Methods and compositions for producing cell-based meat products

Abstract

Methods and compositions for producing cell-based meat products are provided. Disclosed herein are methods and compositions for producing cell-based meat products with improved structural uniformity. In particular, the technology of this disclosure relates to filamentous fungus-yeast biocapsules and their use in methods for producing cell-based meat products.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 304,374, filed January 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present technology relates to methods and compositions for producing cell-based meat products with improved structural uniformity. In particular, the technology relates to the generation of filamentous fungal-yeast biocapsules and their use in methods of producing cell-based meat products. [Background technology]

[0003] The following explanation is provided to aid the understanding of the reader. None of the information provided or references cited are admitted to be prior art to the compositions and methods disclosed herein.

[0004] Livestock production has significant negative impacts on the environment, public health and animal welfare. It is estimated that roughly 30% of the Earth's surface and 70% of all arable land are dedicated to raising animals, with livestock accounting for 20% of the total terrestrial biomass. In addition to its staggering land consumption, livestock production consumes an estimated 8% of the planet's total freshwater supply. Finally, livestock production is responsible for 18% of global greenhouse gas (GHG) emissions and is the single greatest threat to the world's biodiversity.

[0005] Animal husbandry practices and poor animal welfare conditions are the cause of foodborne illnesses. Many pathogenic viruses originate from livestock, including swine flu and avian flu. Furthermore, poor hygiene practices when handling poultry products and raw pork promote the spread of E. coli, Salmonella, Campylobacter, and parasites. Furthermore, 70% of all antibiotics used in the United States are used on livestock, and this overuse of antibiotics is the primary cause of the emergence of antibiotic-resistant bacteria. In the United States alone, these antibiotic-resistant bacteria create an economic burden of roughly $55 billion and overwhelm the healthcare system with roughly 2 million infections, 250,000 hospitalizations, and at least 23,000 deaths annually. Finally, the livestock industry is not only rife with public health threats, but also with bad animal husbandry practices and a blatant disregard for animal welfare.

[0006] An emerging industry poised to combat the negative impacts of animal agriculture is the cell-based meat (also known as clean meat, artificial meat, cultured meat, lab-grown meat, or lab-cultured meat) industry. The cell-based meat industry is estimated to reduce energy usage by 7-45%, greenhouse gas emissions by 78-96%, land usage by 99%, and water usage by 82-96%. Additionally, the sterile production environment of cell-based meat eliminates the need for antibiotic use, thereby preventing the emergence of antibiotic-resistant bacteria.

[0007] Current cell-based meat technology relies heavily on satellite cell culture, a technique in which animal cells are grown in liquid medium suspension and aggregated on microcarriers. These cell-based meat products resemble minced meat and lack the shape and texture of natural animal meat, and their overall structure is derived from a biomaterial scaffold such as the extracellular matrix (ECM). The ECM is composed of oligosaccharides and glycoproteins that impart the biochemical and biomechanical properties of natural meat. In addition to providing structure, the ECM contains a variety of matrix proteins that facilitate cell attachment, proliferation, and determine the overall behavior and composition of the final tissue. Thus, biomaterial scaffolds such as the ECM are critical for the generation of cell-based meat.

[0008] Current methods of generating ECM and similar biomaterial scaffolds are inefficient and costly. Commercially available ECM, such as Matrigel™, is harvested from mouse sarcomas. The production of Matrigel™ is an expensive and inefficient process that requires mouse cancer cell lines and biosafety laboratories. Non-ECM scaffolds can be made from natural biomaterials, such as alginate, silk, and chitosan, as well as synthetic biomaterials, such as polyethylene glycol, polyglycolic acid, and polyacrylamide. However, to generate biomaterial scaffolds suitable for the growth of animal cells, these natural biomaterial substrates must be further functionalized with purified matrix proteins. Purification of matrix proteins is time-consuming and costly. Furthermore, functionalizing biomaterial substrates with purified matrix proteins requires costly and time-consuming techniques, such as ion beam deposition and plasma treatment. Thus, there is a need to develop new approaches to produce inexpensive, robust, customizable, edible biomaterial scaffolds that contain polysaccharide-based structural elements and / or proteins for the growth and proliferation of any given animal cell type into any desired tissue. Summary of the Invention

[0009] In one aspect, the present disclosure provides a biocapsule for use in producing a cell-based meat product, where the biocapsule comprises a biomaterial scaffold comprising (i) a filamentous fungus and (ii) a plurality of yeast cells.

[0010] In some embodiments, the filamentous fungus comprises a mycelium.

[0011] In some embodiments, the plurality of yeast cells are genetically engineered to express one or more heterologous proteins, hi some embodiments, the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or any combination thereof.

[0012] In some embodiments, the filamentous fungus comprises one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucorhynchum and Atractycota).

[0013] In some embodiments, the filamentous fungal strain is from the Ascomycota phylum and comprises one or more strains belonging to a genera selected from the group consisting of Aspergillus, Penicillium, and Neurospora crassa. In some embodiments, the filamentous fungus is from the genus Aspergillus and comprises one or more strains of Aspergillus oryzae. In some embodiments, the filamentous fungus is from the genus Neurospora crassa and comprises one or more strains of Neurospora crassa.

[0014] In some embodiments, the filamentous fungal strain is a strain of Rhizopus oryzae or Rhizopus oligosporus from the Mucoramium.

[0015] In some embodiments, the plurality of yeast cells comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris, hi some embodiments, the plurality of yeast cells comprises one or more strains of Saccharomyces cerevisiae.

[0016] In some embodiments, the one or more heterologous proteins are chordate ECM proteins, growth factor proteins, cell adhesion proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, cell surface proteins, or any combination thereof. In some embodiments, the heterologous chordate protein is a mammalian ECM protein, growth factor protein, adhesion molecule protein, cell signaling protein, protein motifs (synthetic or naturally occurring), flavor proteins, cell surface proteins, or any combination thereof.

[0017] In some embodiments, the one or more heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides.

[0018] In some embodiments, one or more heterologous proteins are solubly expressed, targeted to a specific subcellular localization, or secreted.

[0019] In some embodiments, the plurality of yeast cells is immobilized or decellularized.

[0020] In one aspect, the present disclosure provides a cell-based meat product comprising: (i) an edible biomaterial scaffold comprising a plurality of (a) filamentous fungi and (b) yeast cells; and (ii) a biomaterial scaffold comprising a plurality of one or more cell types.

[0021] In some embodiments, the plurality of one or more cell types comprises one or more cell types selected from the group consisting of myoblasts or their precursor cells, adipocytes or their precursor cells, fibroblasts or their precursor cells, endothelial cells or their precursor cells, smooth muscle cells or their precursor cells, muscle satellite cells, induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and any combination thereof.

[0022] In some embodiments, the plurality of yeast cells are genetically engineered to express one or more heterologous proteins.

[0023] In some embodiments, the plurality of one or more cell types are chordate cells. In some embodiments, the chordate cells are mammalian cells.

[0024] In some embodiments, the chordate cell is a porcine cell, a bovine cell, or a poultry cell.

[0025] In some embodiments, the filamentous fungus comprises a mycelium.

[0026] In some embodiments, the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or any combination thereof.

[0027] In some embodiments, the filamentous fungus comprises one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucorhynchum and Atractycota).

[0028] In some embodiments, the filamentous fungal strain is from the Ascomycota phylum and comprises one or more strains belonging to a genera selected from the group consisting of Aspergillus, Penicillium, and Neurospora crassa. In some embodiments, the filamentous fungus is from the genus Aspergillus and comprises one or more strains of Aspergillus oryzae. In some embodiments, the filamentous fungus is from the genus Neurospora crassa and comprises one or more strains of Neurospora crassa.

[0029] In some embodiments, the filamentous fungal strain is a strain of Rhizopus oryzae or Rhizopus oligosporus from the Mucoramium.

[0030] In some embodiments, the plurality of yeast cells comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris, hi some embodiments, the plurality of yeast cells comprises one or more strains of Saccharomyces cerevisiae.

[0031] In some embodiments, the one or more heterologous proteins are chordate ECM proteins, growth factor proteins, cell adhesion proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or cell surface proteins. In some embodiments, the heterologous chordate protein is a mammalian ECM protein, growth factor protein, adhesion molecule protein, cell signaling protein, protein motifs (synthetic or naturally occurring), flavor protein, or cell surface protein.

[0032] In some embodiments, the one or more heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides.

[0033] In some embodiments, one or more heterologous proteins are solubly expressed, targeted to a specific subcellular localization, or secreted.

[0034] In some embodiments, the plurality of yeast cells is fixed or decellularized.

[0035] In one aspect, the present technology discloses a method for producing a filamentous fungus-yeast biocapsule, the method comprising adding a plurality of yeast cells to a filamentous fungus culture comprising mycelium to form a filamentous fungus-yeast preparation, and culturing the preparation under conditions inducing spontaneous co-immobilization of the filamentous fungus and yeast to form a filamentous fungus-yeast biocapsule.

[0036] In some embodiments, the conditions that induce co-immobilization of filamentous fungi and yeast include the use of a first culture medium and a second culture medium.

[0037] In some embodiments, the plurality of yeast cells are genetically engineered to express one or more heterologous proteins.

[0038] In some embodiments, the first medium comprises a carbon source that can be used by filamentous fungi but not by yeast, the carbon source being selected from the group consisting of gluconic acid, starch, cellulose, inulin, and other similar colloidal molecules.

[0039] In some embodiments, the second culture medium comprises a carbon source preferred by yeast that can also be utilized by the filamentous fungus.

[0040] In some embodiments, the method further comprises contacting the filamentous fungus with an enzyme to modify one or more of the density, porosity, or texture of the filamentous fungus, hi some embodiments, the enzyme is selected from the group consisting of zymolase, lyticase, and glucalase.

[0041] In some embodiments, the method further comprises fixing or decellularizing the yeast cells.

[0042] In some embodiments, the filamentous fungus-yeast biocapsules are used as an edible biomaterial scaffold for cell-based meat production.

[0043] In some embodiments, the method further comprises shaping the fungus-yeast biocapsules, hi some embodiments, the shape of the fungus-yeast biocapsule resembles the shape of a three-dimensional meat product.

[0044] In some embodiments, the method further comprises seeding the filamentous fungus-yeast biocapsules with a plurality of one or more cell types selected from the group consisting of myoblasts or their precursors, adipocytes or their precursors, fibroblasts or their precursors, endothelial cells or their precursors, smooth muscle cells or their precursors, muscle satellite cells, induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and any combination thereof to form seeded biocapsules, and culturing the seeded biocapsules under conditions effective to induce differentiation of the plurality of one or more cell types to produce an edible cell-based meat product.

[0045] In some embodiments, the plurality of one or more cell types are chordate cells.

[0046] In some embodiments, the chordate cell is a mammalian cell.

[0047] In some embodiments, the chordate cell is a porcine cell, a bovine cell, or a poultry cell.

[0048] In some embodiments, the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or any combination thereof.

[0049] In some embodiments, the filamentous fungus comprises one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucorhynchum and Atractycota).

[0050] In some embodiments, the filamentous fungal strain is from the Ascomycota phylum and comprises one or more strains belonging to a genera selected from the group consisting of Aspergillus, Penicillium, and Neurospora crassa. In some embodiments, the filamentous fungus is from the genus Aspergillus and comprises one or more strains of Aspergillus oryzae. In some embodiments, the filamentous fungus is from the genus Neurospora crassa and comprises one or more strains of Neurospora crassa.

[0051] In some embodiments, the filamentous fungal strain is a strain of Rhizopus oryzae or Rhizopus oligosporus from the Mucoramium.

[0052] In some embodiments, the plurality of yeast cells comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris, hi some embodiments, the plurality of yeast cells comprises one or more strains of Saccharomyces cerevisiae.

[0053] In some embodiments, the one or more heterologous proteins are chordate ECM proteins, growth factor proteins, cell adhesion proteins, cell signaling proteins, peptide motifs (synthetic or naturally occurring), flavor proteins, or cell surface proteins. In some embodiments, the heterologous chordate protein is a mammalian ECM protein, growth factor protein, adhesion molecule protein, cell signaling protein, peptide motifs (synthetic or naturally occurring), flavor protein, or cell surface protein.

[0054] In some embodiments, the one or more heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides.

[0055] In some embodiments, one or more heterologous proteins are solubly expressed, targeted to a specific subcellular localization, or secreted. [Brief description of the drawings]

[0056] [Figure 1A] Images showing co-immobilization of yeast (Saccharomyces cerevisiae) and Koji mold (Aspergillus oryzae) mycelium. Images showing a yeast reporter strain expressing cytoplasmic GFP grown with Koji mold under conditions that promote adsorption of the yeast cell wall to the Koji hyphal structures, including the mycelium. Even after extensive washing, a subpopulation of yeast still remains associated with the Koji mycelium hyphae. [Figure 1B]Image showing the co-immobilization of yeast (Saccharomyces cerevisiae) and koji mold (Aspergillus oryzae) mycelium. The co-immobilized yeast and koji mold structures can be grown into flat sheets. [Figure 1C] Image showing the co-immobilization of yeast (Saccharomyces cerevisiae) and koji mold (Aspergillus oryzae) mycelium. The co-immobilized yeast and koji mold structures are allowed to grow into spheres. [Diagram 2] Images showing myoblast attachment to a scaffold consisting of symbiotic growths of Aspergillus oryzae and Saccharomyces cerevisiae. Quail myoblasts (QM7 cell line) were seeded onto flat sheet scaffolds consisting of symbiotic growths of Saccharomyces cerevisiae and Aspergillus oryzae grown as described in Example 7 (Scaffold generation - flat sheets). The cell-laden scaffolds were fixed and stained for actin (visualized with 543-phalloidin), which stains animal cells. [Figure 3A] Images showing the cell-laden scaffold assembled into a structured cultured meat product.Images showing the cell-laden scaffold harvested, stained red, and cross-linked with microbial transglutaminase to produce a "raw" cultured meat product. [Figure 3B] An image showing the cell-laden scaffold assembled into a structured cultured meat product.An image of a cultured meat product cooked in avocado oil. [Figure 4]Plasmid map of the Saccharomyces cerevisiae cytoplasmic expression plasmid encoding the bovine COL1A1 45 kDa fragment. The 45 kDa fragment of the bovine (Bos taurus) COL1A1 fragment has a 6xHIS epitope appended to it ("6xHIS-Bt_COL1A1 45 kDa") and terminated with the 3'UTR from the yeast CYC1 gene ("3'UTR_CYC1"). Expression is driven by the yeast GPD promoter ("P_GPD"). The plasmid contains a kanamycin resistance marker ("Kan_R") for E. coli selection, an origin of replication from a pUC plasmid ("Ori_pUC") for E. coli propagation, a LEU2 auxotrophic marker ("LEU2") for yeast selection, and a 2um element ("2um") for yeast propagation. [Figure 5A] Images showing myoblasts attached to a scaffold composed of symbiotically grown Aspergillus oryzae and Saccharomyces cerevisiae engineered to express bovine collagen COL1A1. Quail myoblasts (QM7 cell line) were seeded onto flat sheet scaffolds composed of symbiotically grown Saccharomyces cerevisiae and Aspergillus oryzae expressing a 45 kDa fragment of bovine COL1A1 grown as described in Example 8 (Scaffold generation - flat sheets). Cell-laden scaffolds were fixed and stained for actin (visualized with 543-phalloidin). [Figure 5B] Image showing myoblasts attached to a scaffold composed of symbiotically grown Aspergillus oryzae and Saccharomyces cerevisiae engineered to express bovine collagen COL1A1. Quail myoblasts (QM7 cell line) were seeded onto flat sheet scaffolds composed of symbiotically grown Saccharomyces cerevisiae and Aspergillus oryzae expressing a 45 kDa fragment of bovine COL1A1 grown as described in Example 8 (Scaffold generation - flat sheets). The cell-laden scaffolds were fixed and nuclei (visualized with DAPI) stained. [Figure 5C]Images showing myoblasts attached to a scaffold composed of symbiotically grown Aspergillus oryzae and Saccharomyces cerevisiae engineered to express bovine collagen COL1A1. Quail myoblasts (QM7 cell line) were seeded onto flat sheet scaffolds composed of symbiotically grown Saccharomyces cerevisiae and Aspergillus oryzae expressing a 45 kDa fragment of bovine COL1A1 grown as described in Example 8 (Scaffold generation - flat sheets). The cell-laden scaffolds were fixed and stained for actin and nuclei and co-visualized. [Figure 6A] Images showing cell-laden engineered scaffolds assembled into a structured cultivated meat product.Top view of a cell-laden scaffold with Saccharomyces cerevisiae engineered to express cytoplasmic collagen, harvested and cross-linked with transglutaminase to produce a "live" quail cell (QM7) cultivated meat product. [Figure 6B] FIG. 6B is a side view of the cultured meat product of FIG. [Figure 6C] An image of a cultured meat product cooked in avocado oil. [Figure 7] Plasmid map of the Saccharomyces cerevisiae secretion expression plasmid encoding the bovine COL1A1 45 kDa fragment. The 45 kDa fragment of the bovine (Bos taurus) COL1A1 fragment ("Bt_COL1A1 45 kDa") was fused to the 3' end of the mating factor alpha leader sequence fused to a 6× histidine epitope ("MFa-6×HIS") followed by a thrombin cleavage site ("Thrombin cleavage"). Expression of the fusion protein was driven from the yeast TEF1 promoter ("P_TEF1") and terminated with a 3× stop codon ("3× stop"). The plasmid contains a kanamycin resistance marker ("Kan_R") for E. coli selection, an origin of replication ("ColE1 ori") for E. coli propagation, a URA3 auxotrophic marker ("LEU2") for yeast selection, and a 2um element ("2um") for yeast propagation. [Figure 8]Plasmid map of the Saccharomyces cerevisiae integrating vector for expression of endoplasmic reticulum-targeted bovine P4HA1. The bovine (Bos taurus) P4HA1 gene with the 3'HDEL peptide sequence appended ("Bt_P4HA1-HDEL") was fused to the 3' end of the Kar2 leader sequence ("Kar2") for targeting to the endoplasmic reticulum. The fusion protein was driven from the yeast TEF1 promoter ("P_TEF1") and terminated with the 3'UTR from the yeast CYC1 gene ("3'UTR_CYC1"). The plasmid contains a kanamycin resistance marker ("Kan_R") for E. coli selection, an origin of replication ("ColE1 ori") for E. coli propagation, a LEU2 auxotrophic marker ("LEU2") for yeast selection, a HIS3 auxotrophic marker ("HIS3") for yeast selection, and a 2um element ("2um") for yeast propagation. [Figure 9] Plasmid map of the Saccharomyces cerevisiae integrating vector for expression of endoplasmic reticulum-targeted bovine P4HB. The bovine (Bos taurus) P4HB gene with the 3'HDEL peptide sequence appended ("Bt_P4HB-HDEL") was fused to the 3' end of the Kar2 leader sequence ("Kar2") for targeting to the endoplasmic reticulum. The fusion protein is driven from the yeast TEF1 promoter ("P_TEF1") and terminates in the 3'UTR of the yeast CYC1 gene ("3'UTR_CYC1"). The plasmid contains a kanamycin resistance marker ("Kan_R") for E. coli selection, an origin of replication ("ColE1 ori") for E. coli propagation, a URA3 auxotrophic marker ("URA3") for yeast selection, a HIS3 auxotrophic marker ("HIS3") for yeast selection, and a 2um element ("2um") for yeast propagation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] It should be understood that certain aspects, modes, embodiments, variations and features of the present technology are described below at various levels of detail to provide a substantial understanding of the present technology. Definitions of certain terms as used herein are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which the present technology belongs.

[0058] I. Definition All terminology used herein is commonly used in biochemistry and molecular biology and therefore would be understood by one of ordinary skill in the art to which this technology pertains. These terms can be found, for example, in the following documents: Molecular Cloning: A Laboratory Manual 3rd ed., vol. 1-3, ed. Sambrook and Russell (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); Current Protocols In Molecular Biology, ed. Ausubel et al., (Greene Publishing Associates and Wiley-Interscience, New York, 1988) (including regular updates); Short Protocols In Molecular Biology: A Compendium Of Methods From Current Protocols In Molecular Biology 5th ed., vol. 1-2, ed. Ausubel et al., (John Wiley & Sons, Inc., 2002); Genome Analysis: A Laboratory Manual, vol. 1-2, ed. Green et al., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997).

[0059] The following terms are used herein and their definitions are provided as a guide.

[0060] As used herein, the singular forms "a," "an," and "the" designate both the singular and the plural, unless expressly stated to designate only the singular.

[0061] The use of the term "about" and ranges generally means that the understood numbers, whether or not limited by the term about, are not limited to the exact numbers described herein and are intended to refer to a range that does not depart from the scope of the present technology but is substantially within the cited range. As used herein, "about" will be understood by those of ordinary skill in the art and will vary to some extent with the context in which it is used. If there are uses of the term that are not clear to those of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0062] As used herein, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," can refer in one embodiment to A only (optionally including elements other than B), in another embodiment to B only (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0063] As used herein, when the term "approximately" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the numerical values. In general, the term "approximately" is used herein to modify a numerical value by a variance of 20%, 10%, 5%, or 1% above and below the stated value. In certain embodiments, the term "approximately" is used to modify a numerical value by a variance of 10% above and below the stated value. In certain embodiments, the term "approximately" is used to modify a numerical value by a variance of 5% above and below the stated value. In certain embodiments, the term "approximately" is used to modify a numerical value by a variance of 1% above and below the stated value.

[0064] As used herein, the term "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, may optionally be present, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements);

[0065] As used herein, the term "biocapsule" or "filamentous fungus-yeast biocapsule" refers to a form of co-immobilization of yeast and filamentous fungus. Due to the combined inherent adhesive properties of yeast and filamentous fungus, biocapsules form spontaneously through hydrophobic interactions when cultured in liquid medium under certain conditions. In some embodiments, the yeast is not genetically engineered. In some embodiments, the yeast is genetically engineered to express one or more heterologous proteins selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof, where the heterologous proteins are soluble expressed, targeted to a specific subcellular localization, or secreted. In some embodiments, the yeast is immobilized or decellularized. In some embodiments, the biocapsules are used as biomaterial scaffolds. In some embodiments, the biocapsules are used as edible biomaterial scaffolds. In some embodiments, the biocapsules are used as edible biomaterial scaffolds for cell-based meat production according to the methods described herein.

[0066] As used herein, the term "embodiment" means that a particular feature, structure, or characteristic is included in at least one or more manifestations, examples, or implementations of the technology. Moreover, particular features, structures, or characteristics can be combined in any suitable manner, as would be apparent to one of ordinary skill in the art. Combinations of features of different embodiments are intended to be within the scope of the technology, and it is not necessary to explicitly describe every possible permutation by example. Thus, any of the claimed embodiments can be used in any combination.

[0067] As used herein, the term "genetic engineering" or "genetically engineered" encompasses any method for introducing nucleic acids or specific mutations into a host organism. For example, yeast cells are genetically engineered when they are transformed with a polynucleotide sequence that expresses a heterologous protein. A cell is genetically engineered when a polynucleotide sequence is introduced that results in the expression of novel and / or heterologous genes in the cell, or increased or decreased levels of a gene product naturally found in the cell, by up- or down-regulation, deletion, or change in copy number.

[0068] "Heterologous nucleic acid" refers to a nucleic acid, DNA, or RNA that is introduced into a cell (or an ancestor of the cell) that is not a copy of a sequence naturally found in the cell into which it is introduced. Such a heterologous nucleic acid can include a segment that is a copy of a sequence naturally found in the cell into which it is introduced, or a fragment thereof.

[0069] As used herein, the term "progenitor cells" refers to cells that can give rise to differentiated cells of multiple lineages. Progenitor cells can include stem cells, such as embryonic stem cells, mesenchymal stem cells, adult stem cells, differentiated embryonic stem cells, differentiated adult stem cells, or induced pluripotent stem cells.

[0070] II. Overview The present technology relates to compositions and methods for the production of edible biocapsules that provide a structure and suitable growth environment for the attachment, proliferation, and morphogenesis of cultured animal cells for the production of cell-based meat products, such as steak-like cultured meat products. The biocapsules include an edible base substrate and yeast cells, such as baker's yeast (Saccharomyces cerevisiae). In some embodiments, the yeast of the biocapsules is not modified or genetically engineered. In some embodiments, the yeast of the biocapsules is genetically engineered to express one or more heterologous proteins, such as one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof. The base substrate includes mycelium of an edible filamentous fungus, which can be grown to any desired shape and size, and serves as the primary structural element of the final cultured meat product. The base substrate is functionalized with yeast and can be optionally genetically engineered. The base substrate can be functionalized with recombinant proteins expressed by genetically engineered baker's yeast, which is composed primarily of edible polysaccharides, for example. The genetically engineered baker's yeast can be decellularized or fixed, rendering the yeast inactive while allowing heterologous proteins that can be solubly expressed, targeted to specific subcellular localization, or secreted to remain structurally intact.

[0071] III. Scaffolds for Animal Tissue Growth Animal tissues are organized arrangements of various cell types grown into three-dimensional structures. For individual cells to grow into organized tissues, the cells may adhere to some kind of supporting scaffold that promotes expansive growth in three dimensions. Scaffold biomaterials have a biocompatible surface that mimics the structure of tissues in three dimensions and may exhibit interconnected porosity to support cell / tissue infiltration. Scaffolds made from natural sources are advantageous due to their innate ability to promote biological recognition and support cell adhesion and function.

[0072] Naturally derived scaffold biomaterials are generally classified into two groups: (1) extracellular matrix (ECM) protein-based biomaterials, such as collagen, silk fibroin, gelatin, fibronectin, keratin, fibrin, and eggshell membrane, and (2) polysaccharide-based biomaterials, such as hyaluronan, cellulose, glucose, alginate, chondroitin, chitin, and its derivative, chitosan. Although both groups of scaffold proteins are important for tissue growth, most of the development has only been in the area of ​​polysaccharide-based biomaterials. Although progress has been made in the development of tunable scaffolds made of food-safe, plant-derived polymers, such as gellan gum, alginate, pectin, and modified cellulose, ECM proteins are currently still mostly harvested from animal sources, which is inefficient and introduces batch-to-batch variability. Furthermore, current methods in the art for producing scaffolds that combine both ECM proteins and polysaccharide-based biomaterials use functionalization techniques that require highly purified ECM proteins, which are very costly and inefficient.

[0073] As described herein, the technology relates to the use of a fungal-based approach to fabricate robust, customizable scaffolds that have polysaccharide-based structural elements and / or proteins (such as ECM proteins) and have the potential to improve the growth and proliferation of any given animal cell type into any desired tissue.

[0074] IV. Fungi Molds and yeasts are fungi that are widespread in nature. A fungus is any member of a group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. Molds or filamentous fungi are presented as long filaments (hyphae) of cells that form a mycelium. Hyphae are the long, branched, filamentous structures of fungi. Filamentous fungi consist of a cross-linked network of hyphae, which expand through polarized tip elongation and branching (an increase in the number of growing tips), which corresponds to cell division in animals and plants. In most fungi, hyphae are the primary mode of vegetative growth and are collectively referred to as "mycelium".

[0075] It should be understood that the mycelium of the present technology can be grown by any method known to one of skill in the art.

[0076] In some embodiments, the mycelial scaffold can be produced by using a perfusion bioreactor system for cell-based meat technology, which can include, for example, a suspension reactor unit for growth, dialysis, oxygenation of bovine muscle cells, pumps for medium circulation and medium supply between the reactor units, and a scaffold bioreactor unit for producing aggregated cell mass with or without mechanical actuation of the aggregated cell mass.

[0077] In some embodiments, the filamentous fungus comprises one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucococci and Trichoderma). In some embodiments, the filamentous fungus is from the phylum Ascomycota and comprises one or more strains belonging to a genus selected from the group consisting of Aspergillus, Penicillium, and Neurospora. In some embodiments, the filamentous fungus is from the genus Aspergillus and comprises one or more strains of Aspergillus oryzae. In some embodiments, the filamentous fungus is from the genus Neurospora and comprises one or more strains of Neurospora crassa. In some embodiments, the filamentous fungus is a strain of Rhizopus oryzae or Rhizopus oligosporus from the phylum Mucomycota.

[0078] Molds such as Penicillium and Aspergillus are aerobic and due to their chemical activity are of great importance in the production of antibiotics, cheese, etc. Penicillium is a genus of ascomycetes important in the natural environment, food spoilage, and food and pharmaceutical production. Aspergillus is a genus consisting of a group of filamentous fungi or common molds, most of which occur asexually and spread across many different environments, reproducing and growing by producing conidia (asexual spores or conidiophores) that can germinate.

[0079] Yeasts are eukaryotic, unicellular microorganisms classified as members of the kingdom Fungi. Saccharomyces cerevisiae (commonly known as brewer's yeast) is a type of anaerobic yeast useful in winemaking, baking, and brewing. Some Saccharomyces cerevisiae may form a veil or flower on the surface of wine once alcoholic fermentation has ended and fermentable sugars have been exhausted, which is a form of spontaneous immobilization.

[0080] Brewer's yeast can be easily cultured in liquid form at room temperature, grows quickly (doubling time of approximately 2 hours), and its genome can be easily manipulated using well-established molecular biology toolsets. As a eukaryotic organism, brewer's yeast is endowed with the cellular machinery to efficiently produce mammalian proteins. Brewer's yeast is edible and is best known for its role in baking and brewing, in alcohol production, and is also consumed as a dietary supplement due to its numerous health benefits.

[0081] Aspergillus oryzae (commonly known as koji mold) is traditionally used in the production of sake, soy sauce, and miso, and is safe for human consumption because it has a natural umami flavor similar to meat. As a sole material or in combination with Saccharomyces cerevisiae, the fibrous material from the mycelium of koji mold can form a fungal vegetative structure composed of branching, thread-like hyphae. Koji mold can be grown to form solid structures of any size.

[0082] Additionally, the density and material properties of the Aspergillus oryzae can be tuned during production, making the filamentous fungus amenable to genetic engineering, if desired. Thus, in some embodiments of the present technology, Aspergillus oryzae constitute the main chitinous structural element of the scaffold, providing the structural backbone of the animal tissue. The addition of artificial brewer's yeast to the Koji scaffold adds customizable properties that can be used to improve the attachment and growth of various animal cells into functional tissues.

[0083] V. Chitin Chitin is the main component of the cell walls of budding yeast and filamentous fungi. Chitin is a naturally abundant polysaccharide and a product of deacetylated chitosan, another naturally abundant polymer. Chitin / chitosan is the main component of the shells of crustaceans such as shrimp, crabs, and lobsters, and is also the main component of the cell walls of various fungi, including Aspergillus oryzae (commonly known as koji mold) and Saccharomyces cerevisiae (commonly known as brewer's yeast, baker's yeast, or budding yeast). Chitin can be easily engineered into three-dimensional scaffolds as substrates for animal tissue culture. In addition to its easy-to-handle chemical properties, animal-free nature, and abundance, chitin / chitosan also contains antibacterial properties and nutritional benefits. As described herein, in some embodiments, chitin / chitosan derived from both baker's yeast and koji mold are used as the main structural components of customized scaffolds engineered specifically for each animal tissue.

[0084] As a sole material or in combination with Saccharomyces cerevisiae, fibrous material from the mycelium of Aspergillus oryzae can form fungal vegetative structures composed of branched, filamentous hyphae. Aspergillus oryzae can be grown to form solid structures of any size using procedures known in the art. Furthermore, its density and material properties can be tuned during manufacture, making this filamentous fungus amenable to genetic engineering, if desired. Thus, as described herein, in some embodiments, Koji constitutes the primary chitinous structural element of the biomaterial scaffold, providing the structural framework (e.g., the shape of a steak) for animal tissues. The addition of engineered brewer's yeast to the Koji scaffold adds customizable properties that can be used to improve the attachment and growth of a variety of chordate and non-chordate cells into functional tissues.

[0085] VI. Yeast and genetically engineered yeast As described herein, the biocapsule compositions and methods of the present technology include yeast cells. These yeast cells can be optionally genetically engineered to express one or more heterologous proteins for animal cell attachment and subsequent cellular activity. In some embodiments, the yeast comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris. In some embodiments, the yeast comprises one or more strains of Saccharomyces cerevisiae (baker's yeast, brewer's yeast).

[0086] In some embodiments, the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof. In some embodiments, the heterologous proteins are selected from one or more chordate or non-chordate marine organism ECM proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof. In some embodiments, the heterologous chordate proteins are mammalian ECM proteins, growth factor proteins, adhesion molecule proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof. In some embodiments, the heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides. Flavor proteins include proteins that affect the flavor of the composition, for example, through binding and / or absorption of flavoring compounds. The range of suitable flavor proteins and how they affect the flavor of the composition will be understood by those of skill in the art. See Zhang, Jian, Kang, Dachend, Zhang, Wangang, and Lorenzoe, Jose, Recent advantage of interactions of protein-flavor in foods: Perspective of theoretical models, protein properties and extrinsic factors. Trends in Food Science and Technology. 2021 May;111:405-425. In some embodiments, the heterologous protein is soluble expressed, targeted to a specific subcellular location, or secreted.

[0087] In some embodiments, yeast cells can be used live or dead, intact, permeabilized, or even emptied of all original cytoplasmic contents (decellularized). In some embodiments, the yeast cells of the present technology are fixed. Yeast cells can be fixed or decellularized by any suitable method known in the art, including, but not limited to, fixation or decellularization by heat shock, treatment with dehydrating agents such as detergents, alcohols, osmotic shock, lyophilization, physical (mechanical) lysis, electrical disruption, enzymatic digestion, or any combination thereof.

[0088] VII. Encapsulation Encapsulation involves incorporating food ingredients, enzymes, cells, or other materials into tiny capsules. Applications of this technique are increasing in the food industry because the encapsulated material can be protected from moisture, heat, or other extreme conditions, thereby improving its stability and maintaining its viability. Various techniques are used to form biocapsules, including spray drying, spray cooling or spray chilling, extrusion coating, fluidized bed coating, liposome encapsulation, coacervation, inclusion complexation, centrifugal extrusion, and rotary suspension separation. A wide variety of foods have been encapsulated, including flavoring agents, acids-bases, artificial sweeteners, colorants, preservatives, leavening agents, antioxidants, and drugs containing undesirable flavors, odors, and nutrients. Microencapsulation, or the process of surrounding tiny particles or droplets with a coating to give tiny capsules, has also been implemented, particularly for the immobilization of enzymes, because many enzymes can be entrapped in synthetic polymers (such as cellulose acetate tallate) without denaturation or chemical modification.

[0089] The combination of immobilized yeast containing heterologous proteins with filamentous fungal-based (e.g., Aspergillus oryzae-based) scaffolds exploits the inherent adhesion properties of both fungi. Brewer's yeast naturally binds to filamentous fungal hyphae through strong hydrophobic interactions.

[0090] The inherent adhesive properties of the fungi used in the process (a division of fungi that includes all fungi (Ascomycota and Basidiomycota) distinct from the Myxomycota) allow the functionalization of the Aspergillus-based substrate with yeast cells. Baker's yeast naturally binds to filamentous fungal hyphae through strong hydrophobic interactions. Furthermore, protocols have been previously described to induce the co-immobilization of filamentous fungi (e.g., Aspergillus oryzae) and brewer's yeast without the addition of chemical crosslinkers or external supports. A technique has been demonstrated to produce large spheres (approximately 2 cm in diameter) containing both microorganisms, i.e. "filamentous fungus-yeast biocapsules", which are extremely stable under many harsh conditions and resemble well-established scaffolds.

[0091] The density and porosity of these biocapsules can be tuned by exploiting the unique properties of glucan, the main component of Saccharomyces cerevisiae cell walls. Glucans are polysaccharides derived from D-glucose linked by glycosidic bonds. Glucans can polymerize to form gels at neutral pH, adding another tunable layer to the system. As described herein, a biomaterial scaffold composed of an edible, structurally robust Aspergillus oryzae-based matrix and brewer's yeast, optionally engineered to express heterologous proteins such as extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof, provides three-dimensional structure and cell growth cues for the generation of muscle / fat-based animal tissues.

[0092] VIII. Multiple Cell Types, Seeding, and Culturing of Cells A number of cell types or cell populations can be cultured with the filamentous fungus-yeast biocapsules of the present technology to form three-dimensional edible cell-based meat products. In some embodiments, the one or more cell types are selected from the group consisting of myoblasts or their precursors, adipocytes or their precursors, fibroblasts or their precursors, endothelial cells or their precursors, smooth muscle cells or their precursors, muscle satellite cells, induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, and any combination thereof.

[0093] In some embodiments, the multiple cell types are progenitor cells. In some embodiments, the progenitor cells are cultured in monoculture. In some embodiments, the progenitor cells are differentiated in monoculture. In some embodiments, the progenitor cells are differentiated in monoculture and then seeded and incubated on the three-dimensional filamentous fungus-yeast biocapsules of the present technology. In some embodiments, the progenitor cells are seeded and incubated on the three-dimensional filamentous fungus-yeast biocapsules of the present technology and differentiated in situ. Methods for culturing progenitor cells and differentiating them into differentiated cells will be readily apparent to one of skill in the art.

[0094] In some embodiments, the multiple cell types are obtained from a live animal and cultured as primary cell lines. In some embodiments, the cells are obtained by biopsy and cultured in vitro. In some embodiments, the cells are obtained from a commercially available cell line. In some embodiments, the cells are immortalized or reprogrammed primary cell lines.

[0095] In some embodiments, the plurality of cell types is derived from a chordate. In some embodiments, the plurality of cell types is derived from a non-human chordate selected from mammals, birds, fish, reptiles, and amphibians. In some embodiments, the plurality of cell types is derived from a non-human mammal. In some embodiments, the plurality of cell types is derived from livestock, including livestock, semi-livestock, and captive wild animals, including, but not limited to, cows, moose, reindeer, bison, horses, deer, sheep, goats, pigs, poultry, llamas, alpacas, and live fish. In some embodiments, the plurality of cell types is derived from a non-chordate marine organism. In some embodiments, the plurality of cell types is derived from a non-chordate marine organism, including, but not limited to, crustaceans (e.g., lobsters, crabs, shrimp, clams, oysters, and mussels), sea urchins, squid, and octopus. In some embodiments, the plurality of one or more cells is derived from the same chordate or non-chordate as the heterologous protein that may be expressed by the yeast cells of the biocapsule of the present technology. In some embodiments, the plurality of one or more cells and heterologous proteins that can be expressed by the yeast cells of the biocapsules of the present technology are derived from a mammal. In some embodiments, the plurality of one or more cells and heterologous proteins that can be expressed by the yeast cells of the biocapsules of the present technology are either homogeneous or heterogeneous combinations of chordates or non-chordates as described herein. In some embodiments, the plurality of one or more cells and heterologous proteins that can be expressed by the yeast cells of the biocapsules of the present technology are either homogeneous or heterogeneous combinations of porcine, bovine, or poultry cell types.

[0096] One of skill in the art will readily recognize that each of the multiple cell types used in the compositions and methods of the present technology has a preferred medium for cell growth and maintenance, as well as a preferred range of cell densities.

[0097] In some embodiments, the biocapsules of the present technology contain about 1 cm of medium suitable for cell growth and maintenance. 2 ~60cm 2 On a scaffold with a total surface area of ​​approximately 1 x 10 3cells ~ approx. 1×10 6 A range of cells are seeded with multiple cell types. In some embodiments, the biocapsules contain approximately 9 cm of scaffold in approximately 3 mL medium. 2 on a total surface area of ​​approximately 1 × 10 5 The cells are seeded.

[0098] In some embodiments, "% coverage" refers to the area or volume of a biocapsule scaffold that is in contact with a plurality of one or more cell types. In another embodiment, % coverage refers to the area or volume of a biocapsule scaffold that is occupied by a plurality of one or more cell types. As used herein, cells in contact with a biocapsule scaffold are on it, within it, or a combination thereof.

[0099] In some embodiments, the % coverage of the plurality of cell types is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%. In some embodiments, the % coverage of the plurality of cells is 5-20%, 15-30%, 25-40%, 35-50%, 45-60%, 55-70%, 65-80%, 75-90%, 85-100%, or any range therebetween.

[0100] One of ordinary skill in the art will appreciate that the seeding and / or culturing of cells is performed in the presence of a cell culture medium. In some embodiments, the cell culture medium comprises growth factors, cytokines, bioactive agents, nutrients, amino acids, antibiotic compounds, anti-inflammatory compounds, or any combination thereof. Appropriate media and compounds suitable for cell viability and growth are known to those of ordinary skill in the art.

[0101] Growth factors or fragments thereof that can be used in the methods and compositions of the present technology include, but are not limited to, platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-1). PDGF and IGF-1 are known to stimulate mitogenic, chemotactic, and proliferation (differentiation) cellular responses. The growth factor can be one or more of the following, but is not limited to: PDGF, e.g., PDGF AA, PDGF β ... BB; IGF, e.g., IGF-I, IGF-II; fibroblast growth factors (FGF), e.g., acidic FGF, basic FGF, beta endothelial growth factor, FGF4, FGF5, FGF6, FGF7, FGF8, and FGF9; transforming growth factors (TGF), e.g., TGF-P1, TGFβ, TGF-p2, TGF-p3, TGF-p5; bone morphogenetic proteins (BMP), e.g., BMP1, BMP2, BMP3, BMP4; vascular endothelial growth factors (VEGF), e.g., VEGF, placental growth factor; epidermal growth factor (EGF); EGF), such as EGF, amphiregulin, betacellulin, heparin-binding EGF; interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14; colony-stimulating factors (CSF), such as CSF-G, CSF-GM, CSF-M; nerve growth factor (NGF); stem cell factor; hepatocyte growth factor, ciliary neurotrophic factor, and neuregulin (NRG1), or fragments thereof.

[0102] In some embodiments, multiple cell types are incubated with an edible biocapsule scaffold. The scaffold may be of a particular shape or form that influences or defines the three-dimensional shape or form that the population of growing cells takes on. In some embodiments, the biocapsule scaffold mimics the three-dimensional shape of a meat product.

[0103] IX. Methods for Producing Filamentous Fungus-Yeast Biocapsules As described herein, the disclosed technology relates to methods for producing filamentous fungus-yeast biocapsules, in some embodiments, the filamentous fungus-yeast biocapsules function as edible biomaterial scaffolds that provide a three-dimensional structure and suitable growth environment for the attachment, proliferation, and morphogenesis of cultured animal cells for the production of cultivated meat products.

[0104] The method of generating biocapsules of the present technology includes inducing spontaneous co-immobilization between filamentous fungi and multiple yeast cells in the absence of chemical binding compounds and external supports, and artificially creating suitable conditions favorable for the corresponding symbiosis. The combined inherent adhesive properties of yeast and filamentous fungi result in spontaneous formation of biocapsules through hydrophobic interactions when cultured under certain conditions in liquid media. In some embodiments, the yeast is genetically engineered to express one or more heterologous proteins selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof, where the heterologous proteins are soluble expressed, targeted to a specific subcellular localization, or secreted. In some embodiments, the yeast is immobilized. In some embodiments, the yeast is decellularized.

[0105] In some embodiments, the filamentous fungus comprises one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucococci and Trichoderma). In some embodiments, the filamentous fungus is from the phylum Ascomycota and comprises one or more strains belonging to a genus selected from the group consisting of Aspergillus, Penicillium, and Neurospora. In some embodiments, the filamentous fungus is from the genus Aspergillus and comprises one or more strains of Aspergillus oryzae (Koji mold). In some embodiments, the filamentous fungus is from the genus Neurospora and comprises one or more strains of Neurospora crassa. In some embodiments, the filamentous fungus is from the genus Rhizopus and comprises one or more strains of Rhizopus oryzae or Rhizopus oligosporus. In some embodiments, the yeast comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris. In some embodiments, the yeast species is Saccharomyces cerevisiae, Saccharomyces bayanus, or Saccharomyces capensis, and the yeast comprises one or more strains of Saccharomyces cerevisiae. It should be understood that specific examples of filamentous fungi and yeasts are not limited to those explicitly listed herein, and other examples are contemplated.

[0106] The method includes creating conditions that induce co-immobilization between filamentous fungi and yeast cells. The conditions for inducing co-immobilization between filamentous fungi and yeast cells include the use of a first culture medium and a second culture medium. The first culture medium includes a carbon source that is available to the filamentous fungi but not to the yeast, the carbon source being selected from gluconic acid, starch, cellulose, inulin, or other similar colloidal molecules. The second culture medium includes a carbon source that is preferred by the yeast that is also available by the filamentous fungi. Agitation can be performed using an orbital agitator, in a thermostat with an agitation and aeration system, sparging, or any other means suitable for agitation.

[0107] The external factors or conditions of the method of producing biocapsules of the present technology may include the following: an incubation temperature of about 20° C. to about 37° C., an agitation speed of about 0 rpm to about 270 rpm, and an incubation time of 24 hours or more. It should be understood that these external factors are provided for illustrative purposes only.

[0108] The biocapsules produced by the methods of the present technology may, in some embodiments, be used to produce a cell-based meat product. Thus, in some embodiments, the method may further include seeding the filamentous fungus-yeast biocapsule with a plurality of one or more cell types selected from the group consisting of myoblasts or their precursors, adipocytes or their precursors, fibroblasts or their precursors, endothelial cells or their precursors, smooth muscle cells or their precursors, muscle satellite cells, induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, and any combination thereof to form a seeded biocapsule. The method may further include culturing the seeded biocapsule under conditions effective to induce differentiation of the plurality of one or more cell types to produce an edible cell-based meat product.

[0109] In some embodiments, the method may also include engineering the yeast cells to express at least one heterologous protein, protein fragment, and / or protein motif to confer one or more properties to the filamentous fungus. In some embodiments, the heterologous protein is soluble expressed, targeted to a specific subcellular localization, or secreted. In some embodiments, the heterologous protein comprises one or more heterologous proteins selected from extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, motif peptides (synthetic or naturally occurring), flavor proteins, or any combination thereof. In some embodiments, the ECM protein is selected from one or more of collagen, laminin, fibronectin, and RGD motif peptide. In some embodiments, the heterologous protein is from a non-human chordate. In some embodiments, the heterologous protein is from a non-human mammal. In some embodiments, the heterologous protein is from a porcine. In some embodiments, the heterologous protein is from a bovine. In some embodiments, the heterologous protein is from a poultry. In some embodiments, the heterologous protein is from a non-chordate marine organism. A non-exhaustive list of examples includes the addition of collagen to provide a jelly structure, and the addition of fibronectin to provide cell attachment.

[0110] Thus, while one or more desired properties of the edible biocapsule scaffolds described herein are incorporated into and intrinsic to the scaffold, others in the art modify scaffolds via chemistry, by the induction, deposition, or injection of exogenous materials or biomaterials such as hormones, minerals, agarose / gelatin, etc., to impart desirable properties to the scaffold. Additionally, the scaffolds of the present technology are tunable and can be engineered to express growth factors that can improve cell proliferation and differentiation, for example, to allow for co-culture of muscle and adipocytes, among others, potentially reducing production costs.

[0111] In some embodiments, the method includes molding the scaffold into any desired shape, hi some embodiments, the shape of the scaffold mimics the shape of a three-dimensional meat product.

[0112] In some embodiments, the method includes introducing an enzyme (e.g., zymolyase, lyticase, and / or glucalase, among others) into the filamentous fungus to modify a parameter of the filamentous fungus, such as density, porosity, and / or texture of the filamentous fungus.

[0113] In some embodiments, the method includes producing a fungus-yeast biocapsule. In some embodiments, the fungus-yeast biocapsule is a hollow capsule of a certain shape (e.g., a shape that mimics the shape of a meat product), whose walls limit the interior space that is partially occupied by free yeast cells and other cells that bind to the hyphae, forming clusters, and leaving the culture medium transparent. In some embodiments, the scaffold has the shape of the final meat product, e.g., a slice of meat, and thus the shape is a lamellar shape with micropores or striations. It should be understood that the shape of the biocapsule is not limited and any shape is contemplated. The size of the biocapsule may depend on several external factors, such as the stirring speed, stirring time, and incubation temperature. The diameter of the biocapsule may vary from a few millimeters to a few centimeters. The three-dimensional structure of the biocapsule is not limited to a spherical shape and can adopt the shape of the container in which it is cultured.

[0114] In some embodiments, the biocapsules described herein are edible biomaterial scaffolds that provide structure and a suitable growth environment for the attachment, proliferation, and morphogenesis of cultured animal cells to produce steak-like cultivated meat products. In some embodiments, the scaffold comprises an edible base substrate (filamentous fungi) functionalized with yeast cells. In some embodiments, the base substrate is functionalized with heterologous proteins expressed by genetically engineered yeast cells (e.g., baker's yeast). In some embodiments, the base substrate comprises mycelium of edible filamentous fungi, which can be grown to any desired shape and size and serves as the primary structural element of the final cultivated meat product. In some embodiments, the base substrate is functionalized with heterologous proteins expressed by genetically engineered yeast (e.g., baker's yeast) that is primarily composed of edible polysaccharides. In some embodiments, the baker's yeast is decellularized or immobilized. EXAMPLES

[0115] The following examples are provided only as examples, not as limitations.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce essentially the same or similar results.The examples should not be interpreted in any way as limiting the scope of the present technology, as defined by the appended claims.

[0116] Example 1: Preparation of biocapsules Yeast is grown in YP + 3% glycerol. Aspergillus is grown on plates and spores are harvested in BFM. BFM (0.67% YNB + 0.5% gluconic acid) is inoculated with various ratios of yeast and spores. Growth occurs with shaking at 30°C until the biocapsules reach the desired size. The biocapsules are then grown in YP + 18% glucose. The biocapsules are then harvested and washed with water.

[0117] Example 2: Ethanol fixation protocol Wash the scaffolds (biocapsules) with 2 × 50 mL water for 10 min, then incubate with 50 mL of 40% ethanol for 60-180 min. Then, transfer the scaffolds to a sterile tube, wash it with sterile water, and store it in sterile PBS.

[0118] Example 3: Seeding, growth and differentiation of C2C12 on scaffolds This example utilizes C2C12, an immortalized mouse myoblast cell line. Developed for the in vitro study of myoblasts isolated from a complex interplay of in vivo conditions, C2C12 cells are useful for biomedical research because they proliferate rapidly under high serum conditions and are capable of differentiating into myotube fibers under low serum conditions.

[0119] C2C12 cells are grown in growth medium (DMEM+10% FBS) to 80-90% confluence. Cells are trypsinized and washed with growth medium. Seeding at 40-50% confluence is then performed directly onto a scaffold, such as the biocapsule scaffold generated in Example 1. Growth occurs in DMEM+FBS for 7 days, where the medium is replenished every 2 days. Optionally, a portion of the sample is sacrificed and examined by phalloidin / nuclear staining to determine cell proliferation. On day 7, switch to differentiation medium (DMEM+2% horse serum). Growth then occurs for 5-7 days, where the medium is replenished every 4 days. Optionally, a portion of the sample is sacrificed and examined by phalloidin / nuclear staining to determine differentiation / myotube formation.

[0120] Example 4: Growth of biocapsules using solid substrates Aspergillus is grown on plates and spores are harvested in BFM. Yeast is grown in YP + 3% glycerol. Various ratios of Aspergillus spores and yeast are suspended in water and spread on BFM agar. Plates are incubated at 28°C for 72 hours. BFM is aspirated and replaced with YPD18 (18% glucose). Incubation occurs for 24 hours at 28°C. Biocapsules on the agar surface are harvested and processed.

[0121] Example 5: Use of fungal-yeast biocapsules for the production of cell-based meat products This example demonstrates that the filamentous fungus-yeast biocapsules of the present technology are useful in a method for producing a cell-based meat product.

[0122] Briefly, genetically engineered Saccharomyces cerevisiae (baker's yeast) cells are added to a mycelium-containing culture of Aspergillus oryzae (koji mold) to form a fungal-yeast preparation. The yeast cells are genetically engineered to express one or more heterologous proteins. The heterologous proteins may be soluble expressed, targeted to a specific subcellular localization, or secreted. The heterologous proteins may include any one or more chordate ECM proteins (e.g., collagen, laminin, fibronectin, RGD motif peptides), growth factor proteins, cell adhesion proteins, cell signaling proteins, peptide motifs (synthetic or naturally occurring), or cell surface proteins. The preparation is then cultured under conditions (as described herein) sufficient to induce spontaneous co-immobilization of Aspergillus oryzae and Saccharomyces cerevisiae, thereby forming a fungal-yeast biocapsule. The filamentous fungus-yeast biocapsules are then fixed, decellularized, or otherwise processed to inhibit further growth while keeping the endogenous protein structure intact.

[0123] The inactive filamentous fungus-yeast biocapsules are then seeded with a plurality of one or more cell types selected from myoblasts or their precursors, adipocytes or their precursors, fibroblasts or their precursors, endothelial cells or their precursors, smooth muscle cells or their precursors, muscle satellite cells, induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, or any combination thereof to form seeded biocapsules. The seeded biocapsules are then cultured under conditions effective to induce the growth and / or differentiation of the plurality of one or more cell types to produce an edible cell-based meat product.

[0124] Thus, these results demonstrate that the filamentous fungus-yeast biocapsules of the present technology are effective in methods for producing cell-based meat products.

[0125] Example 6: Co-immobilization of baker's yeast and koji mycelium Introduction. This experiment demonstrates the generation of a biocapsule / scaffold for use in the production of cell-based meat products. In particular, this example demonstrates the co-immobilization of Saccharomyces cerevisiae (also known as baker's yeast) and the filamentous fungus, Aspergillus oryzae (also known as koji mold).

[0126] Methods. Briefly, spontaneous co-immobilization of filamentous fungi and yeast occurs when both fungi and yeast are grown in a carbon source that can be utilized only by the filamentous fungus. As a result, the yeast binds tightly to the hyphae of the filamentous fungus through strong hydrophobic interactions. The resulting biocapsules can be grown to a variety of shapes and sizes, most commonly into spheres that can be 1-2 centimeters in size. To determine the extent of co-immobilization, co-immobilized Koji / yeast symbiotic growths were washed extensively with excessive agitation in different solvents of varying ionic strength and polarity. Liquid medium containing a carbon source utilized only by Aspergillus oryzae (0.67% yeast nitrogen base with amino acids, 0.5% gluconic acid) was inoculated with an equimolar ratio of Aspergillus oryzae spores and Saccharomyces cerevisiae and grown overnight at 30 °C with vigorous shaking to allow the filamentous fungi to germinate. The next day, an equal volume of 2x rich glucose-containing medium (2% yeast extract, 4% peptone, 32% glucose) was added to the existing co-culture and incubated at 30 °C for 1–2 days with vigorous shaking. Biocapsules composed of both fungi were then harvested and washed with a total excess of water while vortexing. Biocapsules were then incubated with 40% ethanol, 100 mM NaCl, or 300 mM NaCl for 90–120 min with agitation. Biocapsules were then washed extensively with water and examined microscopically for the presence of Saccharomyces cerevisiae on the Aspergillus hyphae.

[0127] Results. The results of this experiment showed that Aspergillus oryzae (Koji mold) and Saccharomyces cerevisiae (Baker's yeast) were co-immobilized. As shown in Figure 1A, some yeast dissociated from the filamentous fungus after extensive washing, but many remained adsorbed / attached to the Koji mycelium when examined under a fluorescent microscope. As shown in Figure 1B and Figure 1C, the scaffolds can be grown into a variety of shapes and densities, from flat sheets (Figure 1B) to spheres (Figure 1C).

[0128] Thus, these results demonstrate that the methods of the present technology are useful for generating biocapsules that can be used in the production of cell-based meat products, where the biocapsules contain filamentous fungi and multiple yeast cells.

[0129] Example 7: Production of structured cultured meat products using non-genetically engineered yeast Scaffold generation - for flat sheets. 5 mL of liquid YPD16 (1% yeast extract, 2% peptone, 16% glucose) was inoculated with a Baker's yeast strain and grown for 16 h with shaking at 30 °C. Aspergillus oryzae spores were grown on BFM plates (0.67% yeast nitrogen base, 0.5% gluconic acid, 2% agar) for 2-3 days. Aspergillus oryzae spores were harvested from the plates using sterile water. An equimolar mixture of yeast and Aspergillus oryzae spores in sterile water was plated onto YPD agar plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar) with a thin film of cellophane overlaid on top of the agar. The plates were incubated at 30 °C for 1–2 days until a blurred layer was visible on top of the overlaid cellophane. The mycelium-yeast symbiotic growth constructs were then peeled off from the cellophane layer and transferred to a sterile flask containing 50 mL of YPD16 and incubated at 30 °C for 16–24 h without shaking. The flasks were then transferred to a shaker and grown for an additional 16–24 h at 30 °C with gentle shaking. The mycelium-yeast symbiotic growth was then harvested by washing with excess sterile water and incubated with 40% ethanol for 90 min. The scaffolds were then stored in PBS. The flattened scaffolds were cut into the desired shapes using a sterile scalpel.

[0130] Scaffold generation - spherical case. 5 mL of liquid YPD16 (1% yeast extract, 2% peptone, 16% glucose) was inoculated with baker's yeast and grown for 16 h with shaking at 30 °C. Aspergillus oryzae spores were grown on BFM plates (0.67% yeast nitrogen base, 0.5% gluconic acid, 2% agar) for 2-3 days. Aspergillus oryzae spores were harvested from the plates using sterile water. 5 mL of YPD16 was inoculated with an equimolar mixture of yeast and Aspergillus oryzae spores in sterile water and grown for 16-24 h with vigorous shaking at 30 °C. Spherical mycelium-yeast symbiotic growth structures were then harvested by washing with excess sterile water and incubated in 40% ethanol for 90 min. The spherical scaffolds were then stored in PBS.

[0131] Cell seeding and growth. Flat scaffolds were cut into 35 mm diameter circles and transferred into wells of a 6-well tissue culture plate. Scaffolds were washed with 3 mL of growth medium (GM: DMEM high glucose, pyruvate free + 10% FBS + 1% penicillin / streptomycin). Then, 4 × 10 cells were seeded in GM. 5 Three mL of a suspension of quail myoblast (QM7) cells at 1000 cells / mL was added dropwise to the scaffolds in tissue culture wells. The cells on the scaffolds were incubated in a humidified incubator at 37 °C and 5% CO2. 3 mL of fresh GM was replaced every other day for 2–3 weeks. The cell-laden scaffolds were sacrificed on day 2, and cell adhesion was assayed by fluorescence microscopy.

[0132] Fluorescence confocal microscopy. The cell-laden scaffolds were transferred to a new 35 mm Petri dish. The cells were fixed in 1 mL of 4% paraformaldehyde for 20 min at room temperature. The cell-laden scaffolds were then incubated in PBS + 0.2% Triton X-100 for 15 min at room temperature. The cell-laden scaffolds were then incubated in a primary antibody solution of 543-phalloidin and DAPI in PBS for 30-60 min at room temperature. The cell-laden scaffolds were washed twice in PBS with incubation at room temperature for 15 min. The stained cell-laden scaffolds were then transferred to a microscope slide, sandwiched between a cover slip, and then imaged by fluorescence confocal microscopy to confirm the adhesion of the animal cells to the scaffolds (Figure 2).

[0133] Assembly of cultured meat products. Cell-laden scaffolds were washed thoroughly with sterile PBS upon harvesting. Some scaffolds were briefly stained with 1% carmine solution to impart red colorant, then washed with PBS to wash away free dye. Red and white cell-laden scaffolds were dusted with a casein + microbial transglutaminase powder mixture (MooGloo RM). The dusted scaffolds were then layered on top of each other to form a three-dimensional shape. The mass was then incubated at 37 °C for 16 hours to allow the transglutaminase to "glue" the pieces together (Figure 3A). The cultured meat products were cooked in a pan with avocado oil (Figure 3B).

[0134] Results. The results of this experiment produced a structured cultured meat product, as shown in Figures 3A and 3B.

[0135] Thus, these results demonstrate that the methods of the present technology are useful for generating cell-based meat products, including edible biomaterial scaffolds that include filamentous fungi and multiple yeast cells, and multiple one or more cell types, such as myoblasts.

[0136] Example 8: Production of structured cultured meat products using engineered yeast expressing cytoplasmic exogenous bovine collagen Cytoplasmic expression plasmid design. A 45 kDa fragment of the bovine COL1A1 gene was cloned into a Saccharomyces cerevisiae cytoplasmic expression plasmid containing a LEU2 auxotrophic selection marker, an ampicillin resistance marker, and a 2 um origin. Expression of COL1A1 was driven from the GPD promoter. The resulting plasmid is shown in Figure 4.

[0137] Generation of yeast strains. Saccharomyces cerevisiae strain s288c (MATα SUC2 gal2 mal2 mel flo1 flo8-1 hap1 ho bio1 bio6) (Mortimer RK, Johnston JR. Genealogy of principal strains of the yeast genetic stock center. Genetics. 1986 May;113(1):35-43. doi:10.1093 / genetics / 113.1.35. PMID:3519363; PMCID:PMC1202798.) was grown in liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose) to an OD600 of 0.4–0.8. Yeast was transformed with the cytoplasmic expression plasmid (Figure 4) using the lithium acetate transformation method (Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nat Protoc. 2007, 2(1):31-4. doi:10.1038 / nprot.2007.13. PMID:17401334.) and plated on synthetic complete leucine dropout (SC-LEU) agar plates (0.67% yeast nitrogen base without amino acids, 2% glucose, synthetic dropout medium supplement without leucine, 2% agar). Auxotrophic transformants were picked and restreaked on new SC-LEU plates. Auxotrophic strains were maintained in / on selective media for all downstream applications.

[0138] Scaffold generation - for flat sheets. 5 mL of liquid SC-LEU (0.67 yeast nitrogen base without amino acids, 2% glucose, synthetic dropout medium supplement without leucine) was inoculated with the transformed yeast strains described above and grown for 16 h with shaking at 30 °C. Aspergillus oryzae spores were grown on BFM plates (0.67% yeast nitrogen base, 0.5% gluconic acid, 2% agar) for 2-3 days. Aspergillus oryzae spores were harvested from the plates using sterile water. An equimolar mixture of yeast and Aspergillus oryzae spores in sterile water was plated on SC-LEU agar plates with a thin film of cellophane overlaid on top of the agar. Plates were incubated at 30 °C for 1-2 days until a fine fuzzy layer was visible on top of the overlaid cellophane. The mycelium-yeast symbiotic growth constructs were then peeled off from the cellophane layer and transferred to a sterile flask containing 50 mL of SC-LEU and incubated at 30 °C without shaking for 16–24 h. The flasks were then transferred to a shaker and grown for an additional 16–24 h at 30 °C with gentle shaking. The mycelium-yeast symbiotic growth was then harvested by washing with excess sterile water and incubated with 40% ethanol for 90 min. The scaffolds were then stored in PBS. The flat scaffolds were cut into the desired shapes using a sterile scalpel.

[0139] Scaffold generation - spherical case. 5 mL of liquid SC-LEU (0.67 yeast nitrogen base without amino acids, 2% glucose, synthetic dropout medium supplement without leucine) was inoculated with the above transformed yeast strain and grown for 16 h with shaking at 30 °C. Aspergillus oryzae spores were grown on BFM plates (0.67% yeast nitrogen base, 0.5% gluconic acid, 2% agar) for 2-3 days. Aspergillus oryzae spores were harvested from the plates using sterile water. 5 mL of SD-LEU was inoculated with an equimolar mixture of yeast and Aspergillus oryzae spores in sterile water and grown for 16-24 h with vigorous shaking at 30 °C. Spherical mycelium-yeast symbiotic growth structures were then harvested by washing with excess sterile water and incubated with 40% ethanol for 90 min. The spherical scaffolds were then stored in PBS.

[0140] Cell seeding and growth. Flat scaffolds were cut into 35 mm diameter circles and transferred into wells of 6-well tissue culture plates. Scaffolds were washed with 3 mL of growth medium (GM: DMEM high glucose, pyruvate free + 10% FBS + 1% penicillin / streptomycin). Then, 3 mL of a suspension of QM7 cells at 4×10^5 cells / mL in GM was added dropwise to the scaffolds in the tissue culture wells. Cells on the scaffolds were incubated in a humidified incubator at 37°C with 5% CO2. 3 mL of fresh GM was replaced every other day for 2–3 weeks. Cell-laden scaffolds were sacrificed on day 2 and cell adhesion was assayed by fluorescence microscopy.

[0141] Fluorescence confocal microscopy. The cell-laden scaffolds were transferred to a new 35 mm Petri dish. The cells were fixed in 1 mL of 4% paraformaldehyde for 20 min at room temperature. The cell-laden scaffolds were then incubated in PBS + 0.2% Triton X-100 for 15 min at room temperature. The cell-laden scaffolds were then incubated in a primary antibody solution of 543-phalloidin and DAPI in PBS for 30-60 min at room temperature. The cell-laden scaffolds were washed twice in PBS with incubation at room temperature for 15 min. The stained cell-laden scaffolds were then transferred to a microscope slide, sandwiched between a cover slip, and imaged with a fluorescence confocal microscope to confirm that the animal cells were attached to the scaffolds. As shown in Figures 5A-5C, the resulting scaffolds were covered with an actin network (Figure 5A) and cells with nuclei (Figure 5B), and the merged view (Figure 5C) shows the intact cell-laden scaffolds.

[0142] Assembly of cultured meat products. QM7 cell-laden scaffolds were washed thoroughly with sterile PBS upon harvesting. The cell-laden scaffolds were dusted with a casein + microbial transglutaminase powder mixture (MooGloo RM). The dusted scaffolds were then stacked on top of each other to form a three-dimensional shape. The mass was then incubated at 37 °C for 16 hours to allow the transglutaminase to "glue" the pieces together (Figure 6A, top view; Figure 6B, side view). The cultured quail meat products were cooked in a pan with avocado oil (Figure 6C).

[0143] Results: As shown in Figures 6A-6C, the results of this experiment successfully produced a cultured meat product with improved structural uniformity compared to the meat product in Figures 3A-3B.

[0144] Thus, these results demonstrate that the methods of the present technology are useful for generating cell-based meat products, including filamentous fungi, multiple engineered yeast cells expressing exogenous proteins such as collagen targeted to a subcellular location, and edible biomaterial scaffolds comprising multiple one or more cell types, such as myoblasts.

[0145] Example 9: Production of structured cultured meat products using engineered yeast expressing secreted exogenous bovine collagen Design of secretory and integrative expression plasmids. A 45 kDa fragment of the bovine COL1A1 gene was cloned into a Saccharomyces cerevisiae secretory expression plasmid containing a URA3 auxotrophic selection marker, a kanamycin resistance marker, a 2 um element for propagation in Saccharomyces cerevisiae, and a ColE1 origin for propagation in E. coli. A mating factor alpha (MFa) leader sequence with a 3' thrombin cleavage site was fused to the 5' end of the 45 kDa fragment of the bovine COL1A1 gene and a 3× stop was added. MFa-COL1A1 45kDaExpression of the fusion was driven from the TEF1 promoter. The resulting secretory expression plasmid is shown in Figure 7. The bovine P4HA1 gene was cloned into a Saccharomyces cerevisiae integrative plasmid containing a dual LEU2 and HIS3 auxotrophic selection marker, a kanamycin resistance marker, and a ColE1 origin of replication for propagation in E. coli. The P4HA1 gene was fused to the 3' end of the Kar2 leader sequence and to the HDEL 3' terminal peptide sequence for targeting to the endoplasmic reticulum. The resulting P4HA1-integrative plasmid is shown in Figure 8. The bovine P4HB gene was cloned into a Saccharomyces cerevisiae integrative plasmid containing a dual URA3 and HIS3 auxotrophic selection marker, a kanamycin resistance marker, and to the ColE1 origin of replication for propagation in E. coli. The P4HB gene was fused to the 3' end of the Kar2 leader sequence and to the HDEL 3' terminal peptide sequence for targeting to the endoplasmic reticulum. The resulting P4HB integrative plasmid is shown in Figure 9.

[0146] Generation of yeast strains. A sequential transformation procedure was performed to generate triple transformants containing P4HA1 and P4HB integrated into the genome, and the MFa-COL1A1 plasmid, which was maintained under auxotrophic selection. Saccharomyces cerevisiae strain s288c (MATα SUC2 gal2 mal2 mel flo1 flo8-1 hap1 ho bio1 bio6) (Mortimer RK, Johnston JR. Genealogy of principal strains of the yeast genetic stock center. Genetics. 1986 May;113(1):35-43. doi:10.1093 / genetics / 113.1.35. PMID:3519363; PMCID:PMC1202798.) was grown in liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose) to an OD600 of 0.4–0.8. Yeast was then transformed with the P4HB integrant plasmid containing Kar2-P4HB-HDEL using the lithium acetate transformation method (Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nat Protoc. 2007, 2(1):31-4. doi:10.1038 / nprot.2007.13. PMID:17401334.) and plated onto synthetic complete uracil dropout (SC-URA) agar plates (0.67% yeast nitrogen base without amino acids, 2% glucose, synthetic dropout medium supplement without leucine, 2% agar). Uracil auxotrophic transformants were picked and restreaked onto new SC-URA plates, then restreaked again onto nonselective rich medium YPD agar (1% yeast extract, 2% peptone, 2% glucose, 2% agar). A single transformant was then grown overnight in liquid YPD, transformed with the P4HA1 integration plasmid using the lithium acetate method, and plated onto SC-LEU plates. Transformants were streaked onto SC-LEU plates and then restreaked onto YPD plates.The P4HA1 and P4HB double transformants were then grown in YPD and transformed with the MFa-COL1A1 secretion plasmid using the lithium acetate transformation method and plated onto SC-HIS plates. Transformants were picked and streaked onto SC-HIS plates. The final auxotrophic strains were maintained in selective dropout HIS medium for all downstream applications.

[0147] Creating scaffolds with secreted COL1A1. 5 mL of liquid SC-HIS (0.67 yeast nitrogen base without amino acids, 2% glucose, synthetic dropout medium supplement without histidine) was inoculated with the triply transformed yeast strain and grown for 16 h with shaking at 30 °C. Aspergillus oryzae spores were grown on BFM plates (0.67% yeast nitrogen base, 0.5% gluconic acid, 2% agar) for 2–3 days. Aspergillus oryzae spores were harvested from the plates using sterile water. An equimolar mixture of yeast and Aspergillus oryzae spores in sterile water was plated on SC-HIS agar plates with a thin film of cellophane overlaid on top of the agar. Plates were incubated at 30 °C for 1–2 days until a fine fuzzy layer was visible on top of the overlaid cellophane. The mycelium-yeast symbiotic growth constructs were then peeled off from the cellophane layer and transferred to a sterile flask containing 50 mL of SC-HIS and incubated at 30 °C without shaking for 16–24 h. The flask was then transferred to a shaker and grown for an additional 16–24 h at 30 °C with gentle shaking. The mycelium-yeast symbiotic growth was then harvested by washing with excess sterile water and incubated with 40% ethanol for 90 min. The scaffolds were then stored in PBS. The flat scaffolds were cut into the desired shapes using a sterile scalpel (data not shown).

[0148] Thus, these results demonstrate that the methods of the present technology are useful for generating edible biomaterial scaffolds comprising filamentous fungi and multiple genetically engineered yeast cells.

[0149] Example 10: Assembly of cultivated meat products using secreted collagen scaffolds Cell seeding and growth. Flat scaffolds generated in Example 9 are cut into 35 mm diameter circles and transferred into wells of a 6-well tissue culture plate. The scaffolds are washed with 3 mL of growth medium (GM: DMEM high glucose, pyruvate free + 10% FBS + 1% penicillin / streptomycin). 3 mL of a suspension of QM7 cells at 4 x 10^5 cells / mL in GM are added dropwise to the scaffolds in the tissue culture wells. The cells on the scaffolds are incubated in a humidified incubator at 37°C with 5% CO2. 3 mL of fresh GM is replaced every other day for 2-3 weeks.

[0150] Assembly of the cultured meat product. The QM7 cell-laden scaffold is washed thoroughly with sterile PBS upon harvesting. The cell-laden scaffold is dusted with a casein + microbial transglutaminase powder mixture (MooGloo RM). The dusted scaffolds are stacked on top of each other to form a three-dimensional shape. The mass is then incubated at 37°C for 16 hours to allow the transglutaminase to "glue" the pieces together. The cultured quail meat product is cooked in a pan with avocado oil.

[0151] Results: The results of these experiments are expected to lead to successful production of cultured meat products.

[0152] Thus, these results demonstrate that the methods of the present technology are useful for generating cell-based meat products, including edible biomaterial scaffolds containing filamentous fungi, secreted exogenous proteins such as collagen, and multiple cell types, such as cells, myoblasts, and the like.

[0153] Equivalent The present technology is not limited with respect to the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It should be understood that the present technology is not limited to specific methods, reagents, compounds compositions, or biological systems, which, of course, can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0154] Each publication and patent mentioned in the above specification is incorporated herein by reference in its entirety for all purposes. Various modifications and variations of the described methods and systems of the present technology will be apparent to those skilled in the art without departing from the scope and spirit of the present technology. Although the present technology has been described in connection with specific embodiments, the present technology as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present technology will be apparent to those skilled in the art and related fields and are intended to be within the scope of the following claims.

Claims

1. 1. A biocapsule for use in the production of a cell-based meat product, said biocapsule comprising: (i) filamentous fungi, and (ii) A biocapsule comprising a biomaterial scaffold containing a plurality of yeast cells.

2. The biocapsule of claim 1 , wherein the filamentous fungus comprises a mycelium.

3. 3. The biocapsule of claim 1 or 2, wherein the plurality of yeast cells are genetically engineered to express one or more heterologous proteins.

4. 4. The biocapsule of claim 3, wherein the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or any combination thereof.

5. The biocapsule of any one of claims 1 to 4, wherein the filamentous fungus comprises one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucococci and Trichoderma).

6. 6. The biocapsule of claim 5, wherein the filamentous fungal strain comprises one or more strains from the phylum Ascomycota and belonging to a genera selected from the group consisting of Aspergillus, Penicillium, and Neurospora.

7. 7. The biocapsule of claim 6, wherein the filamentous fungus is from the genus Aspergillus, including one or more strains of Aspergillus oryzae.

8. 7. The biocapsule of claim 6, wherein the filamentous fungus is from the genus Neurospora and includes one or more strains of Neurospora crassa.

9. 6. The biocapsule according to claim 5, wherein the filamentous fungal strain is a strain of Rhizopus oryzae or Rhizopus oligosporus from the Mucophylum phylum.

10. 10. The biocapsule of any one of claims 1 to 9, wherein the plurality of yeast cells comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris.

11. 11. The biocapsule of claim 10, wherein the plurality of yeast cells comprises one or more strains of Saccharomyces cerevisiae.

12. 4. The biocapsule of claim 3, wherein the one or more heterologous proteins are chordate ECM proteins, growth factor proteins, cell adhesion proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, cell surface proteins, or any combination thereof.

13. 13. The biocapsule of claim 12, wherein the heterologous chordate protein is a mammalian ECM protein, a growth factor protein, an adhesion molecule protein, a cell signaling protein, a protein motif (synthetic or naturally occurring), a flavor protein, a cell surface protein, or any combination thereof.

14. 4. The biocapsule of claim 3, wherein the one or more heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides.

15. 4. The biocapsule of claim 3, wherein the one or more heterologous proteins are solubly expressed, targeted to a specific intracellular localization, or secreted.

16. The biocapsule of any one of claims 1 to 15, wherein the yeast cells are fixed or decellularized.

17. 1. A cell-based meat product comprising: (i) (a) a filamentous fungus, and (b) an edible biomaterial scaffold comprising a plurality of yeast cells; and (ii) a plurality of one or more cell types.

18. 20. The cell-based meat product of claim 17, wherein the plurality of one or more cell types comprises one or more cell types selected from the group consisting of myoblasts or their precursor cells, adipocytes or their precursor cells, fibroblasts or their precursor cells, endothelial cells or their precursor cells, smooth muscle cells or their precursor cells, muscle satellite cells, induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and any combination thereof.

19. 19. The cell-based meat product of claim 17 or 18, wherein the plurality of yeast cells are genetically engineered to express one or more heterologous proteins.

20. 20. The cell-based meat product of any one of claims 17-19, wherein the plurality of one or more cell types are chordate cells.

21. 21. The cell-based meat product of claim 20, wherein the chordate cells are mammalian cells.

22. 21. The cell-based meat product of claim 20, wherein the chordate cell is a porcine cell, a bovine cell, or a poultry cell.

23. 23. The cell-based meat product of any one of claims 17 to 22, wherein the filamentous fungus comprises a mycelium.

24. 20. The cell-based meat product of claim 19, wherein the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or any combination thereof.

25. 25. The cell-based meat product of any one of claims 17 to 24, wherein the filamentous fungi comprise one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucorhynchum and Ornithomycota).

26. 26. The cell-based meat product of claim 25, wherein the filamentous fungal strain comprises one or more strains from the phylum Ascomycota and belonging to a genus selected from the group consisting of Aspergillus, Penicillium, and Neurospora crassa.

27. 27. The cell-based meat product of claim 26, wherein the filamentous fungus is from the genus Aspergillus, including one or more strains of Aspergillus oryzae.

28. 27. The cell-based meat product of claim 26, wherein the filamentous fungus is from the genus Neurospora and includes one or more strains of Neurospora crassa.

29. 26. The cell-based meat product of claim 25, wherein the filamentous fungal strain is a strain of Rhizopus oryzae or Rhizopus oligosporus from the phylum Mucocomycetes.

30. 30. The cell-based meat product of any one of claims 17-29, wherein the plurality of yeast cells comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris.

31. 31. The cell-based meat product of claim 30, wherein the plurality of yeast cells comprises one or more strains of Saccharomyces cerevisiae.

32. 20. The cell-based meat product of claim 19, wherein the one or more heterologous proteins are a chordate ECM protein, a growth factor protein, a cell adhesion protein, a cell signaling protein, a protein motif (synthetic or naturally occurring), a flavor protein, or a cell surface protein.

33. 33. The cell-based meat product of claim 32, wherein the heterologous chordate protein is a mammalian ECM protein, a growth factor protein, an adhesion molecule protein, a cell signaling protein, a protein motif (synthetic or naturally occurring), a flavor protein, or a cell surface protein.

34. 20. The cell-based meat product of claim 19, wherein the one or more heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides.

35. 20. The cell-based meat product of claim 19, wherein the one or more heterologous proteins are solubly expressed, targeted to a specific subcellular localization, or secreted.

36. 36. The cell-based meat product of any one of claims 17-35, wherein the plurality of yeast cells are fixed or decellularized.

37. 1. A method for producing a filamentous fungus-yeast biocapsule, comprising: adding a plurality of yeast cells to a filamentous fungal culture comprising mycelium to form a filamentous fungal-yeast preparation; and culturing said preparation under conditions inducing spontaneous co-immobilization of said filamentous fungus and yeast to form a filamentous fungus-yeast biocapsule.

38. 38. The method of claim 37, wherein the conditions inducing co-immobilization of the filamentous fungus and yeast comprise the use of a first culture medium and a second culture medium.

39. 39. The method of claim 37 or 38, wherein the plurality of yeast cells are genetically engineered to express one or more heterologous proteins.

40. 39. The method of claim 38, wherein the first culture medium comprises a carbon source that can be used by the filamentous fungus but not by the yeast, the carbon source being selected from the group consisting of gluconic acid, starch, cellulose, inulin, and other similar colloidal molecules.

41. 39. The method of claim 38, wherein the second culture medium comprises a carbon source preferred by the yeast that is also utilizable by the filamentous fungus.

42. 38. The method of claim 37, further comprising contacting the filamentous fungus with an enzyme to modify one or more of the density, porosity, or texture of the filamentous fungus.

43. 43. The method of claim 42, wherein the enzyme is selected from the group consisting of zymolase, lyticase, and glucalase.

44. 44. The method of any one of claims 37 to 43, further comprising fixing or decellularizing the yeast cells.

45. 45. The method of any one of claims 37 to 44, wherein the filamentous fungus-yeast biocapsules are used as an edible biomaterial scaffold for cell-based meat production.

46. 46. ​​The method of any one of claims 37 to 45, further comprising shaping the filamentous fungus-yeast biocapsules.

47. 47. The method of claim 46, wherein the shape of the fungus-yeast biocapsule resembles the shape of a three-dimensional meat product.

48. seeding the fungus-yeast biocapsule with a plurality of one or more cell types selected from the group consisting of myoblasts or their precursors, adipocytes or their precursors, fibroblasts or their precursors, endothelial cells or their precursors, smooth muscle cells or their precursors, muscle satellite cells, induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and any combination thereof, to form a seeded biocapsule; and 38. The method of claim 37, further comprising culturing the seeded biocapsules under conditions effective to induce differentiation of one or more cell types of the plurality to produce an edible cell-based meat product.

49. 49. The method of claim 48, wherein the plurality of one or more cell types are chordate cells.

50. 50. The method of claim 49, wherein the chordate cell is a mammalian cell.

51. 50. The method of claim 49, wherein the chordate cell is a porcine cell, a bovine cell, or a poultry cell.

52. 40. The method of claim 39, wherein the one or more heterologous proteins are selected from one or more extracellular matrix (ECM) proteins, growth factor proteins, cell adhesion proteins, cell surface proteins, cell signaling proteins, protein motifs (synthetic or naturally occurring), flavor proteins, or any combination thereof.

53. 53. The method of any one of claims 37 to 52, wherein the filamentous fungi comprise one or more strains belonging to a phylum selected from the group consisting of Ascomycota, Basidiomycota, and Zygomycota (Mucorhynchum and Trichoderma).

54. 54. The method of claim 53, wherein the filamentous fungal strain comprises one or more strains from the phylum Ascomycota and belonging to a genera selected from the group consisting of Aspergillus, Penicillium, and Neurospora crassa.

55. 55. The method of claim 54, wherein the filamentous fungus is from the genus Aspergillus, including one or more strains of Aspergillus oryzae.

56. 55. The method of claim 54, wherein the filamentous fungus is from the genus Neurospora and includes one or more strains of Neurospora crassa.

57. 54. The method of claim 53, wherein the filamentous fungal strain is a strain of Rhizopus oryzae or Rhizopus oligosporus from the Mucophylum division.

58. 58. The method of any one of claims 37-57, wherein the plurality of yeast cells comprises one or more strains selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces capensis, Schizosaccharomyces pombe, and Pichia pastoris.

59. 60. The method of claim 58, wherein the plurality of yeast cells comprises one or more strains of Saccharomyces cerevisiae.

60. 40. The method of claim 39, wherein the one or more heterologous proteins are chordate ECM proteins, growth factor proteins, cell adhesion proteins, cell signaling proteins, peptide motifs (synthetic or naturally occurring), flavor proteins, or cell surface proteins.

61. 61. The method of claim 60, wherein the heterologous chordate protein is a mammalian ECM protein, a growth factor protein, an adhesion molecule protein, a cell signaling protein, a peptide motif (synthetic or naturally occurring), a flavor protein, or a cell surface protein.

62. 40. The method of claim 39, wherein the one or more heterologous proteins comprise one or more ECM proteins selected from the group consisting of collagen, laminin, fibronectin, and RGD motif peptides.

63. 40. The method of claim 39, wherein the one or more heterologous proteins are solubly expressed, targeted to a specific subcellular location, or secreted.

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