Animal proteins in cyanobacteria

Abstract

In some embodiments, the gene is integrated into the genome of the cyanobacteria. In some of these embodiments, the gene is integrated into an NS1 site of the cell. The sequence encoding an animal protein is optimized for expression in the cyanobacteria. In some embodiments, the animal protein is a muscle protein. In some of these embodiments, the muscle protein is a myosin subunit, myoglobin, or alpha-actin. In other embodiments, the animal protein is a subunit of a complex. In some of these embodiments, the complex is a myosin complex. The production of proteins by modified cells may be performed in multiple different cells, in some embodiments, the cyanobacteria cell is S. elongatus is PCC7942 or UTEX2973 or PCC7002, or Synechocystis sp. PCC6803. In some embodiments, multiple genes are incorporated into a cell. In some embodiments, each gene comprises a myosin complex subunit. The myosin complex subunits are MLC1, MLC2, and MYH2.

Description

Docket No.10916-10347 PCT Animal Proteins in Cyanobacteria CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent No.63 / 485,694 filed on February 17, 2023, and entitled Animal Proteins in Cyanobacteria. The entirety of the contents of which are incorporated herein by this reference. TECHNICAL FIELD

[0002] Cellular production of proteins. BACKGROUND

[0003] Traditional production of animal-derived food products requires growing feed crops in open fields (e.g., soy, corn, sorghum etc.), harvesting, transporting, and processing of the crops to different locations where it will be used as feed for animals such as cows, chickens, and pigs. This production chain is resource intensive, inefficient, and most importantly, is not able to support the fast-growing human population demand for food. The paradigm is that animals are grown for their ability to produce milk, eggs, and meat. With those products being collected by farmers, transported to designated food production facilities where it is processed into different products or integrated as ingredients in many different food products. As 50% of the habitable land is already used for food production, and climate change has a dramatic and devastating influence on food production, humanity urgently needs to find an alternative that allows production of food all year round, in any geographical location and without fertilizers, pesticides, or risk of pathogenic bacterial / viral / fungal contaminants.

[0004] These outdated and ancient methods of food production are unsustainable and resource intensive (water, land, antibiotics, gas, and energy) and also raise moral issues about the use of animals as food production “machines.” Furthermore, whether based on animal or crop agriculture, food production requires transportation of the goods from remote locations to designated facilities where the goods are processed and packed into food products. In light of the global energy crisis, and the need to produce food more efficiently and sustainably, a new method of food production has been developed that takes place in a single geographicalDocket No.10916-10347 PCT location, detached from land type, requires just a fraction of the water and electricity, and is not dependent on season of the year. BRIEF SUMMARY

[0005] In a first aspect, the disclosure provides a genetically modified cell. The cell comprises a gene. The gene comprises; a promoter, a sequence encoding an animal protein, and a terminator. The cell is a cyanobacteria cell.

[0006] In some embodiments, the gene is integrated into the genome of the cyanobacteria. In some of these embodiments, the gene is integrated into an NS1 site of the cell. Genetic modification is possible in many organisms. To ensure that the genetic modification results in expression in the target organism, the genetic sequences can be adapted to specific organisms. The sequence encoding an animal protein may be optimized for expression in the cyanobacteria. Many proteins are present in organisms and different proteins provide different functions within the organism. In some embodiments, the animal protein is a muscle protein. In some of these embodiments, the muscle protein is a myosin subunit, myoglobin, or alpha-actin. In other embodiments, the animal protein is a subunit of a complex. In some of these embodiments, the complex is a myosin complex. The production of proteins by modified cells may be performed in multiple different cells. In some embodiments, the cyanobacteria cell is Synechochos elongatus. Multiple strains of different species of organisms can be for the modification of cells. In some embodiments the S. elongatus is PCC7942, PCC6803, PCC7002, or UTEX2973. In some embodiments, the gene is in a vector. In some embodiments, multiple genes are incorporated into a cell and in these embodiments, a second gene is incorporated in the cell and either the first gene or the second gene is in a vector. In some embodiments, a third gene is incorporated, and any of the first gene, second gene, or third gene or any combination thereof is in a vector. In some embodiments, each gene comprises a myosin complex subunit. In these embodiments, the myosin complex subunits are MLC1, MLC2, and MYH2. Proteins are produced when the genes in a cell code for those proteins. The genetically modified cells therefore may include a second gene, wherein the first gene, the second gene, or both genes are integrated into the genome of the cyanobacteria. The modified cell may also include a third gene, wherein any of the first gene, the second gene or the third gene or any combination thereof is integrated into the genome of the cyanobacteria. When the cell includes multiple genes, each gene comprises a myosin complex subunit. The myosin complex subunits which are encoded byDocket No.10916-10347 PCT genes in the modified cell are MLC1, MLC2, and MYH2. In some embodiments, any of the first gene, second gene, or third gene or any combination thereof is in a vector and those genes not in a vector are incorporated into the genome of the cyanobacteria. In some embodiments, the promoter is one of SEQ ID NO: 24 or SEQ ID NO: 25.

[0007] In a second aspect the disclosure provides a method for producing an animal protein from a genetically modified cell. The method comprises inserting a gene into a cell. The gene comprises a promoter, a sequence encoding an animal protein, and a terminator. The cell is a cyanobacteria cell. The method of producing an animal protein from a genetically modified cell may be applied so that any of the cells and proteins previously discussed are applied to the method.

[0008] In a third aspect, the disclosure provides a method of producing an animal protein in cyanobacteria. The method comprises isolating the animal protein from any of the modified cells described herein.

[0009] Further aspects and embodiments are provided in the foregoing drawings, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings are provided to illustrate certain embodiments described herein. The drawings are merely illustrative and are not intended to limit the scope of claimed inventions and are not intended to show every potential feature or embodiment of the claimed inventions. The drawings are not necessarily drawn to scale; in some instances, certain elements of the drawing may be enlarged with respect to other elements of the drawing for purposes of illustration.

[0011] Figure 1 is a schematic representation of the types of tested elements.

[0012] Figure 2A is a pSYN6 annotated vector map with the NS1 integration site.

[0013] Figure 2B is a pSYN6 annotated vector map with the NS2 integration site.

[0014] Figure 2C is a pSYN6 annotated vector map with the NS3 integration site.

[0015] Figure 3A is a PCR analysis confirming the genomic integration of Myoglobin coding sequences into chromosomal DNA, for the Evaluation of Myoglobin Expression in S. elongatus Using Different Codon Optimization Algorithms.Docket No.10916-10347 PCT

[0016] Figure 3B is a Western blot analysis detecting Myoglobin (17KD) in total protein lysates, for the Evaluation of Myoglobin Expression in S. elongatus Using Different Codon Optimization Algorithms.

[0017] Figure 3C is a quantitative ELISA measuring Myoglobin expression levels out of total soluble protein, for the Evaluation of Myoglobin Expression in S. elongatus Using Different Codon Optimization Algorithms.

[0018] Figure 4A is a PCR analysis confirming the genomic integration of Myoglobin coding sequences into chromosomal DNA, to show the Impact of Different Promoters on Myoglobin Expression and Genomic Integration.

[0019] Figure 4B is a quantitative ELISA measuring Myoglobin expression levels out of total soluble protein, to show the Impact of Different Promoters on Myoglobin Expression and Genomic Integration.

[0020] Figure 5A is a PCR analysis confirming the genomic integration of Myoglobin coding sequences into chromosomal DNA, to show the Impact of Neutral Genomic Integration Sites on Myoglobin Expression.

[0021] Figure 5B is a quantitative ELISA measuring Myoglobin expression levels out of total soluble protein, to show the Impact of Neutral Genomic Integration Sites on Myoglobin Expression.

[0022] Figure 6A PCR analysis confirming the genomic integration of Myoglobin coding sequences into chromosomal DNA.

[0023] Figure 6B is a quantitative ELISA measuring Myoglobin expression levels out of total soluble protein.

[0024] Figure 7A is a PCR analysis confirming the genomic integration of alpha-actin coding sequences into chromosomal DNA.

[0025] Figure 7B is a quantitative ELISA measuring alpha-actin expression levels out of total soluble protein.

[0026] Figure 7C is a Western blot analysis detecting alpha-actin (42.1 KD) in total protein lysates.

[0027] Figure 8A is a Schematic representation of the types of tested elements in a polycistronic operon.Docket No.10916-10347 PCT

[0028] Figure 8B is Agar plates with Spectinomycin resistance colonies vs. No-DNA control of polycistronic myosin operon.

[0029] Figure 8C is PCR analysis evaluating Spectinomycin resistance and polycistronic Myosin.

[0030] Figure 8D is a PCR analysis evaluating polycistronic myosin operon genomic integration into chromosomal DNA.

[0031] Figure 9A is a PCR analysis confirming the genomic integration of MLC1.

[0032] Figure 9B PCR analysis confirming the genomic integration of MYH2.

[0033] Figure 9C is a Western blot analysis detecting MLC1 (20.9KD) in total protein lysates.

[0034] Figure 9D is a quantitative ELISA measuring MLC1 expression levels out of total soluble protein.

[0035] Figure 9E is a PCR analysis confirming the genomic integration of MLC1+MLC2.

[0036] Figure 9F is a Western blot analysis detecting MLC2 (19.0KD) in total protein lysates.

[0037] Figure 9G is a PCR analysis confirming the genomic integration of MYH2 coding sequences into chromosomal DNA.

[0038] Figure 10A is a Western blot analysis detecting MYH2 protein.

[0039] Figure 10B is an SDS-PAGE analysis detecting MYH2 relative abundance in different extracted cell fraction lysates.

[0040] Figure 10C is a Western blot analysis detecting MYH2 and MLC2.

[0041] Figure 10D is a Western blot analysis detecting MLC1.

[0042] Figure 10E is an SDS-PAGE analysis detecting the relative abundance of MYH2, MLC1, and MLC2 in different extracted cell fraction lysates.

[0043] Figure 10F is a quantification of soluble MYH2 (ratio of Supernatant induction / pellet induction samples) in the presence or absence of MLC1+MLC2 under two induction temperatures.Docket No.10916-10347 PCT DETAILED DESCRIPTION

[0044] The following description recites various aspects and embodiments of the inventions disclosed herein. No particular embodiment is intended to define the scope of the invention. Rather, the embodiments provide non-limiting examples of various compositions, and methods that are included within the scope of the claimed inventions. The description is to be read from the perspective of one of ordinary skill in the art. Therefore, information that is well known to the ordinarily skilled artisan is not necessarily included. Definitions

[0045] The following terms and phrases have the meanings indicated below, unless otherwise provided herein. This disclosure may employ other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings that they would possess within the context of this disclosure to those of ordinary skill in the art. In some instances, a term or phrase may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart and vice versa, unless expressly indicated to the contrary.

[0046] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like.

[0047] As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. Unless otherwise expressly indicated, such examples are provided only as an aid for understanding embodiments illustrated in the present disclosure and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed embodiment.

[0048] Meat has long been an important part of the human diet. Meat provides high- quality protein. The protein in meat contains all the essential amino acids, which are crucial for growing tissues and repairing damage to tissues. In addition to being a vital source of nutrients for growth and health, humans generally enjoy the taste of meat and many attempts to create meat substitutes from plants do not have the taste of meat.

[0049] Meat production is often expensive and can be environmentally damaging. Finding a way to produce a meat like substance that tastes like meat and is less environmentally impactful would be greatly beneficial. Introducing a gene encoding an animal muscle protein into a photosynthetic prokaryote enables production of a product thatDocket No.10916-10347 PCT tastes like meat. By using a photosynthetic prokaryote, like cyanobacteria, food can be produced that tastes like meat.

[0050] The genetically modified cells of the disclosure comprise a gene having a promoter, a sequence encoding an animal protein, and a terminator. In some embodiments, the gene is polycistronic – encoding more than one animal protein.

[0051] The promoter can be any promoter that is capable of driving expression of the animal protein in the cells. Examples include, but are not limited to constitutive promoters, inducible promoters, viral promoters, CMV promoter, SV40 promoter, and EF-1alpha promoter. Moreover, the promoter may be native to the cell, such as promoters native to cyanobacteria. Examples of such promoters include, but are not limited to, Ppsba1, Ppsba2, Ppsba3, Ptrc10, J23119, Pcpc560, and Pcpcb2. In specific embodiments the promoter may be selected from any one of SEQ ID NOs: 20-26.

[0052] An animal protein includes any protein that is found wild-type in an organism of the kingdom Animalia. Examples include muscles proteins such as myoglobin, myosin (e.g. MLC1, MLC2, MYH1, MYH2), and alpha-actin. Myoglobin is a protein found in the cardiac and skeletal muscle of vertebrates and particularly in mammals. Myoglobin contains pigments responsible for the color of red meat. Myosin proteins are motor proteins. Myosin proteins are found in eukaryotes some myosin proteins have specialized functions while others are generalists. Some myosin proteins assist in muscle contraction. Alpha-actin is a protein found in skeletal and cardiac muscles, as well as non-muscle cells. In particular aspects, the animal protein may be a subunit of a complex. In a more particular aspect, the complex may be a myosin complex. In specific embodiments the animal protein may be selected from the animal proteins encoded by any one of SEQ ID NOs: 9-17, 36-47, and 49- 51.

[0053] The terminator can be any terminator that is capable of stopping a polymerase during transcription of a gene in the cell in which it is located. Examples include, but are not limited to, Rho-independent (e.g. hairpin-loop structures and poly-u tracts) and Rho- dependent terminators. Moreover, the terminator may be native to the cell, such as terminators native to cyanobacteria. Examples of such terminator include, but are not limited to, TL3S2P21, TECK120029600, TpsbA2, TΩgroEL, and TrmB. In specific embodiments the terminator may be selected from any one of SEQ ID NOs: 31-35.Docket No.10916-10347 PCT

[0054] In embodiments, the modified cell may be a cyanobacteria. The cyanobacteria can be any cyanobacteria capable of producing animal protein. Examples include, but are not limited to, Nostoc, Oscillatoria, Spirulina, Rivularia, Microcystis, Anabaena, Trichodesmium, Synechococcus, and Prochlorococcus. In particular embodiments the cyanobacteria is Synechochos elongatus or a subspecies there of such as PCC7942, UTEX2973, PCC6301 and PCC6803.

[0055] In aspects, other organisms may be modified to include the gene encoding an animal protein. Such organisms include Enteric bacteria such as Escherichia coli. Strains of E. coli include K-12, DH5α, DH10b, MC1061, BL21(DE3), BL21 Star (DE3), Rosetta, MG1655, Nissle 1917 (EcN, Mutaflor), and BW25113.

[0056] In aspects, the gene encoding the animal protein may be present in a vector. Vectors for genetic engineering are well known in art and may be used for the creation of, shuttling, and insertion of sequences of interest including the gene described herein. Examples include, but are not limited to, pET30+ and PDUET-1. In particular aspects the gene encoding the animal protein is present in the modified cell on a vector. In other aspects, the gene encoding the animal protein is integrated into the genome of the cell. In such cases, the gene encoding an animal protein has been inserted itself the genome become a part of the cyanobacteria genome.

[0057] In some aspects, the vector is present in the cell and has not integrated into the genome. It is also possible to introduce multiple vectors into a cell, with each vector carrying its own gene. Some vectors may include multiple genes within the same vector. On occasion when multiple vectors are inserted into a cell, one vector will integrate into the genome while another vector will not. In particular aspects, where the modified cell comprises more than one gene as described herein, any combination of the genes described being present in a vector or integrated into the genome is contemplated. Where there are two such genes, both may be in vector(s), both may be integrated into the genome, or one may be present in a vector and one integrated into the genome. By extension, where there are three such genes: all there may be in vector(s), all may be integrated into the genome, or two may be present in a vector and one integrated into the genome.

[0058] Where the gene encoding the animal protein is integrated into the genome, it may be integrated at an integration site such as a neutral integration site. Neutral integration sites are specific locations within a genome where foreign DNA can be inserted withoutDocket No.10916-10347 PCT disrupting the function of existing genes or causing any negative effects on the organism. Examples of neutral integration sites include, but are not limited to, NS1a, NS1b, NS2a, NS2b, NS3a, and NS3b. In specific embodiments the integration site may be selected from any one of SEQ ID NOs: 1-6, 60, and 65-68.

[0059] In certain aspects, the sequence encoding the animal protein may be optimized for expression in the modified cell. For example, the sequence may be codon optimized to make more efficient use of the preferred codons of the modified cell.

[0060] In one aspect, described is a method of genetically modifying a cell. Such methods comprise the insertion of genes described herein so as produce a modified cell. Examples Materials and Methods Cultivation of Cyanobacteria

[0061] The cyanobacterium S. elongatus PCC 7942, S. elongatus UTEX2973. Synechocystis sp. PCC6803, were grown in BG11 (Phytotech labs, cat: B1511) liquid media, with 20mM hepes (PH 7.6). PCC 7002 were grown in medium A+ (UTEX, medium A+, 30991788343386). All strains were cultivated under 6000K LED lights, with intensity of 100μE2, shaking at 180 RPM in 250 ml Erlenmyer flasks in 37°C.

[0062] The cyanobacterium can also be grown in other conditions. For example, cyanobacterium can be grown in many varieties of liquid media. Cyanobacterium can also be grown using natural sunlight. Other growth containers will also grow cyanobacterium. Some embodiments will use flasks, others will use grow tanks or grow vats. The flasks or growing vats can be placed outside to utilize natural conditions for growth. Sunlight is the most critical natural condition, and the grow containers need to take advantage of as much sunlight as possible. Genetic Transformation

[0063] Transformation of PCC7942 and PCC6803 was performed by collecting 1.5 mL of exponentially growing (OD750 1-2) cyanobacteria, followed by centrifugation in 5,000g for 5 minutes at room temperature. Followed by a wash with 1ml in BG11 media,Docket No.10916-10347 PCT cells were resuspended in 600uL of BG11 and incubated with 125ng of DNA and incubated overnight at 28°C in shaker incubator in the dark. Finally, the cyanobacteria were transferred to BG11 agar plates with 0.3% Sodium Thiosulfate and 10ug / ml of spectinomycin and incubated under 6000K LED lights at intensity of approximately 25 μE2 in 34°C until colonies appeared.

[0064] Transformation of PCC 7002 are performed similarly to PCC7942 and PCC6803 with few modifications. 1-2mL of Exponentially growing culture collected to ventilated cap tube and approximately 200 ng plasmid DNA added. The transformation culture was incubated at 30°C with 150 rpm and 60μE2 for 24 hours. Finally, cyanobacteria were concentrated to 100 μL using centrifugation (5000 × g, 5 min) and spread on medium A+ agar plates with 1 mM of Sodium Thiosulfate and 40μg / mL of spectinomycin.

[0065] Transformation of UTEX 2973 by tri-parental conjugation was employed using conjugal plasmid (pRL528) and helper plasmid (pRK 2013). Shuttle vectors carrying the gene of interest were first transformed into competent DH5^ that contained the helper plasmid to form cargo strains. Overnight cultures of UTEX2973 (BG11; OD750: 0.5-0.7) and both E. coli strains (LB) were pre-washed with BG11 and distilled water respectively, then 400µl of cyanobacteria, 100µl of conjugal strain and 100µl of cargo strain mixed and incubated for 2 hours at room temperature. Centrifuge (6000xg) and pellet cells resuspended in the remaining 100µl supernatant, then mixture plated onto BG11+5% LB agar plates containing selective antibiotics. BG11+5% LB agar plates were incubated at 38°C, 100 60μE2 light overnight. The membrane was then transferred to new BG11 agar plates with appropriate antibiotics until colonies observed. PCR Analysis

[0066] Single colonies were picked into 25uL Hy-Taq PCR ready mix (Hylabs, cat: EZ3006) according to manufacturer protocol. The primers were annealed at 55°C. The PCR products were run on 1% Agarose gel, and imaged using Amersham Imager 600 (General Electric, 29083461). Western Blotting

[0067] Cyanobacteria samples were disrupted using acid washed glass beads shaken at 400RPM using amalgamator in ice cold PBS supplemented with protease inhibitor cocktail (Sigma-Aldrich). Subsequently, 20 μg of protein samples were boiled and reduced by 5 minutes incubation in 80°C with loading buffer (Thermofisher) and were loaded on SDS-Docket No.10916-10347 PCT page gel to be finally transferred to a PVDF membrane for Myoglobin, and Nitrocellulose membrane for alpha-actin, MLC1 and MLC2. Myoglobin was detected using polyclonal rabbit anti-Myoglobin antibody (Abcam, cat: ab231530, dilution 1:750), followed by goat anti-rabbit peroxidase conjugated antibody (Jackson, cat: 111-035-003, dilution 1:10,000). Alpha-Actin, MLC1 and MLC2 were detected using monoclonal mouse anti alpha-actin (DSHB:JLA20-s, dilution 1:50), anti MLC1 (DSHB F310-s, dilution 1:70), anti MLC2 (CST- 3672S, dilution 1:750), followed by goat anti-mouse peroxidase conjugated antibody (Jackson, cat: 115-035-003, dilution 1:10,000). Membranes were imaged using Amersham Imager 600 (General Electric, 29083461), and band densitometry was measured using ImageJ. ELISA

[0068] 96 well plate (Greiner) was coated with 5 μg of protein from Cyanobacteria carrying gene expression cassette 2 hours at 37°C, followed by blocking with 5% skim Milk in PBS for 1 hour at 37°C, and washing with PBS+0.05% Tween-20. Samples were incubated over night at 4°C with polyclonal rabbit anti-Myoglobin antibody (Abcam, cat: ab231530, dilution 1:2,500), washed four times with PBS+0.05% Tween-20, and incubated for 1.5 hours at 37°C with goat anti-rabbit Peroxidase conjugated antibody (Jackson, cat: 111-035-003, dilution 1:10,000). Alpha-Actin, MLC1 and MLC2 were incubated overnight at 4°C and detected using monoclonal mouse anti alpha-actin (DSHB:JLA20-s, dilution 1:50) and anti MLC1 (DSHB F310-s, dilution 1:110), followed by goat anti-mouse peroxidase conjugated antibody (Jackson, cat: 115-035-003, dilution 1:7,500). Finally, samples were incubated with TMB / E (Merck Millipore, ES001) and the reaction was stopped with H2SO4. OD450nm was read using Tecean plate reader. Several animal proteins were tested for use in cyanobacteria myoglobin, alpha-actin, and myosin. To produce a high yield of animal proteins in cyanobacteria, otherwise known as Blue-Green Algae, a matrix of operational genetic units that are known to affect the mRNA transcription and protein translation was designed.

[0069] The variety of elements was tested in a combinatorial manner, by stably transforming into two strains of Cyanobacteria species - Synechochos elongatus PCC7942, Synechosystis sp. PCC6803, PCC7002, and UTex2935. Table #1 indicates the names of the genetic elements that were tested.Docket No.10916-10347 PCT Table #1 shows a schematic representation of the tested elements:Docket No.10916-10347 PCTDocket No.10916-10347 PCT

[0070] The process of genetic transformation is known and described in Li et al., 2018 PCC7942, and Yu et al., 2015 for UTEX2973. The vector used for the insertion of animal DNA was pSyn6 by ThermoFisher (cat: A24230). The pSyn6 vector was used in some embodiments without modification. In other embodiments, modifications were made to the pSyn6 vector. In embodiments where modifications were made to the pSyn6 vector, or pSyn6 where homologeous sequences for neutral genomic integration site #1 (SEQ ID NOS: 1, 2 Fig. 2A) were replaced either by neutral genomic integration site #2 left and right (SEQ ID NOS: 3,4 Fig. 2b), or by neutral genomic integration site #3 left and right (SEQ ID NOS:. 5, 6 Fig. 2C). Simultaneously, the antibiotic resistance gene for spectinomycin was replaced with antibiotic resistance gene for Kanamycin (SEQ ID NO: 7) see Fig. 2B or Ampicillin (SEQ ID NO: 8) see Fig. 2C.

[0071] Using pSyn6 vector the effect of nine mRNA optimization algorithms on the expression of Myoglobin in S. elongatus were tested. The Bovine Myoglobin original mRNA sequence (SEQ ID NO: 9, Uniprot: P02192) was compared with mRNA optimized sequences generated by the following algorithms GeneArt (SEQ ID NO: 10), DeNovoDNA (SEQ ID NO: 11), GeneScript (SEQ ID NO: 12), ExpoOptimizer (SEQ ID NO: 13), IDT (SEQ ID NO: 14), JCAT (SEQ ID NO: 15), Codon Harmonizer global codons (SEQ ID NO: 16), and Codon Harmonizer amino acid codons (SEQ ID NO: 17). The various coding sequences (CDS) were subcloned into the pSyn6 vector.

[0072] Next, S. elongatus PCC7942 were transformed with pSyn6 vectors carrying the mRNA optimized sequences. Transformed colonies were selected from agar plate containing 10ug / ml of Spectinomycin, and genomic integration of the area between the left and right neutral integration site #1, was validated using PCR reaction amplifying DNA between neutral integration sites. The reaction was performed using compatible primers (SEQ ID NOS: 18,19), with Hy-Taq PCR ready mix (Hylabs, cat: EZ3006). The resultingDocket No.10916-10347 PCT products of the PCR reaction were analyzed using 1% agarose gel and are presented in Fig. 3A and confirmed the integration of the target DNA. S. elongatus PCC7942 carries 3-8 chromosomal copies per cell (Griese et. al 2011), the lack of an additional low size product corresponding to the space between Neutral Integration site 1 (left and right) confirms the target DNA was integrated to all chromosomal copies of the isolated strains.

[0073] The stably transformed isolated strains were further cultivated in a 250ml Erlenmeyer flask under constant illumination (200uE), 37°C in an incubator shaker at 200RPM. Cultures were harvested and disrupted using acid washed glass beads (Sigma, cat: G4649). Protein quantities were measured using Bradford assay (Thermofisher, cat:23236). 20ug of protein from each sample, were using Western-Blott analysis to confirm the presence of Myoglobin protein using specific antibody (Abcam, cat: ab231530). The results of the analysis showed a detection of Myoglobin in three samples (GeneArt, ExpoOptimizer and Codon Harmonizer), that correspond to the correct size of 17KD of bovine Myoglobin (Fig. 3B), that were absent in the control sample (lacking myoglobin) and from the Bovine Original sequence, DeNovoDNA, GeneScript, IDT and JCAT tested samples.

[0074] To determine the expression level of Myoglobin out of the total soluble protein extracted from the S. elongatus, a quantitative ELISA assay was employed. High protein binding 96 well plate, was coated with 5ug / well of protein samples from the isolated strains carrying Myoglobin expression cassette. The samples were incubated with Myoglobin specific antibody, and the OD450nm measures were compared vs purified Bovine Myoglobin (Worthington Biochemicals, Cat: LS002408). The results of this analysis correspond with the Western Blott analysis (Fig. 3B) and showed three codon optimization algorithms were able to produce Myoglobin (GeneArt, ExpoOptimizer and Codon Harmonizer). Furthermore, the calculated expression level is presented in Fig. 3C.

[0075] The commercial viability for the production of recombinant proteins a tightly bound with the expression levels. In order to achieve higher levels of expression for the recombinant proteins the replacement of genetic promoters on the expression of Myoglobin was examined. A variety of endogenous and exogenous promoters were tested in pSyn6 vector: psbA1 (original vector promoter, SEQ ID NO: 20), psbA2 (SEQ ID NO: 21), psbA3 (SEQ ID NO: 22), TrcSEQ ID NO:10(SEQ ID NO: 23), J23119 (SEQ ID NO: 24), cpc560 (SEQ ID NO: 25), cpcB (SEQ ID NO: 26).Docket No.10916-10347 PCT

[0076] To validate that Myoglobin was integrated into the expected Genomic Site (Neutral Site #1), a PCR reaction was made using compatible primers (SEQ ID NOS: 18, 19) to amplify the relevant genomic region by using Hy-Taq PCR ready mix. As described above, the integration of DNA expression cassette was validated by PCR product analysis (Fig.4A), and the expression levels were measured using an ELISA assay (Fig. 4B). The results of this analysis showed a 13.25-fold increase in protein expression level when psbA1 promoter was replaced with J23119 promoter. When the psbA1 promoter was replaced with the CPC560 promoter, there was a 20.5-fold increase in protein expression. Last, the size of Myoglobin product driven by J23119 was analyzed by Western Blott and was confirmed to correspond to the original bovine 17KD sized protein.

[0077] To quantitatively assess the expression level of Myoglobin out of the total soluble protein extracted from the Blue-Green algae, an ELISA assay was employed. Specific Myoglobin antibody was used to measure the quantity of the recombinant protein extracted from the transgenic algae and the absolute value of expression level was determined by comparing it to a control sample spiked with pure (>90%) Myoglobin extracted from bovine muscle (Worthington Biochemicals, Cat: LS002408). The analysis showed that the protein coded by GeneArt’s mRNA optimization algorithm linked to J23119 resulted in 8.3% expression of Myoglobin out of the total soluble protein (Fig. 4B).

[0078] Assessment of how different neutral genomic integration sites affect the expression levels of Myoglobin under the transcriptional control of CPC560 with the ExpoOptimizer codon optimization algorithm was conducted. Genomic regions may exhibit varying transcriptional activity, consequently influencing mRNA and, subsequently, Myoglobin protein expression. Myoglobin was subcloned into NS2 and NS3 vectors (Figs. 2B and 2C), and the constructs' genomic integration and segregation were validated using PCR with the pair NS2 FW (SEQ ID NO: 27) and NS2 RV (SEQ ID NO: 28) primers, and a second pair of NS3 FW (SEQ ID NO: 29) and NS3 RV (SEQ ID NO: 30) primers (Fig. 5A). Surprisingly, the Myoglobin construct did not integrate into neutral genomic integration site #3. Subsequently, Myoglobin expression levels were measured using a quantitative ELISA assay, revealing a 1.25-fold increase in protein expression when utilizing NS1 compared to NS2 (Fig. 5B).

[0079] Evaluation of how different genetic terminators can affect the expression levels of Myoglobin under transcriptional control of CPC560 directed to genomic integration site 1 (NS1). DNA constructs were generated with synthetic, endogenous, and bacterialDocket No.10916-10347 PCT terminators: TL3S2P21 (SEQ ID NO: 31), TECK120029600 (SEQ ID NO: 32), TpsbA2 (SEQ ID NO: 33), TΩgroEL (SEQ ID NO: 34), TrrnB (original pSyn6 terminator, SEQ ID NO: 35). The constructs’ genomic integration and segregation was validated using PCR with NS1 FW (SEQ ID NO: 18) and NS1 RV (SEQ ID NO: 19) primers (Fig. 6A). Myoglobin expression levels for differing terminators were measured using quantitative ELISA assay Myoglobin using a quantitative ELISA assay, revealing a range of a 2.1 to7.5-fold increase in protein expression when utilizing TrrnB as compared to the other tested terminators (Fig. 6B).

[0080] The relative strength of genetic elements, such as integration sites, promoters, and terminators, is generally preserved across different genes within an organism. Therefore, to examine the repeatability of the highest expressing genetic operon, a second animal protein, alpha-actin (SEQ ID NO: 36, Uniprot: P68138) was evaluated. On the contrary, the efficiency of mRNA optimization algorithms is not predictable. Therefore, the NS1:CPC560: CDS:TrrnB operon was used with eleven mRNA-optimized sequences of alpha-actin (SEQ ID NOs: 37-47) were sub-cloned. The constructs’ genomic integration and segregation was validated using PCR with NS1 FW (SEQ ID NO: 18) and NS1 RV (SEQ ID NO: 19) primers (Fig. 7A). The expression levels were measured using an ELISA assay with alpha-actin specific antibody (DSHB:JLA20-s), demonstrating up to 2.7-fold increase in expression level compared with the bovine original sequence by GeneArt algorithm (Fig. 7B). Western blot analysis, using specific antibodies (HDSHB JLA20-s), confirmed the presence of Alpha-actin in (ATGme, GeneArt, DeNovo, Harmonizer (local) the original Bovine sequence, corresponding to the correct size of 42.1KD for bovine Alpha-actin (Figure 7C).

[0081] Three additional animal proteins were tested, by replacing the coding mRNA sequence of Myoglobin with the bovine original sequence and GeneArt optimized sequence, for Myosin Light Chain 1 (MLC1, SEQ ID NO: 49), Myosin Light Chain 2 (MLC2, SEQ ID NO: 50), Myosin 2 Heavy Chain (MYH2, SEQ ID NO: 51.

[0082] The expression of the skeletal myosin complex, which is composed of the following three proteins: Myosin light chain 1 (Uniprot: A0JNJ5), Myosin light chain 2 (Uniprot: Q0P571), and Myosin heavy chain 2 (Uniprot: Q9BE41). Co-expression of multiple recombinant proteins can be achieved using bacterial operons, which is beneficial when equal stoichiometric ratios of several proteins are desired, as an operon is transcribed as one single mRNA unit. The skeletal Myosin type II hexamer complex is composed of one heavy chain and two light chains (Light chain 1 and Light chain 2) at a 1:1:1 ratio, therefore prokaryoticDocket No.10916-10347 PCT expression such as in cyanobacteria is advantageous for its expression. To express Myosin type II three subunits under one regulatory unit, a polycistronic expression cassette was constructed with the following elements (Fig. 8A), the intergenic region corresponds to SEQ ID NO: 48, while the coding mRNA for Myosin Type 2 subunits, are SEQ ID NO: 49 for MLC1, SEQ ID NO: 50 for MLC2, and SEQ ID NO: 51 for MYH2.

[0083] Four independent transformation trials resulted in colonies that were antibiotic resistant, in contrast to “No DNA” control (Fig. 8B). Protein toxicity may lead to abnormal DNA recombination in such a way that the only variants that are unable to express the transgene are viable. To examine if operon aberrant integration was caused by the three myosin subunit toxicity, a promoter free construct that is unable to result in protein transformation was subcloned. PCR analysis showed that the antibiotic resistance gene (Fig. 8C) (primers SEQ ID NOS: 52-53), but not the promoter less myosin operon (primers SEQ ID NOS: 19 and 54) was integrated into the genome (Fig. 8D).

[0084] The expression of the skeletal myosin complex was attempted. Each of the three genes for the myosin complex (MLC1, MLC2, and MYH2; SEQ ID NOS: 49-51, respectively) was cloned into separate vectors with the operon NS1a_J23119: CDS:Trrnb_NS1b. The transformation resulted in Spectinomycin resistance colonies for MLC1 and MYH2, but not for MLC2. Independent transformation trials for the MLC2 construct did not yield visible colonies, suggesting that the sole expression of MLC2 induced toxicity effects in cyanobacteria. Subsequent PCR analysis confirmed the integration of MLC1 (Fig. 9A) and partial integration for MYH2 at a size of 3.5-4.5kb, which falls short of the expected 7.9kb for this construct (Fig. 9B). Western Blot analysis, using MLC1-specific antibodies (DSHB F310-s), detected a protein band corresponding to the full-length MLC1 at ~20.9KD (Fig. 9C). The expression levels, measured using an ELISA assay, resulted in similar expression to that of Myoglobin under the same operon (Fig. 9D).

[0085] MLC2 expression often would require the presence of MLC1, as these proteins have protein-protein interactions at several positions. Therefore, an MLC1+ MLC2 operon was constructed. Subsequent PCR analysis confirmed the integration of MLC1+MLC2 construct (Fig. 9E). Western Blot analysis, using MLC2-specific antibodies (Cell Signaling: CST-3672S) detected a protein band corresponding to the full-length MLC2 at 19KD (Fig. 9F).Docket No.10916-10347 PCT

[0086] Next, MYH2 was transformed into the MLC1:MLC2 expressing strain. Subsequent PCR analysis with specific primers (SEQ ID NOS: 18-19) confirmed its integration (Fig.9F). Western Blot analysis, using MYH2-specific antibodies (DSHB BF-35- s) is used to detect a protein band corresponding to the full-length MYH2 (220.1KD), together with a quantitative ELISA assay to assess its expression level.

[0087] E. coli, a common model organism for cyanobacteria was used to assess the solubility of MYH2 in the absence or presence of the myosin complex light and regulatory chains MLC1 and MLC2. To this end, MYH2 (SEQ ID NO:51) that was used for expression in Cyanobacteria, was subcloned into a pET-30(a+) expression vector (SEQ ID NO: 55). Western Blott analysis confirmed the expression of tagged MYH2 using anti-his specific antibodies (GenScript, cat: A00186) by detecting a protein band that corresponds to the size of HIS-MYH2 (224KD) (Fig. 10A). Next, SDS-PAGE analysis was performed to evaluate the solubility of MYH2, which was assessed by a protein band corresponding to the size of MYH2 in the transformed bacteria and not control (Fig. 10B), by calculating its soluble / in- soluble proportion. The results of this experiment showed a limited solubility for MYH2 of 5% when expression was induced for 4 hours at 37°C, or 30% when expression was induced for 16 hours at 15°C.

[0088] AN MLC1+MLC2 expression vector was constructed by subcloning of MLC1+MLC2 (SEQ ID NOs:49 and 50) that was used for expression in Cyanobacteria into pETDuet-1 expression vector (SEQ ID NO: 56). Western blot analysis confirmed the expression of HIS-MYH2, and FLAG-MLC2 (Fig. 10C), and HIS-MLC1 (Fig. 10D) using specific antibodies GenScript, cat: A00170 and A00186, respectively. MYH2 solubility was reevaluated when co-expressed with MLC1 and MLC2 and found a MYH2 solubility at to be 10% at 37C° and 90% at 15C° (Figure 10E). The summary of MYH2 solubility under the tested in the absence or presence of MLC1+MLC2 under induction temperatures of 15C° or 37C° shows, an 18X increase in its solubility at 15C° with MLC1+MLC2 compared with expression at 37C° in the absence MLC1+MLC2 (Figure 10F).

[0089] Therefore, the solubility of MYH2, the major structural component of the skeletal Myosin II complex, is dependent on the co-expression of MLC1 and MLC2 in the same bacterial cell. Furthermore, as insoluble proteins are generally expressed in lower yields and can only partially be renatured using complex techniques, it is essential to achieve a high yield of soluble functional Myosin complex to express all of its three subunits simultaneously.Docket No.10916-10347 PCT

[0090] Other animal proteins are also acceptable to grow in the growth system. The proteins used in the embodiments described were all bovine proteins. Other animals commonly used for food products such as chicken, pig, fish, sheep, and goat also have proteins that would function in this system. The coding mRNA sequences for other proteins could also be inserted.

[0091] Another example is the expression of Myoglobin, Actin, and Myosin in PCC6803 marine Cyanobacterial strain. A vector for stable transformation is constructed by replacing NS1a (SEQ ID NO: 1) and NS1b (SEQ ID NO: 2) sites in the pSyn6 vector with NS1a_PCC6803 (SEQ ID NO: 57) and NS1b_PCC6803 (SEQ ID NO: 58) or NS2a (SEQ ID NO: 3) and NS2b (SEQ ID NO: 4) with NS2a_PCC6803 (SEQ ID NO: 59) and NS2b_PCC6803 (SEQ ID NO: 60). Next, we subclone Myoglobin (SEQ ID NO: 13), alpha- actin (SEQ ID NO: 37) with CPC560 promoter and rrnB terminator to NS1_PCC6803. J23119 promoter and rrnB terminator MLC1+MLC2 (SEQ ID NOS: 49-50) is used to subclone to NS2_PCC6803 and MYH2 (SEQ ID NO: 51) to NS1_PCC6803 in the new expression vectors. PCR analysis is performed to confirm the integration of the expression cassette into the genome using specific primers NS1a_PCC6803_FW (SEQ ID NO: 61) and NS1b_PCC6803_RV (SEQ ID NO: 62) for NS1 or with NS2a_PCC6803_FW (SEQ ID NO: 63) and NS2b_PCC6803_RV (SEQ ID NO: 64). Western Blott is used to detect a protein band that corresponds to the full-length size of Myoglobin, Actin, and Myosins, and measure the expression levels with quantitative ELISA, both with specific antibodies for Myoglobin, alpha-actin, and MLC1, MLC2, and MYH2, as indicated in the examples for PCC7942.

[0092] Another example is the expression of Myoglobin, Actin, and Myosin in PCC7002 Cyanobacteria strain. A vector is constructed for stable transformation by replacing NS1a (SEQ ID NO: 1) and NS1b (SEQ ID NO: 2) sites in the pSyn6 vector with NS1a_PCC7002 (SEQ ID NO: 65) and NS1b_PCC7002 (SEQ ID NO: 66) or NS2a (SEQ ID NO: 3) and NS2b (SEQ ID NO: 4) with NS2a_PCC7002 (SEQ ID NO: 67) and NS2b_PCC7002 (SEQ ID NO: 68). Next, we subclone Myoglobin (SEQ ID NO: 13), alpha- actin (SEQ ID NO: 37) with CPC560 promoter and rrnB terminator to NS1_PCC7002. We subclone with J23119 promoter and rrnB terminator MLC1+MLC2 (SEQ ID NOS: 49-50) to NS2_PCC7002 and MYH2 (SEQ ID NO: 51) to NS1_PCC7002 in the new expression vectors. PCR analysis is performed to confirm the integration of the expression cassette into the genome using specific primers NS1a_PCC7002_FW (SEQ ID NO: 69) and NS1b_PCC7002_RV (SEQ ID NO: 70) for NS1 or with NS2a_PCC7002_FW (SEQ ID NO:Docket No.10916-10347 PCT 71) and NS2b_PCC7002_RV (SEQ ID NO: 72). Western Blott is used to detect a protein band that corresponds to the full-length size of Myoglobin, Actin, and Myosins, and measure the expression levels with quantitative ELISA, both with specific antibodies for Myoglobin, alpha-actin, and MLC1, MLC2, and MYH2, as indicated in the examples for PCC7942.

[0093] Another example is the expression of Myoglobin, Actin, and Myosin in UTEX2973 Cyanobacteria strain. Using pSyn6 vectors transform Myoglobin (SEQ ID NO: 13), alpha-actin (SEQ ID NO: 37) with CPC560 promoter and rrnB terminator to NS1_PCC6803. Transformation is done with J23119 promoter and rrnB terminator MLC1+MLC2 (SEQ ID NOS: 49-50) to NS2(SEQ ID NOS: 3-4) and MYH2 (SEQ ID NO: 51) to NS1 (SEQ ID NOS: 1-2).PCR analysis is performed to confirm the integration of the expression cassette into the genome using specific primers NS1FW (SEQ ID NO: 18) and NS1RV (SEQ ID NO:19) for NS1 or with NS2a_PCC6803_FW (SEQ ID NO: 27) and NS2RV (SEQ ID NO: 28). Western Blott is used to detect a protein band that corresponds to the full-length size of Myoglobin, Actin, and Myosins, and measure the expression levels with quantitative ELISA, both with specific antibodies for Myoglobin, alpha-actin, and MLC1, MLC2, and MYH2, as indicated in the examples for PCC7942.

[0094] A first aspect provides a genetically modified cell, the cell comprising: a gene comprising: a promoter; a sequence encoding an animal protein; and a terminator; wherein the cell is a cyanobacteria cell.

[0095] In a second aspect, the gene is integrated into the genome of the cyanobacteria.

[0096] In a third aspect, the gene is integrated into an NS1 site of the cell.

[0097] In a fourth aspect, the sequence encoding an animal protein is optimized for expression in the cyanobacteria.

[0098] In a fifth aspect, the animal protein is a muscle protein.

[0099] In a sixth aspect, the muscle protein is a myosin subunit. [000100] In a seventh aspect, the myosin subunit is MLC1. [000101] In an eighth aspect, the myosin subunit is MLC2. [000102] In a ninth aspect, the myosin subunit is MYH2. [000103] In a tenth aspect, the muscle protein is myoglobin.Docket No.10916-10347 PCT [000104] In an eleventh aspect, the muscle protein is alpha actin. [000105] In a twelfth aspect, the animal protein is a subunit of a complex. [000106] In a thirteenth aspect, the complex is a myosin complex. [000107] In a fourteenth aspect, the myosin subunits in the myosin complex are MLC1 and MLC2. [000108] In a fifteenth aspect, the myosin subunits in the myosin complex are MLC1 and MYH2. [000109] In a sixteenth aspect, the myosin subunits in the myosin complex are MLC2 and MYH2. [000110] In a ninth aspect, the cyanobacteria cell is Synechochos elongatus. [000111] In a tenth aspect, the S. elongatus is PCC7942. [000112] In an eleventh aspect, the cyanobacteria cell is PCC6803. [000113] In a twelfth aspect, the gene is in a vector. [000114] In a thirteenth aspect, the genetically modified cell includes a second gene, wherein either the first gene or the second gene is in a vector. [000115] In a fourteenth aspect, the genetically modified cell includes a third gene wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector. [000116] In a fifteenth aspect, each gene comprises a myosin complex subunit. [000117] In a sixteenth aspect, the myosin complex subunits are MLC1, MLC2, and MYH2. [000118] In a seventeenth aspect, the genetically modified cell includes a second gene, wherein the first gene, the second gene, or both genes are integrated into the genome of the cyanobacteria. [000119] In an eighteenth aspect, the genetically modified cell includes a third gene, wherein any of the first gene, the second gene or the third gene or any combination thereof is integrated into the genome of the cyanobacteria. [000120] In a nineteenth aspect, each gene comprises a myosin complex subunit.Docket No.10916-10347 PCT [000121] In a twentieth aspect, the myosin complex subunits are MLC1, MLC2, and MYH2. [000122] In a twenty-first aspect, any of the first gene, second gene, or third gene or any combination thereof is in a vector and those genes not in a vector are incorporated into the genome of the cyanobacteria. [000123] In a twenty-second aspect, the promoter is one of SEQ ID NO: 24 or SEQ ID NO: 25. [000124] In a twenty-third aspect a method for producing an animal protein from a genetically modified cell is disclosed. The method comprises: inserting into a cell a gene comprising: a promoter; a sequence encoding an animal protein; and a terminator; wherein the cell is a cyanobacteria cell. [000125] A twenty- fourth aspect is the method of aspect twenty-three wherein the gene is integrated into the genome of the cyanobacteria. [000126] A twenty- fifth aspect is the method of aspect twenty-four wherein the gene is integrated into an NS1 site of the cell. [000127] A twenty- sixth aspect is the method of aspect twenty-three wherein the sequence encoding an animal protein is optimized for expression in the cyanobacteria. [000128] A twenty- seventh aspect is the method of aspect twenty-three wherein the animal protein is a muscle protein. [000129] A twenty-eighth aspect is the method of aspect twenty-seven wherein the muscle protein is a myosin subunit, myoglobin, or alpha-actin. [000130] A twenty- ninth aspect is the method of aspect twenty-three wherein the animal protein is a subunit of a complex. [000131] A thirtieth aspect is the method of aspect twenty-nine wherein the complex is a myosin complex. [000132] A thirtieth-first aspect is the method of aspect twenty-three wherein the cyanobacteria cell is from the species Synechochos elongatus. [000133] A thirty- second aspect is the method of aspect thirty-one wherein the S. elongatus is PCC7942.Docket No.10916-10347 PCT [000134] A thirty-third aspect is the method of aspect thirty-two wherein the S. elongatus is PCC6803. [000135] A thirty-fourth aspect is the method of aspect twenty-three wherein the gene is in a vector. [000136] A thirty-fifth aspect is the method of aspect thirty-four further comprising a second gene, wherein either the first gene or the second gene is in a vector. [000137] A thirty- sixth aspect is the method of aspect thirty-five further comprising a third gene wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector. [000138] A thirty-seventh aspect is the method of aspect twenty-three wherein each gene comprises a myosin complex subunit. [000139] A thirty-seventh aspect the method of aspect thirty-seven wherein the myosin complex subunits are MLC1, MLC2, and MYH2. [000140] A thirty-eighth aspect is the method of aspect thirty-seven further comprising a second gene, wherein the first gene, the second gene, or both genes are integrated into the genome of the cyanobacteria. [000141] A thirty-ninth aspect is the method of aspect thirty-eight further comprising a third gene, wherein any of the first gene, the second gene or the third gene or any combination thereof is integrated into the genome of the cyanobacteria. [000142] A fortieth aspect is the method of aspect thirty-nine wherein each gene comprises a myosin complex subunit. [000143] A forty-first aspect is the method of aspect forty wherein the myosin complex subunits are MLC1, MLC2, and MYH2. [000144] A forty-second aspect is the method of aspect forty wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector and those genes not in a vector are incorporated into the genome of the cyanobacteria. [000145] A forty-third aspect is a method wherein the promoter is one of SEQ ID NO: 24 or SEQ ID NO: 25.Docket No.10916-10347 PCT [000146] A forty-third aspect is a method of producing an animal protein in cyanobacteria, the method comprising: isolating the animal protein from the modified cells of any one of claims 1-21. [000147] A forty-fourth aspect is a cyanobacteria cell for producing an animal protein comprising: a first vector inserted into the cell, the vector comprising: a promoter; a first mRNA optimized subunit; and a terminator. [000148] A forty-fifth aspect is the cyanobacteria cell of aspect forty-four wherein the vector comprises a pSyn6 vector. [000149] A forty-seventh aspect is the cyanobacteria cell of aspect forty-four wherein the pSyn6 vector further comprises a genomic integration site targeting sequence. [000150] A forty-eighth aspect is the cyanobacteria cell of aspect forty-seven wherein the genomic integration site targeting sequence targets the NS1 integration site. [000151] A forty-ninth aspect is the cyanobacteria cell of aspect forty-four wherein the cyanobacteria is Synechochos elongatus. [000152] A fiftieth aspect is the cyanobacteria cell of aspect forty-nine wherein the strain of Synechochos elongatus is PCC7942. [000153] A fifty-first aspect is the cyanobacteria cell of aspect forty-nine wherein the strain of Synechochos elongatus is UTEX2973. [000154] A fifty-second aspect is the cyanobacteria cell of aspect forty-four wherein the at least one mRNA optimized subunit codes for an animal protein. [000155] A fifty-third aspect is the cyanobacteria cell of aspect fifty-two wherein the animal protein is myosin. [000156] A fifty-fourth aspect is the cyanobacteria cell of aspect fifty-three wherein expression of the myosin is between about 5% and about 40%. [000157] A fifty-fifth aspect is the cyanobacteria cell of aspect fifty-four wherein expression of the myosin is between about 10% and about 30%. [000158] A fifty-sixth aspect is the cyanobacteria cell of aspect fifty-five wherein expression of the myosin is about 20%. [000159] A fifty- seventh aspect is the cyanobacteria cell of aspect fifty-two wherein the animal protein is myoglobin.Docket No.10916-10347 PCT [000160] A fifty- eighth aspect is the cyanobacteria cell of aspect fifty-two wherein the animal protein is alpha-actin. [000161] A fifty-ninth aspect is the cyanobacteria cell of aspect forty-four wherein the cyanobacteria cell produces multiple animal proteins. [000162] A sixtieth aspect is the cyanobacteria cell of aspect fifty-nine wherein multiple vectors each with its own promoter, first mRNA optimized subunit, terminator, and intergenic region. [000163] A sixty-first aspect is the cyanobacteria cell of aspect sixty wherein each of the first mRNA optimized subunits of each vector codes for a myosin protein. [000164] A sixty-second aspect is the cyanobacteria cell of aspect sixty-one wherein the myosin proteins are selected from the group comprising MLC1, MLC2, MYH2. [000165] A sixty-third aspect is the cyanobacteria cell of aspect sixty-two wherein the multiple vectors comprise three vectors, and wherein each vector includes one of MLC1, MLC2, or MYH2 mRNA optimized subunit. [000166] A sixty-fourth aspect is the cyanobacteria cell of aspect forty-four further comprising a second subunit and a third subunit. [000167] A sixty-fifth aspect is the cyanobacteria cell of aspect sixty-four wherein the first, second, and third subunits are myosin coding mRNA. [000168] A sixty-seventh aspect is the cyanobacteria cell of aspect sixty-five wherein the first subunit is any of MLC1, MLC2, or MYH2. [000169] A sixty-eighth aspect is the cyanobacteria cell of aspect sixty-seven wherein the first subunit is any of MLC1, MLC2, or MYH2 and the second subunit is any of MLC1, MLC2, or MYH2 wherein the first and second subunits are not the same subunit. [000170] A seventieth aspect is the cyanobacteria cell of aspect sixty-seven wherein the first subunit is mRNA that codes for any of MLC1, MLC2, or MYH2 and the second subunit is mRNA that codes for any of MLC1, MLC2, or MYH2, and the third subunit is mRNA that codes for any of MLC1, MLC2, or MYH2 and wherein the first, second, and third subunits are not the same subunit. [000171] A seventy-first aspect is the cyanobacteria cell of aspect forty-four further comprising a first genomic integration site.Docket No.10916-10347 PCT [000172] A seventy-second aspect is the cyanobacteria cell of aspect seventy-one wherein the first genomic integration site is SEQ ID NO: 1. [000173] A seventy-third aspect is the cyanobacteria cell of aspect forty-four further comprising a second genomic integration site. [000174] A seventy-fourth aspect is the cyanobacteria cell of aspect seventy-three wherein the second genomic integration site is SEQ ID NO: 2. [000175] A seventy-fifth aspect is the cyanobacteria cell of aspect forty-four further comprising a second vector. [000176] A seventy-sixth aspect is the cyanobacteria cell of aspect seventy-five wherein the first vector and the second vector are inserted at separate genomic sites. [000177] A seventy-seventh aspect is the cyanobacteria cell of aspect seventy-six wherein the mRNA of the first vector and the mRNA of the second vector are coexpressed. [000178] A seventy-eighth aspect is the cyanobacteria cell of aspect seventy-seven wherein the cell further comprises a third vector. [000179] A seventy-ninth aspect is the cyanobacteria cell of aspect seventy-eight wherein the first vector, the second vector, and the third vector are inserted at separate genomic sites. [000180] An eightieth aspect is the cyanobacteria cell of aspect forty-four wherein the mRNA of the first vector, the mRNA of the second vector, and the mRNA of the third vector are coexpressed. [000181] All patents and published patent applications referred to herein are incorporated herein by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. Nevertheless, it is understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

Docket No.10916-10347 PCT WHAT IS CLAIMED IS:

1. A genetically modified cell, the cell comprising: a gene comprising: a promoter; a sequence encoding an animal protein; and a terminator; wherein the cell is a cyanobacteria cell.

2. The genetically modified cell of claim 1, wherein the gene is integrated into the genome of the cyanobacteria.

3. The genetically modified cell of claim 2, wherein the gene is integrated into an NS1 site of the cell.

4. The genetically modified cell of claim 1, wherein the sequence encoding an animal protein is optimized for expression in the cyanobacteria.

5. The genetically modified cell of claim 1, wherein the animal protein is a muscle protein.

6. The genetically modified cell of claim 5, wherein the muscle protein is a myosin subunit, myoglobin, or alpha-actin.

7. The genetically modified cell of claim 1, wherein the animal protein is a subunit of a complex.

8. The genetically modified cell of claim 7, wherein the complex is a myosin complex.

9. The genetically modified cell of claim 1, wherein the cyanobacteria cell is Synechochos elongatus.Docket No.10916-10347 PCT 10. The genetically modified cell of claim 9, wherein the S. elongatus is PCC7942 or UTEX2973 or PCC7002 or Synechocystis sp. PCC6803.

11. The genetically modified cell of claim 1, wherein the gene is in a vector.

12. The genetically modified cell of claim 1, further comprising a second gene, wherein either the first gene or the second gene is in a vector.

13. The genetically modified cell of claim 13, further comprising a third gene wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector.

14. The genetically modified cell of any of claims 11-14, wherein each gene comprises a myosin complex subunit.

15. The genetically modified cell of claim 15, wherein the myosin complex subunits are MLC1, MLC2, and MYH2.

16. The genetically modified cell of claim 1, further comprising a second gene, wherein the first gene, the second gene, or both genes are integrated into the genome of the cyanobacteria.

17. The genetically modified cell of claim 16, further comprising a third gene, wherein any of the first gene, the second gene or the third gene or any combination thereof is integrated into the genome of the cyanobacteria.

18. The genetically modified cell of any of claims 16-17, wherein each gene comprises a myosin complex subunit.

19. The genetically modified cell of claim 18, wherein the myosin complex subunits are MLC1, MLC2, and MYH2.Docket No.10916-10347 PCT 20. The genetically modified gene of either of claims 13 or 17, wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector and those genes not in a vector are incorporated into the genome of the cyanobacteria.

21. The genetically modified cell of claim 1, wherein the promoter is one of SEQ ID NO: 24 or SEQ ID NO:

25.

22. A method for producing an animal protein from a genetically modified cell, the method comprising: inserting into a cell a gene comprising: a promoter; a sequence encoding an animal protein; and a terminator; wherein the cell is a cyanobacteria cell.

23. The method of claim 22, wherein the gene is integrated into the genome of the cyanobacteria.

24. The method of claim 22, wherein the gene is integrated into an NS1 site of the cell.

25. The method of claim 22, wherein the sequence encoding an animal protein is optimized for expression in the cyanobacteria.

26. The method of claim 22, wherein the animal protein is a muscle protein.

27. The method of claim 26, wherein the muscle protein is a myosin subunit, myoglobin, or alpha-actin.

28. The method of claim 22, wherein the animal protein is a subunit of a complex.

29. The method of claim 28, wherein the complex is a myosin complex.Docket No.10916-10347 PCT 30. The method of claim 22, wherein the cyanobacteria cell is from the species Synechochos elongatus.

31. The method of claim 26, wherein the S. elongatus is PCC7942 or UTEX2973 or PCC7002 or Synechocystis sp. PCC6803.

32. The method of claim 22, wherein the gene is in a vector.

33. The method of claim 32, further comprising a second gene, wherein either the first gene or the second gene is in a vector.

34. The method of claim 33, further comprising a third gene wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector.

35. The method of any of claims 32-34, wherein each gene comprises a myosin complex subunit.

36. The method of claim 35, wherein the myosin complex subunits are MLC1, MLC2, and MYH2.

37. The method of claim 22, further comprising a second gene, wherein the first gene, the second gene, or both genes are integrated into the genome of the cyanobacteria.

38. The method of claim 37, further comprising a third gene, wherein any of the first gene, the second gene or the third gene or any combination thereof is integrated into the genome of the cyanobacteria.

39. The method of any of claims 37-38, wherein each gene comprises a myosin complex subunit.

40. The method of claim 39, wherein the myosin complex subunits are MLC1, MLC2, and MYH2.Docket No.10916-10347 PCT 41. The genetically modified gene of either of claims 34 or 38, wherein any of the first gene, second gene, or third gene or any combination thereof is in a vector and those genes not in a vector are incorporated into the genome of the cyanobacteria.

42. The method of claim 22, wherein the promoter is one of SEQ ID NO: 24 or SEQ ID NO:

25.

43. A method of producing an animal protein in cyanobacteria, the method comprising: isolating the animal protein from the modified cells of any one of claims 1-21.

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