Genetically engineered yeast cells, recombinant proteins produced thereof and applications thereof

Abstract

A method for increasing cell growth and protein expression in an in vitro cell culture using recombinant growth factors produced by a genetically modified yeast strain is provided. Recombinant growth factors are introduced to the culture medium of the cells to be cultured, thereby increasing the rate of growth or protein expression of the cultured cells. In one embodiment, a recombinant growth factor of (i) genetically the same or similar species to the cultured cells and / or (ii) genetically the same genus to the cultured cells is used. In another embodiment, a recombinant growth factor with amino acid sequence modified for improved stability, bioactivity or other characteristics that promote cell growth and / or protein expression of the cultured cells is used.

Description

PCT PATENT APPLICATIONGENETICALLY ENGINEERED YEAST CELLS, RECOMBINANT PROTEINS PRODUCED THEREOF AND APPLICATIONS THEREOFINVENTORS: Po San Mario CHINHong KongKai Yi Carrie CHAN Hong KongChun Hei POON Hong KongLok Hin WONG Hong KongGENETICALLY ENGINEERED YEAST CELLS, RECOMBINANT PROTEINS PRODUCED THEREOF AND APPLICATIONS THEREOFCross Reference to Related Application

[0001] This application claims the priority of US non-provisional patent application serial number 17 / 831 ,351 , filed on June 2, 2022 which is a continuation-in-part application of PCT / IB2020 / 061200 filed on November 27, 2020 claiming the benefits of US provisional patent application serial number 62 / 942,568 filed on December 2, 2019. This application also claims the benefits of US provisional patent application serial number 63 / 423,488 filed on November 7, 2022. All applications identified above are hereby incorporated by reference in their entirety.Technical Field

[0002] Embodiments discussed herein generally relate to genetically engineered strains of yeast and improved methods for producing recombinant proteins, cell products or cultivated meat. Embodiments discussed herein also generally relate to the improved methods for cell growth and protein expression using growth factors.Background

[0003] Animal meat is high in protein, and supplies all the amino acids needed to build the protein used to support body functions. Meat for consumption is traditionally obtained from animals or fish that are reared on farms. However, agriculture and aquaculture for producing animal meat require a large amount of energy and resources and have a high carbon footprint. Meat produced by agriculture or aquaculture may pose a public health risk as the production processes may expose the meat to diseases, pollutants, and toxins. A number of concerns such as a growing population, increasing demand for meat, environmental concerns, limited land and water resources, biodiversity loss, and the negative perception associated with animal slaughter have led scientists to develop techniques to produce meat by alternative processes.

[0004] In vitro meat production is the process by which muscle tissue or organ tissue from animals is grown in laboratories using cell culture techniques to manufacture meat and meat products. Apart from food products such as meat, in vitro cell culture can also be employed to produce active ingredientsof various products or the products themselves (including but not limited to dietary supplements, hair care, skincare, wound care, cosmetic products, supplements, drugs and other medicinal applications). As used herein, in vitro meat and meat products includes animal protein products as well as non-meat products including soluble forms and solid forms. While still in an early stage of development, in vitro meat and meat products may offer a number of advantages over traditional meat products such as health and environmental advantages and benefits to animal welfare. It is a next-generation and emerging technology that operates as part of a wider field of cellular agriculture, or the production of agricultural products from cell cultures.

[0005] Cells for the production of in vitro meat may be cells (e.g., muscle cells, somatic cells, stem cells, etc.) taken from animal biopsies, which may then be grown separately from the animal in culture media in a bioreactor or other type of sterile environment. The cells may grow into a semi-solid or solid form mimicking an animal organ by attaching to an edible three-dimensional scaffold that is placed in the bioreactor. The starter cells may be primary cells directly obtained from the animal’s tissues, or continuous cell lines. If grown under the right conditions in appropriate culture media, primary cells will grow and proliferate, but only a finite number of times that is related to the telomere length at the end of the cell’s DNA. Continuous cell lines, on the other hand, can be cultured in vitro over an extended period. Cell biology research has established procedures on how to convert primary cells into immortal continuous cell lines. Primary cells may be transformed into continuous cell lines using viral oncogenes, chemical treatments, or overexpression of telomerase reverse transcriptase to prevent the telomeres from shortening.

[0006] The culture media may contain components necessary for cell proliferation such as amino acids, salts, vitamins, growth factors, and buffering systems to control pH. Current methods usually add exogenous animal products such as fetal bovine serum (FBS) or recombinant mammalian growth factors or cytokines to the media prior to use to provide vital macromolecules, growth factors, and immune molecules to maintain cell proliferation and increase cellular yields. However, such an approach has a few drawbacks. First, growing the cells in an animal component-free medium is an important factor considered by scientists involved in in vitro meatproduction research. FBS is derived from unborn calves and, therefore, is incompatible with the objective of being free from animal products.

[0007] Secondly, it is common that over 95% of the culture-medium cost is attributed to the protein components. Recombinant human growth factors (e.g. insulin growth factor 1 (IGF-1 )), human serum albumin (HSA), or fetal bovine serum (FBS) are often of research or pharmaceutical grade and are often supplemented in excess amounts to basal media, which increases medium costs and is unnecessary in most industrial applications.

[0008] Thirdly, while these supplemental components effectively promote the growth and differentiation of human cells, they are less bioactive on crossspecies cells (i.e. cells from distant species such as fish, birds). This causes a long culturing period and low cell quality. To compensate for the low bioactivity of non-human cells, excessively high levels of human protein factors or FBS are added to the growth medium, which leads to high costs. The situation for in vitro cell culture of non-mammalian cells (e.g. fish) is worse than that of mammalian cells due to limited commercial sources of nonmammalian growth factors. Using mammalian growth factors to enhance the growth of non-mammalian cells are ineffective as the mammalian growth factors may not bind to or act poorly on cellular receptors on non-mammalian cells; product manufactured in this manner may also raise consumer concerns about safety and potential allergic reactions.

[0009] Fourthly, most industry practices make use of native growth factors to culture cells which may be inefficient as native growth factors may be unstable or inherently have low bioactivity in their native forms. As a result, mega doses or frequent feeding of growth factors are required to compensate for these issues.

[0010] The above insufficiencies not only lead to high costs of in vitro meat production but also increase health risks to consumers of the final products. To address such insufficiencies and to provide improved methods for increasing cell growth and protein expression in an in vitro cell culture in a more cost-effective yet simplified manner, the present invention provides novel yeast strains that can produce recombinant native or modified growth factors for enhancing cell growth. In some embodiments, recombinant growth factors are introduced to the culture medium of the cells to be cultured,thereby increasing the rate of growth or protein expression of the cultured cells.

[0011] In some embodiments, the recombinant growth factor to be supplemented to the cultured cells is of similar species origin as the cultured cell type and / or with its amino acid sequence modified for improved bioactivity and stability profiles as compared to the native sequence.

[0012] By providing growth factors of similar species origin as the cultured cell type and / or modified growth factors with improved bioactivity and stability profiles, the present invention significantly reduces the amount of growth factors required to be supplemented and risks imposed on consumers. The present invention does not require a complex setup and products produced using the present invention can be produced on a large-scale basis and readily used in most applications without extra cumbersome purification procedures. Furthermore, the present yeast strains could be used to produce both mammalian and non-mammalian growth factors of various kinds and are therefore versatile in their applications. Overall, the present invention provides novel yeast strains and improved methods for producing recombinant proteins that promote cell growth in an in vitro culture and enable the production of in vitro meat products and other types of cell products at lowered costs and in a simplified manner. The resulting products are compatible with various applications including cellular agriculture, cosmetics, pharmaceuticals and biotechnology.Summary

[0013] The embodiments of the present disclosure provide genetically engineered strains of yeast and improved methods for increasing cell growth and protein expression in an in vitro cell culture.

[0014] It is an objective of the present invention to provide novel genetically engineered yeast strains and an alternative method to produce recombinant proteins such as recombinant growth factors for use in an in vitro cell culture. In some embodiments, recombinant growth factors are introduced to the culture medium of the cells to be cultured to increase the rate of growth or protein expression of the cultured cells.

[0015] It is an objective of the present invention to provide an alternative method to cultivate cells using species-specific or genus-specific growthfactors. This approach not only decreases medium-cost by lowering growth factor usage, but it also shortens the culturing time and improves cell quality by enhancing cellular responses. Using species-specific or genus-specific growth factors may help to enhance cellular response (for example, growth and differentiation) and break the maximum cellular response encountered when using non-species-specific or non-genus-specific growth factors. It is also an objective of the present invention to provide an alternative method to cultivate cells using growth factors that have their amino acid modified for improved stability and / or bioactivity as compared to the native growth factors.

[0016] It is an objective of the present invention to provide a method to produce recombinant growth factors that can be used in both non-mammalian and mammalian cell cultures; for example, the resulting recombinant growth factors are of fish origin that may be used to cultivate fish cells or cells of related species. It is also an objective of the present invention to provide a method to produce recombinant native growth factors or modified growth factors that have their amino acid sequences modified for improved stability, bioactivity or other characteristics that promote cell growth and / or protein expression of the cultured cells. As efficacies of growth factors of different species and amino acid sequences in stimulating cell growth could differ vastly, cell culture users can use the present invention to generate different constructs of growth factors and evaluate their efficacies and choose the constructs that the concerned cell types respond maximally to for a particular application. Further, cell products produced using the present inventions are of a purity that is generally acceptable for most types of applications and can be readily used without complex purification procedures. In some embodiments where a higher purity is required for a specific application such as pharmaceutical or medical applications, the resulting in vitro cell products may be further purified using conventional purification techniques. Overall, the application of the present invention not only provides flexibility in its application but also significantly reduces production costs for large-scale cultures.

[0017] According to one embodiment of the present disclosure, a genetically modified yeast cell encoding a recombinant growth factor is provided. The recombinant growth factor can be of a native amino acid sequence or amodified amino acid sequence with improved stability and / or bioactivity as compared to the native form.

[0018] According to one embodiment of the present disclosure, a method for increasing the rate of growth or protein expression of a plurality of cells in an in vitro cell culture is provided. The method comprises the steps of growing the plurality of cells in a nutrient medium under growth conditions suitable for initiation of cell division and introducing at least one recombinant growth factor to the nutrient medium. In some embodiments, the recombinant growth factor is (a) optimized for increasing the rate of growth or protein expression of the plurality of cells or (b) (i) of genetically same or similar species to the plurality of cells and / or (ii) of genetically same genus to the plurality of cells.

[0019] According to one embodiment of the present disclosure, a method for meat production by in vitro cell culture includes isolating tissue from an animal or plant source and making a cell suspension of cells is provided. The method further includes introducing growth factors that are (a) optimized for increasing the rate of growth or protein expression of the plurality of cells or (b) (i) of genetically same or similar species to the cells and / or (ii) of the genetically same genus to the cells. Additionally, the method further includes growing the cells on a food-grade scaffold in a culture medium, the cells growing into a solid or semi-solid structure that mimics an animal organ.

[0020] In a further embodiment, the growth factor that supports cell growth can be produced from other microorganisms such as bacteria or fungi containing and expressing a transgene of the growth factor.

[0021] Given the fact that purification of the expressed growth factor from the microorganisms involves a complicated and expensive process, embodiments of the invention develop a process to use the growth factor directly from the yeast culture to culture cells without an elaborated purification procedure. In some embodiments, the recombinant growth factor to be introduced to an in vitro culture is produced by a genetically modified yeast cell comprising a gene of the recombinant growth factor and obtained from a cell culture medium growing the genetically modified yeast cell. In one embodiment, the recombinant growth factor is purified from the yeast cell culture medium before it is introduced to the in vitro culture. In another embodiment, the recombinant growth factor is introduced to the in vitro culture by introducingthe yeast cell culture medium containing the growth factor to the in vitro culture.

[0022] Aspects of the invention may genetically engineer a yeast clone that contains a IGF-1 gene of a specific species such as a fish IFGF-1 gene. In one aspect, the recombinant yeasts may be grown in a yeast culture medium to allow protein expression of the IGF-1 gene. In one aspect, the IGF-1 may be secreted into the yeast culture medium. In another example, the IGF-1 - containing yeast culture medium may be centrifuged at about 1500 g for 5 minutes to separate the yeast from the medium. The supernatant may be collected and centrifuged again at about 7200 g for about 15 minutes at about 4°C. The clarified supernatant may then be filtered with a 2 pm or smaller pore-size filter. The filtered IGF-1 -containing yeast culture medium may be diluted up to 10,000-fold before adding to the cell culture medium to grow fish cells or other types of cells.

[0023] Brief Description of the Drawings

[0024] The disclosure may be better understood by reference to the detailed description when considered in connection with the accompanying drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure.

[0025] FIG. 1 is a flowchart of a method for meat production by in vitro cell culture, according to one embodiment of the present disclosure.

[0026] FIG. 2 is a chart illustrating the respective cell numbers after treating MCF-7 cells with different concentrations (1 pg / ml to 100 ng / ml) of recombinant human IGF-1 (Oryzogen). Cells were harvested on day 10 for direct cell counting.

[0027] FIG. 3 is a chart illustrating the respective relative fluorescence after the treatment of MCF-7 cells with 15 nM of recombinant human IGF-1 (from 3 different suppliers), recombinant mouse IGF-1 , and recombinant fish (tuna, bream) IGF-1 . Cells were harvested on day 7 and subjected to CyQUANT Cell Proliferation Assay.

[0028] FIG. 4 is a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells with 10 nM of three different clones of recombinant fish IGF-1 (Fish IGF-1 (A), IGF-1 (B) and IGF-1 (C)). Cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.

[0029] FIG. 5 is a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells with 1 % of three different recombinant fish IGF-1 -containing yeast culture medium. Cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.

[0030] FIG. 6 is the Western blot analysis of recombinant native and modified grouper IGF-1 in yeast culture using anti-6X His antibody.

[0031] FIG. 7 is the characterization of recombinant native and modified grouper IGF-1 purified from yeast culture using immobilized metal affinity chromatography. Panel (a) shows an analysis by total protein staining and panel (b) shows an analysis by Western blotting using anti-6X His antibody.

[0032] FIG. 8 shows two charts illustrating the results of the resazurin assay on (a) MCF-7 cells and (b) FHM cells treated with purified grouper IGF-1 at 0 nM, 0.1 nM, 1 nM and 10 nM.

[0033] FIG. 9 shows two charts illustrating the results of the resazurin assay on (a) MCF-7 cells and (b) FHM cells treated with grouper IGF-l-containing yeast culture at 1 : 100 and 1 : 1000 dilutions.Detailed Description

[0034] Referring now to the drawings, and with specific reference to FIG. 1 , a method 10 for in vitro meat production is shown. As used herein, “in vitro meat production” refers to a cell-based meat production process or cell-based agriculture process in which tissues from animals and / or plants are grown in laboratories using cell culture techniques to manufacture meat and meat products. At a block 12, tissue from an animal or a plant is isolated. In one embodiment, the tissue is derived from bony fish of the class Osteichthyes including saltwater fish such as a grouper, sea bass, or yellow cocker. In other embodiments, other types of animal tissue, such as cow tissue, may be isolated. In some embodiments, the block 12 may involve collecting organ tissue, such as a swim bladder, from a fish and making a cell suspension.Although the following description primarily describes tissues derived from fish sources, it will be understood that the concepts may be applied to tissues derived from other types of animal sources and / or plant sources to provide other types of in vitro meat and / or animal protein products, and vegetarian meat and / or protein products.

[0035] Many of the isolated cells are adult cells and can be made to proliferate continuously using various established methods in medical research (block 14). For example, specific genes, such as Yamanaka factors, may be used to reprogram adult cells into stem cells, such as induced pluripotent stem cells (iPSCs). Alternatively, the isolated adult cells may be transformed into continuous cell lines by telomerase reverse transcriptase overexpression. In other embodiments, other types of cells may be isolated such as adult stem cells and embryonic stem cells. In this regard, it will be understood that the methods of the present disclosure include all sources of cell lines.

[0036] In a next block 16, the cells are grown into a solid or semi-solid structure mimicking an animal organ, such as a fish organ, by attaching / adhering to a food-grade biocompatible scaffold in a sterile chamber or container, such as a bioreactor. The sterile chamber or container may be temperature controlled and may have inlets and outlets for introducing and removing substances such as chemicals, nutrients, and cells. The food-grade biocompatible scaffold becomes part of the final edible product, and is made of plant-based or fungi-based materials such as, but not limited to, agarose, alginate, chitosan, mycelium, and konjac glucomannan. Alginate is a biopolymer naturally derived from brown algae and is biocompatible. In addition, plant-based chitosan from fungi has antibacterial properties. In some embodiments, the block 16 is carried out in the absence of antibiotics or antimicrobial compounds in the sterile container. A block 18 involves supplying the culture medium to the bioreactor to support cell survival and growth. The culture medium may be a buffered solution containing components such as, but not limited to, inorganic salts (e.g., calcium chloride (CaCl2), potassium chloride (KCI), sodium chloride (NaCI), sodium bicarbonate (NaHCOs), sodium dihydrogen phosphate (NaH2PO4), magnesium sulfate (MgSO4), etc.), amino acids, vitamins (e.g., thiamine, riboflavin, folic acid, etc.), and other components such as glucose, 0- mercaptoethanol, ethylenediaminetetraacetic acid (EDTA), and sodium pyruvate. Non-limiting examples of growth media include, but are not limited to, Leibovitz’s L-15 medium, Eagle’s Minimum Essential Media (MEM), Medium 199, Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F12Nutrient Mix, Ham’s F10 Nutrient Mix, MacCoy’s 5A Medium, Glasgow Modified Eagle Medium (GMEM), Iscove’s Modified Dulbecco’s Medium, and RPMI 1640.

[0037] According to a block 20, food-grade growth factors and cytokines are introduced into the culture medium in the bioreactor to support cell growth and proliferation. The growth factors and cytokines may include, but are not limited to, insulin growth factor 1 (IGF-1 ), insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11 ), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transferrin. In some embodiments, growth factors of (i) genetically the same or similar species to the isolated / cultured cells and / or (ii) genetically the same genus to the isolated / cultured cells (i.e. cells growing at block 16) are used in the present invention. It is found that the use of growth factors of (i) genetically the same or similar species to the isolated / cultured cells and / or (ii) genetically the same genus to the isolated cells exert higher bioactivities to the isolated cells compared to the use of growth factors and serum of genetically distant species to the isolated cells. Due to the enhanced compatibility, there is no need to supply a megadose of “suboptimal” growth factors when culturing these cells using growth factors of (i) genetically same or similar species and / or (ii) genetically same genus. Higher bioactivity could also help reduce the amount of growth factors needed in the culture medium, shortening the culture period and improving cell quality. The cost of the culture medium could be reduced due to the decrease in the levels of growth factors required in the culture medium for the stimulation of cell growth and differentiation.Furthermore, the use of suboptimal growth factors limits the magnitude of the maximum cellular response (growth, differentiation). In some instances, certain responses can never be reached no matter how much of the suboptimal growth factor is supplied. Species-specific and / or genus-specific growth factors could help overcome these limits.

[0038] Species-specific or genus-specific growth factors can effectively act on the receptors of the isolated cells. Compared to the conventional growth medium, which is often supplemented by high levels of human protein growth factors and / or FBS irrespective of the species origin of the isolated cell, the use of species-specific or genus-specific growth factors is better optimized.Species-specific or genus-specific variations in amino acid sequence and post-translational modifications of the growth factor(s) and cell receptor(s) may account for this phenomenon.

[0039] As will be discussed in much greater detail in Example 1 below, to identify which species of a certain growth factor exerts the highest bioactivity on the target cells to be cultured, target cells are first seeded in a complete medium (i.e. basal medium + FBS). Upon reaching the target confluence (around 20%-70%), target cells are treated by the growth factor of different species at a range of concentrations (e.g. 1 pM-1 pM). Target cells are kept in the incubator until reaching the desired time point(s) for the studied parameter (e.g. cell growth, differentiation markers, cellular products). For example, when there are differences in cell confluence between the treatment groups (around 2-10 days), cell growth can be measured by trypan blue exclusion, the CyQUANT assay, or any other appropriate cell proliferation / death assays. The bioactivities of the growth factors of various species are compared based on their EC50 values (half-maximal effective concentration). For cost-effectiveness, target cells should be cultured using the growth factor with the lowest EC50 value. However, if the aim is to attain the shortest culturing time or the highest cell quality, the growth factor which triggers the highest maximum cellular response is preferably selected. The optimal dose of the growth factor is defined as the lowest concentration required to elicit the maximum cellular response.

[0040] In some embodiments, the block 20 may involve co-culturing bioengineered cells with isolated / cultured cells in the absence of fetal bovine serum (FBS). The bioengineered cells are engineered to secrete the above growth factors and cytokines and supply these biomolecules to the isolated cells as needed for growth and proliferation. As used herein, “bioengineered” cells are not equivalent to genetically-modified cells. The bioengineered cells have a specific gene that overexpresses one or more specific proteins. The bioengineered cells may be fish cells, or other types of animal cells, such as cow cells. The bioengineered cells and the isolated cells may be genetically similar or identical species. Also, the bioengineered cells and the isolated cells may belong to the same genus. As non-limiting examples, bioengineered fish cells may be co-cultured with isolated fish cells, or bioengineered cowcells may be co-cultured with isolated cow cells. In some particular examples, the bioengineered cells may be chicken cells or bird cells if chicken cells are used as the isolated cells. In yet another particular example, the bioengineered cells may be yellow crocker cells or other fish cells if yellow cracker cells are used as the isolated cells. The bioengineered cells are not present in the final meat product. The co-culturing method of the present disclosure eliminates the need for animal-derived fetal bovine serum (FBS) in the culture medium. Furthermore, the co-culturing method provides a continuous supply of food-grade specific growth factors and cytokines to the growing isolated cells in situ, and simplifies and reduces the cost of the production process, wherein the growth factors are (i) of genetically same or similar species to the isolated cells and / or (ii) of the same genus to the isolated cells. However, in other embodiments, FBS or other serum or other products containing recombinant growth factors may be used to supply growth factors, cytokines, and other nutrients to support cell growth during the block 16.

[0041] In some embodiments, the block 20 contains recombinant growth factors of genetically the same or similar species to the isolated cells. In yet some embodiments, the recombinant growth factors of the same genus to the isolated cells are used. In some embodiments, the recombinant growth factors are introduced into the growth medium in purified form or in a crudely purified form such as a yeast culture (or the supernatant of the yeast culture) obtained from a culture of recombinant yeast cells expressing the recombinant growth factors. The use of such recombinant growth factors exerts higher bioactivities on the isolated cells than growth factors and serum of distant species on the isolated cells. Bacterial, yeast, insect, mammalian, or any other appropriate protein expression systems may be used to produce such recombinant growth factors. Protein purification is performed by (but not limited to) affinity chromatography, ion-exchange chromatography, size exclusion chromatography, or a combination of these strategies.

[0042] It is anticipated that recombinant growth factors produced using the present invention can be used to support or promote the growth of various kinds of cells and cells of various origins. In some embodiments, the recombinant growth factors are recombinant fish growth factors that may beused to support the growth of fish cells. For example, recombinant growth factors of Epinephelus akaara (fish), which is genetically similar species to the fish muscle cells or swim bladder cells of Epinephelus awoara (fish), are used. The recombinant growth factors for culturing Epinephelus awoara used are Epinephelus akaara’s IGF-1 , insulin and / or transferrin. The concentration of such IGF-1 is ranged from 10ng / ml to 100ng / ml. The concentration of such insulin is ranged from 1 pg / ml - 10pg / ml. The concentration of such transferrin is ranged from 0.5pg / ml - 5pg / ml.

[0043] In some embodiments, the recombinant fish growth factors are used to support or promote the growth of non-fish cells such as human cells or other mammalian cells. In yet another embodiments, the recombinant fish growth factors are used to support or promote the growth of non-mammalian cells other than fish cells.

[0044] In some embodiments, the recombinant growth factors are recombinant bovine growth factors that may be used to support the growth of bovine cells, other mammalian cells or non-mammalian cells.

[0045] In some embodiments, the recombinant growth factors are recombinant avian growth factors that may be used to support the growth of avian cells, other non-mammalian cells or mammalian cells.

[0046] Optionally, according to a block 22, protein expression in the cells may increase the biomass yield in the resulting meat product. As used herein, “biomass yield” refers to the amount of digestible material (e.g., proteins) in the resulting meat product that is available for energy production upon consumption. Again, as an optional feature, the block 22 may involve increasing protein expression by altering micro RNA levels in the cells, with the manipulation of the cells being carried out prior to culturing. Micro RNAs are endogenous, short, non-encoding single-stranded RNA sequences involved in regulating post-transcriptional gene expression. The block 22 may optionally involve increasing the amount of up-regulating micro RNAs that increase protein expression by promoting messenger RNA (mRNA) translation, and / or decreasing the amount of down-regulating micro RNAs that decrease protein expression by suppressing mRNA translation. The micro RNA levels may be increased or decreased by introducing micro RNAs, micro RNA mimics, or micro RNA inhibitors into the cells. The micro RNA mimicshave the same function as micro RNAs, but may be more stable and efficient in modulating protein expression. In some embodiments, electroporation may be used to introduce episomal vectors into the cells that carry instructions to express specific micro RNAs. Alternatively or in combination with this, an adeno-associated virus may be used as a vehicle carrying episomal instructions to express specific micro RNAs. Decreasing the amount of targeted down-regulating micro RNAs may be achieved by introducing inhibitors for the targeted micro RNAs into the cells by transfection. It is noted here that the methods of increasing protein expression / biomass yield according to the present disclosure is carried out without modifying the genome of the cells.

[0047] Example 1 - Identification Of species-specific or genus-specific growth factor

[0048] This example describes a method of identifying species-specific or genus-specific growth factors. It includes two major steps, which are the cell growth stimulation step and the measuring cell growth step.

[0049] Step 1: Cell growth stimulation step

[0050] The MCF-7 human epithelial cell line is cultured in DMEM / F12 complete medium DMEM / F12 , 10% FBS inside a humidified incubator (34°C; 5% CO2; 95% air). Split cells at a ratio of 1 :4 to 1 :8 for routine maintenance.

[0051] Upon reaching about 80% confluence, detach cells by trypsin / EDTA. To study the effect of growth factors on cell growth, cells are seeded at a density of 3 x 104cells / cm2in a complete medium onto 24-well (if cell growth is measured by cell counting) or 96-well plates (if cell growth is measured by the CyQUANT Cell Proliferation Assay Kit). Return the cells to the incubator.

[0052] After 24 hours, remove the medium. Pre-adapt cells to serum-free conditions by adding serum-free medium DMEM / F12, 0.1% human serum albumin. Keep the cells in this medium for at least 16 hours inside the incubator.

[0053] Prepare growth factors (e.g. IGF-1 ) at 10x working concentrations in serum-free medium for each species / genius, which growth-stimulating effect to be examined (e.g. recombinant human IGF-1 , human IGF-1 -LR3, mouse IGF-1 , bream IGF-1 and tuna IGF-1 ). Add the 10x growth factors into the wells such that cells will be treated by 1x growth factors (e.g. add 50 pl 10xgrowth factor to a well containing 450 pl serum-free medium) (e.g. 1 pM-1 pM). Return the cells to the incubator.

[0054] Observe the cells daily under a microscope for signs of cell growth. When there are obvious differences in terms of cell confluence between the treatment groups (usually detected between day 2 - day 10), quantify cell growth either by cell counting, the CyQUANT Cell Proliferation Assay, or any other cell proliferation / death assays.

[0055] Step 2: Measuring cell growth step

[0056] There are two general ways to measure cell growth, namely, trypan blue exclusion and CyQUANT Cell Proliferation Assay Kit.

[0057] Trypan blue exclusion

[0058] Cells should have been treated in 24-well plates. Aspirate the culture medium and detach cells by trypsin-EDTA.

[0059] Stop trypsin activity by adding 1 volume of the complete medium into the well. Ensure that all cells are detached by pipetting 3-5 times inside the well.

[0060] Collect the cell suspension into 1.5 ml tubes. Pellet the cells by centrifuging the tubes at 400 x g for 5 minutes.

[0061] Remove the supernatant without disturbing the cell pellet. Resuspend the cell pellet in 200 pl DMEM / F12 basal medium.

[0062] Mix 10 pl of the cell suspension with 10 pl of 0.4% Trypan Blue solution

[0063] After 2 minutes, add 10 pl of the cell / trypan blue mixture to each chamber of a Countess II FL Disposable Slide or a hemocytometer. If using the Countess II FL system, insert the slide into the slide holder of a Countess II FL Automated Cell Counter and determine the cell concentration and % viability. If using a hemocytometer, count cells with a microscope.

[0064] Calculate the number of cells in each treatment group. The number of viable cells per well equals to viable cell concentration (cells / ml).

[0065] CyQUANT Cell Proliferation Assay Kit

[0066] The CyQUANT Cell Proliferation Assay Kit quantifies cell growth by measuring the nucleic acid content in samples. Cells should have been seeded onto 96-well plates, preferably in triplicate wells per treatment group.

[0067] Remove the culture medium as much as possible by a multichannel pipette. Avoid scratching the well bottom with the pipette tip.

[0068] Freeze the plate in a -80°C freezer. The plate may be stored at -80°C for up to 4 weeks.

[0069] Thaw the plate and assay kit reagents at room temperature.

[0070] Mix the kit reagents according to Table 1 for each well.Table 1Components Volume per well(Ml)Cell lysis buffer stock solution 10 plAutoclaved MilliQ water 189.5 plCyQUANT GR stock solution 0.5 plTotal= 200 pl

[0071] When the plate has completely thawed, add 200 pl of the CyQUANT GR dye / lysis buffer mixture to each sample well and to three empty wells (blank). Incubate the plate at room temperature for 5 minutes, protected from light.

[0072] Using a multichannel pipette, transfer 160 pl from each well of the 96- well plate to the corresponding well of a black 96-well plate.

[0073] Measure the sample fluorescence using a fluorescence microplate reader (e.g. Molecular Devices SpectraMax iD5). Set the excitation and emission wavelengths at 480 nm and 520 nm respectively.

[0074] Average the blank wells fluorescence readings. Subtract this average reading from all sample fluorescence readings to correct for background fluorescence.

[0075] Calculate the mean corrected fluorescence for the vehicle group. Express the treatment group fluorescence readings as fold of control (FOC) by dividing the sample readings (i.e. IGF-1 groups) by the mean vehicle reading.

[0076] Results: IGF-1 stimulated the growth of MCF-7 cells in a dosedependent manner

[0077] To verify that IGF-1 stimulates the growth of human MCF-7 cells, cells were treated with increasing doses of human IGF-1 (0 pg / ml - 100 ng / ml) for 10 days and processed for cell counting. As seen in FIG. 2, while 1-100 pg / ml IGF-1 did not enhance the cell number much, a further increase of IGF-1concentration (1-100 ng / ml) promoted cell growth in a dose-dependent manner. Hence, MCF-7 cells are suitable for evaluating the growth-stimulating activity of IGF-1 .

[0078] Example 2 - Human IGF-1, but not the mouse or fish IGF-1, promoted the growth of human MCF-7 cells

[0079] To investigate whether the growth-stimulating activity of IGF-1 depends on its species origin, we treated MCF-7 cells with 1.5 nM recombinant IGF-1 of various species, i.e. human, mouse, and fish (bream, tuna). After 7 days, cell growth was assessed by the CyQUANT Assay (FIG. 3). While human IGF-1 obtained from multiple sources consistently increased MCF-7 cell growth (~50 - 100% increase), mouse and fish IGF-1 did not (FIG. 3). These findings suggest that human IGF-1 , being the same species as the human MCF-7 cells, is more effective than fish and mouse IGF-1 in promoting cell growth.

[0080] Example 3 - Fish IGF-1 expressed by recombinant yeast cells promoted the growth of fish swim bladder cells

[0081] To investigate the effects of recombinant fish growth factors produced by recombinant yeast cells on the growth of fish cells, fish IGF-1 was produced by and obtained from a recombinant yeast strain that was genetically engineered to carry and express a fish IGF-1 gene, and then added to the culture medium for growing fish swim bladder cells. FIG. 4 shows a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells by 10 nM of three different clones of recombinant fish IGF-1 (Fish IGF-1 (A), Fish IGF-1 (B) and Fish IGF-1 (C)), each of which has a different nucleotide sequence from each other and the native gene of fish IGF-1 . For example, the cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.

[0082] FIG. 5 is a chart illustrating the respective relative fluorescence after the treatment of fish swim bladder cells by 1% (v / v) of three different batches of yeast culture medium, each of which contains exclusively one of the clones of recombinant fish IGF-1 as mentioned in the preceding paragraph (i.e., (Fish IGF-1 (A), Fish IGF-1 (B) and Fish IGF-1 (C)). In this example, the three batches of IGF-1 -containing yeast medium were collected, centrifuged and filtered, and the resulting supernatants were added directly to the fish swimbladder cells. The cultured cells were harvested on day 3 and subjected to CyQUANT Cell Proliferation Assay.

[0083] The results in FIGS. 4 and 5 suggest that the addition of fish IFG-1 obtained from recombinant yeast culture to the culture medium of fish swim bladder cells enhanced the growth of the swim bladder cells. A skilled person in the art would appreciate that apart from IFG-1 , other growth factors and cytokines can also be used in connection with these embodiments and other embodiments of this invention, including but not limited to insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11 ), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transferrin, and the resulting recombinant growth factors or cytokines, no matter in purified form or in a mixture form with the yeast medium culture, can be used to supplement cell culture medium for enhancing the growth of various types of cells. Further, as illustrated in this disclosure and examples herein, growth factors of the same or different origin of the cultured cells can be used, and native or modified growth factors can also be used in order to achieve maximal responsiveness in the cell types in question. A skilled person in the art would appreciate that the cultured cells to be treated with such exogenous growth factors or cytokines are not limited to fish cells and can be non-fish cells including nonmammalian cells and mammalian cells and that the gene of the growth factors or cytokines to be introduced into the recombinant yeast cells as an expression system by way of genetic engineering shall be preferably (i) of genetically the same or similar species to the target cells and / or (ii) of genetically the same genus to the target cells.

[0084] In one embodiment, the concentration of recombinant growth factors or cytokines to be added to the culture medium of the target isolated cells are in the range of 0.1 %-1 % (v / v) or 1 nM-10nM. The present invention shows that it is more effective to apply growth factors of (i) genetically the same or similar species or (ii) genetically the same genus as the cultured cell type. The usage of these growth factors or protein factors may be decreased while achieving the same growth rate. Species-specific growth factors and / or genus-specific growth factors represent a promising direction to reduce media cost especially during large-scale cell production for cultivated meat and other applications using the cultivated cell mass. Using more bioactive growth factors can alsodecrease processing times and improve the quality (e.g. texture, taste, nutritional value) of cultivated meat or cell mass.

[0085] In the following examples, embodiments of genetically engineered yeast cells expressing recombinant fish IGF-1 are described and their applications on promoting the growth of fish and human cells in in vitro culture are provided. A skilled person in the art would appreciate that other growth factors can be produced and applied in a similar manner according to the teaching of the present disclosure.

[0086] Example 4 - Cloning of grouper IGF-1 gene into a yeast expression vector

[0087] Grouper IGF-1 genes having the nucleotide sequences set forth in SEQ ID NOs. 1 -5 encoding the amino acid sequences set forth in SEQ ID NOs. 6-10 were cloned into a PichiaPink™ vector (ThermoFisher) respectively according to the prescribed protocol, resulting in a clone of grouper IGF-1 gene having the native grouper IGF-1 gene sequence and four different clones of modified grouper IGF-1 gene. Details of the clones are summarized in the following Table 1 .

[0088] In some embodiments, the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant IGF-1 selected from the group consisting of SEQ ID NOs. 1 -5. In some embodiments, the resulting recombinant IGF-1 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.

[0089] Additionally, the invention also encompasses polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 6-10. In one embodiment, polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 6-10. In another embodiment, the polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 6-10.Table 1 - Examples of Grouper IGF-1 Gene and Protein Sequences

[0090] Example 5 - Expression of recombinant fish IGF-1 protein from PichiaPink™ strainsA) Pilot expression of recombinant IGF-1 proteinRecombinant yeast cells for expressing recombinant fish IGF-1 protein and the pilot expression of the recombinant fish IGF-1 protein in the resulting recombinant yeast cells were carried out and analyzed as follows:1. Transform the yeast cells (from PichiaPink™ Expression Strain Set, ThermoFisher) with one of the clones of recombinant grouper IGF-1 gene following the manufacturer’s protocol.2. Using a single colony of a transformed PichiaPink™ strain, inoculate 10 mL of BMGY medium (Buffered Glycerol-complex Medium) in a 50 mL centrifuge tube. Grow the cells for 1-3 days at 24-30°C in a shaking incubator set to 200- 300 rpm.3. Centrifuge the yeast culture at 1 ,500* g for 5 minutes at room temperature. Decant the BMGY supernatant and resuspend the cell pellet in 1 mL of BMMY medium to induce expression.4. Return the cells in BMMY medium to the 30°C shaking incubator to continue growth overnight.5. Remove 100 pL from the sample and add 100 pL of 40% methanol every 24 hours to induce expression. Continue to grow the cells at 24-30°C in a shaking incubator set to 200-300 rpm for 24-72 hours.6. Harvest the cells by centrifuging for 10 minutes at 1 ,500 x g.7. Transfer the supernatant to a separate tube and store both the supernatant and the cell pellet at -80°C until use.8. Analyze the supernatants for the best expressing clone and the optimal time of induction by Western blotting.B) Scale-up expression of recombinant IGF-1 proteinScale-up expression of the recombinant fish IGF-1 protein in the recombinant yeast cells was carried out and analyzed as follows:1 . Using a single colony of a transformed PichiaPink™ strain, inoculate 400 mL of BMGY. Grow at 24°C-30°C in a shaking incubator (250-300 rpm) for 2-3 days.2. Harvest the cells by centrifuging in sterile centrifuge bottles at 1 ,500-3,000 x g for 5 minutes at room temperature.3. To induce expression, decant the supernatant and resuspend the cell pellet in 50 mL of BMMY medium. Continue to grow at 24°C-30°C with shaking.4. Add 100% methanol to 0.5% every 24 hours until the optimal time of induction is reached.5. Harvest the cells by centrifuging at 1 ,500 x g for 5 minutes at 4°C.6. Save and centrifuge the supernatant again at 7,197 x g (max. g) for 15 minutes at 4°C.7. Save the supernatant. Store at -80°C until ready to process further.C) Processing of raw IGF-1 containing yeast culture medium for direct usageAfter expression of the fish IGF-1 protein in the yeast cells, the resulting fish IGF- 1 protein was collected together with the yeast culture medium by the following steps to obtain a IGF-1 containing yeast culture medium:1 . Repeat steps 1 to 4 in above section B.2. Harvest the cells by centrifuging at 1 ,500 x g for 5 minutes at 4°C.3. Save and centrifuge the supernatant again at 7,197 x g (max. g) for 15 minutes at 4°C.4. Save the supernatant. Filter the supernatant with a 0.22 pm filter.5. Keep the filtered IGF-1 containing yeast culture medium at 4°C for temporary storage or -80°C for long-term storage.D) Purification of recombinant IGF-1 protein by immobilized metal affinity chromatographyAs an optional and additional step, the recombinant fish IGF-1 protein expressed in the yeast cells was purified using immobilized metal affinity chromatography (HisPur™ Cobalt Resin, ThermoFisher) as follows:1 . Add 1 mL of Cobalt Resin to a 10 mL purification column. Allow storage buffer to drain from the resin by gravity flow. Discard the flow-through.2. Add 2 mL of Equilibration / Wash Buffer and mix until the resin is fully suspended. Allow Equilibration / Wash Buffer to drain from the resin by gravity flow. Discard the flow-through.3. To purify the recombinant IGF-1 protein (which bears a His-tag) from the culture supernatant, first mix the culture supernatant with equilibration buffer in a 1 :1 ratio. Then load the clarified culture supernatant into the purification column. Allow culture supernatant to drain from the purification column by gravity flow. Re-apply the flow-through once to the purification column to maximize binding.4. Wash the resin with 10 mL of Equilibration / Wash Buffer. Allow the Equilibration / Wash Buffer to drain from the purification column by gravity flow. Repeat this wash step two more times.5. Elute bound His-tagged proteins using 1 mL of Elution Buffer. Collect the flow- through. Repeat this step four more times, saving each fraction of the flow- through in a separate tube.6. Dialyzed the eluted protein for a total of 3 rounds with PBS at pH 7.4.7. Store the dialyzed protein at -80°C until use.E) Verification of expression of recombinant IGF-1 protein by Western blottingExpression of the recombinant fish IGF-1 protein in the recombinant yeast cells were verified as follows:1. Mix the recombinant fish IGF-1 protein sample with 2X Tris-Glycine SDS sample buffer supplemented with a reducing agent. Denature the protein sample at 95°C for 5-10 min.2. Load the protein sample and the prestained protein ladder onto a Tris-Glycine polyacrylamide gel.3. Run the polyacrylamide gel in 1X Tris-Glycine SDS Running Buffer at 120V for 75 min.4. Transfer the protein overnight onto a 0.22 urn PVDF membrane.5. Block the PVDF membrane in 5% milk in 1X TBST for 30 min.6. Incubate the PVDF membrane with mouse anti-6X His tag monoclonal antibody (1 :2000 diluted in TBST) for 1 hour at room temperature with constant shaking.7. Wash the membrane for 5 min for three times.8. Incubate the PVDF membrane with goat anti-Mouse IgG, IgM (H+L) Secondary Antibody, HRP (1 :2000 diluted in TBST) for 1 hour at room temperature with constant shaking.9. Wash the membrane for 5 min for three times.10. Mix the ECL components in a 1 : 1 ratio. Incubate the PVDF membrane with the ECL mixture for 1 min.11. Remove excess ECL mixture from the PVDF membrane. Capture the signal using an Azure C300 Imaging System.F) Results:

[0091] The expression of grouper IGF-1 in yeast culture was confirmed by Western blotting. To confirm the expression of recombinant IGF-1 proteins, 20 uL of yeast culture medium was harvested and analyzed by Western blotting. As seen in FIG. 6, expression of recombinant IGF-1 proteins at ~10 kDa in the present recombinant yeast strain was detected and confirmed by Western blotting using anti-6X His antibody. A band between 35 kDa and 55 kDa was likely to be the IGF-1 multimer as it could be detected with anti-6X His antibody, suggesting the presence of His-tag in the protein. No IGF-1 could be detected in the BMMY only (medium control) and the yeast culture only (uninduced control).

[0092] Purified grouper IGF-1 was characterized by total protein staining and Western blotting. To characterize native and modified grouper IGF-1 after immobilized metal affinity chromatography (IMAC), total protein staining and Western blotting were performed. As seen in FIG. 7, expression of recombinant IGF-1 proteins in the present recombinant yeast strain was detected and confirmed at ~10 kDa in total protein staining (panel a) and Western blotting using anti-6X His antibody (panel b). There were two bandsbetween 35 kDa and 70 kDa for all purified IGF-1 proteins in the total protein staining (panel a), which were likely to be the yeast protein as they could not be detected with the anti-6X His antibody, suggesting the absence of His-tag in the proteins. A band between 35 kDa and 55 kDa was detected in the Western blotting (panel b), which was likely to be the IGF-1 multimer as it could only be detected with anti-6X His antibody.

[0093] Example 6 - Cell growth stimulation

[0094] A) MCF-7 human epithelial cell lineThe MCF-7 human epithelial cell line was obtained from ATCC and treated with the recombinant fish IGF-1 obtained from the recombinant yeast cells described in the preceding examples following the below steps:1. Cells are cultured in DMEM / F12 complete medium [DMEM / F12, 10% FBS] inside a humidified incubator (34°C; 5% CO2; 95% air). Split cells at a ratio of 1 : 4 to 1 :8 for routine maintenance.2. Upon reaching about 80% confluence, detach cells by trypsin. To study the effect of growth factors on cell growth, seed cells at a density of 10,000 cells / well onto a 96-well plate with DMEM / F12 complete medium. Return the cells to the incubator.3. After 24 hours, prepare growth factors (e.g. IGF-1 ) at 2x working concentrations in serum-free medium [DMEM / F12, 0.1 % human serum albumin]. Add the 2x growth factors into the wells such that cells will be treated by 1x growth factors (e.g. add 50 uL 2x growth factor to well containing 50 uL serum-free medium). Return the cells to the incubator.4. Observe the cells daily under a microscope for signs of cell growth. When there are obvious differences in terms of cell confluence between the treatment groups (usually detected between day 2 - day 10), quantify cell growth either by resazurin assay (as described in section C in this example) or any other cell proliferation / death assays.

[0095] B) FHM fish epithelial cell lineThe FHM fish epithelial cell line was obtained from ATCC and treated following the below steps:1. Upon cell line arrival, culture cells in DMEM / F12 complete medium inside a humidified incubator (34°C; 5% CO2; 95% air) for passages 1 -3 (P1 -P3). Split cells at a ratio of 1 :4 to 1 :8. From the 4th passage onwards, sequentially adapt FHM cells to reduced serum conditions and finally to serum-free medium. Culture cells in our in-house serum-free medium formulation inside a humidified incubator (34°C; 5% CO2; 95% air). Split cells at a ratio of 1 :4 to 1 :8 for routine maintenance.2. Upon reaching about 80% confluence, detach cells by trypsin. To study the effect of growth factors on cell growth, seed cells at a density of 10,000 cells / well onto a 96-well plate with serum-free medium. Return the cells to the incubator.3. After 24h, prepare growth factors (e.g. IGF-1 ) at 2x working concentrations in treatment medium [DMEM / F12, 0.1 % human serum albumin, 32 uM ethanolamine]. Add the 2x growth factors into the wells such that cells will be treated by 1x growth factors (e.g. add 50 ul 2x growth factor to well containing 50 ul serum-free medium). Return the cells to the incubator.4. Observe the cells daily under a microscope for signs of cell growth. When there are obvious differences in terms of cell confluence between the treatment groups (usually detected between day 2 - day 10), quantify cell growth either by resazurin assay (as described in section C in this example) or any other cell proliferation / death assays.

[0096] C) Measuring cell growth by resazurin assayThe resazurin assay is commonly used to quantify the number of live cells in a sample and to measure the metabolic capacity of live cells.1 . Remove the treatment medium as much as possible by a multichannel pipette. Avoid scratching the well bottom with the pipette tip.2. Mix the resazurin solution in 1 :20 dilution with a culture medium corresponding to each cell line.3. Add 100 uL diluted resazurin solution to each well of the 96-well plate by a multichannel pipette. Incubate the plate in the humidified incubator (34°C; 5% CO2; 95% air).4. When the color of the resazurin solution in the plate (from step 4) becomes pink (usually take 3 - 6 hours), transfer 75 uL of the contents from each well of the 96-well plate to the corresponding well of a black 96-well plate. Add 75 uL diluted resazurin solution (from step 3) to empty wells (blank).5. Measure the sample fluorescence using a fluorescence microplate reader (e.g. Molecular Devices SpectraMax iD5). Set the excitation and emission wavelengths at 550 nm and 590 nm respectively.6. Average the blank well fluorescence readings. Subtract this average reading from all sample fluorescence readings to correct for background fluorescence.7. Calculate the mean corrected fluorescence for the control group. Express the treatment group fluorescence readings as a percentage relative to control by dividing the sample readings (i.e. IGF-1 groups) by the mean control reading.

[0097] D) Results

[0098] Purified grouper IGF-I stimulated cell growth in both mammalian and non-mammalian cells. The stimulatory effect of grouper IGF-1 on cell growth was measured by the resazurin assay. Purified native and modified grouper IGF-1 were treated to MCF7 cells (human) and FHM cells (fish) at 0 nM, 0.1 nM, 1 nM and 10 nM respectively. As seen in FIG. 8, grouper IGF-1 stimulated cell growth in both cell types, indicating their ability to be used after purification. Both cell types also showed different responsiveness to the modified grouper IGF-1.

[0099] Grouper IGF-1 containing yeast culture could be used directly to stimulate cell growth in both mammalian and non-mammalian cells. To examine whether grouper IGF-1 yeast culture could be used directly to stimulate cell growth, a resazurin assay was performed. Grouper IGF-1 yeast cultures after centrifugation and filtering were treated to MCF7 cells (human) and FHM cells (fish) at 1 : 100 and 1 : 1000 dilutions respectively. As seen in FIG. 9, grouper IGF-1 yeast cultures stimulated cell growth in both cell types, while the BMMY only (medium control) and yeast culture only (uninduced control) did not have significant effects on cell growth. These results indicated that the growth stimulatory effect was due to the presence of grouper IGF-1 in the yeast culture but not other components and yeast proteins in the culture medium. Thisresult showed a revolutionary way of using grouper IGF-1 yeast culture with minimal purification steps for cell growth stimulation.

[0100] Recombinant Growth Factors or Proteins

[0101] In various embodiments, recombinant growth factors or proteins are expressed and used for supporting the growth of in vitro cell culture. As provided in the description herein, embodiments of genetically engineered yeast cells may be used to express recombinant growth factors of various origin and the resulting growth factors may be used to supplement in vitro culture for promoting the growth of cells of identical or similar species or genus.

[0102] In some embodiments, the growth factor is IGF-1 such as IGF-1 having the sequence depicted in Table 2 below.

[0103] In some embodiments, the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant IGF-1 selected from the group consisting of SEQ ID NOs. 11 -55. In some embodiments, the resulting recombinant IGF-1 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.

[0104] Additionally, the invention also encompasses polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 56-100. In one embodiment, polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 56-100. In another embodiment, the polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 56-100.Table 2 - Examples of IGF-1 Gene and Protein Sequences

[0105] In some embodiments, the growth factor is fibroblast growth factor 2 (FGF-2) such as FGF2 having the sequence depicted in Table 3 below.

[0106] In some embodiments, the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant FGF-2 selected from the group consisting of SEQ ID NOs. 101 -145. In some embodiments, the resulting recombinant FGF-2 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.

[0107] Additionally, the invention also encompasses polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 146-190. In one embodiment, polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 146-190. In another embodiment, the polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 146- 190.Table 3 - Examples of FGF2 Gene and Protein Sequences

[0108] In some embodiments, the growth factor is epidermal growth factor (EGF) such as EGF having the sequence depicted in Table 4 below.

[0109] In some embodiments, the invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant EGF selected from the group consisting of SEQ ID NOs. 191 -219. In some embodiments, the resulting recombinant FGF2 may be introduced to an in vitro cell culture for increasing the rate of growth or protein expression of the cells in the in vitro cell culture.

[0110] Additionally, the invention also encompasses polypeptide fragments that are substantially similar in sequence to those selected from the group consisting of SEQ ID NOs: 220-248 In one embodiment, polypeptide fragment may have at least 80% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 220-248. In another embodiment, the polypeptide fragment may have at least 90% sequence identity to a polypeptide fragment selected from the group consisting of SEQ ID NOs: 220- 248.

[0111] Table 4 - Examples of EGF Gene and Protein Sequences

[0112] The above description is illustrative and is not restrictive. Many variations of embodiments may become apparent to those skilled in the art upon review of the disclosure. The scope embodiments should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the pending claims along with their full scope or equivalents.

[0113] One or more features from any embodiment may be combined with one or more features of any other embodiment without departing from the scope embodiments. A recitation of "a", "an" or "the" is intended to mean "one or more" unless specifically indicated to the contrary. Recitation of "and / or" is intended to represent the most inclusive sense of the term unless specifically indicated to the contrary.

[0114] While the present disclosure may be embodied in many different forms, the drawings and discussion are presented with the understanding that the present disclosure is an exemplification of the principles of one or more inventions and is not intended to limit any one embodiment to the embodiments illustrated.

[0115] The disclosure, in its broader aspects, is therefore not limited to the specific details, representative system and methods, and illustrative examples shown and described above. Various modifications and variations may be made to the above specification without departing from the scope or spirit of

Claims

the present disclosure, and it is intended that the present disclosure covers all such modifications and variations provided they come within the scope of the following claims and their equivalents.CLAIMSWhat is claimed is:1 . A recombinant growth factor having an amino acid sequence that are identical or substantially similar in sequence to the sequences selected from the group consisting of SEQ ID NOs: 6-10, 56-100, 146-190 and 220-248.

2. The recombinant growth factor of claim 1 , wherein the recombinant growth factor is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 -5, 11 -55, 101 -145 and 191 -219.

3. A genetically modified yeast cell comprising a nucleic acid encoding a recombinant growth factor 1 , wherein the nucleic acid has a sequence selected from the group consisting of SEQ ID NOs. 1 -5, 11 -55, 101 -145 and 191 -219.

4. The genetically modified yeast cell of claim 3, wherein the recombinant growth factor is a insulin growth factor 1 (IGF-1 ), a fibroblast growth factor 2 (FGF-2) or a epidermal growth factor (EGF).

5. The genetically modified yeast cell of claim 3, wherein the amino acid sequence of the recombinant growth factor is optimized for improved stability or bioactivity as compared to the native growth factor.

6. The genetically modified yeast cell of claim 3, wherein the yeast cell is Pichia pastoris.

7. A method for increasing the rate of growth or protein expression of a plurality of cells in an in vitro cell culture, comprising: a) growing the plurality of cells in a nutrient medium under growth conditions suitable for the initiation of cell division; and b) introducing at least one recombinant growth factor to the nutrient medium, wherein the amino acid sequence of the recombinant growth factor is (a) optimized for increasing the rate of growth or protein expression of the plurality of cells or (b) (i) of genetically the same or similar species to the plurality of cells and / or (ii) of genetically the same genus to the plurality of cells.

8. The method of claim 7, wherein the recombinant growth factor is selected from the group consisting of insulin growth factor 1 (IGF-1 ), insulin, interleukin 6 (IL- 6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11 ), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF) and transferrin.

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