a group consisting of: TpsbA, Trps16, TrbcL and TatpA,a pair of flanking sequences comprising a first sequence from a or a portion of a first plastid-encoded gene, and a second sequence from a or a portion of a second plastid-encoded gene, for integrating the sequence encoding at least one collagen α chain into a plastid genome; andoptionally a selectable marker, selected from neo, aadA, nptll, aphA6, badh and npll-aphA6.

22. The nucleic acid construct of claim 20 or 21, wherein the plastid regulatory sequence and plastid terminator comprises or consists of sequences obtained from Chlamydomonas reinhardtii.

23. The nucleic acid construct of any one of claims 20 to 22, wherein the pair of flanking sequences comprising at least one portion of a sequence of two plastid- encoded genes comprise a sequence from two chloroplast-encoded genes.

24. The nucleic acid construct of any one of claims 20 to 23, wherein the plastid regulatory sequence is a promoter selected from rrn, psaA, psbA, rps16, rbcL and atpA and / or 5’ UTR selected from rrn, psaA, psbA, rps16, rbcL and atpA, wherein the promoter of rrn, psaA, psbA, rps16, rbcL and atpA comprises or consists of SEQ ID NO: 14 or 15, 12 or 83, 16, 17, 18 and 81 or functional variant of homolog thereof, respectively, and wherein the 5’UTR of rrn, psaA, and atpA comprises or consists of SEQ ID NO: 13, any one of SEQ ID NO: 84 to 86, and SEQ ID NO: 82 or functional variant of homolog thereof, respectively.15252506-125. The nucleic acid construct of any one of claims 20 to 24, wherein the plastid regulatory sequence is or comprises the promoter of psaA and / or 5’ UTR of psaA derived from Chlamydomonas reinhardtii, wherein preferably the sequence of the promoter of psaA comprises or consists of SEQ ID NO: 12 or 83 or functional variant or homolog thereof and the 5’ UTR of psaA comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

26. The nucleic acid construct of any one of claims 20 to 25, wherein the plastid regulatory sequence is selected from psaA 5’UTR from Chlamydomonas, prfA 5’UTR from Listeria monocytogenes, agsA 5'UTR from Salmonella, ibpA 5 ‘UTR from Vibrio cholerae, PIF7 5’ UTR from Arabidopsis thaliana, and the hsp17 5’UTR from Synechocystis.

27. The nucleic acid construct of any one of claims 20 to 26, wherein the plastid regulatory sequence comprises the psaA 5’UTR from Chlamydomonas reinhardtii and comprises or consists of SEQ ID NO: 84 to 86 or functional variant or homolog thereof.

28. The nucleic acid construct of any one of claims 20 to 27, wherein the plastid terminator comprises TpsbA, TrbcL or Trsp16, wherein TpsbA, TrbcL or Trsp16 comprises or consists of SEQ ID NO: 19, 21 or 92, 20 or 91, and 92 or functional variant or homolog thereof, respectively.

29. The nucleic acid construct of any one of claims 20 to 28, wherein the plastid terminator comprises TrbcL and comprises or consists of SEQ ID NO: 19 or 21 or functional variant or homolog thereof.

30. The nucleic acid construct of any one of claims 20 to 29, wherein the nucleic acid sequence encodes the selection marker aadA, wherein aadA comprises or consists of a sequence defined in SEQ I D NO: 22 or functional variant or homolog thereof.

31. The nucleic acid construct of any one of claims 20 to 30, wherein the nucleic acid construct encodes a nucleic acid sequence encoding at least one collagen a chain, wherein the at least one collagen a chain comprises or consists of a15252506-1sequence defined in any one of SEQ ID NO: 1 or 24 to 52 or 88 to 90 or functional variant or homolog thereof.

32. A genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, wherein the genetically modified organism is a plant, plant part thereof or a plant cell and / or an algae or alga.

33. The genetically modified organism of claim 32, further characterised by the expression of at least one nucleic acid of any of claims 20 to 31 in the plastid.

34. The genetically modified organism of claim 33, wherein the at least one plastid is a chloroplast.

35. The genetically modified organism of any one of claims 32 to 34, wherein the organism is a plant, part thereof or plant cell, and wherein the plant is selected from the group consisting of tobacco (Nicotiana), lettuce, maize (Zea mays), alfalfa (Medicago sativa), rice (Oryza sativa), potato (Solanum tuberosum), soybean (Glycine max), tomato (Solanum lycopersicum), wheat (Triticum), barley (Hordeum vulgare), canola (Brassica napus) and cotton (Gossypium), spinach (Spinacia oleracea) and sugar beet (Beta vulgaris), preferably tobacco (Nicotiana) or lettuce.

36. The genetically modified organism of any one of claims 32 to 34, wherein the organism is an algae or alga, preferably Chlamydomonas reinhardtii.

37. A method of producing a genetically modified organism characterised by the expression of exogenous collagen in at least one plastid, the method comprising introducing and expressing the nucleic acid construct of any of claims 20 to 31, in the plastid of the genetically modified organism.

38. The method of claim 37, wherein the genetically modified organism is a plant, plant part thereof or cell and / or an alga or algae.

39. The method of claim 37 or 38, wherein the organism is a plant, part thereof or plant cell, and wherein the plant is selected from the group consisting of: tobacco15252506-1M& C PC933728W098(Nicotiana), lettuce, maize (Zea mays), alfalfa (Medicago sativa), rice (Oryza sativa), potato (Solanum tuberosum), soybean (Glycine max), tomato (Solanum lycopersicum), wheat (Triticum), barley (Hordeum vulgare), canola (Brassica napus) and cotton (Gossypium), spinach (Spinacia oleracea) and sugar beet (Beta vulgaris), preferably tobacco (Nicotiana), lettuce.15252506-1

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