Cultured meat products using genetically modified cells

Abstract

Provided herein are genetically engineered mammalian cells that endogenously express one or more phytochemicals, vitamins or therapeutic agents and are suitable for culturing meat products. Methods of making and using the genetically engineered mammalian cells and the cultured meat products are also provided.

Description

[0001] Cross - Reference to Related Applications

[0002] This application is a divisional application of a Chinese patent application with application number 202080054515.3, filing date June 1, 2020, and invention title "Cultured Meat Products Using Genetically Modified Cells", and the original application is a national stage application with international application number PCT / US2020 / 035526. This application claims the priority of U.S. Provisional Application No. 62 / 855,698 filed on May 31, 2019 and U.S. Provisional Application No. 62 / 861,606 filed on June 14, 2019, and each of the said provisional applications is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made under government support in accordance with Grant No. EB002520 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Reference to a Sequence Listing Submitted via EFS - WEB

[0006] The content of the ASCII text file of the sequence listing with file name "166118_00970_ST25.txt" and size 114 kb, created on June 1, 2020 and electronically filed herewith with this application via EFS-Web, is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0007] Synthetic biology and metabolic engineering have emerged as important tools across multiple disciplines (Keasling, 2010). However, their widespread use in mammalian systems has developed slowly, and progress within these systems has been largely limited to applications such as therapeutic protein production (e.g., monoclonal antibodies) or the study and treatment of diseases (e.g., metabolic reprogramming) (Davy et al., 2017; Schukur and Fussenegger, 2016). In these efforts, little attention has been paid to the heterologous engineering of mammalian cells using genes from other taxonomic units (i.e., bacteria or plants) in addition to genetic tools such as viral or bacterial editing or regulatory systems (Mojica and Montoliu, 2016; Zhang et al., 2006). In contrast, metabolic engineering efforts in bacteria, yeast, or plants have employed transgenic constructs from multiple taxa for applications ranging from medicine and cosmetics to energy and agriculture (Heider and Wendisch, 2015; Nielsen, 2015; Sack et al., 2015). Recent advances in tissue engineering have expanded the view of the possible applications of animal cell culture in food and nutrition, robotics, and biosensors (Banerjee and Bhunia, 2009; Rubio et al., 2019). This shift presents an opportunity to revisit mammalian metabolic engineering and to strive to expand engineering across taxa for a wide range of applications.

[0008] One such application is the production of meat through cell culture (known by various terms, this article will use “cultured meat”), which has been proposed as a means to address the environmental, ethical, and public health issues of livestock farming (Mattick et al., 2015; Post, 2012). Summary of the Invention

[0009] In a first aspect, provided herein is an engineered cell that endogenously synthesizes phytoene and comprises a heterologous polynucleotide encoding phytoene synthase. In some embodiments, the phytoene synthase has a sequence that is at least 90% identical to SEQ ID NO:1 or SEQ ID NO:_. In some embodiments, the cell endogenously synthesizes lycopene and comprises a heterologous polynucleotide encoding phytoene desaturase. In some embodiments, the phytoene desaturase has a sequence that is at least 90% identical to SEQ ID NO:3. In some embodiments, the cell endogenously synthesizes β-carotene and comprises a heterologous polynucleotide encoding lycopene cyclase. In some embodiments, the lycopene cyclase has a sequence that is at least 90% identical to SEQ ID NO:5 or SEQ ID NO:_. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is Pax7 + mammalian muscle progenitor cells. In some embodiments, the cell is actin + myosin heavy chain (MHC) + mammalian multinucleated myotubes. In some embodiments, the total carotenoid production in the engineered cell is at least 5-fold higher than the total carotenoid production in the cell without the heterologous polynucleotides encoding phytoene synthase, phytoene desaturase, and lycopene cyclase.

[0010] In a second aspect, provided herein is a method of making an engineered cell as described herein, the method comprising introducing into a cell that naturally synthesizes geranylgeranyl pyrophosphate (i) a heterologous polynucleotide encoding phytoene synthase; (ii) a heterologous polynucleotide encoding phytoene desaturase; (iii) a heterologous polynucleotide encoding lycopene cyclase; or (iv) a combination thereof. In some embodiments, the cell is a Pax7+ mammalian muscle progenitor cell.

[0011] In a third aspect, provided herein is an engineered cell that endogenously synthesizes vitamin C and comprises one or more exogenous polynucleotides encoding UDP-glucose 6-dehydrogenase (UGDH), UDP-glucuronosyltransferase 1 (UGT1A1), aldo-keto reductase family 1 (AKR1A1), regucalcin (RGN), and L-gulono-γ-lactone oxidase (GULO).

[0012] In a fourth aspect, the present disclosure provides an engineered cell that endogenously synthesizes curcumin and comprises one or more heterologous polynucleotides encoding prephenate aminotransferase (PTAL), 4-coumarate-CoA ligase (4CL), phenylpropanoyl-diketide-CoA synthase (DCS), and curcumin synthase (CURS3).

[0013] In a fifth aspect, the present disclosure provides an engineered cell that endogenously synthesizes cannabidiol (CBD) and comprises one or more heterologous polynucleotides encoding 3,5,7-trioxododecanoyl-CoA synthase (OLS), oleanolate cyclase (OAC), cannabigerolic acid synthase (CBGAS), and cannabidiolic acid synthase (CBDAS).

[0014] In a sixth aspect, the present disclosure provides an engineered cell that comprises a heterologous polynucleotide encoding a nanobody V565 having a sequence that is at least 95% identical to SEQ ID NO:7. In some embodiments, the polynucleotide encodes the V565 nanobody covalently linked to tumor necrosis factor α (TNFα) via a cleavable polypeptide linker. In some embodiments, the cleavable polypeptide linker comprises SEQ ID NO:9.

[0015] In a seventh aspect, the present disclosure provides an engineered cell that comprises a heterologous polynucleotide encoding an antimicrobial peptide 16 (AMP16) having a sequence that is at least 95% identical to SEQ ID NO:8. In some embodiments, the polynucleotide encodes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 repeats of AMP16, each repeat being separated by a linker polypeptide comprising an Asn-Pro-Val repeat.

[0016] In an eighth aspect, the present disclosure provides a cultured meat product that comprises a confluent serum-free culture comprising the engineered cells as described herein inoculated on a food safety matrix. In some embodiments, the matrix is a membrane. In some embodiments, the matrix is a sponge or a three-dimensional matrix. In some embodiments, the matrix is a chitosan matrix. In some embodiments, the cell culture comprises multinucleated myotubes derived from Pax7+ mammalian satellite cells. In some embodiments, the multinucleated myotubes express myosin heavy chain (MHC) and actin.

[0017] The present application also encompasses the following embodiments:

[0018] 1. An engineered cell that endogenously synthesizes phytoene and comprises a heterologous polynucleotide encoding phytoene synthase.

[0019] 2. The engineered cell according to Embodiment 1, wherein the phytoene synthase has a sequence that is at least 90% identical to SEQ ID NO:1 or SEQ ID NO:10.

[0020] 3. The engineered cell according to Embodiment 1 or 2, wherein the cell endogenously synthesizes lycopene and comprises a heterologous polynucleotide encoding phytoene desaturase.

[0021] 4. The engineered cell according to Embodiment 3, wherein the phytoene desaturase has a sequence that is at least 90% identical to SEQ ID NO:3.

[0022] 5. The engineered cell according to Embodiment 3 or 4, wherein the cell endogenously synthesizes β-carotene and comprises a heterologous polynucleotide encoding lycopene cyclase.

[0023] 6. The engineered cell according to Embodiment 5, wherein the lycopene cyclase has a sequence that is at least 90% identical to SEQ ID NO:5 or SEQ ID NO:11.

[0024] 7. The engineered cell according to any one of Embodiments 1-6, wherein the cell is a mammalian cell.

[0025] 8. The engineered cell according to any one of Embodiments 1-7, wherein the cell is a Pax7 + mammalian muscle precursor cell.

[0026] 9. The engineered cell according to any one of Embodiments 1-7, wherein the cell is an actin + myosin heavy chain (MHC) + mammalian multinucleated myotube.

[0027] 10. The cell according to Embodiment 5 or 6, wherein the total carotenoid yield in the engineered cell is at least 5-fold higher than the total carotenoid yield in the cell without the heterologous polynucleotides encoding phytoene synthase, phytoene desaturase, and lycopene cyclase.

[0028] 11. A method for producing the cell according to any one of Embodiments 1-10, comprising introducing into a cell that naturally synthesizes geranylgeranyl pyrophosphate (i) a heterologous polynucleotide encoding phytoene synthase; (ii) a heterologous polynucleotide encoding phytoene desaturase; (iii) a heterologous polynucleotide encoding lycopene cyclase; or (iv) a combination thereof.

[0029] 12. The method according to embodiment 11, wherein the cell is a Pax7+ mammalian muscle precursor cell.

[0030] 13. An engineered cell that endogenously synthesizes vitamin C and comprises one or more exogenous polynucleotides encoding UDP-glucose 6-dehydrogenase (UGDH), UDP-glucuronosyltransferase 1 (UGT1A1), aldo-keto reductase family 1 (AKR1A1), calreticulin (RGN), and L-gulono-γ-lactone oxidase (GULO).

[0031] 14. An engineered cell that endogenously synthesizes curcumin and comprises one or more heterologous polynucleotides encoding tyrosine ammonia-lyase (PTAL), 4-coumarate-CoA ligase (4CL), phenylpropanoyl-diketide-CoA synthase (DCS), and curcumin synthase (CURS3).

[0032] 15. An engineered cell that endogenously synthesizes cannabidiol (CBD) and comprises one or more heterologous polynucleotides encoding 3,5,7-trioxododecanoyl-CoA synthase (OLS), olivetolic acid cyclase (OAC), cannabigerolic acid synthase (CBGAS), and cannabidiolic acid synthase (CBDAS).

[0033] 16. An engineered cell that comprises a heterologous polynucleotide encoding a nanobody V565 having a sequence that is at least 95% identical to SEQ ID NO:7.

[0034] 17. The engineered cell according to embodiment 16, wherein the polynucleotide encodes the V565 nanobody covalently linked to tumor necrosis factor α (TNFα) via a cleavable polypeptide linker.

[0035] 18. The engineered cell according to embodiment 17, wherein the cleavable polypeptide linker comprises SEQ ID NO:9.

[0036] 19. An engineered cell that comprises a heterologous polynucleotide encoding an antimicrobial peptide 16 (AMP16) having a sequence that is at least 95% identical to SEQ ID NO:8.

[0037] 20. The engineered cell according to embodiment 19, wherein the polynucleotide encodes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 repeat sequences of AMP16, each repeat sequence being separated by a linker polypeptide comprising an Asn-Pro-Val repeat sequence.

[0038] 21. A cultured meat product comprising a confluent serum-free culture comprising cells according to any one of embodiments 1-10 and 13-20 inoculated on a food safety matrix.

[0039] 22. The cultured meat product according to embodiment 21, wherein the matrix is a membrane.

[0040] 23. The cultured meat product according to embodiment 21, wherein the matrix is a sponge or a three-dimensional matrix.

[0041] 24. The cultured meat product according to any one of embodiments 21-23, wherein the matrix is a chitosan matrix.

[0042] 25. The cultured meat product according to any one of embodiments 21-24, wherein the cell culture comprises multinucleated myotubes derived from Pax7+ mammalian satellite cells.

[0043] 26. The cultured meat product according to embodiment 25, wherein the multinucleated myotubes express myosin heavy chain (MHC) and actin. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] This patent or patent application document contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0045] Figure 1 Show gene constructs and their corresponding end products in the carotenoid biosynthetic pathway. All gene constructs contain a puromycin resistance gene and a gene of interest co-initiated by a bidirectional synthetic RBPSA / CMV promoter. All gene of interest regions contain green fluorescent protein (GFP) sequences produced either individually or as part of a polycistronic mRNA transcript. The constructs are named (from top to bottom) pGFP, pCrtB, pCrtB / I, and pCrtB / I / Y.

[0046] Figures 2A-2B show bovine satellite cell characterization. (Figure 2A) Immunofluorescent staining (green) of Pax7, a transcription factor and satellite cell marker, and counterstaining of cellular actin (phalloidin, red) and nuclei (DAPI, blue). (Figure 2B) Immunofluorescent staining (green) of myosin heavy chain (MF20), a skeletal muscle contractile protein, and counterstaining of cellular actin and nuclei. High-objective staining of differentiated cells reveals a striated pattern in actin and myosin heavy chain, indicating skeletal muscle sarcomere formation. Scale bar is 100 μm.

[0047] Figures 3A - 3C show the engineering of cell growth and development. (Figure 3A) Fluorescent images of GFP expression in C2C12 and BSC engineered with all four constructs (carotenoid - producing constructs and control constructs). Due to the structure of the constructs, GFP expression implies the successful expression of other enzymes in the gene constructs. (Figure 3B) Growth curves of C2C12 and BSC engineered with all four constructs over eight days, n = 3 different samples, * indicates significant differences from pGFP of the corresponding color, p < 0.05. (Figure 3C) Immunofluorescent staining of Pax7 (red) and myosin heavy chain (red) in pCrtB / I / Y BSC before (top) and after (bottom) differentiation. DAPI counterstain (blue) shows nuclei, and endogenous GFP (green) shows cell morphology. Scale bar is 100 μm.

[0048] Figures 4A - 4B show the carotenoid production in engineered cells. Carotenoid production in engineered cells. (Figure 4A) Carotenoid production in C2C12 engineered with pCrtB, pCrtB / I, and pCrtB / I / Y. In pCrtB cells, only phytoene is produced. In pCrtB / I cells, most of the phytoene is converted to lycopene. In pCrtB / I / Y cells, the carotenoid accumulates as β - carotene, but the total carotenoid level is significantly reduced compared to pCrtB and pCrtB / I. n = 3 different samples; different letters indicate statistical differences between bars, p < 0.05. (Figure 4B) Carotenoid production in BSC engineered with pCrtB, pCrtB / I, and pCrtB / I / Y. The trend follows that of C2C12, where the total carotenoid content is reduced by approximately 10 - fold compared to C2C12 in all cases. n = 3 different samples; different letters indicate statistical differences between bars, p < 0.05. Carotenoid data are provided as μg / g protein. Individual values and data of carotenoids compared to protein are given in Figures 4C - 4D.

[0049] Figures 4C - 4D show the carotenoid and protein quantification data. Individual carotenoid and protein data points are used to generate μg / g data. (Figure 4C) C2C12 carotenoid and protein quantification per million cells for all cell types and treatments. The trend is the same as in Figures 4 and 5. (Figure 4D) BSC carotenoid and protein quantification per million cells for all cell types and treatments. The trend is the same as in Figures 4A - 4B and 5A - 5C. For all, n = 3 different samples. Within the figure, different letters indicate statistical differences between bars, p < 0.05).

[0050] Figures 5A - 5C show carotenoid optimization. (Figure 5A) The carotenoid biosynthesis pathway in the context of its shared pathway as a precursor in cholesterol synthesis. Possible mechanisms of optimization are shown. Puromycin (purple) acts by selecting cells that provide higher enzyme expression and thus increase carotenoid synthesis flux. Ketoconazole (re(D) acts to increase the level of the carotenoid precursor farnesyl pyrophosphate (FPP) by inhibiting the conversion of lanosterol to ergosterol, by causing upstream accumulation of lanosterol, squalene, and FPP, or by reducing the effect of cholesterol as a feedback inhibitor of the upstream FPP synthesis reaction. (Figure 5B) Images of engineered BSC cell clusters. The coloring effect of carotenoids can be seen, where pGFP cells are slightly green, pCrtB cells are pigment - free, and pCrtB / I and pCrtB / Y cells contain some orange pigments. The optimized pCrtB / I / Y cells are significantly more orange, indicating an increase in carotenoid levels. (Figure 5C) Quantitative HPLC of β - carotene in optimized cells. In BSC, ketoconazole and puromycin treatment conditions increased the accumulation of β - carotene. In C2C12, ketoconazole and puromycin together did not significantly increase β - carotene. n = 3 different samples; *p < 0.05. Carotenoid data are provided as μg / g protein. Individual values and data of carotenoids compared to protein are given in Figures 4C - 4D.

[0051] Figure 6 Thiobarbituric acid - reactive substance (TBARS) analysis of engineered BSC with or without heating is shown. All heated samples showed a significant increase in malondialdehyde (MDA) compared to the raw samples, indicating significant oxidative stress from this "cooking" process. Among the heated samples, pCrtB, pCrtB / I, and pCrtB / I / Y cells showed a significant decrease in malondialdehyde (MDA) compared to the control group. Among the unheated samples, pCrtB / I and pCrtB / I / Y samples showed a significant decrease in MDA compared to the control pGFP cells, and pCrtB did not show a significant change. n = 3 different samples. Bars sharing the same letter are statistically different (p < 0.05).

[0052] Figures 7A-7B show carotenoid optimization. (Figure 7A) Flow cytometry measurements of mean fluorescence intensity in C2C12 and BSC engineered with pCrtB / I / Y (complete flow cytometry data in Figures 8A-8B). Cells were cultured with 2.5 μg / mL puromycin (1×), 5 μg / mL puromycin (2×), or 10 μg / mL puromycin (4×). Higher fluorescence intensity indicates higher mean GFP expression, which corresponds to higher mean enzyme expression. Error is given as mean ± standard deviation. For each condition, n > 1,500 events. (Figure 7B) 8-day cell growth of pCrtB / I / Y C2C12 and BSC cultured in 0, 2.5, 5, 10, or 20 μg / mL ketoconazole. n = 3 different samples. In a and b, * indicates p < 0.05 for both.

[0053] Figures 8A-8B show flow cytometry data. (Figure 8A) BSC data of cells treated with different levels of puromycin. From left to right: non-engineered cells (GFP negative control), pGFP (GFP positive control), pCrtB / I / Y with 2.5 μg / mL puromycin (1× puro), pCrtB / I / Y with 5 μg / mL puromycin (2× puro), and pCrtB / I / Y with 10 μg / mL puromycin (4×). The top row is forward scatter (FSC) and side scatter (SSC) data, showing gating for live cells (determined using the non-engineered control), where the gate value is the percentage relative to the total count (7,500). The bottom row is GFP data, showing gating for GFP-positive cells (determined using the non-engineered control), where the gate value is the percentage relative to live cells (gated in the top row). (Figure 8B) C2C12 data of cells treated with different levels of puromycin. From left to right: non-engineered cells (GFP negative control), pGFP (GFP positive control), pCrtB / I / Y with 2.5 μg / mL puromycin (1× puro), pCrtB / I / Y with 5 μg / mL puromycin (2× puro), and pCrtB / I / Y with 10 μg / mL puromycin (4×). The top row is FSC and SSC data, showing gating for live cells (determined using the non-engineered control), where the gate value is the percentage relative to the total count (7,500). The bottom row is GFP data, showing gating for GFP-positive cells (determined using the non-engineered control), where the gate value is the percentage relative to live cells (gated in the top row).

[0054] Figures 9A - 9B show the carotenoid profiles of optimized cells. (Figure 9A) Lycopene, lycopene, and β - carotene contents of pCrtB / I / Y C2C12 without optimization (top) or with puromycin treatment and ketoconazole treatment (bottom). n = 3 different samples. (Figure 9B) Lycopene, lycopene, and β - carotene contents of pCrtB / I / Y BSC without optimization (upper left), without puromycin treatment but with ketoconazole treatment (upper right), with puromycin treatment but without ketoconazole treatment (lower left), or with puromycin treatment and ketoconazole treatment (lower left). n = 3 different samples. Within the figure, different letters indicate statistical differences between bars, p < 0.05).

[0055] Figure 10 Shows the synthesis pathways of vitamin C, curcumin, and cannabidiol from precursors in BSC.

[0056] Figure 11 Shows an overview of Example 2. In 1, cell composition will be analyzed before and after differentiation. In 2, cells will be engineered to synthesize various compounds, which will be analyzed before and after differentiation. In 3, cell growth and development, nutrient bioavailability, and nutrient function will be analyzed.

[0057] Figure 12 Shows an overview of Example 3. A drug expression vector (A) and an excipient (B) are engineered into bovine muscle and fat cells to explore in vitro drug synthesis. Co - expression of green fluorescent protein (GFP) with the therapeutic component serves as a simple marker of gene expression. The drug expression vectors (V565 and AMP16, each containing a GFP marker) are further engineered to incorporate various disease - responsive mechanisms for drug activity, including a lysine - rich linker for cleavable trypsin, an NPV - linker for cleavable elastase, or a combination of both. Disease - responsive edible therapeutics in bovine muscle cells (proliferating or differentiating) are tested in 3D models of IBD and Salmonella enterica infection, followed by in vitro digestion, with and without heat treatment (i.e., cooking).

[0058] Figures 13A - 13E show the results of bovine satellite cell (BSC) culture. (Figure 13A) Characterization of primary BSCs. Proliferating cells show widespread expression of Pax7, a muscle stem cell marker (scale bar: 100 μm). (Figure 13B) Differentiating cells show myotube formation and expression of myosin heavy chain (MF20), indicating myogenic differentiation (scale bar is 100 μm). (Figure 13C) Four-day growth of BSCs cultured in dilutions of serum-containing growth medium (GM) and serum-free medium (B8). In all cases, including B8 alone (i.e., serum-free, rightmost bar), dilutions containing B8 show significantly improved four-day growth compared to GM alone (leftmost bar). Different letters indicate statistical significance between groups (p < 0.05). (Figure 13D) Adipogenic transdifferentiation of BSCs with free fatty acids (FFAs). Oil Red O staining of BSCs treated with 0 to 150 μM linoleic acid and combinations of linoleic acid, erucic acid, and elaidic acid. Sparse lipid accumulation was observed in BSCs treated with 100 μM to 150 μM linoleic acid. Robust lipid accumulation was observed in BSCs treated with a combination of 75 μM to 125 μM FFAs. At 150 μM, the FFA mixture was detrimental to cell health (scale bar: 250 μm). (Figure 13E) Results of AdipoRed analysis of quantitative analysis of FFA-induced lipid accumulation in BSCs.

[0059] Figures 14A - 14D show the BSC compositional engineering. (Figure 14A) Carotenoid production in BSCs engineered with pCrtB, pCrtB / I, and pCrtB / I / Y. In pCrtB cells, only phytoene is produced. In pCrtB / I cells, most of the phytoene is converted to lycopene. In pCrtB / I / Y cells, the carotenoid accumulates as β - carotene, but the total carotenoid level is significantly reduced compared to pCrtB and pCrtB / I. Different letters indicate statistical differences between bars, p < 0.05. (Figure 14B) In BSCs, ketoconazole and puromycin treatments increase the accumulation of β - carotene by increasing the enzyme supply. * indicates significance (p < 0.05). (Figure 14C) Thiobarbituric acid - reactive substance (TBARS) analysis of engineered BSCs with or without heating. All heated samples showed a significant increase in malondialdehyde (MDA) compared to the raw samples, indicating a significant oxidative challenge from this "cooking" process. Among the heated samples, pCrtB, pCrtB / I, and pCrtB / I / Y cells showed a significant decrease in MDA compared to the control group. Among the unheated samples, pCrtB / I and pCrtB / I / Y samples showed a significant decrease in MDA compared to the control pGFP cells, and pCrtB showed no significant change. Bars sharing the same letter are statistically different (p < 0.05). (Figure 14D) Fluorescence and optical microscopy images of BSCs engineered to express GFP alone (pGFP) or GFP and CBD synthase (pCBD). Green fluorescence indicates gene expression. After puromycin selection, all cells expressed GFP and the enzyme (data not shown) (scale bar: 250 μm).

[0060] Figures 15A - 15H show the 3D intestinal model. Figures 15A - 15D show the intestinal scaffolds 57 . (Figures 15A - 15B) Schematic diagram of the production of silk - based 3D porous scaffolds for constructing patterned and non - patterned model intestines. The scaffolds were prepared by casting a silk solution into a PDMS mold and inserting Teflon - coated wires or nylon screws through the cylinder, followed by freeze - drying and induction of β - sheet formation (scale bar: 4 mm). (Figure 15C) Fluorescent staining of intestinal cell markers (ZO - 1 and MUC2) in the scaffolds and 2D cultures showed increased expression in the 3D scaffolds (scale bar: 200 μm). Optical microscopy observations of toluidine blue staining of mucus deposition in the scaffolds and 2D cultures showed an increase in mucus thickness in the 3D scaffolds compared to 2D, and an increase in mucus thickness in the patterned scaffolds compared to the non - patterned scaffolds (scale bar: 200 μm). Quantification (data not shown) revealed that mucus deposition in the non - patterned and patterned scaffolds increased 2 - fold and 3 - fold, respectively, compared to 2D cultures. Figures 15E - 15H show IBD modeling 55。Figure 15E was induced to inflame by treatment with Escherichia coli (E. coli) O111:B4 LPS and IFNγ. Staining of E-cadherin in the inflammation model showed the difference in epithelial coverage between the inflamed group and the non-inflamed group on day 7 (scale bar: 100 μm). F) MUC2 staining showed positive staining in all groups indicating the presence of goblet cells (scale bar: 100 μm). Figure 15G Cross-section of the model on day 7 showed different epithelial coverages as observed by E-cadherin (scale bar 1 mm). Figure 15H Further observation of the epithelium showed a monolayer in the non-inflamed group and spherical structures in the inflamed group (scale bar: 100 μm).

[0061] Figures 15I - 15L show the infection of a 3D intestinal model with Cryptosporidium parvum 56 。Uninfected cells in Figure 15I showed regular microvilli (scale bar: 2 μm). Cryptosporidium parvum type I meronts (thick arrows) and empty parasitophorous vacuoles (thin arrows) on day 3 in Figure 15J (scale bar: 2 μm). Type I meronts containing eight merozoites on day 3 (magnified view of J; scale bar: 1 μm)) in Figure 15K. Excysting type I meronts on day 2 in Figure 15L (scale bar: 1 μm). The schema was adapted from the following references: A - D 57 , E - H 55 , I - L 56 。

[0062] Figures 16A - 16B show the results of bovine satellite cell (BSC) culture. (Figure 16A) Characterization of primary BSCs. Proliferating cells (left panel) showed widespread expression of Pax7, a muscle stem cell marker. Differentiating cells (right panel) showed myotube formation and expression of myosin heavy chain (MF20), indicating myogenic differentiation. (Figure 16B) BSCs cultured in dilutions of serum-containing growth medium (GM) and serum-free medium (B8) (left panel). In all cases, including B8 alone (i.e., serum-free, rightmost column), dilutions containing B8 showed significantly increased growth over four days compared to GM alone (leftmost column). Dilutions of FGF-2 in B8 medium (right panel) revealed that FGF-2 concentrations greater than or equal to 1.25 ng / mL showed significantly increased growth over four days compared to B8 medium without FGF-2. In the left panel, different letters indicate statistical significance between groups (p < 0.05). In the right panel, * indicates a significant difference (p < 0.05).

[0063] Figures 17A - 17B show the adipogenic trans - differentiation of bovine satellite cells (BSC) with free fatty acids (FFA). (Figure 17A) Oil Red O staining of BSC treated with 0 to 150 μM linoleic acid and combinations of linoleic acid, erucic acid, and elaidic acid. Sparse lipid accumulation was observed in BSC treated with 100 μM to 150 μM linoleic acid. Robust lipid accumulation was observed in BSC treated with a combination of 75 μM to 125 μM FFA. At 150 μM, the FFA mixture was detrimental to cell health. (Figure 17B) Results of AdipoRed analysis for quantitative analysis of FFA - induced lipid accumulation in BSC.

[0064] Figure 18 Shows an overview of Example 4. Formulated media will be explored for various different methods of serum - free and antibiotic - free culture of bovine muscle and adipose tissues. Cell proliferation and differentiation will be analyzed, and nutritional and meat quality characteristics will also be analyzed.

[0065] Incorporated by Reference

[0066] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. Detailed Description

[0067] This disclosure describes engineered cells and their use in cultured meat products. Cells can be engineered to endogenously produce one or more metabolites by expressing one or more exogenous biosynthetic pathway enzymes in the cells. Cells can also be engineered to increase or decrease the endogenous production of one or more metabolites by increasing or decreasing biosynthetic pathway enzymes that the cells naturally have, or by introducing non - native biosynthetic pathway enzymes into the cells to increase the endogenous production of natural metabolites.

[0068] As used herein, "metabolite" refers to a compound that is an intermediate or end - product of primary or secondary metabolism in a cell through one or more biosynthetic pathways. Cellular metabolites have various functions in terms of energy supply, structure, signaling, stimulation, and inhibition of proteins and enzymes, enzyme cofactors or enzyme cosubstrates, defense, and interactions with other organisms or cells (e.g., pigments, odorants, pheromones, quorum sensing, etc.). Metabolites can include, but are not limited to, alcohols, amino acids, nucleotides, antioxidant factors, organic acids, polyols, and vitamins. The cells described herein can be engineered to endogenously produce one or more metabolites, including but not limited to phytochemicals, phyto - nutrients, vitamins, and therapeutic molecules.

[0069] As used herein, “phytochemical” refers to bioactive compounds found in and produced by plants that typically play a role in plant growth or defense against competitors, pathogens, or predators. Phytochemical biosynthetic pathways can be primary or secondary metabolic pathways. Although phytochemicals can be extracted and isolated from source plants, the biosynthetic pathway genes that produce phytochemicals can also be genetically engineered into bacteria, fungi, or other eukaryotic cells, or phytochemicals can be produced synthetically.

[0070] “Phytonutrient” refers to a phytochemical that is an essential nutrient in the mammalian diet or has specific bioactivity that supports mammalian health / wellness. Phytonutrients can include, but are not limited to, polyphenols, terpenoids, resveratrol, flavonoids, isoflavonoids, carotenoids, limonoids, glucosinolates, phytoestrogens, phytosterols, anthocyanins, omega-3 fatty acids, and probiotics. Phytonutrients have specific pharmacological actions such as antimicrobial, antioxidant, anti-inflammatory, non-tolerogenic, antispasmodic, anticancer, anti-aging, hepatoprotective, hypolipidemic, neuroprotective, antihypertensive, CNS-stimulating, analgesic, UVB-induced carcinogenesis-protective, immunomodulatory, and carminative properties. Phytonutrients and their functions in mammalian and human health / wellness are described in the art. See, e.g., Gupta et al. (“Phytonutrients as therapeutic agents,” J. Complement Integr. Med., 2014, 11(3):151-169), which is incorporated herein by reference.

[0071] Generally, one or more heterologous and / or exogenous polynucleotides encoding one or more biosynthetic pathway enzymes necessary to produce one or more metabolites of interest are introduced into mammalian cells to produce genetically engineered cells that endogenously produce one or more metabolites of interest. The cells can be engineered to transiently express the desired biosynthetic pathway enzymes of interest, or the cells can be engineered to stably express the desired biosynthetic pathway enzymes of interest. Any transfection or transduction method known in the art can be used to introduce the heterologous polynucleotide encoding the biosynthetic pathway enzyme into the cells.

[0072] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to the primary, secondary, tertiary, and quaternary amino acid sequences and structures common to the macromolecules described, which are necessary to confer upon them their functions and properties. As used herein, the terms "enzyme" or "biosynthetic pathway enzyme" are used interchangeably and refer to a protein that catalyzes a chemical reaction. A reference to any particular enzyme, whether independent or as part of a biosynthetic pathway, is to be understood as including the enzyme cofactors, coenzymes, and metals necessary for the normal operation of the enzyme.

[0073] As used herein, the terms "polynucleotide," "polynucleotide sequence," "nucleic acid," and "nucleic acid sequence" refer to nucleotides, oligonucleotides, polynucleotides (the terms being used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or antisense strand. The polynucleotide may be cDNA or genomic DNA.

[0074] Also provided are polynucleotides that are homologous to the polynucleotides described herein. Those skilled in the art will understand the degeneracy of the genetic code and that multiple polynucleotides can encode the same polypeptide. In some embodiments, the polynucleotide (i.e., the polynucleotide encoding a biosynthetic pathway enzyme for synthesizing a metabolite of interest) may be codon-optimized for expression in a particular cell, including but not limited to mammalian cells, plant cells, bacterial cells, or fungal cells. In some embodiments, the polynucleotide is codon-optimized for genus- or species-specific expression (e.g., expression in bovine cells). Although examples of specific polynucleotide sequences are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptides described herein may be used. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in a heterologous expression system for a polypeptide or protein. Computer programs may be used to generate degenerate coding sequences and are used for this purpose. Pen, paper, the genetic code, and manual labor may also be used to generate degenerate coding sequences.

[0075] In another aspect of the invention, constructs are provided. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent the antisense or sense strand. A recombinant polynucleotide is a polynucleotide formed by laboratory methods that includes polynucleotide sequences from at least two different natural sources or which may be synthetic. Thus, a construct may include new modifications to an endogenous gene introduced, for example, by genome editing techniques. A construct may also include a recombinant polynucleotide produced using, for example, recombinant DNA methods.

[0076] The constructs provided herein can be prepared by methods available to those skilled in the art. It should be noted that each of the constructs described herein is a recombinant molecule and thus does not exist in nature. Generally, the nomenclature used herein and the laboratory procedures used in this invention include molecular techniques, biochemical techniques, and recombinant DNA techniques that are well known and commonly used in the art. Standard techniques available to those skilled in the art can be used for cloning, DNA and RNA isolation, amplification, and purification. These techniques are well explained in the literature.

[0077] The constructs provided herein can comprise a promoter operably connected to any of the polynucleotides described herein. The promoter can be a heterologous promoter or an endogenous promoter associated with the biosynthetic pathway described herein.

[0078] As used herein, the terms "heterologous promoter", "promoter", "promoter region", or "promoter sequence" generally refer to the transcriptional regulatory region of a gene that is found 5' or 3' to the polynucleotide described herein, or within the coding region of the polynucleotide, or within an intron of the polynucleotide. Generally, a promoter is a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A typical 5' promoter sequence is bounded at its 3' end by a transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary for transcription initiation at a detectable level above background. Within the promoter sequence are a transcription start site (conveniently defined by S1 nuclease mapping) and a protein-binding domain (consensus sequence) responsible for RNA polymerase binding.

[0079] In some embodiments, the polynucleotide encoding the enzyme of the biosynthetic pathway described herein is operably connected to a promoter. As used herein, a polynucleotide is "operably connected / operably linked" when it is in a functional relationship with a second polynucleotide sequence. For example, a promoter is operably connected to a polynucleotide if the promoter is linked to the polynucleotide such that it can affect the transcription of the polynucleotide. In various embodiments, the polynucleotide can be operably connected to at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 promoters.

[0080] Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally-regulated, chemically-regulated, tissue-preferred, and tissue-specific promoters. The heterologous promoter can be a plant, animal, bacterial, fungal, or synthetic promoter. Promoters suitable for expression in plants include, but are not limited to, the 35S promoter of cauliflower mosaic virus, ubiquitin, the tCUP cryptic constitutive promoter, the Rsyn7 promoter, pathogen-inducible promoters, the maize In2-2 promoter, the tobacco PR-1a promoter, glucocorticoid-inducible promoters, estrogen-inducible promoters, and tetracycline-inducible and tetracycline-repressible promoters. Other promoters include the T3, T7, and SP6 promoter sequences, which are commonly used for in vitro transcription of RNA. In mammalian cells, typical promoters include, but are not limited to, the following promoters: Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), Dox-inducible promoters (e.g., Tet response element (TRE)), ubiquitin C (Ubc), CMV early enhancer / chicken β-actin (CAG), human β-actin, phosphoglycerate kinase 1 (PGK1), SV40 virus, etc.; and the translation elongation factor EF-1α promoter or ubiquitin promoter. In insect cells, typical promoters include, but are not limited to, the upstream activating sequence (UAS), actin 5c (Ac5), and polyhedrin. In fish cells, typical promoters include, but are not limited to, the Xenopus laevis elongation factor 1α promoter (XlEef1a1) and the oceanpout antifreeze protein promoter (OP5a). Those skilled in the art are familiar with a wide variety of additional promoters for different cell types. In some embodiments, the heterologous promoter includes a plant promoter that is endogenous or heterologous to the plant host.

[0081] Provide a vector comprising any one of the constructs or polynucleotides described herein. The term "vector" is intended to refer to a polynucleotide capable of transporting another polynucleotide to which it has been ligated. In some embodiments, the vector can be a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors can integrate into the host cell genome after being introduced into the host cell and thus replicate with the host genome, such as some viral vectors or transposons. By way of example, suitable vectors for the compositions and methods of the present disclosure include episomal vectors, viral vectors (e.g., retroviruses, adenoviruses, baculoviruses), plasmids, RNA vectors, or linear or circular DNA or RNA molecules that can comprise or consist of the following: chromosomal, extrachromosomal, semisynthetic, or synthetic nucleic acids. Many suitable vectors are known to those of skill in the art and are commercially available. In some embodiments, the vector is an episomal vector capable of autonomous replication due to the presence of an origin of replication. Also included are plant minichromosomes as vectors. In some embodiments, the vector is a polycistronic vector that contains one or more internal ribosome entry sites (IRESs) and / or one or more 2A peptide sequences, thereby allowing co-expression of multiple polynucleotides from a single construct or vector. The vector can carry genetic elements, such as genetic elements that confer resistance to certain drugs or chemicals.

[0082] In some embodiments, the constructs or vectors described herein can comprise selectable markers. As used herein, a "selectable marker" is a genetic element that allows for the identification and selection of cells containing a construct or vector having the genetic element by expressing the genetic element in the cells. In some embodiments, the selectable marker is a polynucleotide encoding a fluorescent protein such that cells expressing the fluorescent protein can be visually identified or by suitable cell sorting methods (e.g., fluorescence-activated cell sorting (FACS)). In some embodiments, the selectable marker is a polynucleotide that confers antibiotic resistance to cells expressing the selectable marker (e.g., puromycin, penicillin, streptomycin, or hygromycin resistance genes). Cells transduced with a vector having a selectable marker can be exposed to a selection chemical specific for the selectable marker to select cells containing the vector. The selectable marker confers resistance to the selection chemical such that cells containing the vector and the selectable marker survive while cells not containing the vector and the selectable marker are killed.

[0083] As used herein, "introduction" describes the process of introducing an exogenous polynucleotide (e.g., DNA or RNA) into a recipient cell. Methods for introducing polynucleotides into cells are known in the art and can include, but are not limited to, microinjection, transformation, and transfection methods. Transformation or transfection can be carried out under natural or artificial conditions according to various methods well known in the art and can rely on any known method for inserting foreign nucleic acid sequences into a host cell. The method for transformation or transfection is selected based on the type of host cell being transformed and can include, but are not limited to

[0084] In some embodiments, the exogenous polynucleotides, constructs, or vectors described herein are transfected into cells using a suitable carrier. Suitable carriers are known and used in the art and include, but are not limited to, lipid carriers (e.g., Lipofetamine), polymeric nanocarriers,

[0085] A cell is "genome edited" or "genetically modified" if it contains a modification to its genome compared to an unedited cell of the same type. In some cases, the unedited cell is a wild-type cell. As used herein, the terms "genetically modified" and "genetically engineered" are used interchangeably and refer to prokaryotic or eukaryotic cells containing exogenous polynucleotides, regardless of the method used for insertion. In some cases, the cell has been modified to contain a non-naturally occurring nucleic acid molecule generated or modified by artificial means (e.g., using recombinant DNA technology) or derived from such a molecule (e.g., by transcription, translation, etc.). Cells containing exogenous, recombinant, synthetic, and / or otherwise modified polynucleotides are considered engineered or "genome edited" cells. Genome editing or modifying a cell refers to modifying the cellular nucleic acids within the cell, including endogenous and / or exogenous nucleic acids within a genetically modified cell. Genetic modification can include deletions, insertions, integration of foreign DNA, gene correction, and / or gene mutation. For example, genome editing can be carried out using nucleases (e.g., naturally occurring nucleases or artificially engineered nucleases) or transposases. Other methods for genetic modification suitable for use according to the methods provided herein include, but are not limited to, somatic cell nuclear transfer (SCNT) and transgene introduction. Procedures for obtaining recombinant or genetically modified cells are generally known in the art and are described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference.

[0086] In some embodiments, CRISPR / Cas-mediated gene editing (e.g., type II CRISPR / Cas systems) is used to integrate a polynucleotide encoding a biosynthetic pathway enzyme required to produce a metabolite of interest into the genome of a cell. In some cases, a CRISPR / Cas system is used to reduce the protein expression of one or more endogenous genes. In some embodiments, the CRISPR / Cas system can be used to perform site-specific insertion. For example, a nick at the insertion site in the genome can be made by CRISPR / Cas to facilitate the insertion of a transgene at the insertion site. Techniques for CRISPR / Cas-mediated gene editing suitable for host cells are known and as described in the art.

[0087] In some embodiments, a transposon is used to integrate a polynucleotide encoding a biosynthetic pathway enzyme required to produce a metabolite of interest into the genome of a cell. Suitable transposons are known and as described in the art, including but not limited to Sleeping Beauty, PiggyBac, Tol1, Tol2, Minos, Frog Prince, Passport, Hsmar1, and Harbinger3_DR. See, e.g., Ivics et al. (“The expanding universe of transposon technologies for gene and cell engineering,” Mobile DNA, 1(25), 2010), which describes transposon systems and uses in the art.

[0088] In some embodiments, a transgene is used to integrate a polynucleotide encoding a biosynthetic pathway enzyme required to produce a metabolite of interest into the genome of a cell. As used herein, the term “transgene” refers to a gene or genetic material that can be transferred into an organism or its cells.

[0089] In some embodiments, cells are engineered to endogenously produce the carotenoid phytoene. Phytoene is synthesized from two geranylgeranyl diphosphate molecules by the enzyme phytoene synthase. A polynucleotide encoding phytoene synthase is introduced into cells that naturally produce geranylgeranyl diphosphate to generate cells that express phytoene synthase and endogenously synthesize phytoene. In some embodiments, the phytoene synthase is from the bacterium Pantoea ananatis and has the polypeptide sequence of SEQ ID NO:1 or SEQ ID NO:10. In some embodiments, the phytoene synthase has a sequence that is at least 85%, 90%, 95%, 98%, 99% or 99.9% identical to SEQ ID NO:1 or SEQ ID NO:10. In Pantoea ananatis, the phytoene synthase is encoded by the gene crtB having the sequence of SEQ ID NO:2. In some embodiments, the crtB sequence can be codon-optimized for expression in a particular cell of interest. In some embodiments, the crtB sequence is codon-optimized for expression in bovine cells (e.g., SEQ ID NO:38).

[0090] SEQ ID NO:1 Phytoene synthase (Uniprot P21683):

[0091] MNNPSLLNHAVETMAVGSKSFATASKLFDAKTRRSVLMLYAWCRHCDDVIDDQTLGF

[0092] QARQPALQTPEQRLMQLEMKTRQAYAGSQMHEPAFAAFQEVAMAHDIAPAYAFDHLE

[0093] GFAMDVREAQYSQLDDTLRYCYHVAGVVGLMMAQIMGVRDKATLDRACDLGLAFQL

[0094] TNIARDIVDDAHAGRCYLPASWLEHEGLNKENYAAPENRQALSRIARRLVQEAEPYYLS

[0095] ATAGLAGLPLRSAWAIATAKQVYRKIGVKVEQAGQQAWDQRQSTTTPEKLTLLLAASG

[0096] QALTSRMRAHPPRPAHLWQRPL

[0097] SEQ ID NO:10 Phytoene synthase:

[0098] MVNNPSLLNHAVETMAVGSKSFATASKLFDAKTRRSVLMLYAWCRHCDDVIDDQTLG

[0099] FQARQPALQTPEQRLMQLEMKTRQAYAGSQMHEPAFAAFQEVAMAHDIAPAYAFDHL

[0100] EGFAMDVREAQYSQLDDTLRYCYHVAGVVGLMMAQIMGVRDNATLDRACDLGLAFQ

[0101] LTNIARDIVDDAHAGRCYLPASWLEHEGLNKENYAAPENRQALSRIARRLVQEAEPYYL

[0102] SATAGLAGLPLRSAWAIATAKQVYRKIGVKVEQAGQQAWDQRQSTTTPEKLTLLLAAS

[0103] GQALTSRMRAHPPRPAHLWQRPL

[0104] SEQ ID NO:2crtB:

[0105] ATGAATAATCCGTCGTTACTCAATCATGCGGTCGAAACGATGGCAGTTGGCTCGAAA

[0106] AGTTTTGCGACAGCCTCAAAGTTATTTGATGCAAAAACCCGGCGCAGCGTACTGATG

[0107] CTCTACGCCTGGTGCCGCCATTGTGACGATGTTATTGACGATCAGACGCTGGGCTTTC

[0108] AGGCCCGGCAGCCTGCCTTACAAACGCCCGAACAACGTCTGATGCAACTTGAGATGA

[0109] AAACGCGCCAGGCCTATGCAGGATCGCAGATGCACGAACCGGCGTTTGCGGCTTTTC

[0110] AGGAAGTGGCTATGGCTCATGATATCGCCCCGGCTTACGCGTTTGATCATCTGGAAG

[0111] GCTTCGCCATGGATGTACGCGAAGCGCAATACAGCCAACTGGATGATACGCTGCGCT

[0112] ATTGCTATCACGTTGCAGGCGTTGTCGGCTTGATGATGGCGCAAATCATGGGCGTGC

[0113] GGGATAACGCCACGCTGGACCGCGCCTGTGACCTTGGGCTGGCATTTCAGTTGACCA

[0114] ATATTGCTCGCGATATTGTGGACGATGCGCATGCGGGCCGCTGTTATCTGCCGGCAA

[0115] GCTGGCTGGAGCATGAAGGTCTGAACAAAGAGAATTATGCGGCACCTGAAAACCGT

[0116] CAGGCGCTGAGCCGTATCGCCCGTCGTTTGGTGCAGGAAGCAGAACCTTACTATTTG

[0117] TCTGCCACAGCCGGCCTGGCAGGGTTGCCCCTGCGTTCCGCCTGGGCAATCGCTACG

[0118] GCGAAGCAGGTTTACCGGAAAATAGGTGTCAAAGTTGAACAGGCCGGTCAGCAAGC

[0119] CTGGGATCAGCGGCAGTCAACGACCACGCCCGAAAAATTAACGCTGCTGCTGGCCG

[0120] CCTCTGGTCAGGCCCTTACTTCCCGGATGCGGGCTCATCCTCCCCGCCCTGCGCATCT

[0121] CTGGCAGCGCCCGCTCTAG

[0122] In some embodiments, the intracellular endogenous production amount of phytoene is from about 1 μg / g protein to about 800 μg / g protein, about 5 μg / g protein to about 600 μg / g protein, about 10 μg / g protein to about 500 μg / g protein, about 25 μg / g protein to about 300 μg / g protein, about 30 μg / g protein to about 250 μg / g protein, or about 30 μg / g protein to 100 μg / g protein. In some embodiments, compared to a cell that does not contain a polynucleotide encoding phytoene synthase, the cell endogenously produces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold more phytoene.

[0123] In some embodiments, the cell is engineered to endogenously produce the carotenoid lycopene. Lycopene is synthesized from phytoene by phytoene desaturase. A polynucleotide encoding phytoene desaturase is introduced into the cell producing phytoene to produce a cell that expresses phytoene desaturase and endogenously synthesizes lycopene. In some embodiments, the phytoene desaturase is from Pantoea ananatis and has the polypeptide sequence of SEQ ID NO:3. In some embodiments, the phytoene desaturase has a sequence that is at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NO:3. In Pantoea ananatis, the phytoene desaturase is encoded by the gene crtI having the sequence of SEQ ID NO:4. In some embodiments, the crtI sequence can be codon-optimized for expression in a particular cell of interest. In some embodiments, the crtI sequence is codon-optimized for expression in bovine cells (e.g., SEQ ID NO:39).

[0124] SEQ ID NO:3 Phytoene desaturase (Uniprot P21685):

[0125]

[0126] SEQ ID NO:4 crtI:

[0127]

[0128] In some embodiments, the intracellular endogenous production amount is about 1 μg / g protein to about 800 μg / g protein, about 5 μg / g protein to about 600 μg / g protein, about 10 μg / g protein to about 500 μg / g protein, about 25 μg / g protein to about 300 μg / g protein, about 30 μg / g protein to about 250 μg / g protein, or about 30 μg / g protein to 100 μg / g protein of lycopene. In some embodiments, compared to a cell that does not contain polynucleotides encoding phytoene synthase and phytoene desaturase, the cell endogenously produces at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold more lycopene.

[0129] In some embodiments, the cell is engineered to endogenously produce the carotenoid β-carotene. β-carotene is synthesized from lycopene by the enzyme lycopene cyclase. A polynucleotide encoding lycopene cyclase is introduced into the cell that produces lycopene to produce a cell that expresses lycopene cyclase and endogenously synthesizes β-carotene. In some embodiments, the lycopene cyclase is from the bacterium Pantoea ananatis and has the polypeptide sequence of SEQ ID NO:5 or SEQ ID NO:11. In some embodiments, the lycopene cyclase has a sequence that is at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NO:5 or SEQ ID NO:11. In Pantoea ananatis, the lycopene cyclase is encoded by the gene crtY having the sequence of SEQ ID NO:6. In some embodiments, the crtY sequence can be codon-optimized for expression in a particular cell of interest. In some embodiments, the crtY sequence is codon-optimized for expression in bovine cells (e.g., SEQ ID NO:40). Although the sequences listed below are from Pantoea ananatis, those skilled in the art will recognize that homologous sequences can be identified from other species for use in the engineered cells and methods described herein.

[0130] SEQ ID NO:5 Lycopene cyclase (Uniprot P21687):

[0131]

[0132] SEQ ID NO:11 Lycopene cyclase:

[0133]

[0134] SEQ ID NO:6 ctrY:

[0135]

[0136] In some embodiments, the intracellular endogenous production amount is about 1 μg / g protein to about 800 μg / g protein, about 5 μg / g protein to about 600 μg / g protein, about 10 μg / g protein to about 500 μg / g protein, about 25 μg / g protein to about 300 μg / g protein, about 30 μg / g protein to about 250 μg / g protein, or about 30 μg / g protein to 100 μg / g protein of β-carotene. In some embodiments, compared to cells that do not contain polynucleotides encoding phytoene synthase, phytoene desaturase, and lycopene cyclase, the cells endogenously produce at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold more β-carotene.

[0137] In some embodiments, the total carotenoid production in the engineered cells is about 1 μg / g protein to about 800 μg / g protein, about 5 μg / g protein to about 600 μg / g protein, about 10 μg / g protein to about 500 μg / g protein, about 25 μg / g protein to about 300 μg / g protein, about 30 μg / g protein to about 250 μg / g protein, or about 30 μg / g protein to 100 μg / g protein. In some embodiments, the total carotenoid production in the engineered cells is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold more than the total carotenoid production in cells that do not contain polynucleotides encoding phytoene synthase, phytoene desaturase, and lycopene cyclase.

[0138] In some embodiments, the cells are engineered to endogenously produce vitamin C. Although mammalian cells such as naturally produce the vitamin C precursor UDP-glucose and have genes encoding enzymes of the vitamin C biosynthetic pathway, the vitamin C biosynthetic pathway is generally silent in mammalian cells. A schematic diagram of vitamin C biosynthesis, including intermediates, is shown in Figure 10Among them, the enzymes in the vitamin C biosynthesis pathway include UDP-glucose 6-dehydrogenase (UGDH), UDP-glucuronosyltransferase 1 (UGT1A1), aldo-keto reductase family 1 (AKR1A1), calmodulin (RGN), and L-gulono-γ-lactone oxidase (GULO). In some embodiments, a heterologous polynucleotide encoding UGT1A1 is introduced into a cell. In some embodiments, a heterologous polynucleotide encoding UGDH is introduced into a cell. In some embodiments, a heterologous polynucleotide encoding AKR1A1 is introduced into a cell. In some embodiments, a heterologous polynucleotide encoding RGN is introduced into a cell. In some embodiments, a heterologous polynucleotide encoding GULO is introduced into a cell. In some embodiments, one or more heterologous polynucleotides encoding one, two, three, four, or all five of UGT1A1, UGDH, AKR1A1, RGN, and GULO are introduced into a cell. In some embodiments, the amino acid sequences of UGT1A1, UGDH, AKR1A1, RGN, and GULO are endogenous to the cell into which the polynucleotide sequence is introduced. In some embodiments, the amino acid sequences of UGT1A1, UGDH, AKR1A1, RGN, and GULO are exogenous to the cell into which the polynucleotide sequence is introduced. In some embodiments, the UGDH, UGT1A1, AKR1A1, RGN, and GULO enzymes are from Bos taurus, and have the polypeptide sequences of SEQ ID NO:12-16, respectively, and the cell into which the polynucleotide sequence is introduced is a bovine cell. In some embodiments, the UGDH, UGT1A1, AKR1A1, RGN, and GULO enzymes have sequences that are at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NO:12-16, respectively. Although the sequences listed below are from Bos taurus, those skilled in the art will recognize that homologous sequences can be identified from other species for use in the engineered cells and methods described herein.

[0139] SEQ ID NO:12 UGDH (UniProt:P12378):

[0140]

[0141] SEQ ID NO:13 UGT1A1 (UniProt:E1BBB3):

[0142]

[0143] SEQ ID NO:14 AKR1A1 (UniProt:Q3ZCJ2):

[0144]

[0145] SEQ ID NO:15 RGN (UniProt: Q9TTJ5):

[0146]

[0147] SEQ ID NO:16 GULO (UniProt: Q3ZC33):

[0148]

[0149] In some embodiments, cells are engineered to endogenously produce curcumin. Tyrosine, which is naturally produced in mammalian cells, is a precursor for curcumin biosynthesis using the biosynthetic pathway enzymes phenylalanine ammonia-lyase (PTAL), 4-coumarate-CoA ligase (4CL), phenylpropanoyl-diketide-CoA synthase (DCS), and curcumin synthase (CURS3). A schematic representation of the biosynthetic pathway, including intermediates, is shown in Figure 10 . One or more polynucleotides encoding PTAL, 4CL, DCS, and CURS3 are introduced into cells that naturally produce tyrosine to generate cells that express PTAL, 4CL, DCS, and CURS3 and endogenously synthesize curcumin. In some embodiments, the PTAL, 4CL, DCS, and CURS3 biosynthetic enzymes are from rice (Oryza sativa) and turmeric (Curcuma longa) and have polypeptide sequences of SEQ ID NOs: 17 - 20, respectively. In some embodiments, PTAL, 4CL, DCS, and CURS3 have sequences that are at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NOs: 17 - 20, respectively. In some embodiments, only one, two, or three of the biosynthetic pathway enzymes are engineered into the cells to generate pathway intermediates. Although the sequences listed below are from rice and turmeric, those skilled in the art will recognize that homologous sequences can be identified from other species for use in the engineered cells and methods described herein.

[0150] SEQ ID NO:17 PTAL japonica rice (Oryza sativa japonica) (Uniprot: P14717):

[0151]

[0152] SEQ ID NO:18 4CL japonica rice (UniProt: Q6ETN3):

[0153]

[0154] SEQ ID NO:19 DCS Curcuma (UniProt:C0SVZ5):

[0155]

[0156] SEQ ID NO:20 CURS3 Curcuma (UniProt:C6L7V9):

[0157]

[0158] In some embodiments, cells are engineered to endogenously produce curcumin using the biosynthetic pathway enzymes PTAL, 4CL, and bisdemethoxycurcumin synthase. One or more polynucleotides encoding PTAL, 4CL, and bisdemethoxycurcumin synthase are introduced into cells that naturally produce tyrosine to generate cells that express PTAL, 4CL, and bisdemethoxycurcumin synthase and endogenously synthesize curcumin. In some embodiments, PTAL, 4CL, and bisdemethoxycurcumin synthase have the amino acid sequences of SEQ ID NOs: 17, 18, and 21, respectively. In some embodiments, PTAL, 4CL, and bisdemethoxycurcumin synthase have amino acid sequences that are at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NOs: 17, 18, and 21, respectively. Although the sequences listed below are from rice, those skilled in the art will recognize that homologous sequences can be identified from other species for use in the engineered cells and methods described herein.

[0159] SEQ ID NO:21 Bisdemethoxycurcumin synthase Japonica rice (UniProt:Q8LIL0):

[0160]

[0161] In some embodiments, cells are engineered to endogenously produce cannabidiol. Malonyl-CoA, which is naturally produced in mammalian cells, is a precursor for the biosynthesis of cannabidiol (CBD) using the biosynthetic pathway enzymes 3,5,7-trioxododecanoyl-CoA synthase (OLS), olivetolic acid cyclase (OAC), cannabigerolic acid synthase (CBGAS, also known as prenyltransferase 1 or PT1), and cannabidiolic acid synthase (CBDAS). A schematic of the biosynthetic pathway, including intermediates, is shown in Figure 10In the middle. One or more polynucleotides encoding OLS, OAC, CPGAS, and CBDAS are introduced into a cell that naturally produces malonyl-CoA to produce a cell that expresses OLS, OAC, CPGAS, and CBDAS and endogenously synthesizes CBD. In some embodiments, the isoprenyltransferase NphB can be used in place of CPGAS in the CBD biosynthetic pathway. In some embodiments, the OLS, OAC, CPGAS, CBDAS, and NphB biosynthetic enzymes are from Cannabis sativa and have the polypeptide sequences of SEQ ID NOs: 22-26, respectively. In some embodiments, OLS, OAC, CPGAS, CBDAS, and NphB have sequences that are at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NOs: 22-26, respectively. In some embodiments, only one, two, or three of the biosynthetic pathway enzymes are engineered into the cell to produce pathway intermediates. Although the sequences listed below are from Cannabis sativa, those skilled in the art will recognize that homologous sequences can be identified from other species for use in the engineered cells and methods described herein.

[0162] SEQ ID NO:22 OLS (UniProt: B1Q2B6):

[0163]

[0164] SEQ ID NO:23 OAC (UniProt: I6WU39):

[0165]

[0166] SEQ ID NO:24 CBGAS or PT1 (UniProt: A0A455ZIK6):

[0167]

[0168] SEQ ID NO:25 CBDAS (UniProt: A6P6V9):

[0169]

[0170] SEQ ID NO:26 NphB (UniProt: A0A2Z4JFA9):

[0171]

[0172] The following gene sequences are codon-optimized for expression in bovine cells. Those skilled in the art will recognize that the gene sequences can be optimized for expression in cells from anther species, or wild-type sequences from cannabis can be used.

[0173] SEQ ID NO:27OLS:

[0174]

[0175] SEQ ID NO:28OAC

[0176]

[0177] SEQ ID NO:29CBGAS or PT1

[0178]

[0179] SEQ ID NO:30 CBDAS

[0180]

[0181] In some embodiments, the cells are engineered to express the anti-TNFα nanobody V565 (SEQ ID NO:7). The cells are transfected with a heterologous polynucleotide encoding the nanobody V565 to produce cells that express the nanobody V565. In some embodiments, the polynucleotide encodes a polypeptide that is at least 85%, 90%, 95%, 98%, 99% or 99.9% identical to SEQ ID NO:7. In some embodiments, the polynucleotide encoding V565 is codon-optimized for expression in a particular cell of interest. In some embodiments, the polynucleotide encoding V565 is codon-optimized for expression in bovine cells.

[0182] SEQ ID NO:7 nanobody V565:

[0183]

[0184] In some embodiments, V565 is linked to human TNFα via a flexible cleavable linker, and a polynucleotide encoding V565 linked to human TNFα via a flexible cleavable linker is introduced into cells. Without wishing to be bound by any particular theory or mechanism, linking the V565 nanobody to TNFα will inhibit the binding of V565 to the target TNFα in a subject, and cleavage of the flexible cleavable linker under specific conditions will release V565 to bind to the target TNFα in the subject. In some embodiments, the flexible cleavage linker is a trypsin-cleavable linker having at least one, two, three, four, or five lysine residues. In some embodiments, the length of the linker is between about 10 and about 50 amino acids. In some embodiments, the length of the linker is between about 15 and about 40 amino acids. In some embodiments, the linker has the sequence of SEQ ID NO:9 (GGG SGG GSG GGS GGK GGK GGK GGK GGG SGG GSG AQG).

[0185] In some embodiments, cells are engineered to express antimicrobial peptide 16 (AMP16; SEQ ID NO:8). Cells are transfected with a heterologous polynucleotide encoding AMP16 to produce cells that express AMP16. In some embodiments, the polynucleotide encodes a polypeptide that is at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical to SEQ ID NO:8. In some embodiments, the polynucleotide encoding AMP16 is codon-optimized for expression in a particular cell of interest. In some embodiments, the polynucleotide encoding AMP16 is codon-optimized for expression in bovine cells.

[0186] SEQ ID NO:8 AMP16:

[0187] IRPI IRPI IRPI IRPI IRPI IRPI IRPI

[0188] In some embodiments, AMP16 is linked to an elastase-cleavable linker containing an Asn-Pro-Val (NPV) repeat sequence. In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 repeats of AMP16 will be assembled in the construct, with each repeat separated by an NPV linker.

[0189] In some embodiments, cells are engineered to express a serine protease inhibitor (SPI) to inhibit pepsin digestion of V565 or AMP16. In some embodiments, the SPI is Tsp03044, which has the amino acid sequence of SEQ ID NO:31 or a sequence that is at least 85%, 90%, 95%, 98%, 99% or 99.9% identical thereto. In some embodiments, the SPI is TspAd5, which has the amino acid sequence of SEQ ID NO:33 or a sequence that is at least 85%, 90%, 95%, 98%, 99% or 99.9% identical thereto.

[0190] SEQ ID NO:31 Tsp03044 (NCBI Reference Sequence XM_003379333)

[0191]

[0192] SEQ ID NO:32 Tsp03044

[0193]

[0194] SEQ ID NO:33 TspAd5 (GenBank: EU263307.1)

[0195]

[0196] SEQ ID NO:34

[0197]

[0198] In some embodiments, cells are engineered to increase the expression of one or more growth factors selected from FGF-2, TGFβ-3, NRG-1 and IGF-1. In some embodiments, cells are engineered to increase the expression of insulin and transferrin.

[0199] In some embodiments, cells are engineered by introducing one or more polynucleotides encoding phenylalanine ammonia-lyase, 4-coumarate--CoA ligase and cinnamoyl-CoA reductase into the cells to endogenously produce cinnamaldehyde from the precursor phenylalanine.

[0200] In some embodiments, cells are engineered by introducing a polynucleotide encoding (R)-limonene synthase to endogenously produce (R)-limonene from the precursor geranyl diphosphate. In some embodiments, the (R)-limonene synthase has the amino acid sequence of SEQ ID NO:35 or a sequence that is at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical thereto. In some embodiments, cells are engineered by introducing a polynucleotide encoding (S)-limonene synthase to endogenously produce (S)-limonene from the precursor geranyl diphosphate. In some embodiments, the (S)-limonene synthase has the amino acid sequence of SEQ ID NO:36 or a sequence that is at least 85%, 90%, 95%, 98%, 99%, or 99.9% identical thereto.

[0201] SEQ ID NO:35 Citrus limon (UniProt:Q8L5K3):

[0202]

[0203] SEQ ID NO:36 Abies grandis (UniProt:O22340)

[0204]

[0205] With respect to the enzymes and polypeptides described herein, the phrases "percent sequence identity", "percentage identity", or "identity %" are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods for aligning amino acid sequences are well known. Some alignment methods take into account conservative amino acid substitutions. The conservative substitutions generally maintain the charge and hydrophobicity at the site of substitution and thus maintain the structure (and thus the function) of the polypeptide, as explained in more detail below. The percentage identity of amino acid sequences can be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A commonly used and freely available set of sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI website at Bethesda, Md. The BLAST software suite includes various sequence analysis programs, including "blastp" for aligning a known amino acid sequence with other amino acid sequences from multiple databases.

[0206] Polypeptide sequence identity can be measured over the full length of a specific polypeptide sequence, such as defined by a particular SEQ ID number, or can be measured over a shorter length, such as a fragment obtained from a larger specific polypeptide sequence, for example, a length of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. These lengths are merely exemplary, and it should be understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings can be used to describe the length over which the percent identity can be measured.

[0207] Cells suitable for the methods described herein can include, but are not limited to, primary cells, immortalized cells, pluripotent cells, or embryonic cells. Genetically modified or engineered cells can be plant cells or animal cells that are suitable for stable maintenance, growth, and expansion in in vitro culture. Animal cells suitable for the compositions and methods described herein can be mammalian cells, insect cells, fish cells, or avian cells. Methods for using insect cells in cultured meat products are described, for example, in the published number of International Application No. PCT / US2019 / 066452, which is incorporated herein by reference in its entirety.

[0208] Mammalian cells can be any mammalian cells that can be stably maintained, grown, and expanded in in vitro culture. Mammalian cells can be porcine, bovine, deer, goat, rabbit, or murine cells. Mammalian cells can be from ruminant mammals (e.g., cows, sheep, goats, buffalo, deer, elk, etc.). For use in cultured meat products as outlined below, mammalian cells are non-human mammalian cells.

[0209] Mammalian cells can be primary mammalian cells, immortalized mammalian cells, or mammalian cells differentiated from pluripotent cells (e.g., embryonic stem cells, induced pluripotent cells, etc.). Prior to transduction, primary mammalian cells can be grown and proliferated in culture for 1, 2, 3, 4, 5, 6 generations or more. In some embodiments, the mammalian cells are immortalized cells expressing TERT and CDK4.

[0210] In some embodiments, the animal cells are muscle cells. Muscle cells are characterized by the presence of actin, myogenin, and myosin heavy chain (MHC) and the formation of multinucleated myotubes.

[0211] In some embodiments, the mammalian cells are muscle cells. Mammalian muscle cells are characterized by the expression of actin, myogenin, and myosin heavy chain (MHC) and the formation of multinucleated myotubes. In some embodiments, the mammalian cells are MHC + Actin + Bovine muscle cells.

[0212] In some embodiments, the animal cell is a muscle precursor cell expressing paired box protein 7 (Pax7). In some embodiments, the mammalian cell is a muscle precursor satellite cell expressing paired box protein 7 (Pax7). In some embodiments, the mammalian cell is a Pax7+ bovine satellite cell. In some embodiments, the mammalian cell is a Pax7+ primary bovine satellite cell (BSC). In some embodiments, the mammalian cell is from a cow of the European bovine species.

[0213] In some embodiments, mammalian myotube cells are generated by myogenic differentiation of mammalian satellite cells. Mammalian satellite cells can be cultured to confluence in any medium that supports the growth and expansion of mammalian satellite cells. In some embodiments, mammalian satellite cells are cultured to confluence in B8 medium, growth medium containing serum (such as DMEM + 20% fetal bovine serum), or other serum-free media. The confluent mammalian satellite cells are then cultured in a medium comprising Neurobasal / L15 (1:1) basal medium supplemented with epidermal growth factor (EGF; 0.01 - 5 ng / ml; preferably 0.5 ng / mL), insulin-like growth factor 1 (IGF-1; 0.01 - 5 ng / ml; preferably 0.05 ng / mL), and 1% antibiotic-antimycotic. 61 . In some embodiments, the confluent mammalian satellite cells are cultured in DMEM supplemented with approximately 2% fetal bovine serum. The cells will differentiate for about 1 to 2 weeks.

[0214] B8 medium comprises DMEM:F12 (1:1) basal medium supplemented with: L-ascorbic acid 2-phosphate (0.1 μg / ml to 500 μg / ml; preferably approximately 200 μg / mL), insulin (0.1 μg / ml to 100 μg / ml; preferably approximately 20 μg / mL), transferrin (0.1 μg / ml to 100 μg / ml; preferably approximately 20 μg / mL), sodium selenite (0.1 μg / ml to 100 μg / ml; preferably 20 ng / mL), FGF-2 (0.01 μg / ml to 100 μg / ml; preferably 10 ng / mL), neuregulin 1 (NRG-1; 0.001 ng / ml to 50 ng / ml; preferably 0.1 ng / mL), and TGFβ-3 (0.001 ng / ml to 50 ng / ml; preferably 0.1 ng / μL). In some embodiments, B8 medium further contains insulin-like growth factor 1 (IGF-1; approximately 10 ng / mL).

[0215] In some embodiments, the culture medium, in addition to or instead of the antibiotic component, further comprises cinnamon, monolaurin (a derivative of coconut lauric acid), honey, or a combination thereof.

[0216] In some embodiments, instead of serum, the culture medium may comprise plant- or yeast-based extracts. For example, yeast- and plant-based serum replacements include hydrolyzed proteins from soy, yeast, wheat gluten, cottonseed, or corn, as well as rapeseed peptide fractions, maitake mushroom extracts, and sericin derived from silk.

[0217] In some embodiments, the mammalian cell is an adipocyte. Mammalian adipocytes are characterized by the expression of peroxisome proliferator-activated receptor γ (PPARγ) and increased lipid production.

[0218] In some embodiments, mammalian cells are generated from mammalian satellite cells by adipogenic differentiation. Lipid accumulation in mammalian satellite cells can be induced using a differentiation medium containing a combination of free fatty acids (FFAs). Specifically, a 3-FFA mixture of 125 μM equimolar concentrations of linoleic acid, erucic acid, and elaidic acid is used in the culture for about six days to induce intracellular lipid accumulation. Additional details of adipogenic differentiation from mammalian satellite cells are described by Fish et al. (“Prospects and challenges for cell-cultured fat as a novel food ingredient,” Trends Food Sci Technol., 2020, 98:53-67), which is incorporated herein by reference.

[0219] In some embodiments, the yield of the metabolite of interest can be increased or decreased by altering the enzyme expression level or by altering the availability of intermediates or precursor metabolites in the cell.

[0220] In some embodiments, the metabolite of interest is phytoene, lycopene, or β-carotene, and the yield of these metabolites is increased by treating the cells with ketoconazole. Ketoconazole inhibits the synthesis of cellular cholesterol (which competes with carotenoid precursor compounds). To increase carotenoid production, the cells can be cultured with ketoconazole between about 0.5 μg / ml and about 50 μg / ml. In some embodiments, the cells are cultured with ketoconazole between about 1 μg / ml and about 20 μg / ml. In some embodiments, the cells are cultured with about 5 μg / ml of ketoconazole.

[0221] In some embodiments, a vector comprising a polynucleotide encoding a biosynthetic pathway enzyme and an optional marker is transduced into cells, and the expression of the biosynthetic pathway enzyme is increased by increasing the selective pressure on the cells. For example, when the optional marker is an antibiotic resistance gene, increasing the concentration of the antibiotic in the selection medium can impose a selective pressure on the cells to increase the expression of the introduced vector, thereby also increasing the biosynthetic pathway enzyme expression. Similarly, the biosynthetic pathway enzyme expression can be reduced by decreasing the selective pressure on the cells, such as by decreasing the concentration of the selection chemical in the culture.

[0222] As used herein, a "cultured meat product" refers to an edible meat product produced from a cell culture rather than a whole organism. Generally, a cultured meat product is visually identical to farmed meat, acceptable to consumers, and provides nutritional benefits. The cultured meat products described herein comprise mammalian satellite, muscle, and / or fat cell cultures inoculated onto an edible food-safe substrate and cultured to confluence. The mammalian satellite, muscle, and / or fat cell cultures are inoculated onto the food-safe substrate at a density of from about 20,000 cells / cm 2 to about 400,000 cells / cm 2 、about 30,000 cells / cm 2 to about 350,000 cells / cm 2 or about 35,000 cells / cm 2 to about 300,000 cells / cm 2 In some embodiments, the cells are inoculated at a density of about 50,000 cells / cm 2 . In some embodiments, the cells are inoculated at a density of about 40,000 cells / cm 2 、about 50,000 cells / cm 2 、about 60,000 cells / cm 2 、about 70,000 cells / cm 2 、about 80,000 cells / cm 2 、about 100,000 cells / cm 2 、about 150,000 cells / cm 2 、about 200,000 cells / cm 2 、about 250,000 cells / cm 2 、about 300,000 cells / cm 2 or about 350,000 cells / cm 2Density seeding. In some embodiments, once the cells reach confluence, they become non - adherent to the food - safe substrate and are detached from the food - safe substrate without enzymatic dissociation. In some embodiments, the edible food - safe substrate is in the form of a two - dimensional membrane. In some embodiments, the edible food - safe substrate is in the form of a three - dimensional matrix or sponge, and mammalian muscle cells form continuous muscle fibers when cultured in the matrix or sponge matrix. Suitable edible food - safe substrates are known in the art and include, but are not limited to, chitosan matrices, cellulose matrices, silk matrices, alginate matrices, starch matrices, textured vegetable protein matrices, mycelium matrices, and whey matrices.

[0223] Additional methods for culturing meat production are known and described in the art. See, for example, Post(“Cultured meat from stem cells:Challenges and prospects,”Meat Science,2012,92,297 - 301), Warner(“Review:analysis of the process and drivers for cellular meat production,”Animal,2019,13(12):3041 - 3058), U.S. Patent No. 6,835,390, and U.S. Patent No. 7,270,829.

[0224] In some embodiments, cultured meat products are produced using a chitosan matrix. The chitosan matrix can be adjusted to alter the adhesion and growth of mammalian muscle cell cultures. Generally, increasing the chitosan concentration in the matrix decreases the adhesion of mammalian muscle cells. The chitosan concentration in the food - safe substrate of the cultured meat product can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, or 14%. In some embodiments, the chitosan concentration is between about 1% and about 8%. In some embodiments, the chitosan concentration is between about 2% and about 6%. In some embodiments, the chitosan concentration is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, or at least about 8%. Chitosan can be derived from any suitable source. Chitosan sources include, but are not limited to, mushrooms, crustaceans, insects, green algae, and yeast. In some embodiments, the chitosan is mushroom - derived chitosan.

[0225] Three-dimensional chitosan matrices or sponges suitable for forming the muscle fibers and cultured meat products described herein can be formed using methods known in the art. In some embodiments, chitosan sponges are formed using directional freezing of chitosan. To form a chitosan sponge by directional freezing, chitosan is dissolved in a solvent, such as but not limited to acetic acid, and the chitosan solution is poured into a tube. One end of the tube is exposed to liquid nitrogen or another suitable cryogen, such as a slurry of dry ice and ethanol, until the entire solution is frozen. Subsequently, the frozen chitosan is lyophilized to form a chitosan sponge. The mechanical properties of the chitosan sponge can be adjusted by varying the chitosan concentration. Sponges formed using low concentrations of chitosan (e.g., 1%, 2%, 3% chitosan solutions) have a lower elastic modulus, while sponges formed using high concentrations of chitosan (e.g., 6%, 7%, 8% chitosan solutions) have a higher elastic modulus. Chitosan can be sourced from any suitable source. Chitosan sources include but are not limited to mushrooms, crustaceans, insects, green algae, and yeast. In some embodiments, the chitosan is mushroom-derived chitosan.

[0226] As used herein, a "food safety matrix" means a matrix that is edible or safe for human ingestion if at least a portion of the matrix remains attached or associated with the cultured meat product. In some embodiments, the food safety matrix is a chitosan matrix. Chitosan used as a food safety matrix can be sourced from organisms, including but not limited to chitin from mushrooms, crustaceans, insects, green algae, and yeast. In some embodiments, the food safety matrix is a mushroom chitosan matrix. In some embodiments, the food safety matrix is a cellulose-based matrix, such as a matrix formed from decellularized plants (e.g., decellularized spinach or apple).

[0227] In some embodiments, the cultured meat product comprises the engineered cells, plant-based proteins, unmodified (i.e., wild-type) plant or animal cells, or combinations thereof, described herein. In some embodiments, the cultured meat product comprises between about 1% and about 100% of the engineered cells described herein, by weight or by cell count.

[0228] As used herein, "high density" means cells inoculated at a density of greater than 160,000 cells / mL to about 480,000 cells / mL for suspension culture or greater than 100,000 cells / cm 2 to about 300,000 cells / cm 2 for adherent culture.

[0229] As used herein, "medium density" means cells inoculated at a density of greater than 64,000 cells / mL to about 160,000 cells / mL for suspension culture or greater than 40,000 cells / cm 2 to about 100,000 cells / cm2 Cells seeded at a low density.

[0230] As used herein, "low density" refers to cells seeded at a density of from about 16,000 cells / ml to about 64,000 cells / ml for suspension culture or from about 10,000 cells / cm 2 to about 40,000 cells / cm 2 Cells seeded at a density.

[0231] As used herein, "serum-free" refers to culture conditions and media that do not contain serum or serum replacements or that contain essentially no serum or serum replacements. For example, a substantially serum-free medium may contain less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% serum. As used herein, "serum replacement" refers to an animal serum- and animal cell product-based replacement for serum conventionally used in media. Any plant- or yeast-based serum replacement described herein does not fall within the definition of "serum replacement" when determining that a medium is serum-free. In other words, a medium can be serum-free and contain a plant- or yeast-based serum replacement described herein.

[0232] As used herein, terms such as "defined culture medium" indicate that the nature and quantity of each medium component are known. When used with respect to a medium or culture conditions, the term "defined" refers to a medium or culture conditions for which the nature and amounts of substantially all components are known. When using conventional detection methods known to those of ordinary skill in the art, the levels of certain reagents in a culture, composition, and medium, such as signal transduction inhibitors, animal components, or feeder cells, are below detectable levels, or these reagents are not added to the culture, composition, or medium, the culture, composition, or medium is "substantially free" of these reagents.

[0233] As used herein, "effective amount" means an amount of a reagent sufficient to cause a specified cellular effect according to the present invention.

[0234] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that all definitions, as defined and used herein, take precedence over dictionary definitions, definitions in incorporated references by reference, and / or general meanings of defined terms.

[0235] All references, patents, and patent applications disclosed herein are incorporated by reference relative to the subject matter to which each is cited, and in some cases may cover the entire document.

[0236] As used herein in the specification and claims, the indefinite article "a / an" shall be understood to mean "at least one" unless expressly stated to the contrary.

[0237] As used herein in the specification and claims, the phrase "and / or" shall be understood to mean "either or both" of the elements so joined, i.e., the elements exist conjunctively in some cases and disjunctively in other cases. Multiple elements listed by "and / or" shall be construed in the same manner, i.e., "one or more" of the elements so joined. Other elements may optionally exist, whether related or unrelated to the specifically identified elements, in addition to the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", a reference to "A and / or B" may in one embodiment refer only to A (optionally including elements other than B); in another embodiment refer only to B (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so on.

[0238] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating several items in a list, "or" or "and / or" shall be interpreted inclusively, i.e., including at least one of the multiple elements or the elements in the list, but also including more than one element, and optionally additional unlisted items. Only terms expressly stated to the contrary, such as "only one of... " or "exactly one of... " or "consisting of... " when used in the claims will refer to exactly one of the multiple elements or the elements in the list. In general, when used with exclusive terms such as "any one", "one of... ", "only one of... " or "exactly one of... ", the term "or" as used herein shall be interpreted only as indicating an exclusive alternative (i.e., "one or the other, but not both"). "Consisting essentially of... " when used in the claims shall have the ordinary meaning as used in the field of patent law.

[0239] As used herein, unless otherwise stated or otherwise apparent from the context, the terms "about" or "approximately" with respect to a numerical value are generally considered to include numerical values within 5% in either direction (greater than or less than) of the numerical value (unless the numerical value would exceed 100% of the possible value). In the case of a stated range, unless otherwise stated or otherwise apparent from the context, the endpoints are included within the range.

[0240] Examples

[0241] Example 1

[0242] Metabolic engineering efforts in plants, yeast, and bacteria have exploited many pathways across taxa to develop diverse applications (Heider and Wendisch, 2015; Nielsen, 2015; Sack et al., 2015). The same breadth has not been achieved in mammalian systems, where most efforts have focused on biologics (Ceroni and Ellis, 2018; Davy et al., 2017). While these efforts are not inferior to those in other systems, the difference in scope is evident. To date, this difference may be at least partially attributed to the lack of an urgent case for engineering animal cells with metabolic pathways from other taxa; from a bioprocess perspective, the production of recombinant proteins, small molecules, and biofuels is cheaper in bacterial, yeast, or plant systems and mammalian bioprocesses are only preferred when species-specific post-translational modifications are required (Davy et al., 2017; Giddings, 2001). The possibility of cultured meat provides a new framework for mammalian metabolic engineering. In cultured meat production, the product is the cells themselves, and thus the endogenous synthesis of additional compounds becomes a purely additive effort, at least for those cases that are biologically possible and do not interfere with bioprocess parameters or product value. Here, the fact that cultured meat relies only on cell growth rather than intact animals (a limitation inherent to animal transgenesis) enables consideration of a wider range of metabolites that may be difficult to achieve in animals but are harmless or even beneficial in cell culture.

[0243] In this example, we demonstrate the possibility of generating nutritionally enhanced meat products by cell culture through engineering the endogenous production of non-native dietary carotenoids into primary bovine and immortalized murine skeletal muscle cells. While past speculation has suggested that cultured meat could be enhanced nutritionally by adding exogenous nutrients, endogenous production of nutrients is advantageous from a bioprocess cost scale perspective and from the positive impact that endogenous nutrients may have on nutrient bioavailability or growth kinetics (Desmarchelier and Borel, 2017; Le Moal et al., 2017). We show that carotenoids are produced at levels capable of significantly reducing lipid oxidation, can be optimized through a range of strategies, and the optimized levels are substantially higher than levels found to accumulate in beef. These compounds, along with their general nutritional value and specific relevance to meat-related diseases, give them the function of being able to counter the effects of lipid oxidation on shelf life, color stability, and flavor. Thus, these are compelling candidates to demonstrate how metabolic engineering can impact multiple metrics of meat quality.

[0244] In addition, the positive effect of carotenoid synthesis on C2C12 growth suggests exciting commensurate benefits to this approach. Namely, increased cell growth can contribute to substantially reducing the production cost of cultured meat while producing a more nutritious product. A limitation of this study was the reliance on primary bovine satellite cells, as immortalized bovine cells would enable the exploration of this possibility in a cell line more relevant to meat, without the confounding effects of primary cell senescence and antibiotic selection pressure. Additionally, immortalized cell lines would allow the use of less efficient targeted gene insertion techniques (such as CRISPR / Cas9), which would enable the generation of a homogeneous cell population that could more clearly elucidate carotenoid yields before and after achievable optimizations. Industrial cultured meat production processes will likely use targeted engineering of immortalized cells and avoid antibiotic selection, so subsequent exploration of carotenoid synthesis in such a cell population would be valuable future work.

[0245] The embodiments described herein show the incorporation of the biosynthetic pathway of antioxidant phytonutrients (carotenoids) into mammalian cells and the production of cultured meat therefrom, a pathway that is natural for plants and some prokaryotes but not for animals. These nutrients confer multiple benefits through their roles as antioxidant factors, provitamins, and free radical scavengers (Botella-Pavia and Rodriguez-Concepcion, 2006). Using non-viral Sleeping Beauty transposon-mediated transgenes of phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene cyclase (CrtY) from the bacterium Pantoea ananatis, we converted endogenous geranylgeranyl pyrophosphate (GGPP) to phytoene, lycopene, and β-carotene in immortalized mouse myoblasts and primary bovine muscle stem cells (Botella-Pavia and Rodriguez-Concepcion, 2006; Izsvák et al., 2000). This work builds on previous crop engineering efforts and evidence of CrtB efficacy in mammalian cells (Satomi et al., 1995; Ye et al., 2000). We confirmed the endogenous production of all three carotenoids and showed maintenance of cell myogenicity after modification. We then quantified and optimized carotenoid yields by increasing enzyme expression and inducing precursor accumulation, obtaining yields substantially higher than those reported for beef (Simonne et al., 1996). Finally, we verified the in vitro antioxidant capacity of the endogenous carotenoids, which indicates nutritional and food quality functionality. This work represents the first example of cultured meat nutritional engineering and demonstrates how cross-species transgenes can broaden the scope of metabolic engineering in mammalian cells beyond clinical applications.

[0246] Materials and Methods

[0247] C2C12 Cell Culture - C2C12 mouse skeletal myoblasts (ATCC# CRL-1772, Manassas, VA, USA) were grown on tissue culture plastic in DMEM Glutamax (ThermoFisher# 10566024, Waltham, MA, USA) supplemented with 10% FBS (ThermoFisher# 26140079) and 1% antibiotic-antimycotic (ThermoFisher# 15240062) (Yaffe and Saxel, 1977). For routine cell maintenance, cells were cultured to a maximum of 70% confluence and then passaged using 0.25% trypsin-EDTA (ThermoFisher# 25200056) or frozen in FBS with 10% dimethyl sulfoxide (DMSO, Sigma# D2650, St. Louis, MO, USA). Cells were incubated at 37 °C and 5% CO2.

[0248] Primary Bovine Satellite Cell Isolation and Culture - Primary bovine satellite cells (BSCs) were isolated using a previously described method (Simsa et al., 2019). Briefly, a small piece of muscle (approx. 0.5 cm 3 ) was excised from the semitendinosus muscle of <60-day-old Simmental bulls housed at Tufts Cummings School of Veterinary Medicine according to a method approved by the Tufts University IACUC (protocol # G2018-36). Muscle tissue was dissected from fat and connective tissue, minced into a paste, and digested in 0.2% collagenase II (Worthington Biochemical# LS004176; 275 U / mg, Lakewood, NJ, USA) for one hour with regular agitation. The digestion was stopped with growth medium containing DMEM Glutamax supplemented with 20% FBS, 1 ng / mL human FGF-basic (ThermoFisher# 68-8785-63), and 1% Primocin (Invivogen# ant-pm-1, San Diego, CA, USA), and the cells were filtered and seeded at 100,000 cells / cm 2The density was spread onto an uncoated tissue culture flask. After culturing at 37 °C and 5% CO2 for 24 hours, the spreading suspension (containing slow-adhering satellite cells) was transferred to a new flask coated with 1 μg / cm 2 of mouse laminin (Sigma #CC095). These flasks were left standing for three days, at which time the growth medium was changed, and the cells were cultured on tissue culture plastic coated with iMatrix recombinant laminin-511 (Iwai North America #N892021, San Carlos, CA, USA) using standard practices. After culturing for two weeks, Primocin in the growth medium was replaced with 1% antibiotic-antimycotic. Cell passaging and cryopreservation were performed as for C2C12. To induce myogenic differentiation, the cells were cultured to confluence and then cultured for one week without changing the medium.

[0249] Immunofluorescence and cell characterization - To confirm the nature of the isolated primary BSCs, the cells were stained for Pax7 and myosin heavy chain (MHC) before and after differentiation, respectively. These markers confirm the primary and final states of satellite cell myogenesis and are thus used to verify the nature and myogenicity of the isolated cells. Proliferating cells and cells after one week of differentiation were fixed with 4% paraformaldehyde (ThermoFisher#AAJ61899AK) for 30 minutes, washed in PBS, permeabilized with PBS containing 0.5% Triton X (Sigma#T8787) for 15 minutes, blocked with PBS containing 5% goat serum (ThermoFisher#16210064) with 0.05% sodium azide (Sigma#S2002) for 45 minutes, and washed with PBS containing 0.1% Tween-20 (Sigma#P1379). The primary Pax7 antibody (ThermoFisher#PA5-68506) was diluted 1:100 with the blocking solution and added to the proliferating cells. The primary MHC antibody (Developmental studies hybridoma bank#MF-20, Iowa City, IA, USA) was diluted to 4 μg / mL with the blocking buffer containing 1:100 phalloidin 594 (ThermoFisher#A12381) and added to the differentiated cells. The primary antibodies were incubated overnight at 4°C. The next day, the cells were washed with PBS+Tween-20, incubated with the secondary antibodies for Pax7 (ThermoFisher#A-11008, 1:500) and MHC (ThermoFisher#A-11001, 1:1000) for 1 hour at room temperature, washed with PBS+tween-20, and mounted with Fluoroshield mounting medium with DAPI (Abcam#ab104139, Cambridge, UK), followed by imaging.

[0250] Molecular cloning and Sleeping Beauty construct design - Amino acid sequences of CrtB, CrtI, and CrtY from Pantoea ananas were obtained from UniProt (accession numbers P21683, P21685, and P21687, respectively). The gene sequences of these proteins were optimized for expression in European cattle using codon optimization software (IDT, Coralville, IA). A self-cleaving 2A peptide was added to the end of each gene to facilitate polycistronic expression, and all genes were flanked by multiple cloning sites (Szymczak et al., 2004). The final gene construct was ordered through ThermoFisher's GeneArt gene synthesis service (Table 1). Next, three Sleeping Beauty transposon vectors were constructed using the synthesized genes and based on plasmids available through Addgene: pCMV-GFP was a gift from Connie Cepko (Addgene #11153, Watertown, MA, USA), pSBbi-GP and pSBbi-Pur were gifts from Eric Kowarz (Addgene #60511 and #60523), and pCMV(CAT)T7-SB100 was a gift from Zsuzsanna Izsvak (Addgene #34879) (Kowarz et al., 2015; Mátés et al., 2009; Matsuda and Cepko, 2004). Transposon constructs were made using standard cloning techniques. Briefly, CrtB was cloned into pCMV-GFP using EcoRI-HF and XmaI restriction (NEB #R3101S and #R0180S, Ipswich, MA, USA), followed by T4 DNA ligation (NEB #M0202S) to generate pCMV-CrtB-P2A-eGFP, a plasmid for transient bicistronic expression of CrtB and green fluorescent protein (GFP) under the cytomegalovirus (CMV) promoter.

[0251] Table 1: Carotenoid synthesis genes. Gene sequences used in the construct (in uppercase), followed by the 2A linker sequence (SEQ ID NO:37) used after each gene (in lowercase).

[0252]

[0253]

[0254]

[0255] Next, the CMV-CrtB-P2A-GFP portion of this plasmid was cloned into pSBbi-Pur by Gibson assembly (SGI-DNA #GA1100, San Diego, CA, USA), excising the EF1α promoter of the backbone and replacing it with the CMV promoter of the insert. This generated pSBbi-(CMV-CrtB-T2A-GFP)-pur, a Sleeping Beauty transposon vector (Kowarz et al., 2015) carrying the same bicistronic CrtB and GFP expression cassettes under the CMV promoter, and the puromycin resistance gene under a synthetic promoter oriented in the reverse direction to CMV. Subsequently, CrtI and CrtY were Gibson-assembled into this vector to generate three final Sleeping Beauty transposon carotenoid-producing vectors: pSBbi-(CMV-CrtB-T2A-)-pur (pCrtB), pSBbi-(CMV-CrtB-T2A-CrtI-P2A-GFP)-pur (pCrtB / I), and pSBbi-(CMV-CrtY-T2A-CrtB-T2A-CrtI-P2A-GFP)-pur (pCrtB / I / Y)( Figure 1 ). By removing the carotenoid synthase and 2A sequences from pCrtB / I / Y, leaving only GFP under the CMV promoter, a control Sleeping Beauty transposon vector containing only GFP (pGFP) was generated( Figure 1 ). All constructs were maintained in 5-α competent chemically competent Escherichia coli (NEB #C2988J), verified by Sanger sequencing (Genewiz, Cambridge, MA, USA), and purified by GeneJet miniprep (ThermoFisher #K0503). For Gibson assembly, polymerase chain reaction was performed using Q5 high-fidelity polymerase (NEB #M0492S), run through 1% agarose gel electrophoresis, and purified by GeneJet gel extraction (ThermoFisher #K0692).

[0256] Transfection and Selection - C2C12 and BSC were cultured in 6-well plates to 80% to 90% confluence and transfected with Lipofectamine 3000 (ThermoFisher #L3000015) according to the manufacturer's protocol adapted for co-transfection of two plasmids. Briefly, 2.5 μg of the carotenoid-producing vector or pGFP control vector was combined with 0.25 μg of pCMV(CAT)T7-SB100 in a solution of 250 μL of Opti-MEM medium (ThermoFisher #31985088), 7.5 μL of Lipofectamine 3000 reagent, and 5 μL of p3000 reagent. This mixture was incubated for 15 minutes at room temperature. During incubation, the cells were rinsed once with PBS and covered with 2 mL of Opti-MEM before addition of the Lipofectamine mixture. After 4 hours at 37 °C, 2 mL of growth medium was added to the wells and the cells were incubated overnight at 37 °C. The next morning, the medium was replaced with appropriate growth medium and the cells were cultured for two days before observing GFP expression using a fluorescence microscope. After two days, the medium was changed to growth medium with 2.5 μg / mL puromycin to initiate selection. Within a week, the cells were passaged and the medium was changed as needed due to cell death resulting from selection. Once all cells were observed to express GFP, the culture was expanded and cryopreserved for subsequent use.

[0257] Growth and development of engineered cells - On days 1, 2, 4, 6, and 8 of culture, the growth of engineered BSCs and C2C12s was analyzed using a dsDNA quantification kit (ThermoFisher #F2962). Cells were plated at a density of 1000 cells / well in a 96-well plate at each time point. At each time point, the medium was removed from the wells and the plate was frozen at -80 °C. After freezing at all time points, the plate was thawed to room temperature and 100 μL of ultrapure water (ThermoFisher #10977023) was added to the wells. The plate was incubated at 37 °C for one hour, refrozen to -80 °C, and thawed again to room temperature. Each well was stained with 100 μL of Hoechst 33258 working solution. Fluorescence was measured using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA) with excitation and emission filters centered at 360 and 460 nm, respectively. In parallel, 96-well plates were inoculated with a series of known cell densities, incubated for 12 hours, and measured using the same protocol to obtain a standard curve and convert fluorescence values to cell numbers. To analyze the effect of transfection on cell phenotype, as previously described, BSCs transfected with pCrtB / I / Y were differentiated and stained for Pax7 and MHC, except that a red (594 nm) fluorophore-conjugated antibody was used to detect Pax7 (ThermoFisher #A-11072) and MF20 (Abcam #ab150116), and no cytoskeletal counterstaining was performed.

[0258] Carotenoid extraction - Cells were cultured to confluence in 175 cm 2 flasks, harvested, and counted using a hemocytometer. The harvested cells (about 0.3 - 10 million for BSCs and about 1 - 20 million for C2C12s) were washed once with PBS and resuspended in 1 mL of a 50:25:25 solution of hexane (Sigma #270504), acetone (ThermoFisher #A18-500), and ethanol (Sigma #459836), shaken for one minute, and incubated on an orbital shaker plate at room temperature for 30 minutes. After 30 minutes, 150 μL of deionized water was added and the suspension was shaken again for one minute. The solution was centrifuged at 3,000 g for 5 minutes at 4 °C, and the carotenoid-containing upper layer was collected in a Pyrex tube. Another 1 mL of hexane:acetone:ethanol solution was added to the remaining pellet, and the extraction was repeated. The two carotenoid solutions were pooled in one tube. The extract was dried under a nitrogen stream at room temperature, capped, and stored at -80 °C before HPLC analysis.

[0259] Quantitative HPLC - carotenoids were analyzed by HPLC following the published technique on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA) using a C 30 stationary phase (YMC#CT99S03 - 1546WT, 3μm, 4.6×150mm, Allentown, PA, USA) and mobile phase solvents A - methanol (ThermoFisher#A454 - 1) containing 1% ammonium acetate (ThermoFisher) and B - methyl - tert - butyl ether (MTBA, ThermoFisher#AC378720010) (Melendez - Martinez et al., 2013). Briefly, the carotenoid extract was suspended in 150 μL of ethanol, vortexed, and injected (50 μL) into the HPLC system. The mobile phase solvents were pumped through the system at 1 mL / min with the following time - dependent gradient: t = 0 min, 95% A, 5% B; t = 15 min, 85% A, 15% B; t = 30 - 37 min, 40% A, 60% B; t = 40 - 55 min, 30% A, 70% B; t = 59 - 64 min, 90% A, 10% B; t = 70 min, 95% A, 5% B. Carotenoid standard curves were generated by dissolving (E / Z) - phytoene (Sigma#78903), lycopene (Sigma#SMB00706), and β - carotene (ThermoFisher#AAH6010603) standards in hexane at a concentration of approximately 0.5 mg / mL. The absorbance of phytoene, lycopene, and β - carotene was measured at 286, 472, and 453 nm, respectively, in a 1 - cm path - length quartz absorption cell by spectrophotometry (SpectraMax M2, Molecular Devices, San Jose, CA, USA) to determine the exact concentration, and using A 1％Calculate the exact concentration for 915, 3450, and 2592. The standards were dried under nitrogen and stored at -80 °C until the samples were analyzed after serial dilution. Since FBS may contain trace amounts of carotenoids (from animal feed), the endogenously produced carotenoid content was obtained by subtracting any peak areas found in the extracts from pGFP cells (which do not have the mechanism for endogenous carotenoid production) (Chitchumroonchokchai et al., 2017). If the calculation returned a negative value or no peak was detected, the endogenous carotenoid content was set to zero. The final values were converted to ng / cell × 10 6 .

[0260] Protein quantification - To normalize the cellular carotenoid content under a more food-relevant metric than cell number, the cellular protein content was measured. Briefly, engineered cells were cultured as previously described, collected, and counted. One million cells were pelleted, washed once in PBS, and treated with 100 μL of RIPA buffer (Thermo Fisher #89900) containing 1% Halt protease inhibitor cocktail without EDTA (ThermoFisher #78425) according to the manufacturer's instructions. The resulting cell lysates were analyzed by Pierce BCA protein assay (ThermoFisher #23225) according to the manufacturer's instructions. The absorbance at 562 nm was compared to a standard curve of known bovine serum albumin (BSA) concentrations and used to determine the cellular protein / million cells (Supplementary Figure 1 ). These values were then used together with the carotenoid quantification / million cells (Figure 4C - Figure 4D) to obtain the carotenoid content / protein mass.

[0261] Carotenoid Optimization - Two methods for optimizing carotenoid production in pCrtB / I / Y cells were explored. The first method focused on increasing gene expression by increasing puromycin selection pressure. The second method focused on increasing the level of the carotenoid pathway precursor farnesyl pyrophosphate (FPP), which was achieved by using the small molecule ketoconazole to inhibit a downstream reaction in one of the alternative metabolic pathways of FPP, namely cholesterol synthesis (Sun et al., 2007). By exploring these two methods, the likelihood that a single method could not address the rate-limiting steps in carotenoid production was reduced. Specifically, if the FPP level was completely limiting (i.e., there were sufficient enzymes to completely deplete the FPP level), then increasing enzyme expression would not increase the carotenoid level. Similarly, if the enzyme level was completely limiting, then ketoconazole treatment would not provide an increase in carotenoid production. To determine the appropriate puromycin concentration for increasing enzyme expression, engineered cells were cultured in media containing 2.5 μg / mL puromycin (1×puro), 5 μg / mL puromycin (2×puro), or 10 μg / mL puromycin (4×puro), and relative GFP expression was measured by flow cytometry as an approximate measure of enzyme expression. Briefly, cells were cultured, harvested, and analyzed for GFP using a BD FACSCalibur (BD Sciences) or Attune NxT (ThermoFisher) flow cytometer. Appropriate gating was set using pGFP and unmodified cells as positive and negative controls, and the mean fluorescence intensity was determined. Data were analyzed using FlowJo software (v10). To determine the appropriate ketoconazole concentration for metabolic flux regulation, growth curves were obtained as previously described using a dsDNA quantification kit and media treated with 0, 2.5, 5, 10, or 20 μg / mL ketoconazole (KTZ, ThermoFisher #455470010) dissolved in DMSO starting on day 1. The final DMSO concentration was kept constant (0.3%) in all conditions. After determining the appropriate puromycin and ketoconazole concentrations for carotenoid optimization, pCrtB / I / Y cells were cultured in media containing 1×puro plus 5 μg / mL ketoconazole, 4×puro without ketoconazole, or 4×puro plus 5 μg / mL ketoconazole, and carotenoid analysis was performed as previously described.

[0262] Lipid oxidation - To analyze the antioxidant functionality of carotenoids in cells, a thiobarbituric acid reactive substances (TBARS) assay was performed. This assay measures malondialdehyde (MDA), which is a byproduct of the oxidation of unsaturated fatty acids and thus serves as a measure of lipid oxidation in cells. Because lipid oxidation is typically catalyzed by processes that generate oxidative stress, such as meat cooking, the TBARS assay was performed on both "raw" cells and cells that had been heated to simulate cooking. Briefly, engineered BSCs were cultured as previously described, harvested, and counted. 1.5 million cells were aliquoted into tubes for heated or unheated assays, washed once in PBS, pelleted, and the supernatant PBS removed. The harvested cell pellets were stored overnight at 4 °C. The next day, the samples designated for heating were incubated in a 100 °C heating block for 10 minutes, cooled to room temperature, and placed on ice. All cells (heated and unheated) were resuspended in 500 μL of PBS, incubated on ice for 2 hours, and sonicated on ice using a probe sonicator (Branson, St. Louis, MO, USA) at 15% intensity for 5 × 15 second pulses with 20 second intervals between pulses. Next, following the manufacturer's protocol, a TBARS assay (Cayman Chemical #700870, Ann Arbor, MI, USA) was performed on 100 μL of the sonicated cell lysate, and MDA was determined by measuring the absorbance at 535 nm and comparing it to a standard curve of known MDA concentrations. The remaining cell lysate was frozen at -80 °C until protein quantification was performed following the manufacturer's protocol by Bradford assay (ThermoFisher #23200). Briefly, 5 μL of cell lysate was incubated with 250 μL of Coomassie reagent for 10 minutes, and the absorbance was measured at 595 nm and compared to a BSA standard curve. The TBARS measurements were normalized by the protein content of each sample in order to obtain corrected cellular MDA (mg / g protein).

[0263] Statistical analysis - Statistical analysis was performed using GraphPad Prism 8.0 software (San Diego, California, USA). Carotenoid content, flow cytometry, and ketoconazole growth were analyzed by one-way ANOVA. Growth curves and TBARS analysis were analyzed by two-way ANOVA. Multiple comparisons for all analyses were performed using Tukey's HSD post-hoc test. In the process of converting carotenoid and MDA quantification to per-protein values, the following was used taking into account error propagation: σ_(A / B) = (A / B)√((σ_(A / A)^2+(σ_(B / B)^2)), where σ_A is the standard deviation of data A. P values < 0.05 were considered significant. Unless otherwise stated, errors were given as ± standard deviation.

[0264] Results and discussion

[0265] Primary bovine skeletal muscle stem cells - Immunofluorescent staining for early markers of satellite cell properties and terminal markers of myogenic differentiation was used to characterize primary bovine satellite cells (BSCs). Proliferating cells showed widespread staining for Pax7, a marker of satellite cell phenotype (Figure 2A), indicating that the cell isolation protocol was successful in generating a highly homogeneous population of myogenic stem cells. After one week of differentiation, myosin heavy chain (MHC) staining of the cells showed long multinucleated myotubes (Figure 2B). Actin and MHC staining of these myotubes revealed a striated architecture indicative of sarcomere formation, and the observation of spontaneous contractions in the differentiated cultures further supported the presence of contractile machinery. These data together indicated the isolation of a highly myogenic population of bovine skeletal muscle precursors and the ability of these cells to undergo robust differentiation in vitro.

[0266] Stable Transgene Expression in Cells - Multicistronic vectors were engineered to modify BSC and C2C12 immortalized murine progenitor cells to produce a series of protein conversations. These were the single green fluorescent protein (GFP) that does not produce any carotenoids (pGFP), CrtB and GFP that produce phytoene (pCrtB), CrtB, CrtI, and GFP that produce phytoene and lycopene (pCrtB / I), or CrtB, CrtI, CrtY, and GFP that produce phytoene, lycopene, and β-carotene (pCrtB / I / Y) (Figure 2A - Figure 2B). The multicistronic nature of these vectors enabled the use of GFP expression as an indicator of vector integration and gene expression. Specifically, the 2A sequence between genes allowed for near-stoichiometric expression of the enzyme and GFP (Szymczak et al., 2004). After transfection, after one week of selection in puromycin-containing medium, all cells stably expressed their corresponding gene cassette, as visible by GFP fluorescence (Figure 3A). The fluorescence intensity was not uniform, which may be attributed to the randomness of transposition. No obvious morphological differences were found between control pGFP cells and cells expressing carotenoid synthases.

[0267] Growth and Development of Engineered Cells - Because antioxidant factors are known to affect muscle progenitor cell proliferation, the growth rates of cells producing all three carotenoids were analyzed over eight days (Le Moal et al., 2017). For BSC, the size of the inserted gene construct was negatively correlated with the growth rate (Figure 3B). In contrast, C2C12 engineered with the two largest constructs (pCrtB / I and pCrtB / I / Y) showed higher growth over eight days compared to cells engineered with pGFP or pCrtB. This difference may be attributed to the effects of insertion efficiency and subsequent selection on primary cells, but does not affect immortalized cells. That is, cells transfected with larger constructs have lower insertion efficiency and thus require more doublings to produce an equally sized population of engineered cells (Izsvák et al., 2000). In primary cells, growth slows with increasing doublings and eventually reaches cellular senescence (Ding et al., 2018). However, in immortalized cells, doubling does not impede growth. Instead, antioxidant capacity has been found to increase cell proliferation (Le Moal et al., 2017), and immortalized cells pCrtB / I and pCrtB / I / Y cells with increased antioxidant capacity showed improved growth compared to the control group. It is possible that immortalized bovine cells will show similar relative kinetics.

[0268] To determine whether genetic modification affects myogenic potential, pCrtB / I / Y BSCs were stained for Pax7 and MHC (Figure 3C). Staining showed maintenance of Pax7 expression and MHC-positive myotube formation. Striations were not observed in differentiated cells, which could indicate less robust differentiation compared to non-engineered cells. This may be attributed to reduced myofiber maturation by antioxidant effects; however, the ability of the cells to form myotubes indicates that these cells have the potential to produce differentiated cultured meat products. These data together point to the ability of the cells to maintain their proliferation and differentiation capabilities after carotenoid synthase modification; however, this proliferation and differentiation ability may be reduced in primary cells.

[0269] Carotenoid production in engineered cells - Quantitative HPLC confirmed the production of phytoene in pCrtB cells, phytoene and lycopene in pCrtB / I cells, and phytoene, lycopene, and β-carotene in pCrtB / I / Y cells. In all cells, carotenoids accumulated as end compounds, but precursors were also present (i.e., phytoene and lycopene in pCrtB / I / Y cells), indicating high but non-exhaustive conversion efficiency throughout the pathway (Figures 4A - 4D). In pCrtB / I cells, most of the phytoene was converted to lycopene, and the total carotenoid content was highly maintained compared to cells that only produced phytoene. In pCrtB / I / Y cells, carotenoids accumulated as the end compound (β-carotene), but the yield was significantly reduced compared to pCrtB or pCrtB / I cells. The carotenoid production of all three constructs was greater in C2C12 compared to BSCs. The total carotenoids of C2C12 pCrtB, pCrtB / I, and pCrtB / I / Y were 505.3, 254.8, and 63.19 μg / g protein, respectively. For BSCs, these were 35.8, 31.2, and 3.7 μg / g protein, respectively. The significantly reduced total carotenoid content of BSCs compared to C2C12, and of pCrtB / I / Y cells compared to pCrtB or pCrtB / I cells, can be attributed to increased cellular burden of selection and protein production and / or endogenous enzymatic degradation of β-carotene (Geurts et al., 2003; Lindqvist et al., 2005).

[0270] Optimization of carotenoid production - Since the carotenoid production in BSCs was relatively lower compared to C2C12, and in pCrtB / I / Y cells compared to pCrtB or pCrtB / I cells, we hypothesized that the production in these cells could be significantly increased. We explored two ways to achieve this. In the first approach, enzyme expression was increased by increasing the selection pressure of the transgene. Puromycin was shown to induce a significant increase in GFP expression in C2C12 and BSCs at 4× concentration (10 μg / mL) (Figure 7A, Figures 9A - 9B), so this concentration was used for optimization. In the second approach, the accumulation of carotenoid precursors was induced by using the small molecule ketoconazole to inhibit cholesterol synthesis (cholesterol competes with carotenoid precursors) (Figure 5A) (Sun et al., 2007). A ketoconazole concentration of 5 μg / mL was found not to significantly affect growth in C2C12 and BSCs (Figure 7B), so this concentration was used.

[0271] To test how these treatments affected carotenoid production individually and synergistically, pCrtB / I / Y BSCs were cultured with 1× puromycin + / - ketoconazole and 4× puromycin + / - ketoconazole. A significant increase in orange pigmentation was observed in cells cultured with 4× puromycin + ketoconazole (Figure 5B), and for these cells, quantitative HPLC showed a significant increase in β - carotene production (Figure 5C). Specifically, the optimized BSCs produced 10 - fold more β - carotene compared to unoptimized BSCs (2.08 vs. 22.6 μg / g protein, respectively). Notably, this level was approximately 7 - fold higher than the literature value of β - carotene (μg / g protein) in beef from pasture - fed cattle and approximately 14 - fold higher than that from feedlot - fed cattle (Simonne et al., 1996). Subsequently, the treatment with the best effect (4× puromycin + ketoconazole) was used to optimize pCrtB / I / Y C2C12. The average β - carotene concentrations in unoptimized and optimized C2C12 were 35.44 and 42.43 μg / g protein, respectively, but this difference was not statistically significant. Analysis of the total carotenoid profiles of the optimized cells revealed a tendency to accumulate in the form of the end - compound (β - carotene) in all cases (Figures 8A - 8B).

[0272] Elevated levels of enzymes and precursors can increase carotenoids, indicating that both enzymes and precursors are limiting in the system, which may be attributed to the non-uniform nature of enzyme expression in cells. Specifically, in cells with high basal enzyme levels, the precursor is the rate-limiting step, and in cells with low basal enzyme levels, the enzyme capacity is the rate-limiting step. The efficacy of technologies to optimize both suggests multiple ways to seek optimization when engineering cultured meat. While using ketoconazole to induce precursor accumulation was useful in proof-of-concept, achieving the same effect in the production process of nutritionally enhanced cultured meat is likely to rely on genetic strategies.

[0273] Lipid oxidation and antioxidant functionality - Lipid oxidation is a key mechanistic link between red or processed meat intake and colorectal cancer, and a major cause of non-microbial meat quality deterioration during storage (Falowo et al., 2014; Wolk, 2017). We therefore analyzed cellular malondialdehyde (MDA) levels by thiobarbituric acid reactive substances (TBARS) assay to explore the ability of endogenous carotenoids to reduce lipid oxidation in vitro, i.e., in "raw" unheated cells and in "cooked" heated cells ( Figure 6 ). In pGFP BSC, unheated and heated samples showed 1.9 and 4.6 mg MDA / g protein, respectively, indicating a significant effect of the heating method in promoting lipid oxidation. BSC engineered with pCrtB showed reduced lipid oxidation in unheated and heated samples (1.7 and 3.6 mg / g protein, respectively), but this difference was not statistically significant in unheated samples. Cells engineered with pCrtB / I and pCrtB / I / Y showed a significant reduction in lipid oxidation compared to control unheated and heated samples (1.0 and 1.1 mg / g protein for CrtB / I and CrtB / I / Y unheated samples, respectively, and 1.7 and 2.3 mg / g protein for heated samples, respectively).

[0274] It is noteworthy that cells containing lycopene and / or β-carotene have more antioxidant capacity than cells containing only phytoene, even though the latter are pCrtB cells with the highest total carotenoid levels. This may be attributed to the different antioxidant capacities of these carotenoids, with phytoene having the weakest capacity (Martínez et al., 2014). Similarly, pCrtB / I / Y cells and pCrtB / I cells exhibit almost equivalent antioxidant activities, even though the latter have approximately ten times more total carotenoids, which may be attributed to the synergistic effect shown by carotenoids when present simultaneously in the system (Kotíková et al., 2011). Finally, measuring lipid oxidation alone can affect the results because cholesterol can enhance membrane antioxidant capacity (cholesterol synthesis competes with carotenoid synthesis) (López-Revuelta et al., 2006). Therefore, it is worthwhile to measure total oxidation (including proteins). Nevertheless, the simplicity and universality of TBARS analysis in meat science applications make this technique very important, and the approximately two-fold reduction in lipid oxidation in heated and unheated samples containing endogenously produced lycopene and / or β-carotene indicates that these nutrients will have a significant impact on the nutrition and food quality of engineered cultured meat products.

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[0316] Example 2

[0317] Due to its ability to provide valuable nutrients in high density, meat is a key nutritional source for many populations. Meat provides a substantial protein content (about 30% by weight when cooked), a well-proportioned amino acid profile for human health, a range of essential fatty acids, vitamins A, B, D, and E, and key minerals in highly bioavailable forms 5-9 . The impact of these characteristics on human health is amplified by the current and projected prevalence of meat in the global diet. In fact, in some Western diets, meat constitutes up to 40% of daily protein intake, and a recent report from the United Nations Food and Agriculture Organization (FAO) indicates that global meat production will increase by 76% from 2012 to 2050, up to 455 million tons 5,10 .

[0318] However, despite this value, a growing body of research has highlighted the fact that the global impact of meat as a source of nutrition and a mass commodity extends beyond its nutritional value. From a health perspective, the consumption of large amounts of red and processed meat has been associated with an increased risk of many conditions, including type II diabetes, cardiovascular disease, and colorectal cancer. 11-16 . While the epidemiological studies behind these correlations have difficulty providing firm dietary advice (and are therefore often controversial), for colorectal cancer, there is sufficient meta-analytical evidence that is very persuasive to most nutritionists, and the International Agency for Research on Cancer (IARC) of the World Health Organization has classified red meat and processed meat as 'probably carcinogenic' and 'carcinogenic', respectively. 17,18 . From a production perspective, livestock farming causes considerable use of natural resources, greenhouse gas emissions, and environmental degradation. Specifically, it is estimated that livestock farming generates approximately 15% of all anthropogenic greenhouse gases (CO2 equivalent), accounts for approximately 29% of the global human water footprint, contributes to approximately 71% of deforestation in some regions, and is a major source of eutrophication. 10,19 . At the same time, concentrated livestock farming has raised concerns about animal welfare and antibiotic resistance. 10 . In the context of a predicted substantial increase in meat production by 2050, these problems are likely to intensify.

[0319] Recently, the possibility of producing meat by cell culture (referred to as 'cultured meat') has been proposed to address the environmental and ethical issues of livestock farming. 20 . Producing cultured meat involves expanding muscle and fat cells from a relevant species in vitro and inoculating them onto an edible scaffold to form 3D tissue. 21 . By producing meat in a situation that excludes the competing energy demands present in a whole animal, cultured meat is expected to significantly reduce the land use, water use, and greenhouse gas emissions of meat. 22 . Cultured meat is also expected to reduce the animal welfare and antibiotic resistance issues of conventional meat.

[0320] However, despite these predicted benefits, it is not yet clear exactly how cultured meat reproduces the nutritional characteristics of meat, and what its nutritional advantages or disadvantages might be. On the one hand, due to the frequent immaturity of in vitro cultured skeletal muscle fibers and the importance of sarcomeric proteins for the nutritional value of meat, cultured meat may provide a poorer protein profile than conventional meat. 23 . In addition, the content of many nutrients, such as minerals and some vitamins that are not endogenously synthesized in muscle tissue, including essential vitamins such as B 12 . will depend entirely on the absorption of cells from the culture medium components, and may therefore be reduced or absent in bioprocesses with strict constraints on culture medium costs. 24On the other hand, some harmful nutrients that are present in muscle tissue but not endogenously synthesized, such as trans-saturated fatty acids, will be easily excluded from cultured meat products. 25 .

[0321] Along with the inherent differences, the cell-level control inherent in cultured meat provides opportunities for modulating the composition towards various targets. This can be modulating natural nutrients, such as amino acid or lipid profiles, or adding compounds that are not endogenously synthesized in meat tissue, such as vitamins or other phytochemicals. These modifications can be targeted at addressing potential nutritional deficiencies in cultured meat, enhancing its nutritional advantages, or adding entirely new nutritional functionalities. Looking beyond nutrition, the composition also has the potential to be modulated towards: sensory and food functionality targets, such as flavor, aroma, color, or storage stability; or therapeutic activity, thus allowing the production of 'therapeutic foods' that have favorable bioactivity upon ingestion.

[0322] One promising technology for influencing the composition of cultured meat is through metabolic engineering, or the genetic and regulatory control of cellular metabolism, towards various targets. Metabolic engineering efforts in plants, yeast, and bacteria have targeted applications ranging from pharmaceuticals and cosmetics to energy and agriculture, leveraging many pathways from diverse taxonomic units. 26-28 . The same breadth has not been achieved in mammalian systems, with most efforts focused on biopharmaceuticals. 29-31 . While these efforts are not inferior to other systems, the difference in scope is evident. So far, this difference may be at least partially attributed to the lack of an urgent case for engineering animal cells with metabolic pathways from other taxa; from a bioprocess perspective, the production of recombinant proteins, small molecules, and biofuels is cheaper in bacterial, yeast, or plant systems and mammalian bioprocesses are only preferred when species-specific post-translational modifications are required. 29,32 . The possibility of cultured meat provides a new framework where the product is the cells themselves, and thus the endogenous synthesis of additional compounds becomes a mere adjunct task, at least for those cases that are biologically possible and do not impede the bioprocess or product value. Here, the fact that cultured meat depends only on cell growth rather than on a whole animal for survival (which is an inherent limitation of animal transgenesis) enables the consideration of a wider range of metabolites that may be difficult to achieve in animals but are harmless or even beneficial in cell culture. Compelling functionalities that can be explored include nutrition, bioactivity, and food functionality, flavor, or therapeutic activity.

[0323] Example 1 presents mammalian cells engineered with a metabolic pathway for carotenoid synthesis, a pathway that is natural for many plants and some prokaryotes but not for animals. Specifically, using non-viral Sleeping Beauty transgenes, we engineered the production of phytoene, lycopene, and β-carotene from natural precursors in primary bovine and immortalized C2C12 murine muscle cells. For several reasons, these nutrients are attractive targets. First, each provides unique nutritional value; phytoene has been proposed to act as a UV light protectant, lycopene has been shown to reduce hepatocellular carcinoma and fatty liver disease, and β-carotene is an essential vitamin A precursor in the human diet. 33-35 . Second, all three compounds are antioxidant factors 36 . This is particularly relevant for red meat, as the key mechanistic link between red or processed meat intake and colorectal cancer is lipid oxidation 17 . The antioxidant properties of these compounds also provide value in extending the shelf life of meat, as lipid oxidation is the primary cause of non-microbial deterioration over time 37 . Finally, these compounds are red pigments, and thus their synthesis can affect the color of cultured meat products. We demonstrated the successful synthesis of all three dietary carotenoids, the phenotypic integrity of the modified cells, and the ability to modulate carotenoid yields through multiple strategies.

[0324] The potential for a metabolite to have diverse effects on cultured meat products, such as antioxidant factors conferring both ancillary meat quality benefits as well as nutritional benefits, is a common feature of many potential targets. In addition to the aforementioned meat quality effects, antioxidant factors can also affect cell expansion, where unregulated levels of reactive oxygen species induce cell cycle arrest and inhibit proliferation 38-40 . Indeed, in our previous studies, C2C12 with synthetic carotenoids having increased antioxidant capacity showed higher proliferation compared to the control group, but this was not found in engineered primary cells. Other interesting cases of the proliferative effects of phytochemicals include the cases of vitamin C in teleost myoblasts, cyanidin (a polyphenolic nutrient) in C2C12, and D-limonene (a flavor and aroma compound found in citrus) in lymphocytes 41,42 . In contrast, phytochemicals such as the polyphenol resveratrol have been shown to have anti-proliferative but pro-differentiating effects in myoblasts, suggesting its potential applicability during the differentiation stage of cultured meat production 43 . Similarly, there is evidence indicating that cannabinoid receptor 1 (CB1) antagonists, such as cannabidiol, a therapeutic phytochemical, can enhance the in vitro differentiation of C2C12 and primary human myoblasts 44 . Depending on the dose, some phytochemicals, such as the nutrient curcumin, can exhibit both proliferative and differentiating effects on primary myoblasts45 Given these roles, it makes sense to consider how engineering goals can enhance both cultured meat products and processes simultaneously.

[0325] This embodiment described herein demonstrates (1) a baseline nutritional analysis of in vitro cultured bovine skeletal muscle cells (proliferating and differentiating cells will be analyzed) to better understand the inherent nutritional differences between cultured meat and conventional meat, (2) testing various engineering goals to enhance nutrition, food quality, or bioactivity to outline the scope of possibilities for modulating the composition of cultured meat, and (3) analyzing the effects of modifications on food functionality, such as oxidation and nutrient bioavailability, and on bioprocess parameters, such as cell growth and differentiation. This work will help elucidate the inherent nutritional characteristics of cultured meat and establish a basis for the broad use of metabolic engineering to modulate composition and bioprocess parameters.

[0326] Cell isolation and culture - Bovine satellite cells (BSCs) were isolated from biopsies of live animals collected at Tufts University Cummings School of Veterinary Medicine using methods previously reported by our group. 46 Briefly, a small piece of tissue (approx. 0.25 cm 3 ) was excised from the semitendinosus muscle of <30-day-old Simmental bulls, minced, and digested in DMEM GlutaMAX (Gibco) with 0.2% collagenase II (Worthington). The resulting cell suspension was diluted in growth medium containing DMEM GlutaMAX with 20% fetal bovine serum (FBS), 1% Primocin (Sigma), and 1 ng / mL FGF-2 (Thermo Fisher), filtered, and plated onto tissue culture flasks. After 24 hours, non-attached cells were transferred to new tissue culture flasks coated with 0.25 μg / cm2 recombinant laminin-511 (Iwai) and left to settle for three days. This pre-coating separated the slowly adhering BSCs from the rapidly adhering fibroblasts. After three days, the BSCs were replenished with growth medium every 2-3 days and passaged onto laminin-coated flasks at 70% confluence. After two weeks, 1% antibiotic-antimycotic (Thermo Fisher) replaced Primocin in the medium. For BSC differentiation, the cells were cultured to confluence and then cultured for one week without medium change. To confirm BSC identity, immunocytochemistry was performed against the satellite cell marker Pax7 (during proliferation) and the myogenic marker myosin heavy chain (MHC; after differentiation).

[0327] Protein analysis - Cells were harvested both before and after differentiation in normal medium, medium with 1×, 2×, and 4× levels of essential amino acids, and medium formulated for an amino acid profile very compliant with human nutritional requirements. 1 Bulk protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer's instructions. Amino acid composition was quantified by HPLC as previously described. 47,48 Briefly, cells were homogenized by sonication, plus an internal standard of known concentration of norvaline, hydrolyzed with HCl, and after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate column, quantified by HPLC relative to amino acid standards (also plus norvaline). Samples were separated on a Nova-Pak C18 (Waters) column and analyzed by fluorescence detection excited at 250 nm and emitted at 395 nm. Bulk protein and amino acid composition were correlated with DNA content (CyQuant assay; Thermo Fisher) to compare undifferentiated and differentiated cells. Conventional meat samples were analyzed and compared with cultured cells.

[0328] Lipid analysis - Lipid analysis was performed using liquid chromatography-mass spectrometry (LC-MS) as previously described. 49 Briefly, undifferentiated and differentiated BSCs were homogenized by sonication, and a lipid internal standard was added to the homogenate (a mixture of components including phosphatidylcholine (PC), phosphatidylethanolamine (PE), ceramide (Cer), phosphatidylserine (PS), phosphatidic acid (PA), and monoacylglycerol, diacylglycerol, and triacylglycerol (MG, DG, and TG)). Cellular lipids were extracted with a solution of chloroform and methanol (2:1, v / v), centrifuged, and the lower phase was collected and combined with an external standard (including isotopically labeled TG and PC). Samples were separated by LC-MS on a BEH C18 column (Waters) relative to lipid standards, followed by quadrupole-time-of-flight detection for quantification. Fatty acid profiles were normalized using internal and external standards and relative to DNA and protein quantification. Again, samples were analyzed at different levels of differentiation and compared with store-bought products.

[0329] The bulk protein in bovine cells was expected to be similar to the bulk protein found in store-bought beef. Specifically, for the bulk protein in mammalian muscle cells, previous reports have suggested an expected value of approximately 20% protein by wet cell weight, which is similar to the concentration found in beef. 9。Meanwhile, reduced extracellular protein accumulation and myotube maturation can lead to a decrease in protein levels. The expected amino acid profile is a combination of the amino acid profile of the cell growth medium (i.e., the input profile) and the amino acid profile of the store-bought product (i.e., the native tissue profile). Among them, a significant but non-stoichiometric increase in cellular amino acids is expected after supplementing the medium with essential amino acids.

[0330] Lipids account for approximately 10% of the dry mass or approximately 2 - 5% of the wet mass of cultured myoblasts (a rough estimate based on 70% water). 50 。This corresponds to 20 to 50 mg / g of protein. Since store-bought samples will contain skeletal muscle cells, extracellular matrix, and intramuscular adipocytes, it is difficult to predict how the lipid content will compare. On the one hand, the higher protein content of store-bought samples may result in relatively less lipid per protein; however, intramuscular adipocytes may produce relatively more lipid per protein. Nevertheless, store-bought meat presents approximately 4% by mass lipid and approximately 20% by mass protein (wet), and the cellular lipid content is expected to be similar to that of conventional meat. 9 。The expected specific lipid ratios will represent a combination of endogenously produced fatty acids and lipids present in the medium. Since medium lipids will be provided by fetal bovine serum and the cells are bovine in nature, these ratios are expected to closely match those of store-bought beef samples. Lipidomics analysis can also be performed on bovine adipocytes instead of BSCs, or in combination with BSCs.

[0331] Genetic modification - Genetic modification of the cell pathway will be achieved by two mechanisms: transposon-mediated transgenesis or CRISPR-dCas9 gene activation. For constitutive expression, transposon-mediated gene insertion will be used due to its non-viral nature, high efficiency, and proven utility in previous carotenoid work. Specifically, depending on the insertion size, the requirement for multiple insertions, and the ease of cloning, the Sleeping Beauty, PiggyBac, or Tol2 transposon systems will be considered. 54-56 。For gene activation, the CRISPR-dCas9 system will be used due to its high specificity, efficiency, and ease of use. 57 。Among them, guide RNAs targeting specific genes will be introduced into the cells together with a plasmid encoding deactivated Cas9 (dCas9) protein linked to an activation domain. These will trigger transcription, thus activating the native gene.

[0332] Molecular cloning will be performed using standard techniques. For gene insertion, the transposable element will contain the gene of interest and a gene resistant to a selectable antibiotic (e.g., puromycin) to enable selection of successfully transfected cells. Cell transfection will be performed using Lipofectamine 3000 reagent (Thermo Fisher). Briefly, cells will be cultured to 85% confluence, washed with OptiMEM medium, and incubated with the transfection reagent containing the purified plasmid. After 6 hours, growth medium will be added to the cells, and after 42 hours, a selection pressure (e.g., puromycin) will be added to the medium. Cells will be cultured under selective pressure for one week, after which expression will be verified by immunocytochemistry, using fluorescent tags, or polymerase chain reaction (PCR). When polycistronic expression is required, genes will be linked by 2A peptide sequences to allow translation of multiple proteins from a single RNA strand encoding multiple genes. 58 . In most cases, the 2A sequence will link the green fluorescent protein (GFP) to the expression cassette such that expression can be screened by simple fluorescence microscopy. Throughout these studies, cell health and myogenic potential will be verified by observing cell proliferation and differentiation.

[0333] Vitamin C: Genes for vitamin C synthesis are present in bovine muscle cells but are silent. From readily available precursors (UDP-glucose) in BSC, this pathway involves five enzymes ( Figure 1 ): UDP-glucose 6-dehydrogenase (UGDH), UDP-glucuronosyltransferase 1 (UGT1A1), aldo-keto reductase family 1 (AKR1A1), regucalcin (RGN), and L-gulono-γ-lactone oxidase (GULO) 59-61 . RNA sequencing data from steer muscle shows UGT1A1 expression to be the lowest of these, two orders of magnitude lower. 62 . Therefore, UGT1A1 will be the first target to be targeted. The second least expressed enzyme is GULO, followed by RGN, and thus if engineering of UGT1A1 proves ineffective, these enzymes will be targeted. It should be noted that this transcriptomics approach to target selection has the drawback that RNA production is not necessarily related to protein production or enzyme activity. This limitation should be borne in mind throughout the process.

[0334] Methods for engineering UGT1A1, RGN, and GULO expression in both cell types will be explored. In the first method, CRISPR-dCas9 gene activation will be performed. Transposon-mediated insertion and constitutive expression of these genes can also be performed as previously described for carotenoids. Gene modification will be performed with Lipofectamine 3000, and vitamin C levels will be analyzed by enzyme assay (Abcam) according to the manufacturer's instructions.

[0335] The bypass biosynthetic pathway is also relevant for vitamin C production. For example, D-threoascorbate 1-dehydrogenase (CDS) and L-galactono-1,4-lactone dehydrogenase (GLDH), which are natural to bacteria and synthesize vitamin C from L-galactose, can be expressed in mammalian cells to increase vitamin C production. 59 The CDS and GLDH enzymes can be constitutively expressed in cells as a bypass for endogenous vitamin C production.

[0336] Curcumin: From the readily available precursor (tyrosine) in mammalian cells, curcumin synthesis involves four enzymes ( Figure 10 ): phenylalanine ammonia-lyase (PTAL), 4-coumarate-CoA ligase (4CL), phenylpropanoyl-diketide-CoA synthase (DCS), and curcumin synthase (CURS3) 63,64 . These genes were codon-optimized, synthesized, and cloned into a polycistronic tetracycline-responsive expression vector. This expression vector was stably inserted into BSC by transfection with Lipofectamine 3000 as previously mentioned. Cells will be cultured and treated with tetracycline during proliferation or after the onset of differentiation. Curcumin will be detected by HPLC based on the previously reported method 65-67 . Briefly, cells will be collected and homogenized by sonication. Berberine will be added to the homogenate as an internal standard, and the solution will be acidified with 6N HCl. Curcumin and berberine will be extracted with ethyl acetate:propanol (9:1, v / v), centrifuged, and the top layer will be collected for HPLC analysis by separation on a Zorbax Eclipse XDB-C18 column (Agilent) and detection at 420 nm. Standard curves of pure curcumin and berberine will be used to accurately quantify the sample curcumin level, which will subsequently be normalized to cellular protein. To increase cellular curcumin levels, cells can be cultured in a medium containing a higher level of tyrosine.

[0337] A bypass pathway using caffeoyl-CoA and feruloyl-CoA as intermediates can be used for endogenous curcumin production in mammalian cells. Similarly, phenylalanine can be used instead of tyrosine as a precursor for p-coumaroyl-CoA.

[0338] Cannabidiol: From the readily available precursor (malonyl-CoA) in mammalian cells, cannabidiol synthesis involves four enzymes ( Figure 10 ): 3,5,7-trioxododecanoyl-CoA synthase (OLS), olivetolic acid cyclase (OAC), cannabigerolic acid synthase (CPGAS), and cannabidiolic acid synthase (CBDAS) 68. In some embodiments, CPGAS can be replaced by the prenyltransferase NphB, which will have the same activity and improved expression. These genes are cloned into a polycistronic expression vector and inserted into the BSC as with other vectors. Cells will be cultured, harvested, and homogenized as in the case of curcumin and vitamin C, and cannabidiol will be analyzed by HPLC using the method described previously. 69 . Briefly, the homogenate will be spiked with a known concentration of the internal standard 4,4'-dichlorodiphenyltrichloroethane (DDT), proteins will be precipitated with cold acetonitrile, and the organic fraction will be separated with hexane. The solution will be centrifuged, the upper layer will be separated and dried under nitrogen and resuspended in acetonitrile. Cannabidiol and DDT will be separated on a C18-PFP HPLC column (ACE) and detected at 220 nm. As with other nutrients, the readings will be normalized relative to a standard curve, internal standard, and cellular protein content.

[0339] Additional targets of interest include D-limonene (a flavor compound that only requires the enzyme and shows increased cell proliferation in some cell types), vitamin B 12 , flavor compounds (such as limonene or cinnamaldehyde), and other therapeutic compounds such as antibiotics.

[0340] Previous carotenoid data showed approximately 35 μg / g protein in the BSC. At least this level of vitamin C (which is natural for mammalian cells) and similar levels of curcumin or cannabidiol are expected in vitro. The carotenoid yield in immortalized mouse muscle cells C2C12 (which works with the BSC) reaches up to 500 μg / g protein, and thus it is expected that appropriate optimization could potentially increase the nutrient levels by an order of magnitude

[0341] Bovine liver presents approximately 0.3 mg / 100 g of vitamin C (and is approximately 20 wt% protein), and thus it is expected that a protein level of at least approximately 15 μg / g in the BSC will not provide the drawbacks of cytotoxicity or negative cellular effects 9 . While there is no data available to predict the potential cytotoxicity of curcumin or cannabidiol synthesis in the BSC, previous studies have shown that a range of curcumin from approximately 0.005 to approximately 1 μg / mL in the culture medium has no negative in vitro effects on myoblasts 45 . Similarly, studies using a range of cell types have explored treatment with cannabidiol in the range of 1 - 50 μM (0.313 - 15.65 μg / mL) without mention of excessive cell death 70 . These correspond to levels significantly lower compared to those produced for carotenoids, and thus it is likely that curcumin and cannabidiol synthesis will need to be controlled in vitro. However, these results do not definitively indicate the cytotoxicity of the endogenous nutrients, and thus higher levels are feasible.

[0342] Growth and Differentiation of Engineered Cells: To understand how metabolic engineering affects growth kinetics, cell growth curves were generated using a dsDNA-based fluorescence assay (Molecular Probes) for engineered cells, control cells, and cells treated with pure compounds (i.e., vitamin C, curcumin, and cannabidiol). Additionally, cell cycle analysis was performed on a NC-3000 image cytometer (ChemoMetec). To explore cellular senescence in engineered cells, quantitative PCR was performed for Pax7, a marker of BSC stemness, throughout the culture of engineered cells, control cells, and compound-treated cells. 73 . To understand the effect on cell differentiation, cells (engineered cells, control cells, and cells treated with pure compounds) were differentiated as described previously and stained for myosin heavy chain (MHC). For further analysis, quantitative PCR was performed for the intermediate and terminal myogenic markers myogenin and MHC.

[0343] Bioavailability: Nutrient bioavailability depends on the surrounding food matrix 4,74 . Therefore, bioavailability analysis of raw and cooked samples of all engineered cells (including carotenoid-producing cells) was performed by in vitro digestion followed by enterocyte absorption analysis. 4,75 . Briefly, cells producing nutrients were cultured, harvested, and the cell pellets were kept "raw" or "cooked" by heating the cell pellets to 100 °C for ten minutes. Samples were added to a saline solution containing 140 mM NaCl, 5 mM KCl, and 150 μM butylated hydroxytoluene. To simulate gastric digestion, HCl was added to a final pH of 2.0, the samples were mixed 36:1 with a solution of 4% w / v pepsin in 0.1 M HCl, and the samples were incubated in an oscillating water bath at 37 °C for one hour. The pH of the digest was raised to 6.9 with NaHCO3, and further intestinal digestion was performed by mixing the samples 7.4:1 with a pancreatin-bile solution (1.2 w / v bile extract and 0.2% pancreatin in 0.1 M NaHCO3) and incubating the samples in an oscillating water bath at 37 °C for two hours.

[0344] Caco-2 intestinal cells (ATCC) will be maintained in a growth medium of DMEM GlutaMAX, 5% FBS, and 1% antibiotic-antimycotic, and cells will be passaged at 70% confluence. To analyze the bioavailability of nutrients in the digest, Caco-2 cells will be cultured to confluence in a porous plate. The digestion solution will be centrifuged, and the supernatant will be diluted 1:4 in Williams' Medium E (Gibco), and the resulting solution will be added to the Caco-2 cell culture after rinsing with PBS. The cells will be incubated at 37 °C for 12 hours, after which the cells will be washed 3 times with PBS, and nutrient analysis will be performed on Caco-2 cells as previously described and on the nutrients under discussion as needed. Similar bioavailability analysis will be performed on control cells in DMSO and pure compounds added to Williams' Medium E.

[0345] Lipid Oxidation: Vitamin C, curcumin, and cannabidiol have all been reported to provide antioxidant activity 76-78 . Since lipid peroxidation is a significant contributing factor to meat quality deterioration during storage, the antioxidant capacity of the additives may have a specific relevance to the food quality of cultured meat 79 . Therefore, lipid peroxidation analysis will be performed using thiobarbituric acid reactive substances (TBARS) assay (Cayman Chemical) according to the manufacturer's instructions, before and after storage at 4 °C for one week and as in the bioavailability analysis, before and after "cooking".

[0346] Previous studies have shown that many phytochemicals have higher bioavailability when derived from non-plant sources because the compounds are not trapped in the cellulose network 80 . Therefore, high bioavailability of endogenously expressed nutrients, including carotenoids, vitamin C, curcumin, and cannabidiol, is expected. An increase in cellular iron bioavailability for vitamin C production is expected 81 . A reduction in lipid oxidation is expected for all cell samples producing vitamin C, curcumin, and cannabidiol 76-78 .

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[0436] Example 3

[0437] Oral administration is an ideal form of drug delivery due to its convenience, non-invasiveness, and safety. However, oral delivery, especially of protein therapeutics, poses significant challenges in terms of drug stability, release kinetics, and permeability. 1 To address these challenges, highly engineered complex drug formulations have been designed, but these are costly to develop and manufacture and still have efficacy issues. To solve these problems, the approach described in this example shows a method based on mammalian cell engineering and the use of in vitro tissue-engineered muscle and fat to produce food for human consumption (termed "cultured meat") that builds on recent advances. Specifically, the cell-level control available in cultured meat production offers the potential to tailor food composition to achieve different outcomes, from personalized nutrition and improved public health to personalized medicine and drug delivery. We have recently demonstrated this possibility by engineering the endogenous synthesis of antioxidant phytonutrients (carotenoids) in bovine muscle stem cells to confer novel micronutrient and functional value to cell-cultured foods. 2See Example 1. This example describes engineering animal muscle cells to synthesize therapeutic agents in a disease-responsive edible formulation. These "therapeutic foods" will provide a novel form of oral (food-based) drug delivery that offers improved drug absorption, patient compliance, drug activity, and cost savings. Therapeutic foods can provide significant benefits in terms of patient compliance, reduced drug costs, and improved pharmacokinetic / pharmacodynamic outcomes.

[0438] Cell engineering. (a) Drug production in mammalian cells: We will engineer bovine muscle cells grown in a defined serum-free medium to synthesize three types of drugs to demonstrate a wide range of approaches. These will be: 1) the physiochemically stable anti-TNFα nanobody V565, which targets IBD, and 2) the proteolytically stable antimicrobial peptide (AMP), herein called AMP16, which is effective against Salmonella infection. 3,4 The cells will be engineered using the methods described herein and molecular biology techniques known in the art. The yield, stability, and activity / biological availability of the cell-encapsulated drug will be analyzed with or without heat treatment (e.g., cooking). The result will be the production of therapeutic compounds in mammalian cells. (b) Optimization of cell-encapsulated therapeutic agents: The drug yield will be adjusted by metabolic engineering. To optimize delivery, the cells will be engineered to produce adjuvants for increasing the stability of protein drugs. Specifically, engineering to produce the serpin protease inhibitor ecotin will be introduced into the drug-producing cells using recombinase-mediated cassette exchange (RMCE) to mitigate drug degradation in the gastric environment. 22 The result will be the control of drug synthesis levels and the improvement of the pharmacokinetic profile.

[0439] Engineering a disease-responsive drug formulation: The drug activity will be made disease-responsive by taking advantage of the elevated levels of specific digestive enzymes present during the disease. 5,6 Specifically, for V565, the previously reported "antibody masking" technique will be employed, in which V565 will be linked to its epitope via a flexible peptide linker rich in trypsin cleavage sites lysine and arginine. 7 When exposed to the trypsin-rich gut (a feature of IBD), the linker will be cleaved and V565 will be activated; however, in the non-inflamed gut, the drug activity will be reduced. The efficacy of the unmasking will be analyzed by in vitro digestion in a trypsin-rich condition nanobody binding affinity assay. 7 For AMP16, a similar approach will express a peptide in the form of a long chain containing repeats of AMP16 linked by an elastase-cleavable domain NPV. 8When exposed to increased levels of elastase, a feature of Salmonella enterica infection, increased linker cleavage will release more AMP16, thereby increasing activity. The cleavability and antibiotic action of the linked AMP16 peptide will be analyzed by in vitro digestion in elastase-rich conditions and application to Salmonella enterica cultures. To explore the possibility of a single therapeutic food acting on multiple pathologies in a disease-responsive manner, cells will be engineered to produce two disease-responsive drugs. In vitro digestion will be performed in an enzyme-rich environment, and the activities of the two drugs will be tested to determine the specificity of the engineered disease-responsive system. The result will be a multi-drug disease-responsive formulation capable of responding to multiple pathologies and demonstrating the flexibility of these systems.

[0440] In vitro evaluation of drug bioactivity: The drug bioactivity will be analyzed using the previously described 3D intestinal tissue model engineered to mimic an inflammatory disease (i.e., IBD) and allowing incorporation of gut-colonizing bacteria (i.e., Salmonella enterica) and immune cells (i.e., macrophages, the proposed target of V565). 9-12 Thus, the actions of anti-inflammatory and antibiotic drugs will be assessable using this system. Trypsin and elastase levels will be regulated to mimic the protease profiles of IBD and Salmonella enterica gastroenteritis, respectively, and drug activity will be analyzed in the inflamed tissue model and in the model inoculated with Salmonella enterica compared to a standard oral formulation as a control group. The relative effects of individual or combinatorial engineering strategies will be evaluated.

[0441] In terms of patient compliance, comfort, safety, and convenience, oral therapeutics offer significant advantages over alternative options (e.g., parenteral delivery). Thus, it is the preferred method of drug delivery and in fact represents more than half of the new drugs recently approved by the FDA. 13 However, oral drug delivery of biologic (e.g., protein or peptide) therapeutics faces significant challenges, meaning that less than 5% of FDA-approved peptide therapeutics can be delivered via the oral route, despite increasing interest in their potential. 14,15 These challenges include the harsh gastrointestinal environment and limited intestinal absorption, resulting in limited bioavailability (<1%). 13 To reconcile the promise of therapeutic biologics with the challenges of their oral delivery, novel drug formulations are needed. Biologics and small molecule therapeutics can complicate clinical prescribing in addition to the fact that drug stability and bioavailability can be significantly affected by co-ingested food. 16。Thus, drug formulations incorporated into a defined food matrix can provide improved pharmacological consistency and predictability, thus improving patient outcomes. Finally, the promising possibility of disease-responsive "intelligent" drug delivery systems that can selectively act on active diseases has attracted interest in recent years 17,18. These drugs will become active only when needed (i.e., when presented to the diseased environment), and can thus improve pharmacological outcomes by reducing off-target effects, avoiding excessive drug exposure, and regulating release to steady-state levels. The edible therapeutic systems described herein will explore the potential of therapy cell-based foods for all three of these drug targets, and both biological and small molecule therapeutics have improved bioavailability and disease specificity.

[0442] Scientific premise - The bioavailability of many oral drugs is significantly affected by the concomitantly ingested food 19-21 。This causes considerable pharmacokinetic uncertainty 22 。The possibility of delivering drugs as part of a defined food matrix provides tight control over food-drug interactions and thus drug delivery. Second, the ability of mammalian cells to synthesize a variety of proteins and metabolites allows complex formulations to address the obstacles associated with oral delivery, while also eliminating the expensive purification steps used for extracting, stabilizing, and formulating oral therapeutics 23 。For example, through metabolic engineering, food-based delivery systems can utilize the simultaneous synthesis of drug-stabilizing formulations, digestive enzyme inhibitors (to improve the intestinal stability of the ingested drug), and permeation enhancers within mammalian cells to improve absorption 3 。Meanwhile, the "natural liposomes" of the cell lipid bilayer provide further control over stability and delivery 24 。Finally, when engineered as an environment-responsive system, these therapeutic foods can provide high specificity for diseases 5 。For example, many lesions cause alterations in the intestinal enzyme profile, and thus protease-triggered drug activity can enhance drug release / bioavailability to achieve disease-responsive activity against a range of lesions 5,6 。

[0443] Therapeutic foods - In addition to the aforementioned benefits, edible cultured meat-based therapeutic formulations will also provide a completely novel class of drug delivery systems that can be of significant use to patients, clinicians, etc. For example, the most common form of drug non-compliance in the elderly is underdosing, mainly due to forgetfulness and polypharmacy 25,26It is possible to directly combine medicines and dietary inseparable drugs into a more ingrained and pleasant daily ritual, which will help alleviate these problems. Additionally, the possibility of formulating foods with complex combinations of disease-responsive drugs can alleviate the problems presented by polypharmacy. Finally, there are solid oral drug swallowing disorders in different age groups, and ad hoc oral drug alterations (i.e., crushing, cutting, dispersing in liquids, etc.) can increase variability and pose risks to patients. 27 Edible formulations will help alleviate these problems by making drug intake simply a matter of chewing food. Cultured meat is a promising emerging technology that has the potential to reduce the environmental, ethical, and public health problems associated with meat production. Specifically, speculative life cycle analyses estimate that cultured meat can reduce greenhouse gas emissions by 78 - 96%, land use by 99%, water use by 82 - 96%, and energy use by 7 - 45% compared to a range of livestock species. 28 。

[0444] Recently, the production of drugs in plants has been proposed as a means of generating edible therapeutics, including antibodies or vaccines. 23,32 For example, vaccines against hepatitis B, Norwalk virus, rabies, and HIV have been expressed in tobacco plants, potatoes, lettuce, tomatoes, and corn, and human clinical trials have confirmed the efficacy of edible vaccines in transgenic potatoes. 33,34 The proposed benefits of these products have largely centered on cost reduction, achieved by eliminating purification steps and leveraging the large scale of agricultural production. However, despite the promising early results for a variety of drug types, the variability in drug concentrations and plant-based differences relative to human glycosylation patterns have contributed to limitations in further development. 32 Edible therapeutics based on mammalian cells offer a potential way to overcome these obstacles. Specifically, the highly controlled conditions present in cultured meat production can circumvent this variability, as cells will grow in a controlled culture environment without variables that affect crops (such as weather patterns). Moreover, the use of animal cells will facilitate proper glycosylation. Finally, the encapsulation and delivery of therapeutics within whole animal cells is an idea that has not been explored, leaving a vast blank space for innovation in targets and applications.

[0445] The inherent cell-level control in cultured meat (at the gene, transcript, protein, and metabolite levels) provides opportunities to regulate composition for various targets, enabling the production of completely novel foods with regulated meat quality and added functionality. To demonstrate this, we recently engineered mammalian cells with metabolic pathways to synthesize three different carotenoids, which are essential micronutrients natural to many plants and some prokaryotes but not animals. 2Ultimately, the novel food functionality in cultured meat products can be applied to nutrition (general and personalized), food quality (i.e., flavor, texture, and other sensory characteristics), diet-related diseases (e.g., diabetes or colorectal cancer), therapeutic agents (as explored in this proposal), edible vaccines, or other enhancements not considered herein.

[0446] Cultivation of bovine muscle and fat cells - So far, our group has explored several topics directly related to this proposal. From the perspective of the culture system, we have established a protocol to generate a population of primary bovine satellite cells (BSCs) that are pure (universal expression of the satellite cell marker paired-box seven (Pax7) in imaged cells) (Figure 13A) and myogenic, capable of generating long multinucleated myotubes that express myosin heavy chain (MHC) and form an actin / myosin contractile apparatus (Figure 13B). 42,43 In addition, we have confirmed a serum-free, growth factor-based medium formulation (B8) for BSC culture based on a previously reported recipe. 44 Moreover, superior BSC growth was observed in B8 medium compared to serum-containing growth medium (GM) (Figure 13C). We have also established an adipogenic transdifferentiation method to convert myogenic BSCs into lipid-accumulating adipocytes by treating the cells with a mixture of free fatty acids (FFAs) (Figure 13D). We have used this method to demonstrate robust and adjustable lipid accumulation (Figure 13E), indicating that a single cell population (i.e., bovine satellite cells) can be used to generate the muscle and fat of cultured meat, which can significantly simplify the industrial production process. 45 This work demonstrates extensive experience in the culture of relevant bovine muscle progenitor cells.

[0447] Metabolic engineering of bovine satellite cells - From the perspective of synthetic biology and metabolic engineering, we engineered BSCs to endogenously synthesize the carotenoids phytoene, lycopene, and β-carotene. 2 For several reasons, these nutrients are attractive targets. First, they each provide unique nutritional value; phytoene has been proposed to act as a UV light protectant, lycopene has been shown to reduce hepatocellular carcinoma and fatty liver disease, and β-carotene is an essential vitamin A precursor in the human diet. 46-48 Second, all three compounds are antioxidant factors. 49 This is particularly relevant for red meat because the key mechanistic link between red meat or processed meat intake and colorectal cancer is lipid oxidation. 50 The antioxidant properties of these compounds also provide value in extending the shelf life of meat because lipid oxidation is the primary cause of non-microbial deterioration over time. 51 .

[0448] Cells were genetically engineered by Sleeping Beauty transposon-mediated transformation to express a single green fluorescent protein (GFP) that does not produce any carotenoids (pGFP), CrtB and GFP that produce phytoene (pCrtB), CrtB, CrtI and GFP that produce phytoene and lycopene (pCrtB / I), or CrtB, CrtI, CrtY and GFP that produce phytoene, lycopene and β-carotene (pCrtB / I / Y). The polycistronic nature of these vectors enabled the use of GFP expression as a marker for vector integration and gene expression. All cells also expressed a puromycin resistance gene to enable selection of engineered cells. We first confirmed the successful synthesis of all three carotenoids in the cells (Figure 14A). Next, we showed that carotenoid levels could be modulated by increasing the puromycin concentration (i.e., increasing the selection pressure to select cells with the transgene localized to more transcriptionally active loci) or by adding ketoconazole, a small molecule that inhibits cholesterol synthesis and competes with carotenoids for a metabolic pathway sharing a precursor, to the medium (Figure 14B). We demonstrated the food functionality of reducing lipid oxidation in vitro (Figure 14C). Along with previous carotenoid work, we have engineered cells to express enzymes involved in cannabidiol synthesis, a small molecule anti-inflammatory and pain-mediating therapeutic agent 52,53 . BSCs were genetically engineered by Sleeping Beauty to express 3,5,7-trioxododecanoyl-CoA synthase (OLS), oleanolate cyclase (OAC), cannabigerolic acid synthase (CPGAS), cannabidiolic acid synthase (CBDAS) and GFP (plasmid name pCBD) to convert malonyl-CoA to CBD 54 . Cells showed successful gene expression (Figure 14D).

[0449] 3D enteropathy model - This model was formed by casting fibrin around a central wire used to generate geometrically engineered hollow lumens in a polydimethylsiloxane (PDMS) mold and then inducing β-sheet formation (Figures 15A - 15B). We have shown that these scaffolds show robust cell development when seeded with human intestinal cells (e.g., organoids), including the production of microvilli, expression of enterocyte markers and production of a mucus layer (Figures 15C, 15D and 15I). When additionally seeded with macrophages and treated with Escherichia coli O111:B4 lipopolysaccharide (LPS) and interferon γ (IFNγ), the cultures were able to mimic enteritis, showing increased secretion of pro-inflammatory cytokines associated with IBD (i.e., CXCL10, IL-1β, IL-6, MCP-2 and MIP-1β) 55When inoculated with oocysts of the apicomplexan parasite Cryptosporidium parvum that infect the intestine, the 3D model shows the ability to support infection for over two weeks. In addition, the parasite shows physiological development, progression, and pathology in vitro, indicating the ability of this system to accurately mimic intestinal infection. 56 Finally, our preliminary data support the use of this model for in vitro testing of the activity of a range of drugs against inflammatory and infectious diseases. Among them, the use of human cells makes this proof-of-concept particularly relevant for edible therapeutics.

[0450] Drug production and optimization in mammalian cells - Since the bioavailability of drugs can vary significantly with the composition of the surrounding food matrix, and since muscle cells have considerable compositional changes during differentiation, drug delivery in undifferentiated and differentiated muscle tissues will be explored. 58 Specifically, we explore drug production in undifferentiated bovine satellite cells (BSCs) and BSCs that have differentiated into multinucleated myotubes (Figure 13A). Non-viral transposon-mediated gene editing technology will be used due to its non-viral nature, high efficiency, and proven utility in previous work. 59,60

[0451] Drug production in mammalian cells

[0452] Isolation and Culture of Bovine Satellite Cells - Bovine satellite cells (BSCs) will be isolated from the Cummings School of Veterinary Medicine at Tufts University using a protocol approved by the Tufts University Institutional Animal Care and Use Committee (IACUC protocol number G2018-36) and reported by our laboratory. 43 Briefly, approximately 0.5 cm of muscle is excised from the semitendinosus muscle of 60-day-old Simmental bulls 3 and transferred to DMEM + GlutaMAX with 1× penicillin / streptomycin in the tissue culture laboratory. The tissue will be minced and digested in a 0.2% collagenase II solution for one hour under regular grinding. The digestion will be suspended with growth medium containing DMEM GlutaMAX supplemented with 20% fetal bovine serum (FBS), 1% Primocin, and 1 ng / mL human FGF-2, and the cells will be filtered and plated onto uncoated tissue culture flasks at a density of 100,000 cells / cm 2 After culturing for 24 hours at 37 °C and 5% CO2, the non-attached cells (which contain slow-adhering satellite cells) will be moved to flasks coated with 0.375 μg / cm 2New tissue culture flasks with recombinant laminin-511. Satellite cells will be left static for three days, after which the cells will have the growth medium replenished every 2-3 days and passaged at 70% confluence. The medium will be supplemented with Primocin only during the first two weeks of culture. Due to the importance of using serum-free medium in cultured meat production, the experiments will be conducted in serum-free growth medium using the previously reported medium (B8), which contains DMEM:F12 (1:1) supplemented with L-ascorbic acid 2-phosphate (200 μg / mL), insulin (20 μg / mL), transferrin (20 μg / mL), sodium selenite (20 ng / mL), FGF-2 (10 ng / mL), neuregulin 1 (NRG-1; 0.1 ng / mL), and TGFβ-3 (0.1 ng / μL) (Figure 13A - Figure 13E) 44 To verify cell properties, immunocytochemistry will be performed on satellite cell markers. The cells will be fixed with 4% paraformaldehyde, permeabilized with Triton-X solution, blocked, and stained for paired box 7 (Pax7). Positive staining for these markers will be considered definitive of the BSC phenotype as previously reported and as visible in Figure 2 43 .

[0453] Myogenic Differentiation - To differentiate satellite cells into myotubes (under serum-free conditions), the cells will be cultured to confluence in B8 and the medium will be changed to the previously reported differentiation medium, which contains Neurobasal / L15 (1:1) basal medium supplemented with epidermal growth factor (EGF; 0.5 ng / mL), insulin-like growth factor 1 (IGF-1; 0.05 ng / mL), and 1% antibiotic-antimycotic 61 . The cells will be differentiated for 1 to 2 weeks, fixed as previously described, and stained for myosin heavy chain (MHC) as previously shown (Figure 2) 43 .

[0454] Gene Modification Techniques - Molecular cloning will be performed using standard techniques. For gene insertion, the transposable element will contain the gene of interest (controlled by the CMV promoter) and a gene resistant to the selection antibiotic (puromycin) to enable selection of successfully transfected cells. Additionally, to simplify future engineering efforts, the puromycin resistance cassette will be engineered to have flippase-recombinase target (FRT) sequences flanking it to allow future gene insertion by recombinase-mediated cassette exchange (RMCE) 62,63Cell transfection will be performed using Lipofectamine 3000 reagent (Thermo Fisher). Briefly, cells will be cultured to 85% confluence, washed with OptiMEM medium, and incubated with the transfection reagent containing the purified plasmid. After 6 hours, growth medium will be added to the cells, and after 42 hours, selection pressure (e.g., puromycin) will be added to the medium. Cells will be cultured under selective pressure for one week, after which expression will be verified by immunocytochemistry, using a fluorescent tag, or polymerase chain reaction (PCR). When polycistronic expression is required, genes will be linked by a 2A peptide sequence to allow translation of multiple proteins from a single RNA strand encoding multiple genes. 64 In most cases, the 2A sequence will link the green fluorescent protein (GFP) to the expression cassette so that expression can be screened by simple fluorescence microscopy. Throughout these studies, cell health and myogenic potential will be verified by observing cell proliferation and differentiation.

[0455] V565 Production and Activity -V565 amino acid sequence (DVQLVESGGGLVQPGGSLKLSCAASGFDFSSHWMYWVRQAPGKELEWLSEINTNGLIT HYGDSVKGRFTVSRNNAANKMYLELTRLEPEDTALYYCARNQHGLNKGQGTQVTVSS; SEQ ID NO:7) 3,65 It will be codon-optimized for bovine expression, modified with a 6xHis-tag sequence for quantification, and cloned into the polycistronic expression vector of V565 and GFP as described above. Cells will be transfected, selected, and gene expression will be verified by fluorescence imaging. Cells will be cultured, harvested (10 million cells, as proliferating BSCs or differentiated myotubes), and V565 production will be quantified by competitive His-tag ELISA on cell lysates. As an orthogonal measure of gene expression, GFP will be quantified from cell lysates using a GFP quantification kit (abcam#ab235672). The 2A sequence allows stoichiometric expression of multiple proteins and should allow GFP quantification to act as an accurate representative of V565 levels. 66 Readouts will be normalized relative to a standard curve, cell count, and cell protein content (determined by Bradford assay).

[0456] To analyze V565 in an edible form, cells (10 million cells) will be harvested and kept "raw" or "cooked" by heating the cell pellet to 100 °C for 1 to 10 minutes. Samples will be lysed using standard cell lysis buffer or by in vitro digestion based on previously reported methods. 67,68Briefly, the sample was added to a saline solution containing 140 mM NaCl, 5 mM KCl, and 150 μM butylated hydroxytoluene. To simulate gastric digestion, HCl was added to a final pH of 2.0, the sample was mixed 36:1 with a solution of 4% w / v pepsin in 0.1 M HCl, and the sample was incubated for one hour in an oscillating water bath at 37 °C. The pH of the digest was raised to 6.9 with NaHCO3, and further intestinal digestion was carried out by mixing the sample 7.4:1 with a pancreatin-bile solution (1.2 w / v bile extract and 0.2% pancreatin with 550 U / mL trypsin and 3.3 U / mL chymotrypsin in 0.1 M NaHCO3) and incubating the sample for two hours in an oscillating water bath at 37 °C. After in vitro digestion, the sample was analyzed for binding activity by His-tag quantification and by competitive ELISA as previously described 69 Briefly, plates were coated with TNFα, washed, blocked, and treated with the digest. Anti-His secondary antibody and horseradish peroxidase (HRP)-conjugated tertiary antibody were added, followed by treatment with 3,3′,5,5′-tetramethylbenzidine (TMB). Absorbance at 450 nm was used to quantify binding. Results were compared between cooked, uncooked, digested, and undigested samples

[0457] AMP16 Production and Activity -AMP16 peptide amino acid sequence (IRPI IRPI IRPI IRPI IRPI IRPI IRPI; SEQ ID NO:8) 4 It was codon-optimized for bovine expression and cloned into two polycistronic expression vectors containing or not containing a 6xHis-tag for AMP16 and GFP. Cells were transfected, selected, and gene expression was verified by fluorescence imaging. As with V565, cells were cultured, differentiated into muscle and fat, harvested (10 million cells), and expression levels were analyzed using His-tag quantification and GFP quantification. The use of both methods would allow quantification of the untagged peptide, as the 6xHis-tag could ultimately confound peptide activity. As with other drugs, the readings were normalized relative to a standard curve, cell count, and cell protein content

[0458] To analyze the antimicrobial activity of AMP16 in an edible form, cells were harvested (10 million cells), treated, and "digested" as with V565 cells. After in vitro digestion, paper disks were soaked in the digest and placed on agar plates previously inoculated with Salmonella enterica and incubated at 37 °C. After incubation for 1 to 24 hours, the zones of inhibition were measured and compared between samples

[0459] It has previously been shown that while V565 is largely stable in the gastric environment, it is sensitive to pepsin. 3 Similarly, degradation of small peptide therapeutics in gastric juice has been shown to be largely related to pepsin. 70 Thus, delivery of both V565 and AMP16 can be greatly improved by inhibiting pepsin activity during digestion.

[0460] Enhanced Drug Yield - The drug yield in cells will be enhanced by increasing gene expression in culture and increasing the levels of drug precursors. To increase gene expression, cells will be cultured in the presence of an increased level of the selection antibiotic puromycin, such that cells in which the transgene has inserted into a more transcriptionally active locus are selected. Our previous carotenoid work has shown that this can increase gene expression (as shown by GFP expression) by approximately two-fold, and subsequently increase carotenoid levels by approximately three-fold (Figure 14B). To increase the levels of the drug precursors of V565 and AMP16, the culture medium will be supplemented with the most prevalent amino acids in these two drugs, as amino acid depletion is a potential bottleneck in therapeutic protein production. 71 Specifically, glycine, leucine, and serine are the three most common amino acids in V565 (representing more than 30% of the total sequence), and thus the culture medium will be supplemented with additional G, L, and S (100 - 200% of the standard medium levels). For AMP16, isoleucine, arginine, and proline together represent 100% of the total sequence, and thus the culture medium will be supplemented with these amino acids (100 - 200% of the standard medium levels). The drug yield will be analyzed as previously described.

[0461] Reduced Gastric Degradation - As previously described, pepsin inhibition will be engineered into cells by generating serpin and using the Flp-In system (ThermoFisher). 72Briefly, the serpin Tsp03044 sequence (Genbank accession number XM_003379333) was codon-optimized and cloned into the pcDNA5 / FRT plasmid (ThermoFisher). This plasmid and the pOG44 plasmid encoding the Flp recombinase (ThermoFisher) were co-transfected into engineered cells expressing V565 or AMP16. Flp-mediated RMCE will facilitate the insertion of Tsp03044 and the hygromycin resistance gene (from the pcDNA5 / FRT plasmid) into cells producing V565 and AMP16, and thus treating the cells with puromycin and hygromycin will ensure the production of V565 or AMP16 as well as Tsp03044. Tsp03044 expression will be verified by RT-PCR. The efficacy of the serpin in maintaining drug activity will be analyzed by in vitro digestion and activity assays as previously described. The results will be compared with undigested cell lysates and cell lysates treated only with pepsin.

[0462] Alternative IBD-targeting drugs can also be expressed in mammalian cells, including the peptide PTG-200 73 or a microbial anti-inflammatory molecule (MAM) or a constitutive peptide thereof, which has been shown to provide anti-inflammatory activity in a murine colitis model 74 Alternative antibiotics can include other small peptide antibiotics or small molecule antibiotics such as penicillin 75 In addition, small molecule therapeutic agents can be explored, including cannabidiol or anti-inflammatory compounds such as curcumin or the immunosuppressant FK506 53 ,76 ,77 If puromycin treatment proves ineffective in increasing drug yield, then the protein copy number can be increased by engineering the plasmid to contain multiple copies of the drug sequence, in a polycistronic form separated by 2A sequences, or each copy having a different promoter. If protein stability is insufficient after serpin synthesis, then the cells can be encapsulated in alginate or chitosan scaffolds to slow down enzyme degradation, which are promising scaffolds for cultured meat production 13,78 Finally, alternative cell types can be considered, such as trans-differentiated bovine adipocytes, immortalized bovine muscle cells, or immortalized mouse muscle cells (C2C12).

[0463] Engineering of Disease - Responsive Drug Formulations - Many lesions cause alterations in the intestinal enzyme profile. Specifically, fecal profiling has shown a >5-fold increase in trypsin-like protease in patients with IBD compared to healthy patients, and Salmonella enterica infection in the gut has been shown to increase elastase levels by approximately 2-fold 5,6In view of this, protease-triggered drug release / activity can be used to tune the drug to achieve disease-responsive activity in an edible therapeutic agent. Disease responsiveness will be engineered into the V565 and AMP16 systems for IBD and Salmonella enterica infection, respectively.

[0464] Engineering of the IBD Response of V565 - The disease response to IBD will utilize the previously reported epitope masking strategy 7 Specifically, V565 will be synthesized conjugated to a fragment of its target antigen, TNFα, such that the ability of V565 to bind TNFα in tissues is masked. The linker used will be readily cleaved by trypsin, such that when this construct is exposed to a trypsin-rich intestinal environment (as seen in IBD), the antigen will be released and V565 will be free to bind tissue TNFα. To engineer these systems, the human TNFα sequence will be codon-optimized for bovine expression and attached to the V565 sequence along with a flexible linker (GGG SGG GSG GGS GGKGGK GGK GGK GGG SGG GSG AQG; SEQ ID NO:9), which has numerous lysine residues to facilitate trypsin cleavage. The cloned sequences will be engineered into cells as previously described (including RMCE-mediated serpin integration). To analyze the drug response to trypsin, cells will be cultured, harvested (10 million cells, before or after differentiation), heated or not heated, lysed, and treated with a series of trypsin concentrations. Drug activity will be analyzed by competitive ELISA as previously described. Meanwhile, cells will undergo harvesting, cooking, and in vitro digestion in different levels of trypsin, and the activity will be evaluated.

[0465] Engineering of the Salmonella Response of AMP16 - The response to Salmonella enterica infection will similarly utilize the altered enzyme levels in the disease. Specifically, it has been demonstrated that Salmonella enterica infection is associated with increased enteric elastase. Thus, the elastase-cleavable linker NPV will be engineered into the AMP16 peptide sequence 8 Specifically, repeats of AMP16 with the NPV sequence between every 10× 28-mer peptides will be assembled and cloned into a plasmid. Cells will be transfected, modified by RMCE to contain Tsp03044, cultured, harvested (10 million cells, before or after differentiation), heated or not heated and lysed as previously described. The cell lysates will be treated with different levels of elastase, and the drug activity will be analyzed by inhibition zone analysis as previously described. Meanwhile, cells will undergo harvesting, cooking, and in vitro digestion in different levels of elastase, and the drug activity will be evaluated.

[0466] Combining Drugs into a Disease - Responsive System- To engineer a truly "intelligent" system that can target multiple lesions in a disease-responsive manner for engineering modification, we combined the above components into a single cell line. Specifically, the V565-epitope, 10xAMP16, and GFP will be linked by a 2A sequence to form a single polycistronic fragment. This fragment will be cloned into the Sleeping Beauty transposon vector as described previously and transfected into cells. The cells will undergo transfection and RMCE to generate a single cell line containing all the previously described disease-responsive drug components. The cells will be cultured, collected (10 million cells, before or after differentiation), heated or not heated, and processed by in vitro digestion at a series of trypsin and / or elastase concentrations to represent patients with IBD, Salmonella enterica infection, both, and neither. The V565 and AMP16 drug activities will be analyzed as described previously.

[0467] Alternatively, copies of V565 with a protease-cleavable linker can be explored as a way to mask the binding domains of these nanobodies. Alternatively, if the ligation of the AMP16 peptide proves ineffective in conferring disease responsiveness during Salmonella enterica infection, then antimicrobial nanobodies with activity against Escherichia coli infection can be explored, where enzyme-cleavable epitope masking is similar to that explored for V565 79 . Additionally, alternative enzyme-cleavable linkers or environmental means (such as pH changes) can be utilized to engineer disease responsiveness 3,80-83 . If synthesizing two-drug systems in a single cell proves challenging, then two single-drug system cell populations can be developed and subsequently combined into a multi-drug food.

[0468] In Vitro Evaluation of Drug Bioactivity - Functional in vitro 3D human intestinal models are a promising option for early validation of edible drug formulations, due to their use of human cells and the ability to mimic relevant tissue characteristics and geometries 84 . For this purpose, the 3D intestinal tissue model (Figures 15A - 15L) was previously developed by this group. This model will be applied to evaluate the efficacy of engineered therapeutic foods in vitro.

[0469] Cells - Human colonoids previously isolated and cultured by this group will be cultured in Matrigel droplets and appropriate media as described previously 55 . Human monocytes previously isolated and cultured by this group will be cultured in appropriate media as described previously 55 . Monocytes will be differentiated into macrophages by treatment with 50 ng / mL macrophage colony-stimulating factor; for M1 polarization, macrophages will subsequently be cultured in media supplemented with lipopolysaccharide (LPS) and interferon interferon γ (IFNγ).

[0470] IBD 3D Model - An intestinal model will be fabricated and seeded with human cells as previously described. 55 Briefly, a fibroin solution extracted from Bombyx mori cocoons will be poured into a PDMS cylindrical mold containing a 2-mm diameter Teflon-coated steel wire that is inserted through the cross-section of the mold to form a channel. The solution will be frozen, lyophilized, and treated under high pressure to induce β-sheet formation. The scaffolds will be immersed in water and cut into 8-mm segments. A biopsy punch will be used to cut out an “inner scaffold” (8 mm in length, 6 mm in outer diameter, 2 mm in inner lumen) and an “outer scaffold” (8 mm in length, 10 mm in outer diameter, 6 mm in inner diameter). For scaffold seeding, following the method reported previously, human colon organoids will first be seeded into the 2-mm lumen. 55 Briefly, the silk scaffolds will be coated with collagen gel, human colon organoids will be dissociated in trypsin-EDTA, and the cell suspension will be pipetted to coat the inner lumen of the inner scaffold as well. These scaffolds will be cultured in an appropriate medium for one week as previously reported. Subsequently, monocytes will be suspended in collagen gel and seeded throughout the outer scaffold body. Monocytes will differentiate into uncommitted macrophages through a six-day culture in an appropriate medium as previously reported. 55 The outer scaffold will then be cultured in macrophage medium with LPS and IFNγ to induce polarization. The outer scaffold seeded with cells will be combined with the inner scaffold seeded with cells to form a complete IBD model. As previously described, the model will be validated by immunofluorescence, cytokine analysis, and mucus thickness measurement and subsequently used to test the efficacy of therapeutic foods. 55 .

[0471] Salmonella Infection 3D Model - The scaffolds will be prepared and seeded as described above, but without LPS or IFNγ treatment. The model will be validated by immunofluorescence to ensure accurate intestinal recapitulation and to obtain an understanding of the number of cells on the lumen. With this information, the inner lumen of the scaffold will be infected by inoculating Salmonella enterica serovar Typhimurium at a multiplicity of infection of 10 to 100, and the dose will be adjusted according to the epithelial cell response in culture for up to one week. 85 .

[0472] Drug Delivery in Disease Models- All forms of drug delivery will be explored in the 3D model, including pure compounds, cell-encapsulated therapeutic agents that are not disease-responsive and have or do not have excipients, and cell-encapsulated therapeutic agents that are disease-responsive and have excipients (either alone or in engineered cells with all drug components). The cells will be cultured, collected (10 million cells, before or after differentiation) and heated or not heated, as previously described. The samples will be dissolved or digested in vitro with a series of trypsin and / or elastase concentrations. The medium will be removed from the scaffolds and replaced with the digest for 2, 3, or 4 hours to obtain a series of viable intestinal retention times, after which the digest will be removed and replaced with cell medium. One hour, two hours, six hours, and 24 hours after treatment, the drug efficacy of the scaffolds will be analyzed.

[0473] Analysis of Drug Action - The effect of the drug on IBD will be analyzed by profiling inflammatory cytokines in treated and untreated scaffolds. Specifically, interleukin 1β and 6 (IL-1β and IL-6), monocyte chemoattractant protein 1 and 2 (MCP-1 and MCP-2), and macrophage inflammatory protein 1β (MIP-1β / CCL4) will be measured by commercially available chemiluminescence arrays. Additionally, epithelial integrity will be analyzed by immunohistochemistry for mucin-2, CD68, and ZO-1. The effect of the drug on Salmonella enterica serovar Typhimurium infection will be analyzed by homogenizing the scaffolds in a lysis solution containing Triton-X, serially diluting the homogenate, and determining the colony-forming units of Salmonella enterica serovar Typhimurium by plating on LB agar plates. 86 The results will be compared between all drug types, samples, and conditions.

[0474] If the 3D disease model proves inapplicable to this study, then a 2D study can be performed using Caco-2 intestinal cells and a Transwell system. If single dosing proves ineffective in generating a response, then dosing can be performed with a larger cell harvest, or the edible therapeutic agent can be redosed (i.e., two to three times daily at "meal times" for one week). Finally, if the in vitro model confirms ineffectiveness, then ex vivo analysis can be performed using biopsies obtained during routine endoscopy of consenting IBD patients. Specifically, the biopsies can be cultured ex vivo, treated with the in vitro digest of the therapeutic food sample, and analyzed for inflammatory and drug activity markers as previously reported. 3,87 Finally, in vivo animal models can be considered.

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[0565] Example 4

[0566] The embodiments described herein demonstrate

[0567] Preliminary Data - So far, our team has explored several topics directly related to this proposal. From the perspective of the culture system, we have adapted serum - free growth of invertebrate muscle cells for use in cultured meat and established a protocol for generating a population of highly myogenic primary bovine satellite cells (BSCs) (Figures 16A - 16B) 22,23 . Based on a previously reported formulation, we have further confirmed a serum - free, growth - factor - based medium formulation (B8) for BSC culture (Figure 16A) 24 . In the case of this formulation, we have observed superior BSC growth compared to serum - containing media and shown that growth is concentration - dependently dependent on fibroblast growth factor 2 (FGF - 2) (Figure 16B).

[0568] Furthermore, we have established an adipogenic trans - differentiation method for converting myogenic BSCs into lipid - accumulating adipocytes by treating the cells with a mixture of free fatty acids (FFAs) (Figure 17A). We have used this method to show robust lipid accumulation, indicating that a single cell population (i.e., satellite cells) can be used to produce both the muscle and fat of cultured meat, which can significantly simplify the industrial production process (Figure 17B) 25 .

[0569] From a nutritional perspective, we have genetically engineered BSCs to endogenously synthesize the carotenoids phytoene, lycopene, and β - carotene and shown a reduction in the resulting lipid oxidation (a key mechanistic driver linking red meat intake and colorectal cancer; Figure 3) 19,26 .

[0570] Finally, from a quality perspective, we have shown that exogenous heme proteins in the medium can improve the color similarity of cultured meat compared to conventional beef (Table 2) 23 .

[0571] Table 2: Color analysis of 3D BSC tissue cultures grown with or without 3 mg / mL hemoglobin (Hb) or myoglobin (Mb). Data for L* (lightness), a* (red / green spectrum), and b* (blue / yellow spectrum) were used to generate ΔE, a measure of color similarity to raw or cooked beef. A low ΔE indicates more similarity.

[0572]

[0573] Methods

[0574] Cell culture

[0575] Bovine satellite cells - Bovine satellite cells (BSCs) will be isolated from the Cummings School of Veterinary Medicine at Tufts University using a protocol approved by the Tufts University Institutional Animal Care and Use Committee (IACUC protocol number G2018-36) and reported by our laboratory 23 . Briefly, approximately 0.5 cm of muscle will be excised from the semitendinosus muscle of a <60-day-old Simmental bull and transferred to DMEM + glutamax with 1× penicillin / streptomycin in the tissue culture laboratory. The tissue will be minced and digested in a 0.2% collagenase II solution for one hour under regular agitation. The digestion will be stopped with growth medium containing DMEM glutamax supplemented with 20% fetal bovine serum (FBS), 1% Primocin, and 1 ng / mL human FGF-2, and the cells will be filtered and plated onto uncoated tissue culture flasks at a density of 100,000 cells / cm 3 . After incubation at 37 °C and 5% CO2 for 24 hours, the non-adherent cells (which include slowly adherent satellite cells) will be moved to a new tissue culture flask coated with 0.375 μg / cm 2 of recombinant laminin-511. The satellite cells will be allowed to settle for three days, after which the cells will be fed with growth medium every 2-3 days and passaged at 70% confluence. The medium will be supplemented with Primocin only for the first two weeks of culture. 2 To verify cell identity, immunocytochemistry will be performed on satellite cell markers. Cells will be fixed with 4% paraformaldehyde, permeabilized with Triton-X solution, blocked, and stained for paired box 7 (Pax7). Positive staining for these markers will be considered definitive of the BSC phenotype as previously reported and as visible in

[0576] Figure 1 as shown 23 .

[0577] ​Myogenic differentiation - To differentiate satellite cells into mature myotubes (under serum-containing conditions), cells were cultured to confluence and the medium was changed to a differentiation medium containing DMEM GlutaMAX supplemented with 2% FBS and 1% antibiotic-antimycotic. To differentiate satellite cells under serum-free conditions, the previously reported differentiation medium contained Neurobasal / L15 (1:1) basal medium supplemented with epidermal growth factor (EGF; 0.5 ng / mL), insulin-like growth factor 1 (IGF-1; 0.05 ng / mL), and 1% antibiotic-antimycotic. 54 Cells were differentiated for 1 to 2 weeks, fixed as previously described, and stained for myosin heavy chain (MHC) as previously reported and as visible in Figures 16A - 16B. 23 .

[0578] Adipogenic transdifferentiation - Although satellite cells are preferably myogenic, we successfully induced lipid accumulation using a differentiation medium containing a combination of free fatty acids (FFAs) (Figures 17A - 17B). Specifically, cells were treated with a 3-FFA mixture of equal concentrations of linoleic acid, erucic acid, and elaidic acid (125 μM) for six days to induce robust intracellular lipid accumulation. This adipogenic transdifferentiation protocol will be further optimized by testing various concentrations of oleic acid, myristoleic acid, linoleic acid, erucic acid, and elaidic acid through a fractional factorial experimental design. Lipid accumulation will be qualitatively examined by Oil Red O staining and quantitatively analyzed using an AdipoRed plate reader (Lonza). Throughout this proposal, transdifferentiating satellite cells into mature adipocytes will enable the generation of the muscle and fat components of cultured meat from a single cell source, thereby increasing protocol efficiency and impact.

[0579] Serum-free medium

[0580] Media formulation with serum substitutes - A growth factor-dependent serum-free medium (B8) will be generated based on a previously reported formulation and as successfully demonstrated by our group (preliminary data; Figures 16A - 16B). 24 Briefly, B8 medium will contain DMEM:F12 (1:1) supplemented with L-ascorbic acid 2-phosphate (200 μg / mL), insulin (20 μg / mL), transferrin (20 μg / mL), sodium selenite (20 ng / mL), FGF-2 (10 ng / mL), neuregulin 1 (NRG-1; 0.1 ng / mL), TGFβ-3 (0.1 ng / μL), and insulin-like growth factor 1 (IGF-1; 10 ng / mL). Formulations lacking one or more growth factors will be generated simultaneously, as well as a formulation lacking all growth factors but containing a previously reported serum substitute composed of rapeseed peptide fraction, maitake extract, and sericin.

[0581] A culture medium containing rapeseed peptide moieties will be produced as previously described 41 . Briefly, rapeseed protein concentrate will be hydrolyzed with Alcalase 2.4L (Novozymes), an alkaline protease, at 60 °C and a pH of 9.0 for five hours. The components will be precipitated at a pH of 4.0 and centrifuged. The supernatant will undergo 3 kDa membrane ultrafiltration and 0.5 kDa membrane nanofiltration. The nanofiltration retentate will again undergo ultrafiltration (1 kDa), and the retentate of this final filtration step will be added to a growth factor-free basal medium at a concentration of 10 mg / mL. A culture medium containing shiitake mushroom extract will be produced as previously described 55 . Briefly, dried shiitake mushrooms will be incorporated in a growth factor-free basal medium at a concentration of 0.2% w / v and sterilized through a 0.2 μm filter. Finally, a culture medium containing sericin will be produced by diluting purified sericin, which can be obtained from silk (i.e., as a by-product of the textile industry) or produced recombinantly, at a concentration of 30 μg / mL in a growth factor-free basal medium

[0582] Formulations with different growth factor and serum replacement concentrations and combinations will be produced by combining the culture media. BSCs will be cultured in these culture medium formulations as previously described

[0583] Bioprocess kinetics - To understand how the culture medium formulations affect bioprocess growth kinetics, cell growth curves will be generated using dsDNA-based fluorescence assay analysis of engineered and primary BSCs cultured in serum control medium and medium with serum and antibiotic replacements. To understand the effect of these culture systems on mitotic activity, cell cycle analysis will be performed simultaneously on a NC-3000 image cytometer (ChemoMetec). To investigate cellular senescence in engineered cells, quantitative PCR will be performed on Pax7, a marker of BSC stemness, throughout the culture of control cells and cells treated with serum and antibiotic replacements 59 . Based on preliminary data for B8, we expect to obtain cell growth similar to or better than that of serum-containing media

[0584] In conjunction with the cell analysis mentioned above, bioprocess kinetics will be tested throughout the culture by analyzing nutrient uptake and by-product accumulation in the culture medium. Specifically, glucose uptake, glutamine uptake, lactate production, and ammonia production will be analyzed for all culture medium formulations and all cell types using enzyme assay kits throughout the culture 60 .

[0585] Cell differentiation analysis - To understand the effect of medium composition on cell differentiation, cells cultured in all medium formulations will be differentiated (myogenic and adipogenic) as described previously and analyzed for relevant markers. For myogenic differentiation, myogenin and myosin heavy chain (MHC) expression will be evaluated by immunostaining and quantitative polymerase chain reaction (qPCR), and myotube formation will be analyzed by determining the fusion index or the percentage of nuclei within fused myotubes. 63 For adipogenic differentiation, peroxisome proliferator-activated receptor γ (PPARγ) expression will be analyzed by immunostaining and qPCR, and lipid accumulation will be analyzed by Oil-Red O staining, extraction, and quantification. 64 We expect comparable differentiation capabilities of cells cultured in serum-containing or serum-free medium formulations.

[0586] Genetic modification of serum substitutes - To explore cost-effective serum-free medium development orthogonal to the aforementioned serum substitutes, BSCs will be engineered to endogenously produce growth factors and growth factor receptors. These "self-signaling" cells will require less medium input and can thus further reduce culture costs. The goal will correspond to B8. Specifically, cells will be engineered to produce the growth factors FGF-2, TGFβ-3, NRG-1, and IGF-1, as well as the signaling and transport proteins insulin and transferrin.

[0587] Constitutive gene expression will be achieved by Sleeping Beauty transposon-mediated gene insertion due to the non-viral nature, high efficiency, and utility demonstrated in our preliminary work of this system. 56,57 Molecular cloning will be performed using standard techniques. For gene insertion, the transposon element will contain the gene of interest (e.g., FGF-2) and a gene resistant to a selection antibiotic (i.e., puromycin) to enable selection of successfully transfected cells. Cell transfection will be performed using Lipofectamine 3000 reagent (Thermo Fisher, #L3000008). Briefly, cells will be cultured to 85% confluence, washed with OptiMEM medium, and incubated with the transfection reagent and purified plasmid. After 4 to 6 hours, growth medium will be added to the cells, and after 48 hours, a selection pressure (i.e., puromycin) will be added to the medium. Cells will be cultured under selective pressure for one week, after which expression will be verified by immunocytochemistry, using fluorescent tags, or polymerase chain reaction (PCR). When polycistronic expression is required (i.e., for simultaneous expression of all growth factors), genes will be linked by 2A peptide sequences to allow translation of multiple proteins from a single RNA strand encoding multiple genes. 58. In most cases, the 2A sequence will ligate the green fluorescent protein (GFP) to the expression cassette such that expression can be screened by simple fluorescence microscopy. Finally, the transgene will be inserted under the control of an inducible promoter that can be "switched off" for cell differentiation to halt the expression of proliferation-promoting factors. The successfully engineered cells will be cultured in the various growth factor-free media formulations described herein.

[0588] Antibiotic-free medium

[0589] Medium formulations of antibiotic alternatives - Cinnamon, monolaurin (a derivative of coconut lauric acid), and honey are natural compounds that have shown potential utility as food safety antimicrobials 46 ,65 ,66 . Each of these substances will be explored as a medium additive to prevent microbial contamination, both individually and in combination. It will first be added to B8 serum-free medium with growth factors and used to culture BSC. A dose-response curve (DRC) will be generated to determine the effect of each additive on cell health and to identify the maximum concentration that can be used without hindering growth, survival, and differentiation. After evaluating the antimicrobial activity, the successful candidates will be incorporated into the serum-free and growth factor-free media described herein and the effect on culture performance will be evaluated again.

[0590] Analysis of antimicrobial action in cell cultures - The antimicrobial activity of media supplemented with cinnamon, monolaurin, and honey will be tested using the agar well diffusion assay. Bacterial and yeast extracts will be spread across the entire surface of an agar plate, and a biopsy punch will be used to remove 6 mm diameter agar cylinders. Subsequently, 100 μL of media containing various concentrations of the antimicrobial agent will be added to the wells, and the zone of inhibition will be measured after an 18 to 24-hour incubation period 69 . The efficacy of alternative antimicrobials is compared to the common cell culture antimicrobials penicillin-streptomycin (Pen-Strep) and antibiotic-antimycotic (Anti-Anti).

[0591] Analysis of antimicrobial activity in cooked products - To further evaluate the efficacy of alternative antimicrobials and their utility in the commercial production of cultured meat, we will test the ability of the target antimicrobials to inactivate Escherichia coli and / or enhance heat destruction of E. coli in cell-based and conventional meat samples. Antimicrobials used as media additives (cinnamon, fatty acids, honey) will be tested. Additionally, cell-based samples containing engineered cells will be tested without the addition of exogenous compounds. All samples will be inoculated with a mixture of E. coli strains isolated from meat and dairy products, treated with various concentrations of each antimicrobial as appropriate, and then stored at 4 °C for 5 days. The E. coli O157:H7 content will be quantified by surface plating of homogenized samples and colony counting before and after cooking at multiple time points.

[0592] Analysis of cell growth and differentiation - The effect of each alternative antimicrobial on cell proliferation (i.e., growth curve analysis by dsDNA-based fluorescence assay and nutrient / waste uptake / production analysis by enzymatic assay) will be evaluated. The effects on myogenic and adipogenic differentiation will be evaluated by immunostaining for myosin heavy chain (MHC) or Oil-O Red staining and qPCR for MHC and PPARγ. These analyses will be used to determine the concentrations of cinnamon, honey, and monolaurin that can be added to the medium without having an adverse effect on performance.

[0593] Genetic modification of antibiotic alternatives - As a secondary strategy for media additives described herein, metabolic engineering of BSCs will be used to achieve constitutive endogenous production of food safety compounds with antimicrobial activity. Specifically, as utilized in our previous work, the metabolic pathways for synthesizing cinnamaldehyde and limonene from common precursor molecules will be engineered into BSCs using Sleeping Beauty transposon-mediated gene insertion. The synthesis of cinnamaldehyde from the precursor phenylalanine will be achieved by inserting a three-enzyme pathway: phenylalanine ammonia-lyase, 4-coumarate--CoA ligase, and cinnamoyl-CoA reductase. The synthesis of (+)-limonene from the precursor geranyl pyrophosphate will be achieved by inserting a single enzyme: (R)-limonene synthase. The concentrations of cinnamaldehyde and limonene secreted into the medium will be evaluated by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GM-MS), respectively 67,68 . Cell health and development will be analyzed as previously described.

[0594] Food quality analysis

[0595] Proteins and amino acids - Engineered and primary cells will be collected after culturing in all medium formulations and before and after myogenic and adipogenic differentiation. Bulk protein will be quantified using the Pierce BCA protein assay. Amino acid composition will be quantified by HPLC as previously described70,71 Briefly, cells will be homogenized by sonication, spiked with a known concentration of the internal standard norvaline, hydrolyzed with HCl, and quantified by HPLC relative to amino acid standards (also spiked with norvaline) after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Samples will be separated on a Nova-Pak C18 (Waters) column and analyzed by fluorescence detection excited at 250 nm and emitting at 395 nm. The bulk protein and amino acid composition will be correlated with DNA content (CyQuant assay; Thermo Fisher) to compare undifferentiated and differentiated cells. Conventional meat samples will be analyzed and compared to cultured cells.

[0596] We expect the bulk protein in bovine cells to be similar to that found in store-bought beef, but reduced extracellular protein accumulation may result in lower protein levels. We expect the amino acid profile to be a combination of the amino acid profile of the cell growth medium and that of the store-bought product.

[0597] Lipid and fatty acid - lipid analyses will be performed using liquid chromatography - mass spectrometry (LC - MS) as previously described 72 Briefly, undifferentiated and differentiated (myogenic and adipogenic) BSCs from all culture conditions will be homogenized by sonication, and a lipid internal standard will be added to the homogenate (containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), ceramide (Cer), phosphatidylserine (PS), phosphatidic acid (PA), and monoacylglycerol, diacylglycerol, and triacylglycerol (MG, DG, and TG)). Cellular lipids will be extracted with a solution of chloroform and methanol (2:1, v / v), centrifuged, and the lower phase will be collected and combined with an external standard (containing isotopically labeled TG and PC). Samples will be separated by LC - MS on a BEH C18 column (Waters) relative to lipid standards, followed by quadrupole time - of - flight detection for quantification. Fatty acid profiles will be standardized using internal and external standards and relative to DNA and protein quantification. Again, samples will be analyzed at different levels of differentiation and compared to store - bought products. Based on previous data comparing murine adipose tissue and murine adipose progenitors before and after differentiation, we expect the pre - differentiation bovine lipid profile to be significantly different from store - bought beef but similar after differentiation 73 .

[0598] Cholesterol will be analyzed by fluorometric assay (Abcam, #ab65390) and compared to total lipids measured by fluorometric assay (Abcam, #ab242307). Samples will be analyzed at different levels of differentiation and compared to store - bought products before and after cooking again. Based on published data, we expect cholesterol to represent approximately 1 mg / g of lipid in bovine adipocytes74 。

[0599] Micronutrients - Water - soluble and fat - soluble vitamins (vitamins A, B1, B2, B3, B 5、 B6, B 9、 B 12 , E, K, D, E, C) will be analyzed using the core services provided by the Nutritional Evaluation Laboratory (NEL) of the Jean Mayer USDA Human Nutrition Research Center on Aging (HNRCA) at Tufts University. Techniques will include HPLC, spectrophotometric measurement of enzyme activity, radiometric analysis, or colorimetric analysis, as needed and as previously described and performed by the NEL 75,76 . Iron and zinc will be quantified using an iron assay kit (Abcam, #ab83366) and a zinc assay kit (Abcam, #ab102507), respectively. Again, micronutrient results will be compared with those obtained from store - bought beef products

[0600] Although the bovine genome contains the synthetic pathways for vitamins B3, D, and C, these functions occur mainly in the liver or skin, respectively, and tissue concentrations of niacin are generally liver - controlled 77,78 . Therefore, we expect the vitamin composition to be mainly defined by the medium. Similarly, since minerals can only be obtained exogenously, we expect zinc and iron to be similarly defined by the medium. This may not be the case when some plant extracts (such as maitake mushroom extract) are added to the medium, as it is likely to contain some micronutrient components that will be incorporated by the cells

[0601] Myoglobin content - Myoglobin content will be analyzed using a modified Warriss method 82 . BSCs and store - bought beef before and after differentiation will be mechanically homogenized in PBS, centrifuged at 5,000 xg for 30 minutes, and filtered (Whatman No. 1 filter paper) to obtain extracts. The extracts will be oxidized with 60 mM K3F(III)(CN)6 and then treated with 80 mM NaCN to induce a color change from yellow to red. Absorbance will be measured at 540 nm, and the myoglobin concentration will be determined using the molar extinction coefficient of cyanmetmyoglobin (11300)

[0602] We expect the myoglobin content in BSCs to be significantly lower than that in store - bought beef 23 We expect the myoglobin content to be higher after differentiation 83。The average myoglobin content in beef is 8 mg / g, which is associated with the "cherry red" color. The values for lamb and pork are 6 mg / g (light red) and 2 mg / g (pink), respectively 84 。Strategies for modulating myoglobin content in vitro include induced hypoxia, lipid supplementation, and direct exogenous myoglobin supplementation and can be explored in cases of insufficient myoglobin content 23,85 。

[0603] Gelation properties - Thermal gelation properties will be evaluated using actomyosin extraction followed by gel strength and dynamic rheology measurements. Actomyosin (actin / myosin complex) will be extracted from pre - and post - differentiated bovine satellite cells and store - bought beef by a modified Ogawa method 86 。Samples will be mechanically homogenized in a cold isolation buffer (50 mM KCl, 20 mM K2HPO4 / KH2PO4) and filtered (0.9 mm mesh). The suspension will be centrifuged at 10,000 x g for 5 minutes, and the pellet will be homogenized in 0.6 M KCl buffer. After another round of centrifugation and filtration, the supernatant will be mixed with 20 mM potassium phosphate buffer, and the precipitate will be collected by centrifugation and resuspended in the isolation buffer. The final centrifugation step will provide actomyosin fragments; their concentration will be determined by the biuret reaction. Gel strength will be determined by texture analysis. Actomyosin concentration will be normalized, and samples will be sealed in beakers and heated to 80 °C for 20 minutes, then cooled and refrigerated overnight (4 °C). The gels will be equilibrated to ambient temperature and evaluated with a penetration test (1 mm / s) on a texture analyzer. Actomyosin gels will also be evaluated by dynamic rheological analysis during heating from ambient temperature to 80 °C (1 °C / min) 87 。

[0604] Food quality optimization - Depending on the results of nutritional and meat quality analyses, the culture system will be adjusted for deficiencies. For example, in cases of nutrient deficiencies (e.g., insufficient essential amino acids), the culture medium will be supplemented with the relevant nutrients to increase cell concentration. Additionally, metabolic engineering techniques, such as those used in our preliminary carotenoid work, can be used to increase nutrient levels through endogenous production (e.g., incorporation into essential amino acid biosynthetic pathways). In cases where meat science metrics in cultured samples are insufficient compared to conventional meat, several techniques may be used to modulate cultured meat characteristics. For example, as previously mentioned, myoglobin content can be adjusted by exogenous medium supplementation or culturing cells under hypoxic conditions 44,85 。In cases where metabolite profiles are significantly different, key metabolic processes can be modulated by metabolic pathway engineering (as shown in the nutritional engineering of our group's BSC) or by treatment with small molecules 19,88,89Finally, in cases where the gelling properties are significantly different from those of conventional meat, various mechanical means (e.g., via 3D tissue constructs) can be employed to increase myofiber formation and myosin expression 90 。

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[0721] The present invention has been described in terms of one or more preferred embodiments, and it should be understood that many equivalents, alternatives, variations, and modifications other than those explicitly stated are possible and within the scope of the present invention.

Claims

1. An engineered cell that endogenously synthesizes one or more antioxidant factors, wherein, The engineered cell comprises a heterologous polynucleotide encoding the one or more antioxidant factors.

2. The engineered cell according to claim 1, wherein The one or more antioxidant factors comprise antioxidant carotenoids.

3. The engineered cell according to claim 1, wherein, The one or more antioxidant factors comprise antioxidant phytochemicals.

4. The engineered cell according to claim 1, wherein, The one or more antioxidant factors comprise antioxidant plant metabolites.

5. The engineered cell according to claim 1, wherein, The one or more antioxidant factors comprise antioxidant metabolites not naturally produced by animal cells.

6. The engineered cell according to claim 1, wherein, The one or more antioxidant factors comprise phytoene and the cell comprises a heterologous polynucleotide encoding phytoene synthase.

7. The engineered cell according to claim 6, wherein the phytoene synthase has a sequence that is at least 90% identical to SEQ ID NO:1 or SEQ ID NO:

10.

8. The engineered cell according to claim 1, wherein the cell endogenously synthesizes lycopene and comprises a heterologous polynucleotide encoding phytoene desaturase.

9. The engineered cell according to claim 8, wherein the phytoene desaturase has a sequence that is at least 90% identical to SEQ ID NO:

3.

10. The engineered cell according to claim 8, wherein the cell endogenously synthesizes β-carotene and comprises a heterologous polynucleotide encoding lycopene cyclase.

11. The engineered cell according to claim 10, wherein the lycopene cyclase has a sequence that is at least 90% identical to SEQ ID NO:5 or SEQ ID NO:

11.

12. The engineered cell according to claim 1, wherein the cell is a mammalian cell.

13. The engineered cell according to claim 1, wherein the cell is Pax7 + a mammalian muscle precursor cell.

14. The engineered cell according to claim 1, wherein the cell is actin + myosin heavy chain (MHC) + mammalian multinucleated myotube.

15. The cell according to claim 10, wherein the total carotenoid yield in the engineered cell is at least 5-fold higher than the total carotenoid yield in the cell without the heterologous polynucleotides encoding phytoene synthase, phytoene desaturase, and lycopene cyclase.

16. A method of making the cell according to claim 1, comprising introducing a heterologous polynucleotide encoding one or more antioxidant factors into a cell that naturally synthesizes geranylgeranyl pyrophosphate.

17. The method according to claim 16, wherein the cell is a Pax7+ mammalian muscle progenitor cell.

18. A cultured meat product comprising a confluent serum-free culture comprising the cell according to claim 1 inoculated on a food safety matrix.

19. The cultured meat product according to claim 18, wherein the matrix is a membrane.

20. The cultured meat product according to claim 18, wherein the matrix is a sponge or a three-dimensional matrix.

21. The cultured meat product according to claim 18, wherein the matrix is a chitosan matrix.

22. The cultured meat product according to claim 18, wherein the cell culture comprises multinucleated myotubes derived from Pax7+ mammalian satellite cells.

23. The cultured meat product according to claim 22, wherein the multinucleated myotubes express myosin heavy chain (MHC) and actin.

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