RECOMBINANT YEAST STRAINS

Cross Reference to Related Applications

This application is related to U.S. Patent Application No. 15/433,566 entitled

Biofabricated Material Containing Collagen Fibrils, 15/433,650 entitled Method for Making a Biofabricated Material Containing Collagen Fibrils, 62/526,912 entitled Recombinant Yeast Strains, and 62/539,213 entitled Yeast Strains and Method for Controlling Hydroxylation of Recombinant Collagen, and claims priority to 62/562,109, filed September 22, 2017, entitled Recombinant Yeast Strains. The entire contents of these applications are incorporated by reference.

Field of the Invention

This invention herein provides genetically engineered strains of yeast and methods for producing recombinant proteins and carbohydrates. In an embodiment, the invention herein provides genetically engineered strains of yeast and methods for producing recombinant collagen that can be used to produce biofabricated leather materials, articles comprising biofabricated materials, and/or a material having leather-like properties containing recombinant or engineered collagen. The yeast strains are engineered to produce increased amounts of hydroxylated protein (e.g., collagen) and carbohydrates by improving the hydroxylation reaction through the use of an a ketoglutarate transporter (kgtP) and/or one or more polypeptides that enable an ascorbate synthesis pathway to function such as, GDP-L-Gal phosphorylase, inositol-phosphate phosphatase, GDP-Mannose-3,5-epimerase and/or L-gulono-l,4-lactone oxidase,

aldonolactonase, glucurono lactone reductase, D-glucuronate reductase, uronolactonase, D- glucurono kinase, glucuronate-1 -phosphate uridylyltransferase, UDP-D-glucose dehydrogenase, UTP- glucose- 1 -phosphate uridylyltransferase, phosphoglucomutase, and/or hexokinase.

Description of Related Art

Leather is used in a vast variety of applications, including furniture upholstery, clothing, shoes, luggage, handbag and accessories, and automotive applications. The estimated global trade value in leather is approximately US $100 billion per year {Future Trends in the World Leather Products Industry and Trade, United Nations Industrial Development Organization, Vienna, 2010) and there is a continuing and increasing demand for leather products. Traditional leather production is wasteful with respect to animal hide and chemicals. The ever increasing demand for animal hides to keep up with the continuing and increasing demand for leather products puts additional pressure on our increasingly strained food sources, while the chemical waste continues to contribute to our eroding environmental conditions. Thus, new ways to meet this demand are required in view of the economic, environmental and social costs of producing leather. To keep up with technological and aesthetic trends, producers and users of leather products seek new materials exhibiting superior strength, uniformity, processability and fashionable and appealing aesthetic properties that incorporate natural components.

Given population growth and the global environment, there will be a need for alternative materials that have leather-like aesthetics and improved functionalities. Leather is animal hide and consists almost entirely of collagen. There is a need for new sources of collagen that can be incorporated into biofabricated materials.

Production of biofabricated materials using recombinantly-expressed collagen faces a number of challenges including a need for a method for efficiently producing collagen in forms and quantity needed for diverse commercial applications. For some applications a softer and more permeable collagen component is desired; in others, a harder, more resistant and durable collagen component is needed.

The inventors sought to address these challenges by engineering recombinant yeasts with a kgtP gene which provides an a ketoglutarate transporter and/or with one or more genes that enable an ascorbate synthesis pathway to function such that the recombinant yeast cell produces ascorbate.

Summary of the Invention

One aspect of the invention is directed to modified yeast cells which contain an a ketoglutarate transporter. Modification includes transforming a kgtP gene into the yeast which enables the yeast to make the a ketoglutarate transporter. The yeast cells may be further modified to express and/or overexpress a protein which hydroxylates proline and/or lysine residues to yield hydroxyproline, 3-hydroxyproline (Hyp) and hydroxylysine (Hyl), and glycosylation of the hydroxylysyl residues. In an embodiment of the present invention, the present invention provides a method for improving production of proteins such as hydroxylated protein (e.g., collagen) or carbohydrates in yeast. The method includes inserting (e.g., transforming or genomically integrating) a kgtP gene, DNA for a protein or carbohydrate and DNA for promoters, terminators, and markers into the yeast to enable the yeast to produce an a ketoglutarate transporter and the protein or carbohydrate. The method also includes inserting genes expressing a protein(s) which hydroxylates proline and/or lysine residues.

Another aspect of the invention is directed to modified yeast cells which produce ascorbate. Modification includes inserting genes which enable an ascorbate synthesis pathway to function. In an embodiment, the present invention provides a method for producing modified yeast cells which produce ascorbate. The method includes inserting genes necessary to complete a functioning ascorbate synthesis pathway. In this method, it is envisioned that it is only necessary to insert one or more of the genes encoding proteins for the portion of the ascorbate synthesis pathway downstream from the ascorbate pathway precursor fed to the yeast cells. In another embodiment, the present invention provides a method of producing intracellular ascorbate in a yeast cell including growing a modified yeast cell in media. In another embodiment, the present invention provides a modified yeast cell with a constant amount of intracellular ascorbate.

Yet another aspect of the invention is directed to modified yeast cells which contain an a ketoglutarate transporter and which produce ascorbate. The yeast cells may be further modified to express and/or overexpress a protein which hydroxylates proline and/or lysine residues to yield hydroxyproline, 3-hydroxyproline (Hyp) and hydroxylysine (Hyl), and glycosylation of the hydroxylysyl residues. The method includes inserting genes necessary to complete a functioning ascorbate synthesis pathway and an a ketoglutarate transporter into the yeast. The method may also include inserting genes expressing a protein(s) which hydroxylates proline and/or lysine residues. In another embodiment, the present invention provides a method of producing intracellular ascorbate in a yeast cell including growing a modified yeast cell in media. In an embodiment of the present invention, the present invention provides a method for improving production of proteins such as hydroxylated collagen or carbohydrates in yeast that contain an a ketoglutarate transporter and which produce ascorbate.

Detailed Description of the Invention

As described and exemplified herein, yeast cells (e.g., Pichia pastoris) can be used to express recombinant Type ΠΙ bovine collagen with different degrees of hydroxylation.

Hydroxylation of recombinant collagen is accomplished by co-expression of bovine P4HA and bovine P4HB which respectively encode the alpha and beta subunits bovine prolyl-4- hydroxylase. However, the invention is not limited to products and expression of Type ΙΠ collagen and may be practiced with other proteins or carbohydrates, polynucleotides encoding the subunits of other kinds of collagens as well as with enzymes that hydroxylate proline residues, lysine residues, or both proline and lysine residues. Type ΙΠ tropocollagen is a homotrimer. However, in some embodiments a collagen will form a heterotrimer composed of different polypeptide chains, such as Type I collagen which is initially composed of two pro- al(I) chains and one pro-a2(I) chain.

Collagen. Collagen is the main component of leather. Skin, or animal hide, contains significant amounts of collagen, a fibrous protein. Collagen is a generic term for a family of at least 28 distinct collagen types; animal skin is typically Type I collagen, although other types of collagen can be used in forming leather including type III collagen. The term "collagen" encompasses unprocessed (e.g., procollagens) as well as post-translationally modified and proteolysed collagens having a triple helical structure.

Collagens are characterized by a repeating triplet of amino acids, -(Gly-X-Y)n- and approximately one-third of the amino acid residues in collagen are glycine. X is often proline and Y is often hydroxyproline, though there may be up to 400 possible Gly-X-Y triplets. Different animals may produce different amino acid compositions of the collagen, which may result in different properties and in differences in the resulting leather.

The structure of collagen can consist of three intertwined peptide chains of differing lengths. Collagen triple helices (or monomers) may be produced from alpha-chains of about 1,050 amino acids long, so that the triple helix takes the form of a rod of about approximately 300 nm long, with a diameter of approximately 1.5 nm. Collagen fibers may have a range of diameters depending on the type of animal hide. In addition to type I collagen, skin (hides) may include other types of collagen as well, including type IIΙ collagen (reticulin), type IV collagen, and type VII collagen. Various types of collagen exist throughout the mammalian body. For example, besides being the main component of skin and animal hide, Type I collagen also exists in cartilage, tendon, vascular ligature, organs, muscle, and the organic portion of bone. Successful efforts have been made to isolate collagen from various regions of the mammalian body in addition to the animal skin or hide. Decades ago, researchers found that at neutral pH, acid-solubilized collagen self-assembled into fibrils composed of the same cross-striated patterns observed in native tissue; Schmitt F.O. J. Cell. Comp Physiol. 1942;20: 11). This led to use of collagen in tissue engineering and a variety of biomedical applications. In more recent years, collagen has been harvested from bacteria and yeast using recombinant techniques.

Collagens are formed and stabilized through a combination of physical and chemical interactions including electrostatic interactions such as salt bridging, hydrogen bonding, Van der Waals interactions, dipole-dipole forces, polarization forces, hydrophobic interactions, and covalent bonding often catalyzed by enzymatic reactions. Various distinct collagen types have been identified in vertebrates including bovine, ovine, porcine, chicken, and human collagens.

Generally, the collagen types are numbered by Roman numerals, and the chains found in each collagen type are identified by Arabic numerals. Detailed descriptions of structure and biological functions of the various different types of naturally occurring collagens are available in the art; see, e.g., Ayad et al. (1998) The Extracellular Matrix Facts Book, Academic Press, San Diego, CA; Burgeson, R E., and Nimmi (1992) "Collagen types: Molecular Structure and Tissue Distribution" in Clin. Orthop. 282:250-272; Kielty, C. M. et al. (1993) "The Collagen Family: Structure, Assembly And Organization In The Extracellular Matrix," Connective Tissue And Its Heritable Disorders, Molecular Genetics, And Medical Aspects, Royce, P. M. and B. Steinmann eds., Wiley-Liss, NY, pp. 103-147; and Prockop, DJ- and K.I. Kivirikko (1995) "Collagens: Molecular Biology, Diseases, and Potentials for Therapy," Annu.Rev. Biochem., 64:403-434.) Type I collagen is the major fibrillar collagen of bone and skin comprising approximately

80-90% of an organism's total collagen. Type I collagen is the major structural macromolecule present in the extracellular matrix of multicellular organisms and comprises approximately 20% of total protein mass. Type I collagen is a heterotrimeric molecule comprising two al(I) chains and one a2(I) chain, encoded by the COL1 Al and COL1 A2 genes, respectively. In vivo, assembly of Type I collagen fibrils, fibers, and fiber bundles takes place during development and provides mechanical support to the tissue while allowing for cellular motility and nutrient transport. Other collagen types are less abundant than type I collagen and exhibit different distribution patterns. For example, type II collagen is the predominant collagen in cartilage and vitreous humor, while type ΙΠ collagen is found at high levels in blood vessels and to a lesser extent in skin.

Type II collagen is a homotrimeric collagen comprising three identical al(II) chains encoded by the COL2A1 gene. Purified type II collagen may be prepared from tissues by, methods known in the art, for example, by procedures described in Miller and Rhodes (1982) Methods In Enzymology 82:33-64.

Type III collagen is a major fibrillar collagen found in skin and vascular tissues. Type III collagen is a homotrimeric collagen comprising three identical αΙ(ΠΙ) chains encoded by the COL3 Al gene. The COL3 Al gene may be optimized for expression in the host cell, for example Pichia pastoris (SEQ ID NO: 10). Methods for purifying type III collagen from tissues can be found in, for example, Byers et al. (1974) Biochemistry 13:5243-5248; and Miller and Rhodes, supra.

Type IV collagen is found in basement membranes in the form of sheets rather than fibrils. Most commonly, type IV collagen contains two al(IV) chains and one a2(IV) chain. The particular chains comprising type IV collagen are tissue-specific. Type IV collagen may be purified using, for example, the procedures described in Furuto and Miller (1987) Methods in Enzymology, 144:41-61, Academic Press.

Type V collagen is a fibrillar collagen found in, primarily, bones, tendon, cornea, skin, and blood vessels. Type V collagen exists in both homotrimeric and heterotrimeric forms. One form of type V collagen is a heterotrimer of two al(V) chains and one a2(V) chain. Another form of type V collagen is a heterotrimer of al(V), a2(V), and a3(V) chains. A further form of type V collagen is a homotrimer of al(V). Methods for isolating type V collagen from natural sources can be found, for example, in Elstow and Weiss (1983) Collagen Rel. Res. 3:181-193, and Abedin et al. (1982) Biosci. Rep. 2:493-502.

Type VI collagen has a small triple helical region and two large non-collagenous remainder portions. Type VI collagen is a heterotrimer comprising al(VI), a2(VI), and a3(VI) chains. Type VI collagen is found in many connective tissues. Descriptions of how to purify type

VI collagen from natural sources can be found, for example, in Wu et al. (1987) Biochem. J. 248:373-381, and Kielty et al. (1991) J. Cell Sci. 99:797-807.

Type VII collagen is a fibrillar collagen found in particular epithelial tissues. Type VH collagen is a homotrimeric molecule of three al(VII) chains. Descriptions of how to purify type

VII collagen from tissue can be found in, for example, Lunstrum et al. (1986) J. Biol. Chem. 261:9042-9048, and Bentz et al. (1983) Proc. Natl. Acad. Sci. USA 80:3168-3172.Type VIII collagen can be found in Descemet's membrane in the cornea. Type I collagen is a heterotrimer comprising two a 1 (VIII) chains and one a2(VIII) chain, although other chain compositions have been reported. Methods for the purification of type VIII collagen from nature can be found, for example, in Benya and Padilla (1986) J. Biol. Chem. 261:4160-4169, and Kapoor et al. (1986) Biochemistry 25:3930-3937.

Type IX collagen is a fibril-associated collagen found in cartilage and vitreous humor. Type IX collagen is a heterotrimeric molecule comprising al(IX), a2(IX), and α3 (IX) chains. Type IX collagen has been classified as a FACIT (Fibril Associated Collagens with Interrupted Triple Helices) collagen, possessing several triple helical domains separated by non-triple helical domains. Procedures for purifying type IX collagen can be found, for example, in Duance, et al. (1984) Biochem. J. 221:885-889; Ayad et al. (1989) Biochem. J. 262:753-761; and Grant et al. (1988) The Control of Tissue Damage, Glauert, A. M., ed., Elsevier Science Publishers,

Amsterdam, pp. 3-28.

Type X collagen is a homotrimeric compound of al(X) chains. Type X collagen has been isolated from, for example, hypertrophic cartilage found in growth plates; see, e.g., Apte et al. (1992) Eur J Biochem 206 (l):217-24.

Type XI collagen can be found in cartilaginous tissues associated with type II and type IX collagens, and in other locations in the body. Type XI collagen is a heterotrimeric molecule comprising al(XI), a2(XI), and a3(XI) chains. Methods for purifying type XI collagen can be found, for example, in Grant et al., supra.

Type XII collagen is a FACIT collagen found primarily in association with type I collagen. Type XII collagen is a homotrimeric molecule comprising three α1(ΧΠ) chains.

Methods for purifying type ΧΠ collagen and variants thereof can be found, for example, in Dublet et al. (1989) J. Biol. Chem. 264:13150-13156; Lunstrum et al. (1992) J. Biol. Chem. 267:20087-20092; and Watt et al. (1992) J. Biol. Chem. 267:20093-20099. Type XIII is a non- fibrillar collagen found, for example, in skin, intestine, bone, cartilage, and striated muscle. A detailed description of type XIII collagen may be found, for example, in Juvonen et al. (1992) J. Biol. Chem. 267: 24700-24707.

Type XIV is a FACIT collagen characterized as a homotrimeric molecule comprising al(XIV) chains. Methods for isolating type XIV collagen can be found, for example, in Aubert- Foucher et al. (1992) J. Biol. Chem. 267:15759-15764,and Watt et al., supra.

Type XV collagen is homologous in structure to type XVIII collagen. Information about the structure and isolation of natural type XV collagen can be found, for example, in Myers et al. (1992) Proc. Natl. Acad. Sci. USA 89:10144-10148; Huebner et al. (1992) Genomics 14:220- 224; Kivirikko et al. (1994) J. Biol. Chem. 269:4773-4779; and Muragaki, J. (1994) Biol. Chem. 264:4042-4046.

Type XVI collagen is a fibril-associated collagen, found, for example, in skin, lung fibroblast, and keratinocytes. Information on the structure of type XVI collagen and the gene encoding type XVI collagen can be found, for example, in Pan et al. (1992) Proc. Natl. Acad. Sci. USA 89:6565-6569; and Yamaguchi et al. (1992) J. Biochem. 112:856-863.

Type XVII collagen is a hemidesmosal transmembrane collagen, also known at the bullous pemphigoid antigen. Information on the structure of type XVII collagen and the gene encoding type XVII collagen can be found, for example, in Li et al. (1993) J. Biol. Chem.

268(12):8825-8834; and McGrath et al. (1995) Nat. Genet. l l(l):83-86.

Type XVm collagen is similar in structure to type XV collagen and can be isolated from the liver. Descriptions of the structures and isolation of type XVIII collagen from natural sources can be found, for example, in Rehn and Pihlajaniemi (1994) Proc. Natl. Acad. Sci USA 91:4234- 4238; Oh et al. (1994) Proc. Natl. Acad. Sci USA 91:4229-4233; Rehn et al. (1994) J. Biol. Chem. 269:13924-13935; and Oh et al. (1994) Genomics 19:494-499.

Type XIX collagen is believed to be another member of the FACIT collagen family, and has been found in mRNA isolated from rhabdomyosarcoma cells. Descriptions of the structures and isolation of type XIX collagen can be found, for example, in Inoguchi et al. (1995) J.

Biochem. 117:137-146; Yoshioka et al. (1992) Genomics 13:884-886; and Myers et al., J. Biol. Chem. 289:18549-18557 (1994).

Type XX collagen is a newly found member of the FACIT collagenous family, and has been identified in chick cornea. (See, e.g., Gordon et al. (1999) FASEB Journal 13:A1119; and Gordon et al. (1998), IOVS 39:S1128.)

The term "collagen" refers to any one of the known collagen types, including collagen types I through XX described above, as well as to any other collagens, whether natural, synthetic, semi-synthetic, or recombinant. It includes all of the collagens, modified collagens and collagen-like proteins described herein. The term also encompasses procollagens and collagenlike proteins or collagenous proteins comprising the motif (Gly-X-Y)n where n is an integer. It encompasses molecules of collagen and collagen-like proteins, trimers of collagen molecules, fibrils of collagen, and fibers of collagen fibrils. It also refers to chemically, enzymatically or recombinantly-modified collagens or collagen-like molecules that can be fibrillated as well as fragments of collagen, collagen-like molecules and collagenous molecules capable of assembling into a nanofiber. Recombinant collagen molecules whether native or engineered will generally comprise the repeated -(Gly-X-Y)n- sequence described herein. The collagen in a collagen composition may homogenously contain a single type of collagen molecule, such as 100% bovine Type I collagen or 100% Type ΙΠ bovine collagen, or may contain a mixture of different kinds of collagen molecules or collagen-like molecules, such as a mixture of bovine Type I and Type ΙII molecules. Such mixtures may include >0%, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or <100% of the individual collagen or collagen-like protein components. This range includes all intermediate values. For example, a collagen composition may contain 30% Type I collagen and 70% Type ΙΠ collagen, or may contain 33.3% of Type I collagen, 33.3% of Type II collagen, and 33.3% of Type ΙΠ collagen, where the percentage of collagen is based on the total mass of collagen in the composition or on the molecular percentages of collagen molecules.

"Collagen fibrils" are nanofibers composed of tropocollagen (triple helices of collagen molecules). Tropocollagens also include tropocollagen-like structures exhibiting triple helical structures. The collagen fibrils of the invention may have diameters ranging from 1 nm and 1 μm. For example, the collagen fibrils of the invention may have an average or individual fibril diameter ranging from 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nm (1 μm). This range includes all intermediate values and subranges. In some of the embodiments of the invention collagen fibrils will form networks. Collagen fibrils can associate into fibrils exhibiting a banded pattern and these fibrils can associate into larger aggregates of fibrils. In some embodiments the collagen or collagen-like fibrils will have diameters and orientations similar to those in the top grain or surface layer of a bovine or other conventional leather. In other embodiments, the collagen fibrils may have diameters comprising the top grain and those of a corium layer of a conventional leather. A "collagen fiber" is composed of collagen fibrils that are tightly packed and exhibit a high degree of alignment in the direction of the fiber. It can vary in diameter from more than 1 μm to more than 10 μm, for example >1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 μm or more. Some embodiments of the network of collage fibrils of the invention do not contain substantial content of collagen fibers having diameters greater than 5 μm. The composition of the grain surface of a leather can differ from its more internal portions, such as the corium which contains coarser fiber bundles.

"Fibrillation" refers to a process of producing collagen fibrils. It may be performed by raising the pH or by adjusting the salt concentration of a collagen solution or suspension. In forming the fibrillated collagen, the collagen may be incubated to form the fibrils for any appropriate length of time, including between 1 min and 24 hrs and all intermediate values.

The fibrillated collagen described herein may generally be formed in any appropriate shape and/or thickness, including flat sheets, curved shapes/sheets, cylinders, threads, and complex shapes. These sheets and other forms may have virtually any linear dimensions including a thickness, width or height greater of 10, 20, 30, 40, 50, 60, 70,80, 90 mm; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 500, 1,000, 1,500, 2,000 cm or more.

The fibrillated collagen may lack any or any substantial amount of higher order structure. In a preferred embodiment, the collagen fibrils will be unbundled and not form the large collagen fibers found in animal skin and provide a strong and uniform non-anisotropic structure to the biofabricated leather.

In other embodiments, some collagen fibrils can be bundled or aligned into higher order structures. Collagen fibrils in a biofabricated leather may exhibit an orientation index ranging from 0, >0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, or 1.0, wherein an orientation index of 0 describes collagen fibrils that lack alignment with other fibrils and an orientation index of 1.0 describes collagen fibrils that are completely aligned. This range includes all intermediate values and subranges. Those of skill in the art are familiar with the orientation index which is also incorporated by reference to Sizeland, et al., J. Agric. Food Chem. 61 : 887-892 (2013) or Basil- Jones, et al., J. Agric. Food Chem. 59: 9972-9979 (2011).

A biofabricated leather may be fibrillated and processed to contain collagen fibrils that resemble or mimic the properties of collagen fibrils produced by particular species or breeds of animals or by animals raised under particular conditions.

Alternatively, fibrillation and processing conditions can be selected to provide collagen fibrils distinct from those found in nature, such as by decreasing or increasing the fibril diameter, degree of alignment, or degree of crosslinking compared to fibrils in natural leather.

A crosslinked network of collagen, sometimes called a hydrogel, may be formed as the collagen is fibrillated, or it may form a network after fibrillation; in some variations, the process of fibrillating the collagen also forms gel-like network. Once formed, the fibrillated collagen network may be further stabilized by incorporating molecules with di-, tri-, or multifunctional reactive groups that include chromium, amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides, acrylates, epoxides, or phenols.

The fibrillated collagen network may also be polymerized with other agents (e.g.

polymers that are capable of polymerizing or other suitable fibers), which could be used to further stabilize the matrix and provide the desired end structure. Hydrogels based upon acrylamides, acrylic acids, and their salts may be prepared using inverse suspension

polymerization. Hydrogels described herein may be prepared from polar monomers. The hydrogels used may be natural polymer hydrogels, synthetic polymer hydrogels, or a combination of the two. The hydrogels used may be obtained using graft polymerization, crosslinking polymerization, networks formed of water soluble polymers, radiation crosslinking, and so on. A small amount of crosslinking agent may be added to the hydrogel composition to enhance polymerization.

Average or individual collagen fibril length may range from 100, 200, 300, 400, 500,

600, 700, 800, 900, 1,000 (1 μm); 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 μm (1 mm) throughout the entire thickness of a biofabricated leather. These ranges include all intermediate values and subranges.

Fibrils may align with other fibrils over 50, 100, 200, 300, 400, 500 μm or more of their lengths or may exhibit little or no alignment. In other embodiments, some collagen fibrils can be bundled or aligned into higher order structures.

Collagen fibrils in a biofabricated leather may exhibit an orientation index ranging from 0, >0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, or 1.0, wherein an orientation index of 0 describes collagen fibrils that lack alignment with other fibrils and an orientation index of 1.0 describes collagen fibrils that are completely aligned. This range includes all intermediate values and subranges. Those of skill in the art are familiar with the orientation index which is also incorporated by reference to Sizeland, et al., J. Agric. Food Chem. 61 : 887-892 (2013) or Basil- Jones, et al., J. Agric. Food Chem. 59: 9972-9979 (2011).

Collagen fibril density of a biofabricated leather may range from about 1 to 1,000 mg/cc, preferably from 5 to 500 mg/cc including all intermediate values, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1,000 mg/cc.

The collagen fibrils in a biofabricated leather may exhibit a unimodal, bimodal, trimodal, or multimodal distribution, for example, a biofabricated leather may be composed of two different fibril preparations each having a different range of fibril diameters arranged around one of two different modes. Such mixtures may be selected to impart additive, synergistic or a balance of physical properties on a biofabricated leather conferred by fibrils having different diameters.

Natural leather products may contain 150-300 mg/cc collagen based on the weight of the leather product. A biofabricated leather may contain a similar content of collagen or collagen fibrils as conventional leather based on the weight of the biofabricated leather, such as a collagen concentration of 100, 150, 200, 250, 300 or 350 mg/cc.

The fibrillated collagen, sometimes called a hydrogel, may have a thickness selected based on its ultimate use. Thicker or more concentrated preparations of the fibrillated collagen generally produce thicker biofabricated leathers. The final thickness of a biofabricated leather may be only 10, 20, 30, 40, 50, 60, 70, 80 or 90% that of the fibril preparation prior to shrinkage caused by crosslinking, dehydration and lubrication.

"Crosslinking" refers to formation (or reformation) of chemical bonds within between collagen molecules. A crosslinking reaction stabilizes the collagen structure and in some cases forms a network between collagen molecules. Any suitable crosslinking agent known in the art can be used including, without limitation, mineral salts such as those based on chromium, formaldehyde, hexamethylene diisocyanate, glutaraldehyde, polyepoxy compounds, gamma irradiation, and ultraviolet irradiation with riboflavin. The crosslinking can be performed by any known method; see, e.g., Bailey et al., Radiat. Res. 22:606-621 (1964); Housley et al., Biochem. Biophys. Res. Commun. 67:824-830 (1975); Siegel, Proc. Natl. Acad. Sci. U.S.A. 71:4826-4830 (1974); Mechanic et al., Biochem. Biophys. Res. Commun. 45:644-653 (1971); Mechanic et al., Biochem. Biophys. Res. Commun. 41:1597-1604 (1970); and Shoshan et al., Biochim. Biophys. Acta 154:261-263 (1968) each of which is incorporated by reference.

Crosslinkers include isocyantes, carbodiimide, poly(aldehyde), poly(azyridine), mineral salts, poly(epoxies), enzymes, thiirane, phenolics, novolac, resole as well as other compounds that have chemistries that react with amino acid side chains such as lysine, arginine, aspartic acid, glutamic acid, hydroxylproline, or hydroxylysine.

A collagen or collagen-like protein may be chemically modified to promote chemical and/or physical crosslinking between the collagen fibrils. Chemical crosslinking may be possible because reactive groups such as lysine, glutamic acid, and hydroxyl groups on the collagen molecule project from collagen' s rod-like fibril structure. Crosslinking that involve these groups prevent the collagen molecules from sliding past each other under stress and thus increases the mechanical strength of the collagen fibers. Examples of chemical crosslinking reactions include but are not limited to reactions with the ε-amino group of lysine, or reaction with carboxyl groups of the collagen molecule. Enzymes such as transglutaminase may also be used to generate crosslinks between glutamic acid and lysine to form a stable γ-glutamyl-lysine crosslink. Inducing crosslinking between functional groups of neighboring collagen molecules is known in the art. Crosslinking is another step that can be implemented here to adjust the physical properties obtained from the fibrillated collagen hydrogel-derived materials.

Still fibrillating or fibrillated collagen may be crosslinked or lubricated. Collagen fibrils can be treated with compounds containing chromium or at least one aldehyde group, or vegetable tannins prior to network formation, during network formation, or network gel formation.

Crosslinking further stabilizes the fibrillated collagen leather. For example, collagen fibrils pre- treated with acrylic polymer followed by treatment with a vegetable tannin, such as Acacia Mollissima, can exhibit increased hydrothermal stability. In other embodiments, glyceraldehyde may be used as a cross-linking agent to increase the thermal stability, proteolytic resistance, and mechanical characteristics, such as Young's modulus and tensile stress, of the fibrillated collagen.

A biofabricated material containing a network of collagen fibrils may contain 0, >0, 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 15, 20% or more of a crosslinking agent including tanning agents used for conventional leather. The crosslinking agents may be covalently bound to the collagen fibrils or other components of a biofabricated material or non-covalently associated with them.

Preferably, a biofabricated leather will contain no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of a crosslinking agent.

"Lubricating" describes a process of applying a lubricant, such as a fat or other hydrophobic compound or any material that modulates or controls fibril-fibril bonding during dehydration to leather or to biofabricated products comprising collagen. A desirable feature of the leather aesthetic is the stiffness or hand of the material. In order to achieve this property, water-mediated hydrogen bonding between fibrils and/or fibers is limited in leather through the use of lubricants. Examples of lubricants include fats, biological, mineral or synthetic oils, cod oil, sulfonated oil, polymers, organofunctional siloxanes, and other hydrophobic compounds or agents used for fatliquoring conventional leather as well as mixtures thereof. While lubricating is in some ways analogous to fatliquoring a natural leather, a biofabricated product can be more uniformly treated with a lubricant due to its method of manufacture, more homogenous composition and less complex composition.

Other lubricants include surfactants, anionic surfactants, cationic surfactants, cationic polymeric surfactants, anionic polymeric surfactants, amphiphilic polymers, fatty acids, modified fatty acids, nonionic hydrophilic polymers, nonionic hydrophobic polymers, poly acrylic acids, poly methacrylic, acrylics, natural rubbers, synthetic rubbers, resins, amphiphilic anionic polymer and copolymers, amphiphilic cationic polymer and copolymers and mixtures thereof as well as emulsions or suspensions of these in water, alcohol, ketones, and other solvents.

Lubricants may be added to a biofabricated material containing collagen fibrils.

Lubricants may be incorporated in any amount that facilitates fibril movement or that confers leather-like properties such as flexibility, decrease in brittleness, durability, or water resistance. A lubricant content can range from about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60% by weight of the biofabricated leather.

Other additives may be added to modify the properties of biofabricated leather or material. Suitable additives include but are not limited to dyes, pigments, fragrances, resins, and microparticles. Resins may be added to modify the stretchability, strength, and softness of the material. Suitable resins include but are not limited to elastomers, acrylic copolymers, polyurethane, and the like. Suitable elastomers include but are not limited to styrene, isoprene, butadiene copolymers such as KRAYTON® elastomers, Hycar ® acrylic resins. Resins may be used at from about 5% to 200%, or from about 50% to 150% (based on the weight of collagen), these ranges including all values and subranges there between such as 10, 15, 20, 25, 30, 35, 40, 60, 70, 75, 80, 90, 100, 125, 140 etc.

"Dehydrating" or "dewatering" describes a process of removing water from a mixture containing collagen fibrils and water, such as an aqueous solution, suspension, gel, or hydrogel containing fibrillated collagen. Water may be removed by filtration, evaporation, freeze-drying, solvent exchange, vacuum-drying, convection-drying, heating, irradiating or microwaving, or by other known methods for removing water. In addition, chemical crosslinking of collagen is known to remove bound water from collagen by consuming hydrophilic amino acid residues such as lysine, arginine, and hydroxylysine among others. The inventors have found that acetone quickly dehydrates collagen fibrils and may also remove water bound to hydrated collagen molecules. Water content of a biofabricated material or leather after dehydration is preferably no more than 60% by weight, for example, no more than 5, 10, 15, 20, 30, 35, 40, 50 or 60% by weight of the biofabricated leather. This range includes all intermediate values. Water content is measured by equilibration at 65% relative humidity at 25°C and 1 atm.

"Grain texture" describes a leather-like texture which is aesthetically or texturally the similar to the texture of a full grain leather, top grain leather, corrected grain leather (where an artificial grain has been applied), or coarser split grain leather texture. Advantageously, the biofabricated material of the invention can be tuned to provide a fine grain, resembling the surface grain of a leather.

The articles in the invention ma include foot wear, garments, gloves, furniture or vehicle upholstery and other leather goods and products. It includes but is not limited to clothing, such as overcoats, coats, jackets, shirts, trousers, pants, shorts, swimwear, undergarments, uniforms, emblems or letters, costumes, ties, skirts, dresses, blouses, leggings, gloves, mittens, shoes, shoe components such as sole, quarter, tongue, cuff, welt, and counter, dress shoes, athletic shoes, running shoes, casual shoes, athletic, running or casual shoe components such as toe cap, toe box, outsole, midsole, upper, laces, eyelets, collar, lining, Achilles notch, heel, and counter, fashion or women's shoes and their shoe components such as upper, outer sole, toe spring, toe box, decoration, vamp, lining, sock, insole, platform, counter, and heel or high heel, boots, sandals, buttons, sandals, hats, masks, headgear, headbands, head wraps, and belts; jewelry such as bracelets, watch bands, and necklaces; gloves, umbrellas, walking sticks, wallets, mobile phone or wearable computer coverings, purses, backpacks, suitcases, handbags, folios, folders, boxes, and other personal objects; athletic, sports, hunting or recreational gear such as harnesses, bridles, reins, bits, leashes, mitts, tennis rackets, golf clubs, polo, hockey, or lacrosse gear, chessboards and game boards, medicine balls, kick balls, baseballs, and other kinds of balls, and toys; book bindings, book covers, picture frames or artwork; furniture and home, office or other interior or exterior furnishings including chairs, sofas, doors, seats, ottomans, room dividers, coasters, mouse pads, desk blotters, or other pads, tables, beds, floor, wall or ceiling coverings, flooring; automobile, boat, aircraft and other vehicular products including seats, headrests, upholstery, paneling, steering wheel, joystick or control coverings and other wraps or coverings.

Physical Properties of a biofabricated network of collagen fibrils or a biofabricated leather may be selected or tuned by selecting the type of collagen, the amount of concentration of collagen fibrillated, the degree of fibrillation, crosslinking, dehydration and lubrication.

Many advantageous properties are associated with the network structure of the collagen fibrils which can provide strong, flexible and substantially uniform properties to the resulting biofabricated material or leather. Preferable physical properties of the biofabricated leather according to the invention include a tensile strength ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more MPa, a flexibility determined by elongation at break ranging from 1, 5, 10, 15, 20, 25, 30% or more, softness as determined by ISO 17235 of 4, 5, 6, 7, 8 mm or more, a thickness ranging from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3. 1,4, 1,5, 1.6, 1.7, 1.8, 1.9, 2.0 mm or more, and a collagen density (collagen fibril density) of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 mg/cc or more, preferably 100- 500 mg/cc. The above ranges include all subranges and intermediate values. Thickness. Depending on its ultimate application a biofabricated material or leather may have any thickness. Its thickness preferably ranges from about 0.05 mm to 20 mm as well as any intermediate value within this range, such as 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 mm or more. The thickness of a biofabricated leather can be controlled by adjusting collagen content.

Elastic modulus. The elastic modulus (also known as Young's modulus) is a number that measures an object or substance's resistance to being deformed elastically {i.e., non-permanently) when a force is applied to it. The elastic modulus of an object is defined as the slope of its stress- strain curve in the elastic deformation region. A stiffer material will have a higher elastic modulus. The elastic modulus can be measured using a texture analyzer.

A biofabricated leather can have an elastic modulus of at least 100 kPa. It can range from 100 kPa to 1,000 MPa as well as any intermediate value in this range, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 MPa. A biofabricated leather may be able to elongate up to 300 % from its relaxed state length, for example, by >0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 % of its relaxed state length.

Tensile strength (also known as ultimate tensile strength) is the capacity of a material or structure to withstand loads tending to elongate, as opposed to compressive strength, which withstands loads tending to reduce size. Tensile strength resists tension or being pulled apart, whereas compressive strength resists compression or being pushed together.

A sample of a biofabricated material may be tested for tensile strength using an Instron machine. Clamps are attached to the ends of the sample and the sample is pulled in opposite directions until failure. Good strength is demonstrated when the sample has a tensile strength of at least 1 MPa. A biofabricated leather can have a tensile strength of at least 1 kPa. It can range from 1 kPa to 100 MPa as well as any intermediate value in this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500kPA; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 MPa.

Tear strength (also known as tear resistance) is a measure of how well a material can withstand the effects of tearing. More specifically however it is how well a material

(normally rubber) resists the growth of any cuts when under tension, it is usually measured in kN/m. Tear resistance can be measured by the ASTM D 412 method (the same used to measure tensile strength, modulus and elongation). ASTM D 624 can be used to measure the resistance to the formation of a tear (tear initiation) and the resistance to the expansion of a tear (tear propagation). Regardless of which of these two is being measured, the sample is held between two holders and a uniform pulling force applied until the aforementioned deformation occurs. Tear resistance is then calculated by dividing the force applied by the thickness of the material. A biofabricated leather may exhibit tear resistance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% more than that of a conventional top grain or other leather of the same thickness comprising the same type of collagen, e.g., bovine Type I or Type III collagen, processed using the same crosslinker(s) or lubricants. A biofabricated material may have a tear strength ranging from about 1 to 500 N, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 as well as any intermediate tear strength within this range.

Softness. ISO 17235:2015 specifies a non-destructive method for determining the softness of leather. It is applicable to all non-rigid leathers, e.g. shoe upper leather, upholstery leather, leather goods leather, and apparel leather. A biofabricated leather may have a softness as determined by ISO 17235 of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or more.

Grain. The top grain surface of leather is often regarded as the most desirable due to its soft texture and smooth surface. The top grain is a highly porous network of collagen fibrils. The strength and tear resistance of the grain is often a limitation for practical applications of the top grain alone and conventional leather products are often backed with corium having a much coarser grain. A biofabricated material as disclosed herein which can be produced with strong and uniform physical properties or increased thickness can be used to provide top grain like products without the requirement for corium backing.

Content of other components. In some embodiments, the collagen is free of other leather components such as elastin or non-structural animal proteins. However, in some embodiments the content of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins in a biofabricated leather may range from 0, 1, 2, 3,4, 5, 6, 7, 8, 9 to 10% by weight of the biofabricated leather. In other embodiments, a content of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagen structural proteins, and/or noncollagen nonstructural proteins may be incorporated into a biofabricated leather in amounts ranging from >0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% or more by weight of a biofabricated leather. Such components may be introduced during or after fibrillation, cross-linking, dehydration or lubrication.

Content of collagen. The biofabricated material or leather in accordance with the present invention contains an increased collagen content as compared to conventional leather. Within the present invention, the collagen content in the biofabricated material or leather ranges from 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80% or more to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or less, including all ranges and sub-ranges bound by the recited lower and upper limits.

A "leather dye" refers to dyes which can be used to color leather or biofabricated leather. These include acidic dyes, direct dyes, lakes, sulfur dyes, basic dyes and reactive dyes. Dyes and pigments can also be incorporated into a precursor of a biofabricated leather, such as into a suspension or network gel comprising collagen fibrils during production of the biofabricated leather.

"Fillers". In some embodiments a biofabricated leather may comprise fillers, other than components of leather, such as microspheres. One way to control the organization of the dehydrated fibril network is to include filling materials that keep the fibrils spaced apart during dehydration. These filler materials include nanoparticles, microparticles, or various polymers such as syntans commonly used in the tanning industry. These filling materials could be part of the final dehydrated leather material, or the filling materials could be sacrificial, that is they are degraded or dissolved away leaving open space for a more porous fibril network. The shape and dimension of these fillers may also be used to control the orientation of the dehydrated fibril network.

In some embodiments a filler may comprise polymeric microsphere(s), bead(s), fiber(s), wire(s), or organic salt(s). Other materials may also be embedded or otherwise incorporated into a biofabricated leather or into a network of collagen fibrils according to the invention. These include, but are not limited to one fibers, including both woven and nonwoven fibers as well as cotton, wool, cashmere, angora, linen, bamboo, bast, hemp, soya, seacell, fibers produced from milk or milk proteins, silk, spider silk, other peptides or polypeptides including recombinantly produced peptides or polypeptides, chitosan, mycelium, cellulose including bacterial cellulose, wood including wood fibers, rayon, lyocell, vicose, antimicrobial yarn (A.M. Y.), Sorbtek, nylon, polyester, elastomers such as lycra®, spandex or elastane and other polyester- polyurethane copolymers, aramids, carbon including carbon fibers and fullerenes, glass including glass fibers and nonwovens, silicon and silicon-containing compounds, minerals, including mineral particles and mineral fibers, and metals or metal alloys, including those comprising iron, steel, lead, gold, silver, platinum, copper, zinc and titanium, which may be in the form of particles, fibers, wires or other forms suitable for incorporating into biofabricated leather. Such fillers may include an electrically conductive material, magnetic material, fluorescent material, bioluminescent material, phosphorescent material or other photoluminescent material, or combinations thereof. Mixtures or blends of these components may also be embedded or incorporated into a biofabricated leather, for example, to modify the chemical and physical properties disclosed herein.

Various forms of collagen are found throughout the animal kingdom. The collagen used herein may be obtained from animal sources, including both vertebrates and invertebrates, or from synthetic sources. Collagen may also be sourced from byproducts of existing animal processing. Collagen obtained from animal sources may be isolated using standard laboratory techniques known in the art, for example, Silva et. Al., Marine Origin Collagens and its Potential Applications, Mar. Drugs, 2014 Dec, 12(12); 5881-5901).

The collagen described herein also may be obtained by cell culture techniques including from cells grown in a bioreactor.

Collagen may also be obtained via recombinant DNA techniques. Constructs encoding non-human collagen may be introduced into yeast to produce non-human collagen. For instance, collagen may also be produced with yeast, such as Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and the like as the host. Recombinant expression of collagen and collagen-like proteins has been reported by Bell, EP 1232182B1, Bovine collagen and method for producing recombinant gelatin; Olsen, et al., U.S. Patent No. 6,428,978, Methods for the production of gelatin and full-length triple helical collagen in recombinant cells; VanHeerde, et al., U.S. Patent No. 8,188,230, Method for recombinant microorganism expression and isolation of collagen-like polypeptides, the disclosures of which are hereby incorporated by reference. However, recombinant collagens have not been used to produce leather.

Materials that are useful in the present invention include but are not limited to biofabricated leather materials natural or synthetic woven fabrics, non-woven fabrics, knitted fabrics, mesh fabrics, and spacer fabrics.

Any material that retains the collagen fibrils can be useful in the present invention. In general, fabrics that are useful have a mesh ranging from 300 threads per square inch to 1 thread per square foot or a pore size greater than or equal to about 11 μm in diameter. Spun lace materials may also be useful. In some embodiments, water soluble fabrics are useful. When utilized, the portion of the fabric exposed to the solution of collagen dissolves forming a void or hole in the fabric, and the collagen fills the void or hole. Water soluble fabrics are typically formed from polyvinyl alcohol fibers and coated with a resin such as polyvinyl alcohol, polyethylene oxide, hydroxyalkylcellulose, carboxymethylcellulose, polyacrylamide, polyvinyl pyrrolidone, polyacrylate and starch. Alternatively, the void or hole may be covered with a secondary material such as, natural or synthetic woven fabrics, non-woven fabrics, knitted fabrics, mesh fabrics, and spacer fabrics.

Alternatively, biofabricated leather material may be used to plug a void or hole cut into fabric. The size of the void or hole may vary depending on the design to be imparted. The shape of the void or hole may vary depending on the design. Suitable dimensions of void or holes may range from about 0.1 inches to about 5 meters. Suitable shapes include but are not limited to circles, squares, rectangles, triangles, elliptical, ovals and brand logos.

Some materials lend themselves to pretreatment to improve bonding of biofabricated leather materials. Pretreatment may include collagen coating, resin coating, devore of the fabric (also known as burn-out method), chemical or combinations thereof. For example, a chemical pretreatment for materials made from cellulose fibers, may include periodate (an oxidant) solution treatment. Suitable cellulose fabrics are selected from the group consisting of viscose, acetate, lyocell, bamboo and combinations thereof. The oxidant opens sugar rings in the cellulose and enables the collagen to bind to the open rings. The concentration of the oxidant in the solution depends on the extent of oxidation desired. In general, higher the concentration of oxidant or longer the reaction time, higher degree of oxidation is achieved. In an embodiment of the present invention, the oxidation reaction may be carried out for a desired amount of time to achieve the desired level of oxidation. The oxidation reaction can be carried out at various temperatures, depending on the type of oxidant used. The inventors have preferred using controlled oxidation at room (20°C to 25°C, preferably 23°C) or ambient temperature over a time range of 15 minutes-24 hours. However, it is also envisioned that temperatures ranging from 17. 5°C to 30°C may also be used. With respect to time, it is envisioned that it is possible that controlled oxidation may be from 5 minutes, 10 minutes, 15, minutes, 20, minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3, hours, 4, hours, 5 hours, 6 hours, 12 hours to 36 hours, 30 hours, 24, hours, 20 hours, 18 hours, 15 hours, including all ranges and sub -ranges permissible herein. The amount of sodium periodate ranges from 25% to 100% offers on weight of the fabric. As used herein, offer means the amount of an additive based on the weight % of cellulose. Other chemical pretreatments are taught in Bioconjugate Techniques by Greg

Hermanson, which is hereby incorporated by reference.

The biofabricated leather (e.g., bioleather) solution as described herein may include any appropriate non-human collagen source in addition to and in combination with the recombinant collagen produced by the present yeast strains as discussed herein.

As an initial step in the formation of the collagen materials described herein, the starting collagen material may be placed in solution and fibrillated. The collagen concentration may range from approximately 0.5 g/L to 10 g/L, for example 0.75 g/L to 8 g/L, or 1 g/L to 7 g/L, or 2 g/L to 6 g/L, or 2.5 g/L to 5 g/L, or 3 g/L to 4 g/L. Collagen fibrillation may be induced through the introduction of salts to the collagen solution. The addition of a salt or a combination of salts such as sodium phosphate, potassium phosphate, potassium chloride, and sodium chloride to the collagen solution may change the ionic strength of the collagen solution. Collagen fibrillation may occur as a result of increasing electrostatic interactions, through greater hydrogen bonding, Van der Waals interactions, and covalent bonding. Suitable salt

concentrations may range, for example, from approximately 10 mM to 5M, for example 50 mM to 2.5 M, or 100 mM to 1 M, or 250 mM to 500mM.

A collagen network may also be highly sensitive to pH. During the fibrillation step, the pH may be adjusted to control fibril dimensions such as diameter and length. Suitable pH may range from approximately 5.5 to 10, for example 6 to 9, or 7 to 8. After the fibrillation step prior to filtration, the pH of the solution is adjusted to a pH range from approximately 3.5 to 10, for example 3.5 to 7, or 3.5 to 5. The overall dimensions and organization of the collagen fibrils will affect the toughness, stretch-ability, and breathability of the resulting fibrillated collagen derived materials. This may be of use for fabricating fibrillated collagen derived leather for various uses that may require different toughness, flexibility, and breathability.

One way to control the organization of the dehydrated fibril network is to include filling materials that keep the fibrils spaced apart during drying. These filler materials could include nanoparticles, microparticles, microspheres, microfibers, or various polymers commonly used in the tanning industry. These filling materials could be part of the final dehydrated leather material, or the filling materials could be sacrificial, that is they are degraded or dissolved away leaving open space for a more porous fibril network.

The collagen or collagen-like proteins may be chemically modified to promote chemical and physical crosslinking between the collagen fibrils. Collagen-like proteins were taught in the United States patent application US 2012/0116053 Al, which is hereby incorporated by reference. Chemical crosslinking may be possible because reactive groups such as lysine, glutamic acid, and hydroxyl groups on the collagen molecule project from collagen' s rod-like fibril structure. Crosslinking that involve these groups prevent the collagen molecules from sliding past each other under stress and thus increases the mechanical strength of the collagen fibers. Examples of chemical crosslinking reactions include but are not limited to reactions with the ε-amino group of lysine, or reaction with carboxyl groups of the collagen molecule.

Enzymes such as transglutaminase may also be used to generate crosslinks between glutamic acid and lysine to form a stable γ-glutamyl-lysine crosslink. Inducing crosslinking between functional groups of neighboring collagen molecules is known in the art. Crosslinking is another step that can be implemented here to adjust the physical properties obtained from the fibrillated collagen hydrogel-derived materials. Once formed, the fibrillated collagen network may be further stabilized by incorporating molecules with di-, tri-, or multifunctional reactive groups that include chromium, amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides, acrylates, epoxides, or phenols.

The fibrillated collagen network may also be polymerized with other agents (e.g.

polymers that are capable of polymerizing or other suitable fibers) that form a hydrogel or have fibrous qualities, which could be used to further stabilize the matrix and provide the desired end structure. Hydrogels based upon acrylamides, acrylic acids, and their salts may be prepared using inverse suspension polymerization. Hydrogels described herein may be prepared from polar monomers. The hydrogels used may be natural polymer hydrogels, synthetic polymer hydrogels, or a combination of the two. The hydrogels used may be obtained using graft polymerization, crosslinking polymerization, networks formed of water soluble polymers, radiation crosslinking, and so on. A small amount of crosslinking agent may be added to the hydrogel composition to enhance polymerization.

The viscosity of the collagen solution can range from 1 cP to 1000 cP at 20°C, including all values and ranges there between such as 10, 20, 30, 50, 75, 90, 100, 150, 225, 300, 400, 450, 500, 525, 575, 600, 650, 700, 800, 900 etc. The solutions can be poured, sprayed, painted, or applied to a surface. The viscosity may vary depending on how the final material is formed. Where a higher viscosity is desired, known thickening agents such as carboxymethylcellulose and the like can be added to the solution. Alternatively, the amount of collagen in the solution can be adjusted to vary the viscosity.

The flexibility in the collagen solution enables the production of new materials made entirely through the deposition of said collagen solution, for example the creation of biofabricated leather lace materials or 3-dimensional materials. In a sense, the collagen solution may function as a liquid leather to form, for example, biofabricated leather or bioleather. The liquid leather can be poured, pipetted, sprayed through a nozzle, or robotically applied or dip a secondary material into the liquid leather. A textured surface can be achieved through utilizing an apertured material in the formation process of the material. Liquid leather also enables the use of masking, stenciling and molding techniques. The application of the biofabricated leather solution also enables modifying the properties of the material to which it is applied. For example, the biofabricated leather solution can make the end material stronger, more supple, more rigid, more flexible, more elastic or softer.

As mentioned, a biofabricated leather material derived from the methods described above may have similar gross structural and physical characteristics as leathers produced from animal hides. In general, in addition to collagen produced by the present yeast cells, the biofabricated leather materials described herein may be derived from sources other than sheets or pieces of animal hide or skin, although animal hide or skin may be the source of the collagen used in preparing the fibrillated collagen. The source of the collagen or collagen-like proteins may be isolated from any animal (e.g. mammal, fish), or more particularly cell/tissue cultured, source (including in particular microorganism).

The biofabricated leather material may include agents that stabilize the fibril network contained therein or may contain agents that promote fibrillation. As mentioned in previous sections, cross-linking agents (to provide further stability), nucleating agents (to promote fibrillation), and additional polymerizing agents (for added stability) may be added to the collagen solution prior to fibrillation (or after) to obtain a fibrillated collagen material with desired characteristics (e.g. strength, bend, stretch, and so forth). As mentioned, following dehydration or drying, the engineered biofabricated leather materials derived from the methods discussed above have a water content of 20% or less by weight, or 17.5 % or less by weight, or 15% or less by weight, or 12.5% or less by weight, or 10% or less by weight. The water content of the engineered biofabricated leather materials may be fine-tuned in the finishing steps to obtain leather materials for differing purposes and desired characteristics.

As mentioned, any of these biofabricated leathers may be tanned (e.g., using a tanning agent including vegetable (tannins), chromium, alum, zirconium, titanium, iron salts, or a combination thereof, or any other appropriate tanning agent). Thus, in any of the resulting biofabricated leather materials described herein, the resulting material may include a percent (e.g., between 0.01% and 10%, or between 0.025% and 8%, or between 0.05% and 6%, or between 0.1% and 5%, or between 0.25% and 4%, or between 0.5% and 3%, or between 0.75% and 2.5%, or between 1% and 2%) of a residual tanning agent (e.g. tannin, chromium, etc.). Thus, the collagen fibrils in the resulting biofabricated leather material are modified to be tanned, e.g., cross-linked to resist degradation.

The biofabricated leather materials may be treated to provide surface textures. Suitable treatments include but are not limited to embossing, debossing, and vacuum forming with an apertured plate below the material. As is known in the art, the pressure and temperature at which the embossing and debossing are performed may vary depending on the desired texture and design. Surface coating and finishes known in the leather industry may be applied to the biofabricated leather materials.

As mentioned above, in any of the variations for making the biofabricated leathers described herein, the material could be tanned (cross-linked) as the collagen is fibrillated and/or separately after fibrillation has occurred, prior to dehydration. For example, tanning may include crosslinking using an aldehyde (e.g., Relugan GTW) and/or any other tanning agent. Thus in general a tanning agent includes any collagen fibril cross-linking agent such as aldehydes cross linkers, chromium, amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine,

Some methods for making a material including a biofabricated leather material include providing a material, pretreating the material to make it suitable for bonding with collagen, applying collagen solution to the material and drying. Drying may include removing water through a vacuum, heated air drying, ambient air drying, heated pressing and pressure drying. Where pretreatment is required, the pretreatment is either cutting voids or holes into the material, chemically removing certain fibers and treating the material with a chemical or collagen solution. Other methods do not require a pretreatment of the material. Where pretreatment is not required, the material is either partially water soluble or retains collagen but allows water to pass through. Suitable mesh sizes range from 300 threads per square inch to 1 thread per square foot. As used herein, the term bonded or bonding to the fabric mean attached such that the biofabricated leather does not easily peel away from the fabric when pulled by hand. A suitable method for testing the efficacy of bonding is a peel strength test performed on an instrument such as an Instron ® material testing machine. Jaws of the machine are attached to the biofabricated leather material and the material which it was bonded to, and the jaws are pulled apart until the materials tear or separate. The force to tear is reported in N/mm. Suitable peel strengths range from about 0.5 N/mm N/cm ² to 100 N/mm, including 0.75 N/mm to 75 N/mm, or 1 N/mm to 50 N/mm, 1.5 N/mm to 25 N/mm, including all ranges and sub-ranges defined by the recited upper and lower limits. Hydroxylation of proline and lysine residues in a protein (e.g., collagen). The principal post- translational modifications of the polypeptides of proteins that contain proline and lysine, including collagen, are the hydroxylation of proline and lysine residues to yield 4- hydroxyproline, 3-hydroxyproline (Hyp) and hydroxylysine (Hyl), and glycosylation of the hydroxylysyl residues. These modifications are catalyzed by three hydroxylases—prolyl 4- hydroxylase, prolyl 3 -hydroxylase, and lysyl hydroxylase— and two glycosyl transferases. In vivo these reactions occur until the polypeptides form the triple-helical collagen structure, which inhibits further modifications.

Prolyl-4-hydroxylase. This enzyme catalyzes hydroxylation of proline residues to (2S,4R)-4-hydroxyproline (Hyp). Gorres, et al., Critical Reviews in Biochemistry and Molecular Biology 45 (2): (2010) which is incorporated by reference. The Examples below employ tetrameric bovine prolyl-4-hydroxylase (2 alpha and 2 beta chains) encoded by P4HA (SEQ ID NO: 8 (optimized for Pichia) or SEQ ID NO: 15 (native)) and P4HB (SEQ ID NO: 9 (optimized for Pichia) or SEQ ID NO: 16 (native)), however, isoforms, orthologs, variants, fragments and prolyl-4-hydroxylase from non-bovine sources (e.g., human prolyl-4-hydroxylase) may also be used as long as they retain hydroxylase activity in a yeast host cell. Another example of P4HA1 is further described by http://www.omim.org/entry/176710 (Cytogenetic location: 10q22.1; Genomic coordinates (GRCh38): 10:73,007,216-73,096,973 (from NCBI)) and another example of P4HB1 is further described by http://www.omim.org/entry/176790 (Cytogenetic location: 17q25.3; Genomic coordinates (GRCh38): 17:81,843,157-81,860,667 (from NCBI)) both of which are incorporated by reference.

In the context of the present application a "variant" includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence identity, or similarity to a reference amino acid, such as a P4HA and P4HB amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences, and

BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect

Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are described by and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&a mp;PAGE_TYPE=BlastSearch&LINK _LOC=blasthome (last accessed August 14, 2017). Within the present invention, the "variant" retains prolyl-4-hydroxylase activity.

In an embodiment of the present invention, yeast cells are engineered to overproduce the prolyl-4-hydroxylase. A non-limiting example is the incorporation of a polynucleotide encoding the prolyl-4-hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses prolyl-4-hydroxylase activity into an expression vector. In an embodiment of the present invention, the expression vector containing the polynucleotide encoding the prolyl-4-hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses prolyl-4-hydroxylase activity is under the control of an inducible promoter. Suitable yeast cells, expression vectors, and promoters are described below.

Prolyl 3-hydroxylase. This enzyme catalyzes hydroxylation of proline residues. 3- hydroxylase 1 precursor [Bos taurus] is described by NCBI Reference Sequence:

P_001096761.1 (SEQ ID NO: 4) or by NM 001103291.1 (SEQ ID NO: 5). For further description see Vranka, et al., J. Biol. Chem. 279: 23615-23621 (2004) or

http://_www.omim.org/entry/610339 (last accessed July 14, 2017) which is incorporated by reference. This enzyme may be used in its native form. However, isoforms, orthologs, variants, fragments and prolyl-3 -hydroxylase from non-bovine sources may also be used as long as they retain hydroxylase activity in a yeast host cell.

In the context of the present application a "variant" includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence identity, or similarity to a reference amino acid, such as a prolyl-3 -hydroxylase amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are described by and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&a mp;PAGE_TYPE=BlastSearch&LINK _LOC=blasthome (last accessed August 14, 2017). Within the present invention, the "variant" retains prolyl-4-hydroxylase activity.

In an embodiment of the present invention, yeast cells are engineered to overproduce the prolyl -3 -hydroxylase. A non-limiting example is the incorporation of a polynucleotide encoding the prolyl-3 -hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses prolyl-3 -hydroxylase activity into an expression vector. In an embodiment of the present invention, the expression vector containing the polynucleotide encoding the prolyl-3 -hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses prolyl-3 -hydroxylase activity is under the control of an inducible promoter. Suitable yeast cells, expression vectors, and promoters are described below.

Lysyl hydroxylase. Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylysine by the hydroxylation of lysine residues in X-lys-gly sequences. The enzyme is a homodimer consisting of subunits with a molecular mass of about 85 kD. No significant homology has been found between the primary structures of lysyl hydroxylase and the 2 types of subunits of prolyl -4- hydroxylase (176710, 176790) despite the marked similarities in kinetic properties between these 2 collagen hydroxylases. The hydroxylysine residues formed in the lysyl hydroxylase reaction in, for example, collagen have 2 important functions: first, their hydroxy groups serve as sites of attachment for carbohydrate units, either the monosaccharide galactose or the disaccharide glucosylgalactose; and second, they stabilize intermolecular collagen crosslinks.

An exemplary lysyl hydroxylase is PLOD1 procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 [ Bos taurus (cattle)] is described by Gene ID: 281409 (SEQ ID NO: 6), updated on 25-May-2017 and incorporated by reference to https://www.ncbi.nlm.nih.gov/gene/281409 (last accessed July 14, 2017). Another example is described by SEQ ID NO: 7 which describes Bos taurus lysyl oxidase (LOX). Yet another example is described in SEQ ID NO: 14, which is a Bos taurus bifunctional arginine demethylase and lysyl-hydroxylase JMJD6. The lysyl hydroxylase enzyme may be used in its native form. However, isoforms, orthologs, variants, fragments and lysyl hydroxylase from non-bovine sources may also be used as long as they retain hydroxylase activity in a yeast host cell.

In the context of the present application a "variant" includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence identity, or similarity to a reference amino acid, such as a lysyl hydroxylase amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are described by and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&a mp;PAGE_TYPE=BlastSearch&LINK _LOC=blasthome (last accessed August 14, 2017). Within the present invention, the "variant" retains prolyl-4-hydroxylase activity.

In an embodiment of the present invention, yeast cells are engineered to overproduce the lysyl hydroxylase. A non-limiting example is the incorporation of a polynucleotide encoding the lysyl hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses lysyl hydroxylase activity into an expression vector. In an embodiment of the present invention, the expression vector containing the polynucleotide encoding the lysyl hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses lysyl hydroxylase activity is under the control of an inducible promoter.

Suitable yeast cells, expression vectors, and promoters are described below.

a ketoglutarate transporter. The kgtP gene exists in Escherichia coli and encodes an a ketoglutarate transporter which is responsible for uptake of a ketoglutarate across the boundary membrane and concomitant import of a cation (Membrane Topology Model of Escherichia coli oL-Ketoglutarate Permease by PhoA Fusion Analysis, WONGI SEOLt AND AARON J.

SHATKIN, JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 565-567). The gene has been transformed into cyanobacteria (Uptake of 2-Oxoglutarate in Synechococcus Strains

Transformed with the Escherichia coli kgtP Gene, MARI A FE LIX VA ZQUEZ-BERMU DEZ, ANTONIA HERRERO, AND ENRIQUE FLORES, JOURNAL OF BACTERIOLOGY, Jan. 2000, p. 211-215).

The inventors have found that transforming the kgtP gene into yeast provides a healthier organism. Furthermore, if the yeast produces a protein such as collagen or a carbohydrate, the transformed yeast have increased protein or carbohydrate production and improved

hydroxylation of the collagen by making more a ketoglutarate available at the endoplasmic reticulum, where proteins or carbohydrates are made and hydroxylation occurs.

An exemplary kgtP gene is set forth in SEQ ID NO: 2, while an exemplary a

ketoglutarate transporter is set forth in SEQ ID NO: 1. This enzyme may be used in its native form. However, isoforms, orthologs, variants, fragments and a ketoglutarate transporter from non-E.coli sources may also be used as long as they retain a ketoglutarate transporter activity in a yeast host cell. Further, the kgtP gene may be optimized for expression in yeast. For example, an example of an optimized a ketoglutarate transporter gene (kgtP) for expression in Pichia is set forth in SEQ ID NO: 3.

In the context of the present application a "variant" includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence identity, or similarity to a reference amino acid, such as a a ketoglutarate transporter amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are described by and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&a mp;PAGE_TYPE=BlastSearch&LINK _LOC=blasthome (last accessed August 14, 2017). Within the present invention, the "variant" retains prolyl-4-hydroxylase activity.

In an embodiment of the present invention, yeast cells are engineered to overproduce the a ketoglutarate transporter. A non-limiting example is the incorporation of a polynucleotide encoding the a ketoglutarate transporter, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses a ketoglutarate transporter activity into an expression vector. In an embodiment of the present invention, the expression vector containing the polynucleotide encoding the a ketoglutarate transporter, an isoform thereof, an ortholog thereof, a variant thereof, or a fragment thereof which expresses a ketoglutarate transporter activity is under the control of an inducible promoter. Suitable yeast cells, expression vectors, and promoters are described below.

Polynucleotide(s) encoding one or more polypeptides that enable an ascorbate synthesis pathway to function

L-ascorbic acid, ascorbate biosynthetic pathway in yeast, plants and animals is described, e.g., in Smirnoff (2001) Vitamins & Hormones 61-241-266 and Vitamin C. Functions and biochemistry in animals and plants Asard et al (ed.). Garland Science/BIOS Scientific

Publishers, 2004. Various genes, individually or in combination may be used to enable the ascorbate synthesis in yeast. Those include, for example, for a plant pathway, the genes or polynucleotides encoding GDP-L-Gal phosphorylase which converts GDP-L-Galactose to L- Galactose-l-P, Inositol -phosphate phosphatase which converts L-Galactose-l-P to L-Galactose, GDP-Mannose-3,5-epimerase which converts GDP-D-Mannose to GDP-L-Galactose may be transformed into the yeast. Pichia already has the other genes necessary to complete the pathway. Those genes provide the following enzymes: hexokinase, Glucose-6-phosphate- i somerase, Mannose-6-phosphate-i somerase, Phosphomannomutase and Mannose- 1 -phosphate- guanyly transferase .

For an animal pathway, the following genes can be added: L-gulono-l,4-lactone oxidase which converts L-Gulono-l,4-lactone to L-Ascorbate, aldonolactonase which converts L- Gulonic acid to L-Gulono-l,4-lactone, glucurono lactone reductase which converts D- Glucuronic acid to D-Glucurono lactone, D-glucuronate reductase which converts D-Glucuronic acid to L-Gulonic acid, uronolactonase which converts D-Glucuronic acid to D-Glucurono lactone, D-glucurono kinase which converts D-Glucuronic acid-l-P to D-Glucuronic acid, glucuronate-1 -phosphate uridylyltransferase which converts UDP-Glucuronic acid to D- Glucuronic acid-l-P, UDP-D-glucose dehydrogenase which converts UDP-D-Glucose to UDP- Glucuronic acid, UTP- glucose- 1 -phosphate uridylyltransferase which converts D-Glucose-l-P to UDP-D-Glucose, phosphoglucomutase which converts D-Glc-6-P to D-Glucose-l-P, and/or hexokinase which converts D-Glucose to D-Glc-6-P.

It is not necessary to transform all of the genes for all of the enzymes to enable the yeast to make ascorbate. Indeed, in the present invention, it is envisioned that it is only necessary to insert one or more of the genes encoding proteins for the portion of the ascorbate synthesis pathway downstream from the ascorbate pathway precursor fed to the yeast cells. For example, one can insert the gene for the enzyme Inositol-phosphate phosphatase which converts L- Galactose- 1-P to L-Galactose in to the yeast and then feed L-Galactose-l-P to the yeast, which allows the yeast to produce L-ascorbate.

GDP-L-Gal phosphorylase may be provided from Arabidopsis thaliana and include all or a part of the nucleotide sequence of SEQ ID NO: 21.

A representative amino acid sequence of GDP-L-Gal phosphorylase is as set forth in SEQ ID NO: 22.

Inositol-phosphate phosphatase (EC 3.1.3.25) is a known class of enzymes and is a phosphatase acting on L-galactose 1 -phosphate (L-Gal 1-P), D-myoinositol 3 -phosphate (D-Ins 3-P) and D-myoinositol 1 -phosphate (D-Ins 1-P). Can also use beta-glycerophosphate (glycerol 2-P) and, to a lesser extent, D-galactose 1 -phosphate (D-Gal 1-P), alpha-D-glucose 1 -phosphate (a-D-Glc 1-P), D-manitol 1 -phosphate and adenosine 2'-monophosphate as substrates. No activity with D-fructose 1 -phosphate (D-Fru 1-P), fructose 1,6-bisphosphate (Fru 1,6-bisP), D- glucose 6-phosphate (D-Glc 6-P), D-alpha-glycerophosphate (glycerol 3-P), D-sorbitol 6- phosphate and D-myoinositol 2-phosphate. The CI phosphate position in a six-member ring substrate is important for catalysis. Amino acid positions from Arabidopsis thaliana at 71, 91, 93, 94, and 221 contribute to metal binding and amino acid positions 71 and 213 contribute to substrate binding. A representative nucleotide sequence is shown in SEQ ID NO: 23.

One isoform of Inositol -phosphate phosphatase is shown in SEQ ID NO: 24.

GDP-Mannose-3,5-epimerase (EC 5.1.3.18) is a known class of enzymes and catalyzes a reversible epimerization of GDP-D-mannose that precedes the committed step in the

biosynthesis of vitamin C (L-ascorbate), resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl linkage. Able to catalyze 2 distinct epimerization reactions and can release both GDP-L-galactose and GDP-L-gulose from GDP-mannose. In Arabidopsis thaliana, regions 143-145, 216-21 and/or 241-243 are involved in substrate binding with one or more positions 145, 174, 178, 217 and/or 306 involved in enzymatic activity and one or more of positions 58, 78, 174, and/or 178 involved in N AD binding, one or more of positions 103, 203, 225, 306 and/or 356 involved in substrate biding.

The polynucleotide used herein may encode all or a part of the amino acid sequence of SEQ ID NO: 25.

L-gulono-l,4-lactone oxidase (EC 1.1.3.8) is a known class of enzymes and is involved in the biosynthesis of ascorbic acid. In Arabidopsis thaliana, position 156 is involved in activity and preferably only contains amino acids 102 to 610 or a fragment thereof with amino acids 123- 258 involved in FAD binding.

The polynucleotide used herein may encode all or a part of the amino acid sequence of at least one isoform, for example of SEQ ID NO: 26. A second isoform from the above sequence at 494-512 in which the sequence is changed from KSPISPAFSTSEDDIFSWV (SEQ ID NO: 27) and WYNHVPPDSRPSPEKGHHR (SEQ ID NO. 28) and is missing 513-610.

Glucurono lactone reductase (EC 1.1.1.20) is a known class of enzymes and catalyzes a reaction of L-gulono-l,4-lactone + NADP(+) <=> D-glucurono-3,6-lactone + NADPH and is known in the art, e.g., from a number of organisms as outlined below from the publically accessible UniProt database:

Aldonolactonase is known in the art and in some instances is also called a

a gluconolactonase (EC 3.1.1.17) and is known from a number of organisms as outlined below from the publically accessible UniProt database:

D-glucuronate reductase (EC 1.1.1.19) is a known class of enzymes and, e.g., from a number of organisms as outlined below from the publically accessible UniProt database:

Uronolactonase is is a known class of enzymes (EC 3.1.1.19) and catalyzes the reaction of

g!ucuronolactonase or D-glucurono-6,2-Iactone lactonohydrolase and a number of enzymes associated with this enzymatic activity are known, see EC 3.1.1.19. D-glucuronokinase (EC 2.7,1,43) is a known class of enzymes and for instance from Arabidopsis thaliana is involved in the biosynthesis of UDP-glucuronic acid (UDP-GlcA) with amino acids 126-136 involved in binding. The polynucleotide used herein may encode all or a part of the amino acid sequence of SEQ ID NO: 29.

Glucuronate-1 -phosphate uridylyltransferase is a known class of enzymes (EC 2.7.7.44) that catalyzes the chemical reaction UTP + 1-phospho-alpha-D-glucuronate to diphosphate + UDP-glucuronate and is known in the art, e.g., from a number of organisms as outlined below from the publically accessible UniProt database:

UDP-D-glucose dehydrogenase (EC 1.1.1.22) is a known class of enzymes that hat catalyzes the chemical reaction UDP-glucose + 2 NAD ⁺ + H ₂ O UDP-glucuronate + 2 NADH + 2 H ⁺ (E C. 1.1.1.22) and may include all or a part of SEQ ID NO. 30.

Other sequences from the UniProt publically available database are:

UTP- glucose- 1 -phosphate uridylyltransferase (EC 2.7.7.9) is a known class of enzymes and is also known as glucose- 1 -phosphate uridylyltransferase (or UDP -glucose

py rophosphoryl ase) is an enzyme involved in carbohydrate metabolism. It synthesizes UDP- glucose from glucose- 1 -phosphate and UTP. There are hundreds of known sequences from various species associated with this class in the art.

Phosphoglucomutase (EC 5.4.2.2) is a known class of enzymes and is an enzyme that transfers a phosphate group on an a-D-glucose monomer from the Γ to the 6' position in the forward direction or the 6' to the Γ position in the reverse direction. There are hundreds of known sequences from various species associated with this class in the art.

Hexokinase (EC 2.7.1.1) is a known class of enzymes that phosphorylates hexoses (six- carbon sugars), forming hexose phosphate. There are hundreds of known sequences from various species associated with this class in the art.

Assay of degree of hydroxylation of proline residues in recombinant protein (e.g., collagen). The degree of hydroxylation of proline residues in recombinant protein (e.g., collagen) produced in accordance with the present invention may be assayed by known methods, including by liquid chromatography-mass spectrometry as described by Chan, et al., BMC Biotechnology 12:51 (2012) which is incorporated by reference.

Assay of degree of hydroxylation of lysine residues in recombinant protein (e.g., collagen). Lysine hydroxylation and cross-linking of collagen is described by Yamauchi, et al., Methods in Molecular Biology, vol. 446, pages 95-108.; Humana Press (2008) which is incorporated by reference. The degree of hydroxylation of lysine residues in recombinant protein (e.g., collagen) produced in accordance with the present invention may be assayed by known methods, including by the method described by Hausmann, Biochimica et Biophysica Acta (BBA) - Protein Structure 133(3): 591-593 (1967) which is incorporated by reference.

Degree of hydroxylation. The degree of hydroxylation of proline or lysine residues in protein (e.g., collagen) produced in accordance with the present invention is preferably at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%. Also within the scope of the present invention the degree of hydroxylation of proline plus lysine residues is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.

Collagen Melting Point. The degree of hydroxylation of proline, lysine or proline and lysine residues in collagen may be estimated by melting temperature of a hydrated collagen, such as a hydrogel compared to a control collagen having a known content of hydroxylated amino acid residues. Collagen melting temperatures can range from 25-40°C with more highly hydroxylated collagens generally having higher melting temperatures. This range includes all intermediate subranges and values including subranges that are bound on the lower and upper end by a temperature selected from 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40°C.

Codon-modification. Within the scope of the present invention it is envisioned that the gene sequence introduced into the yeast host cell is modified from its native sequence. This process includes alteration of a polynucleotide sequence encoding the protein of interest (e.g. collagen, such as collagen DNA sequence found in nature), to modify the amount of recombinant protein (e.g., collagen) expressed by a yeast, such as Pichia pastoris, to modify the amount of recombinant protein (e.g., collagen) secreted by the recombinant yeast, to modify the speed of expression of recombinant protein (e.g., collagen) in the recombinant yeast, or to modify the degree of hydroxylation of lysine or proline residues in the recombinant protein (e.g., collagen). Codon modification may also be applied to other proteins such as hydroxylases for similar purposes or to target hydroxylases to particular intracellular or extracellular compartments, for example to target a proline hydroxylase to the same compartment, such as the endoplasmic reticulum, as recombinant protein (e.g., collagen) molecule. Codon selections may be made based on effect on RNA secondary structure, effect on transcription and gene expression, effect on the speed of translation elongation, and/or the effect on protein folding.

Codons encoding collagen or a hydroxylase may be modified to reduce or increase secondary structure in mRNA encoding recombinant collagen or the hydroxylase or may be modified to replace a redundant codon with a codon which, on average, is used most frequently by a yeast host cell based on all the protein-coding sequences in the yeast {e.g., codon sampling), is used least frequently by a yeast host cell based on all the protein-coding sequences in the yeast {e.g., codon sampling), or redundant codons that appear in proteins that are abundantly-expressed by yeast host cells or which appear in proteins that are secreted by yeast host cells {e.g., a codon selection based on a High Codon Adaptation Index that makes the gene "look like" a highly expressed gene or gene encoding a secretable protein from the expression host).

Codon-modification may be applied to all or part of a protein-coding sequence, for example, to at least one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth 10% of a coding-sequence or combinations thereof. It may also be applied selectively to a codon encoding a particular amino acid or to codons encoding some but not all amino acids that are encoded by redundant codons. For example, only codons for leucine and phenylalanine may be codon-modified as described above. Amino acids encoded by more than one codon are described by the codon table, which is well-known in the art.

Codon-modification includes the so-called codon-optimization methods described by https://www.atum.bio/services/genegps (last accessed July 13, 2017), by

https://www.idtdna.com/CodonOpt or Villalobos, Alan et al. BMC Bioinformalics 7 (2006): 285.

PMC. Web. 17 Aug. 2017, incorporated herein by reference.

Codon-modification also includes selection of codons so as to permit formation of mRNA secondary structure or to minimize or eliminate secondary structure. An example of this is making codon selections so as to eliminate, reduce or weaken secondary structure strong secondary structure at or around a ribosome-binding site or initiation codon.

In an embodiment of the present invention the a ketoglutarate transporter gene sequence is optimized for use in yeast, in particular Pichia. An example of a optimized a ketoglutarate transporter gene (kgtP) is set forth in SEQ ID NO: 3.

Collagen Fragments. A recombinant collagen molecule can comprise a fragment of the amino acid sequence of a native collagen molecule capable of forming tropocollagen (trimeric collagen) or a modified collagen molecule or truncated collagen molecule having an amino acid sequence at least 70, 80, 90, 95, 96, 97, 98, or 99% identical or similar to a native collagen amino acid sequence (or to a fibril forming region thereof or to a segment substantially comprising [Gly-X-Y]n), such as those of amino acid sequences of CollAl, CollA2, and Col3Al, described by Accession Nos. NP_001029211.1 (SEQ ID NO: 11)

(https://www.ncbi.nlm.nih.gov/protein/77404252, last accessed August 23, 2017), NP_776945.1 (SEQ ID NO: 12) (https://www.ncbi.nlm.nih.gov/protein/27806257 last accessed August 23, 2017) and P_001070299.1 (SEQ I NO: 13) (https://www.ncbi.nlm.nih.gov/protein/116003881 last accessed February 9, 2017), respectively, which are incorporated by reference.

A gene encoding collagen or a hydroxylase may be truncated or otherwise modified to add or remove sequences. Such modifications may be made to customize the size of a polynucleotide or vector, to target the expressed protein to the endoplasmic reticulum or other cellular or extracellular compartment, or to control the length of an encoded protein. For example, the inventors found that constructs containing only the Pre sequence often work better than those containing the entire Pre-pro sequence. The Pre sequence was fused to P4HB to localize P4HB in the ER where collagen localizes as well.

Modified coding sequences for collagens and hydroxylases. A polynucleotide coding sequence for collagen or a hydroxylase, or other proteins, may be modified to encode a protein that is at least 70, 80, 90, 95, 96, 97, 98, or 100% identical or similar to a known amino acid sequence and which retains the essential properties of the unmodified molecule, for example, the ability to form tropocollagen or the ability to hydroxylase proline or lysine residues in collagen. Glycosylation sites in a collagen molecule may be removed or added. Modifications may be made to facilitate collagen yield or its secretion by a yeast host cell or to change its structural, functional, or aesthetic properties. A modified collagen or hydroxylase coding sequence may also be codon-modified as described herein.

BLASTN may be used to identify a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100% sequence identity to a reference polynucleotide such as a polynucleotide encoding a collagen, one or more

hydroxylases described herein, or signal, leader or secretion peptides or any other proteins disclosed herein. A representative BLASTN setting modified to find highly similar sequences uses an Expect Threshold of 10 and a Wordsize of 28, max matches in query range of 0, match/mismatch scores of 1/-2, and linear gap cost. Low complexity regions may be filtered or masked. Default settings of a Standard Nucleotide BLAST are well-known in the art.

BLASTP can be used to identify an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100% sequence identity, or similarity to a reference amino acid, such as a collagen amino acid sequence, using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences, and BLOSUM80 for more distantly related sequences. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity or similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A representative BLASTP setting that uses an Expect Threshold of 10, a Word Size of 3,

BLOSUM 62 as a matrix, and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional compositional score matrix adjustment. Other default settings for BLASTP are well known in the art.

The term "variant", "modified sequence" or "analog" as applied to the polypeptides disclosed herein, refers to a polypeptide comprising an amino acid sequence that is at least 70, 80, 90, 95, or 99% identical or similar to the amino acid sequence of a biologically active molecule. In some embodiments, the derivative comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a native or previously engineered sequence. The derivative may comprise additions, deletions, substitutions, or a combination thereof to the amino acid sequence of a native or previously engineered molecule. For example, a derivative may incorporate or delete 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proline or lysine residues compared to a native collagen sequence. Such selections may be made to modify the looseness or tightness of a recombinant tropocollagen or fibrillated collagen.

A derivative may include a mutant polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-15, 16- 20, 21-25, or 26-30 additions, substitutions, or deletions of amino acid residues. Additions or substitutions also include the use of non-naturally occurring amino acids or modified amino acids. A derivative may also include chemical modifications to a polypeptide, such as crosslinks between cysteine residues, or hydroxylated or glycosylated residues.

Yeast strains. The present invention utilizes yeast to produce collagen or other proteins or carbohydrates. In particular, the present invention utilizes modified yeast to produce collagen or other carbohydrates with an increased degree of hydroxylation. The yeast may be engineered to produce or overproduce a ketoglutarate and/or ascorbic acid by inserting one or more of a gene expression a kgtP gene, a gene expressing a hydroxylase, or gene(s) necessary to complete a functioning ascorbate synthesis pathway.

Suitable yeast includes, but is not limited to, those of the genus Pichia, Candida, Komatagaella, Hansenula, Cryptococcus, Saccharomyces and combinations thereof. The yeast may be modified or hybridized. Hybridized yeast is mixed breeding of different strains of the same species, different species of the same genus or strains of different genera. Examples of yeast strains that may be used according to the invention include Pichia pastoris, Pichia membranifaciens, Pichia deserticola, Pichia cephalocereana, Pichia eremophila, Pichia myanmarensis, Pichia anomala, Pichia nakasei, Pichia siamensis, Pichia heedii, Pichia barke i, Pichia norvegensis, Pichia thermomethanolica, Pichia stipites, Pichia subpelliculosa, Pichia exigua, Pichia occidentalis, Pichia cactophila, Saccharomyces cerevisiae, Saccharomyces pombe, and the like.

Pichia pastoris is a yeast species that has been used to recombinantly express

biotherapeutic proteins, such as human interferon gamma, see Razaghi, et al., Biologicals 45: 52- 60 (2017). It has been used to express type ΠΙ collagen and prolyl -4-hydroxylase, see Vuorela, et al., EMBO J. 16:6702-6712 (1997). Collagen and prolyl-4-hydroxylase have also been expressed in Escherichia coli to produce a collagenous material, see Pinkas, et al., ACS Chem. Biol. 6(4):320-324 (2011).

In one embodiment, the invention is directed to Pichia pastoris strains that have been engineered to express codon-optimized collagen, hydroxylase(s), a ketoglutarate transporter, and/or gene(s) necessary to complete a functioning ascorbate synthesis pathway. Useful Pichia pastoris host strains include, but are not limited to, wild type (Strain PPS-9010); aoxlA

(MutS)(Strain PPS-9011) which is a slow methanol utilization derivative of PPS-9010; and pep4A, prblA (Strain PPS-9016) which is protease deficient. These strains are publically available and may be obtained from ATUM at https://www._atum.bio/products/cell-strains.

Polypeptide secretion sequences for yeast. In some embodiments, a polypeptide encoded by a yeast host cell is fused to a polypeptide sequence that facilitates its secretion from the yeast. For example, a vector may encode a chimeric gene comprising a coding sequence for collagen fused to a sequence encoding a secretion peptide. Secretion sequences which may be used for this purpose include Saccharomyces alpha mating factor Prepro sequence, Saccharomyces alpha mating factor Pre sequence, PHOl secretion signal, a-amylase signal sequence from Aspergillus niger, Glucoamylase signal sequence from Aspergillus aw amor i, Serum albumin signal sequence from Homo sapiens, Inulinase signal sequence from

Kluyveromcyes maxianus, Invertase signal sequence from Saccharomyces cerevisiae, Killer protein signal sequence from Saccharomyces cerevisiae and Lysozyme signal sequence from Gallus gallus. Other secretion sequences known in the art may also be used.

Yeast promoters and terminators. In some embodiments one or more of the following yeast promoters may be incorporated into a vector to promoter transcription of mRNA encoding the protein of interest (e.g., collagen), hydroxylase(s), a ketoglutarate transporter, and/or gene(s) necessary to complete a functioning ascorbate synthesis pathway. Promoters are known in the art and include pAOXl, pDasl, pDas2, pPMP20, pCAT, pDF, pGAP, pFDHl, pFLDl, pTALl, pFBA2, pAOX2, pRKIl, pRPE2, pPEX5, pDAKl, pFGHl, pADH2, pTPIl, pFBPl, pTALl, pPFKl, pGPMl, and pGCW14.

In some embodiments a yeast terminator sequence is incorporated into a vector to terminate transcription of mRNA encoding the protein of interest (e.g., collagen), hydroxylase(s), a ketoglutarate transporter, and/or gene(s) necessary to complete a functioning ascorbate synthesis pathway. Terminators include but are not limited to AOX1 TT, Dasl TT, Das2 TT, AOD TT, PMP TT, Catl TT, TPI TT, FDH1 TT, TEF1 TT, FLD1 TT, GCW14 TT, FBA2 TT, ADH2 TT, FBP1 TT, and GAP TT.

Peptidases other than pepsin. Pepsin may be used to process collagen into tropollagen by removing N-terminal and C-terminal sequences. Other proteases, including but not limited to collagenase, trypsin, chymotrypsin, papain, ficain, and bromelain, may also be used for this purpose. As used herein, "stable collagen" means that after being exposed to a particular concentration of pepsin or another protease that at least 20, 30, 40, 50, 60, 75, 80, 85, 90, 95 or 100% of the initial concentration of collagen is still present. Preferably, at least 75% of a stable collagen will remain after treatment with pepsin or another protease as compared to an unstable collagen treated under the same conditions for the same amount of time. Prior to post- translational modification, collagen is non-hydroxylated and degrades in the presence of a high pepsin concentration (e.g., a pepsin:protein ratio of 1:200 or more).

Once post-translationally modified a collagen may be contacted with pepsin or another protease to cleave the N-terminal and the C-terminal propeptides of collagen, thus enabling collagen fibrillation. Hydroxylated collagen has better thermostability compared to non- hydroxylated collagen and is resistant to high concentration pepsin digestion, for example at a pepsin:total protein ratio of 1:25 to 1:1. Therefore, to avoid premature proteolysis of recombinant collagen it is useful to provide hydroxylated collagen.

Alternative expression systems. Collagen and other proteins can be expressed in other kinds of yeast cells besides Pichia pastoris, for example, in may be expressed in another yeast, methylotrophic yeast or other organism. Saccharomyces cerevisiae can be used with any of a large number of expression vectors. Commonly employed expression vectors are shuttle vectors containing the 2P origin of replication for propagation in yeast and the Col El origin for E. coli, for efficient transcription of the foreign gene. A typical example of such vectors based on 2P plasmids is pWYG4, which has the 2P ORI-STB elements, the GALl-10 promoter, and the 2P D gene terminator. In this vector, an Ncol cloning site is used to insert the gene for the polypeptide to be expressed and to provide an ATG start codon. Another expression vector is pWYG7L, which has intact 2aORI, STB, REP1 and REP2, and the GALl-10 promoter, and uses the FLP terminator. In this vector, the encoding polynucleotide is inserted in the polylinker with its 5' ends at a BamHI or Ncol site. The vector containing the inserted polynucleotide is transformed into S. cerevisiae either after removal of the cell wall to produce spheroplasts that take up DNA on treatment with calcium and polyethylene glycol or by treatment of intact cells with lithium ions.

Alternatively, DNA can be introduced by electroporation. Transformants can be selected, for example, using host yeast cells that are auxotrophic for leucine, tryptophan, uracil, or histidine together with selectable marker genes such as LEU2, TRP1, URA3, HIS3, or LEU2-D.

There are a number of methanol responsive genes in methylotrophic yeasts such as Pichia pastoris, the expression of each being controlled by methanol responsive regulatory regions, also referred to as promoters. Any of such methanol responsive promoters are suitable for use in the practice of the present invention. Examples of specific regulatory regions include the AOX1 promoter, the AOX2 promoter, the dihydroxy acetone synthase (DAS), the P40 promoter, and the promoter for the catalase gene from P. pastoris, etc.

The methylotrophic yeast Hansenula polymorpha is also mentioned. Growth on methanol results in the induction of key enzymes of the methanol metabolism, such as MOX (methanol oxidase), DAS (dihydroxyacetone synthase), and FMHD (formate dehydrogenase). These enzymes can constitute up to 30-40% of the total cell protein. The genes encoding MOX, DAS, and FMDH production are controlled by strong promoters induced by growth on methanol and repressed by growth on glucose. Any or all three of these promoters may be used to obtain high- level expression of heterologous genes inH. polymorpha. Therefore, in one aspect, a

polynucleotide encoding animal collagen or fragments or variants thereof is cloned into an expression vector under the control of an inducible H. polymorpha promoter. If secretion of the product is desired, a polynucleotide encoding a signal sequence for secretion in yeast is fused in frame with the polynucleotide. In a further embodiment, the expression vector preferably contains an auxotrophic marker gene, such as URA3 or LEU2, which may be used to

complement the deficiency of an auxotrophic host.

The expression vector is then used to transform H. polymorpha host cells using techniques known to those of skill in the art. A useful feature of H. polymorpha transformation is the spontaneous integration of up to 100 copies of the expression vector into the genome. In most cases, the integrated polynucleotide forms multimers exhibiting a head-to-tail arrangement. The integrated foreign polynucleotide has been shown to be mitotically stable in several recombinant strains, even under non-selective conditions. This phenomena of high copy integration further ads to the high productivity potential of the system.

Foreign DNA is inserted into the yeast genome or maintained episomally to produce collagen. The DNA sequence for the collagen is introduced into the yeast via a vector. Foreign DNAs are any non-yeast host DNA and include for example, but not limited to, mammalian, Caenorhabditis elegans and bacteria. Suitable mammalian DNA for collagen production in yeast include, but is not limited to, bovine, porcine, kangaroo, alligator, crocodile, elephant, giraffe, zebra, llama, alpaca, lamb, dinosaur and combinations thereof.

The DNA for enabling the production of ascorbate can also be inserted on a single or multiple vectors. For a plant pathway the DNA for the genes GDP-L-Gal phosphorylase, Inositol-phosphate phosphatase, GDP-Mannose-3,5-epimerase are inserted via a vector into the yeast cell. Pichia is already known to contain the remaining genes in the pathway for producing ascorbate from glucose transformed via a vector into the yeast cell.

The DNA for the ascorbic pathway may be inserted by itself or combined with DNA for proteins. The ascorbic pathway enables the production of healthy yeast strains that are suitable for producing most proteins.

The DNA for enabling the production of the a ketoglutarate transporter can also be inserted on a single vector. The yeast optimized kgtP gene is transformed via a vector into the yeast cell.

The DNA for the a ketoglutarate transporter may be inserted by itself or combined with DNA for proteins or carbohydrates. The a ketoglutarate transporter enables healthy, fast growing yeast strains that are suitable for producing most proteins and carbohydrates. The transporter also enables increased production of hydroxylated collagen.

DNA can be inserted into a vector. Vectors useful for expressing proteins in yeasts are known, see Ausubel et al., supra, Vol. 2, Chapter 13; Grant et al. (1987) Expression and

Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, Acad. Press, N.Y. 153:516-544; Glover (1986) DNA Cloning, Vol. Π, IRL Press, Wash., D.C., Ch. 3; Bitter (1987) Heterologous Gene Expression in Yeast, in Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. 152:673-684; and The Molecular Biology of the Yeast

Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II (1982), the disclosures of which are hereby incorporated by reference. Other suitable vectors include, but are not limited to, pHTXl- BiDi-P4HA-Pre-P4HB hygro, pHTXl- BiDi-P4HA-PH01-P4HB hygro, pGCW14-pGAPl- BiDi-P4HA-Prepro-P4HB G418, pGCW14-pGAPl- BiDi-P4HA-PH01- P4HB Hygro, pDF- Col3 Al modified Zeocin, pCAT-Col3 Al modified Zeocin, pDF-Col3 Al modified Zeocin with AOX1 landing pad, pHTXl - BiDi-P4HA-Pre-Pro-P4HB hygro. The vectors typically included at least one restriction site for linearization of DNA.

A select promoter may improve the production of a recombinant protein and may be included in a vector comprising sequences encoding the protein of interest (e.g., collagen) or hydroxylases. Suitable promoters for use in the present invention include, but are not limited to, AOX1 methanol induced promoter, pDF de-repressed promoter, pCAT de-repressed promoter, Dasl-Das2 methanol induced bi-directional promoter, pHTXl constitutive Bi-directional promoter, pGCW14-pGAPl constitutive Bi-directional promoter and combinations thereof. Suitable methanol induced promoters include but are not limited to AOX2, Das 1, Das 2, pDF, pCAT, pPMP20, pFDHl, pFLDl, pTAL2, pFBA2, pPEX5, pDAKl, pFGHl, pRKIl, pREP2 and combinations thereof.

In accordance with the foregoing, it is also envisioned that a vector can be engineered to be an all-in-one vector which contains each of the genes selected from at least one of, including any combination or sub-combination of the same or the entirety of all, collagen, hydroxylase(s), a ketoglutarate transporter, and/or gene(s) necessary to complete a functioning ascorbate synthesis pathway.

In the vectors according to the invention, including the all-in-one vector, a terminator may be placed at the end of each open reading frame utilized in the vectors incorporated into the yeast. The DNA sequence for the terminator is inserted into the vector. For replicating vectors, an origin of replication is necessary to initiate replication. The DNA sequence for the origin of replication is inserted into the vector. One or more DNA sequences containing homology to the yeast genome may be incorporated into the vector to facilitate recombination and incorporation into the yeast genome or to stabilize the vector once transformed into the yeast cell.

A vector according to the invention will also generally include at least one selective marker that is used to select yeast cells that have been successfully transformed. The markers sometimes are related to antibiotic resistance and markers may also be related to the ability to grow with or without certain amino acids (auxotrophic markers). Suitable auxotrophic markers included, but are not limited to ADE, HIS, URA, LEU, LYS, TRP and combinations thereof. To provide for selection of yeast cells containing a recombinant vector, at least one DNA sequence for a selection marker is incorporated into the vector.

In some embodiments of the invention, amino acid residues, such as lysine and proline, in a recombinant yeast-expressed protein, including collagen or collagen-like protein, may lack hydroxylation or may have a lesser or greater degree of hydroxylation than a corresponding natural or unmodified protein, e.g., collagen or collagen-like protein. In other embodiments, amino acid residues in a recombinant yeast-expressed protein, including collagen or collagen- like protein, may lack glycosylation or may have a lesser or greater degree of glycosylation than a corresponding natural or unmodified protein, e.g., collagen or collagen-like protein.

Hydroxylated collagen, for example, has a higher melting temperature (>37°C) than under hydroxylated collagen (<32°C) and also fibrillates better than under hydroxylated collagen and form stronger structure for material purpose. The melting temperature of a collagen preparation may be used to estimate its degree of hydroxylation and can range, for example, from 25 to 40°C, as well as all intermediate values such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, and 40°C, as well as subranges that are bound on the lower and upper end by a temperature selected from the forgoing values can be used to select a population of collagen for use.

Under hydroxylated collagen may only form a jello-like material not suitable for durable items such as shoes or bags but which can be formulated into softer or more absorbent products. By increasing the degree of hydroxylation, it is possible to improve thermal stability of collagen and may also improve strength of the biofabricated material produced from the same. These biofabricated materials may be more suitable for items requiring greater durability including shoes, bags, seats, etc. As a means to modulate the degree of hydroxylation it is envisioned that the number of copies or expression of the respective enzymes may be increased, also envisioned is modification of the temperature, pH, and carbon source of cell culture.

The engineered yeast cells described above can be utilized as hosts to produce collagen or other proteins or carbohydrates. In order to do so, the cells are placed in media within a fermentation chamber and fed dissolved oxygen and a source of carbon, under controlled pH conditions for a period of time ranging from twelve hours to 1 week. Suitable media include but are not limited to buffered glycerol complex media (BMGY), buffered methanol complex media (BMMY), and yeast extract peptone dextrose (YPD). Due to the fact that protein is produced in the yeast cell and using collagen as an example, in order to isolate the collagen, one must either use a secretory strain of yeast or lyse the yeast cells to release the collagen. The collagen may then be purified through conventional techniques such as centrifugation, precipitation, filtration, chromatography, and the like.

EXAMPLES

The following non-limiting Examples are illustrative of the present invention. The scope of the invention is not limited to the details described in these Examples. EXAMPLE 1

Modification of yeast to enable a functional plant synthesis pathway for

ascorbate. Pichia pastoris strain BG10 (wild type) was obtained from ATUM (formerly DNA 2.0). A vector containing DNA sequences of GDP-L-Gal phosphorylase (SEQ ID NO: 19), Inositol-phosphate phosphatase (SEQ ID NO: 20) and GDP-Mannose-3,5-epimerase (SEQ ID NO: 18) was inserted into wild type Pichia pastoris to generate a modified strain. The DNA was digested by Pme I and transformed into PP1 (Wild Type Pichia pastoris strain). This was suitable as a host strain.

EXAMPLE 2

The host strain from example 1 is modified as described below:

DNA encoding native Type ΠΙ bovine collagen is sequenced and the sequence is amplified by polymerase chain reaction "PCR" protocol to create a linear DNA sequence.

DNA encoding P4HA/B enzymes is sequenced (SEQ ID NO: 8 and SEQ ID NO: 9) and the sequence is amplified by polymerase chain reaction "PCR" protocol to create a linear DNA sequence.

The DNA is transformed into the strain from example 1 using a Pichia Electroporation Protocol (Bio-Rad Gene Pulser XcellTM Total System #1652660). Yeast cells are transformed with P4HA/B co-expression plasmid and transformants selected on Hygro plate (200 ug/ml).

A single colony of the resulting strain from example 1 is inoculated in 100 ml YPD medium and grown at 30 degrees overnight with shaking at 215 rpm. The next day when the culture reaches an OD600 ~3.5 (~3-5 X 10 ⁷ cells/OD600) it is diluted with fresh YPD to OD600 -1.7 and grown for another hour at 30 degree with shaking at 215 rpm.

Cells are spun down at 3,500g for 5 min; washed once with water and resuspend in 10 ml lOmM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT (added fresh), and 0.6 M Sorbitol.

For each transformation, 8 X 10 ⁸ cells are aliquoted into 8 ml lOmM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT, 0.6 M Sorbitol and allowed to incubate at room temperature for 30 min.

Cells are spun down at 5000g for 5 mins and washed with ice cold 1.5 ml 1M Sorbitol 3 times and resuspend in 80 ul ice cold 1M Sorbitol.

Various amounts (about 5 ug) of linearized DNA are added to the cells and mixed by pipetting.

Cells and DNA mixture (80-100ul) are added into a 0.2 cm electroporation cuvette and pulsed using Pichia - protocol (1500 v, 25 uF, 200 Ω).

The cells are immediately transferred into 1ml mixture of YPD and 1M Sorbitol (1:1) and incubated at 30 degree for > 2 hour.

These cells are plated at different densities and incubated at 30 °C for 2 days.

Single colonies that appeared on the plate after two days are inoculated into 2 mL BMGY media in a 24 deep well plate and grown out for at least 48 hours at 30 degree Celsius with shaking at 900 rpm. The resulting cells are tested for collagen using cell lysis, SDS-page and pepsin assay following the procedure below.

Yeast cells are lysed in lx lysis buffer using a Qiagen Tissue Lyser at a speed of 30 Hz continuously for 14 mins. Lysis buffer is made from 2.5 ml 1 M HEPES (final concentration 50 mM); 438.3 mg NaCl; final concentration 150 mM; 5 ml Glycerol; final concentration 10%; 0.5 ml Triton X-100; final concentration 1%; and 42 ml Millipure water.

The lysed cells are centrifuged at 2,500 rpm at 4°C for 15 mins on a tabletop centrifuge. The supernatant is retained and pellet discarded.

SDS-PAGE in the presence of 2-mercaptoethanol is performed on the supernatant, molecular weight markers, negative control and positive control. After electrophoresis the gel is removed and stained with Commassie Blue and then destained in water. A pepsin assay is performed with the following procedure:

Before pepsin treatment perform BCA assay to obtain the total protein of each sample per Thermo Scientific protocol. Normalize the total protein to the lowest concentration for all samples. (Note: if lowest total protein concentration is less than 0.5mg/mL do not use that concentration for normalization).

Put 100 uL of ly sate in a microcentrifuge tube. Create a master mix containing the following: 37% HC 1 (0.6μL of acid per 100 μL) and Pepsin (stock is lmg/mL in deionized water, and final addition of pepsin should be at a 1 :25 ratio pepsin:total protein (weigh weight). After addition of pepsin, mix 3X with pipet and allow the samples to incubate for an hour at room temperature for the pepsin reaction to take place. After an hour, add 1 : 1 volume of LDS loading buffer containing β-mercaptoethanol to each sample and allow to incubate for 7 minutes at 70°C. (In this situation lOOuL of LDS should be added). Then spin at 14,000 rpm for 1 minute to remove the turbidity.

Add 18uL from the top of sample onto 3-8% TAE buffer and run gel for lhr 10 minutes at 150V.

EXAMPLE 3

Yeast producing ascorbate

Cells from Example 1 are inoculated into glucose containing medium and grown at 30 degrees C with shaking. Samples are collected at different time points (for example 24, 48, 72, etc hours) and analyzed for ascorbic acid for both intracellular and extracellular by using a commercially available kit. The amount of ascorbic acid is constant over time. EXAMPLE 4

Yeast producing ascorbate and hydrox lated collagen

Cells from Example 2 are inoculated into glucose containing medium and grown at 30 degrees C with shaking. Samples are collected at different time points (for example 24, 48, 72, etc hours) and analyzed for ascorbic acid for both intracellular and extracellular by using a commercially available kit. Collagen expression level and hydroxylation are also analyzed by known methods.

EXAMPLE 5

Modification of yeast to include an a ketoglutarate transporter. Pichia pastoris strain (wild type) was obtained from ATUM (formerly DNA 2.0).

A vector containing DNA sequences of kgtP (SEQ ID NO: 3) was inserted into wild type Pichia pastoris to generate a modified strain. The DNA was digested by Bam HI and transformed into PP1 (Wild Type Pichia pastoris strain). This is suitable as a host strain.

The kgtP gene was tagged with HA tag. The recombinant gene was detected by Western Blot using anti HA antibody. The band shown on the blot had the expected molecular weight for the kgtP gene.

EXAMPLE 6

Modification of yeast to express a ketoglutarate transporter and to produce hydroxylated collagen.

The host strain from example 5 is modified as described below:

DNA encoding native Type IIΙ bovine collagen is sequenced (SEQ ID NO: 10) and the sequence is amplified by polymerase chain reaction "PCR" protocol to create a linear DNA sequence.

DNA encoding P4HA/B enzymes is sequenced (SEQ ID NO: 8 and 9 {Pichia optimized) or SEQ ID NO: 15 and 16 (native)) and the sequence is amplified by polymerase chain reaction "PCR" protocol to create a linear DNA sequence.

The linear DNA containing the collagen sequence is inserted into a pichia genome and the linear DNA containing the P4HA/B sequences are inserted into the pichia genome.

The DNA is transformed into the strain from example 5 using a Pichia Electroporation Protocol (Bio-Rad Gene Pulser XcellTM Total System #1652660). Yeast cells are transformed with P4HA/B co-expression plasmid and transformants selected on Hygro plate (200 μg/ml).

A single colony of the resulting strain from example 1 is inoculated in 100 ml YPD medium and grown at 30 degrees overnight with shaking at 215 rpm. The next day when the culture reaches an OD600 -3.5 (-3-5 X 10 ⁷ cells/OD600) it is diluted with fresh YPD to OD600 -1.7 and grown for another hour at 30 degree with shaking at 215 rpm.

Spin down the cells at 3,500g for 5 min; wash once with water and resuspend in 10 ml lOmM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT (add fresh), 0.6 M Sorbitol

For each transformation, aliquot 8 X 10 ⁸ cells into 8 ml lOmM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT, 0.6 M Sorbitol and incubate at room temperature for 30 min.

Spin down the cells at 5000g for 5 mins and wash with ice cold 1.5 ml 1M Sorbitol 3 times and resuspend in 80 μΐ ice cold 1M Sorbitol

Add various amount (about 5 μg) of linearized DNA to the cells and mix by pipetting.

Add cells and DNA mixture (80-100 μΐ) into 0.2 cm cuvette and pulse using Pichia - protocol (1500 v, 25 uF, 200 Ω) Immediately transfer the cells into 1ml mixture of YPD and 1M Sorbitol (1:1) and incubate at 30 degree for > 2 hour

Plate the cells at different densities.

Inoculate single colonies into 2 mL BMGY media in a 24 deep well plate and grew out for at least 48 hours at 30 degree Celsius with shaking at 900 rpm. The resulting cells are tested for collagen using cell lysis, SDS-page and pepsin assay following the procedure below.

Yeast cells are lysed in lx lysis buffer using a Qiagen TissueLyser at a speed of 30 Hz continuously for 14 mins. Lysis buffer was made from 2.5 ml 1 M HEPES (final concentration 50 mM); 438.3 mg NaCl; final concentration 150 mM; 5 ml Glycerol; final concentration 10%; 0.5 ml Triton X-100; final concentration 1%; and 42 ml Millipure water.

The lysed cells are centrifuged at 2,500 rpm at 4°C for 15 mins on a tabletop centrifuge. The supernatant is retained and pellet discarded.

SDS-PAGE in the presence of 2-mercaptoethanol is performed on the supernatant, molecular weight markers, negative control and positive control. After electrophoresis the gel is removed and stained with Commassie Blue and then destained in water.

A pepsin assay is performed with the following procedure:

Before pepsin treatment perform BCA assay to obtain the total protein of each sample per Thermo Scientific protocol. Normalize the total protein to the lowest concentration for all samples. (Note: if lowest total protein concentration is less than 0.5mg/mL do not use that concentration for normalization)

Put 100 μL, of lysate in a microcentrifuge tube. Create a master mix containing the following: 37% HC 1 (0.6 μL of acid per 100 μL) and Pepsin (stock is lmg/mL in deionized water, and final addition of pepsin should be at a 1 :25 ratio pepsin:total protein (weigh weight). After addition of pepsin, mix 3X with pipet and allow the samples to incubate for an hour at room temperature for the pepsin reaction to take place. After an hour, add 1 : 1 volume of LDS loading buffer containing β-mercaptoethanol to each sample and allow to incubate for 7 minutes at 70°C. (In this situation 100 μΐ, of LDS should be added). Then spin at 14,000 rpm for 1 minute to remove the turbidity.

Add 18 μΙ_, from the top of sample onto a 3-8% TAE and run gel for lhr 10 minutes at 150V. Amino acid analysis was performed to determine the percentage of hydroxylation. Expression of kgtP enables transport of alpha ketoglutarate into the endoplasmic reticulum which results in increased collagen and hydroxylated collagen level.

EXAMPLE 7

The yeast from Example 1 with the ascorbate pathway are modified with procedure of Example 5 to transform the transporter into the yeast providing Pichia with both the ascorbate pathway and the kgtP transporter.

EXAMPLE 8

The yeast from Example 7 with the ascorbate pathway and the transporter for kgtP are modified with the procedure of Example 2 to provide Pichia with the ascorbate pathway, the transporter for kgtP and hydroxylated collagen.

EXAMPLE 9

The gene for L-gulono-l,4-lactone oxidase (purchased from Eurofins Genomics) was codon optimized for expression in Pichia (SEQ ID NO 17). The gene was cloned under the constitutive promoter pDF and the antibiotic marker used was zeocin. The plasmid was transformed into Pichia cells using the same electroporation method as in Example 2 and plated on YPD plates with 50μg/ml of zeocin. The plates were incubated at 30 °C for 2 days or until the transformants started to appear on the plate.

Six transformants from the plate were picked and grown in 2 ml of BMGY media in 24 well plates for 18 hours with constant shaking at 30 °C. After 18 hours, varied concentration of the substrate (L-gulono-l,4-lactone) was added to the growing culture (0, 0.1 and 1 g/L final concentration). Wild type Pichia cells were used as a control for the experiment and the substrate was also added to the control culture. The cultures were harvested after 20 hours after the addition of the substrate. The optical density was normalized and the cells were lysed in water by mechanical shearing. Two methods were used to quantify ascorbic acid concentration in the cell lysate. In the first method, an abeam kit (Cat# ab65356) was used, where fluorescence intensity of the reaction mix was measured. The second method is known as the Sullivan and Clarke method, where absorbance for different cell lysate samples was recorded as the measure of the ascorbic acid production. Cells lysates of the transformants having L-gulono-l,4-lactone oxidase gene showed higher fluorescence intensity and higher absorbance than the controls (wild type Pichia and Transformants grown without the substrate). Preliminary data indicated that ascorbic acid was made when 1 g/L of the substrate was added to the cells and -10 nmol of ascorbic acid was produced at later time points (36 hours post addition of the substrate).