Lysates of engineered yeast as cheap and sustainable alternative to fetal bovine serum in cultivated meat production

Abstract

The present invention relates to lysates of Yarrowia cells, which recombinantly produce bovine growth-associated factors. Also provided is a fetal bovine serum (FBS) replacement composition comprising said lysates and methods for producing such.

Description

[0001] P7124PC00

[0002] Lysates of Engineered Yeast as Cheap and Sustainable Alternative to Fetal Bovine Serum in Cultivated Meat Production.

[0003] Technical field

[0004] The present invention relates to lysates of Yarrowia cells, which recombinantly produce bovine growth-associated factors. Also provided is a fetal bovine serum (FBS) replacement composition comprising said lysates and methods for producing such.

[0005] Background

[0006] Cellular agriculture (Cell Ag) is rapidly emerging as a more environmentally sustainable alternative to traditional farming. In the context of animal-sourced dietary protein, Cell Ag circumvents the need to grow a whole animal to obtain animal-derived food products.

[0007] Various LCA reports have indicated that with its better substrate-to-meat conversion efficiency, meat production by Cell Ag (cultivated meat) promises a more efficient land use over conventional meat (Sinke et al., 2023; Tuomisto et al., 2022). Furthermore, cultivated meat production takes place in sealed vats under a carefully controlled environment. This offers better control of effluent and by-products, thereby resulting in less environmental pollution.

[0008] While cultivated meat offers better environmental and animal welfare, reliance on fetal bovine serum (FBS) represents a major drawback. FBS use in large-scale industrial cell-cultured meat production is cost-prohibitive and results in a counterproductive vicious cycle of continuous dependency on animals to produce cell-cultured meat. Ethical concerns and the risk of zoonosis also plague the use of FBS.

[0009] Summary

[0010] Alternatives to FBS are chemically defined media formulations such as TESR, B8, Beefy-9, Beefy-R, Tribasal 2.0, etc., consisting of purified recombinant growth factors such as FGF2, IGF, PDGF, TGF-beta, etc. While these media formulations represent promising alternatives, the need to recombinantly express and extensively purify growth factors makes them expensive alternatives, with the cost of purification accounting for up to 40-60% of the cost of growth factors. The current growth factor market largely supplies high-value biologics-grade growth factors that are subjected to medical-grade purification - aP7124PC00

[0011] stark contrast to the food industry where cheap substrates are crucial for competitiveness.

[0012] Here we report the use of lysates of recombinant food-safe yeast, Yarrowia lipolytica, producing the growth factors and accessory proteins FGF2-G3, TGF-beta3, serotransferrin, serum albumin, and fetuin-A as a cheap and scalable semi-defined FBS substitute for cultivated meat production. The use of recombinant lysates eliminates the need for post-expression purification of recombinant growth factors and accessory proteins and will potentially drive down the cost of culture media for cultivated meat production. Since Yeast extracts and yeast hydrolysates have been reported to have growth-promoting effects on CHO cells (Mosser et al., 2013; Skrivergaard et al., 2023) at low concentrations, we hypothesize that engineering the yeast to produce growth factors found in culture media could result in a synergistic effect that drives the pro-proliferative effect of yeast lysates on mammalian cells and fish cells to a desired level.

[0013] Yarrowia lipolytica is a particularly attractive host for this purpose because it offers advantages such as: (1) Ubiquity in food such as cheeses, yogurt, and meat (Sorensen et al., 2023); (2) Generally regarded as Safe (GRAS) status; (3) Its extensive use in the production of food ingredients and additives such as citrate, erythritol, and b-carotene, etc., and (4) rich metabolic pathway allowing for cheap diverse substrate and feedstock utilization (Sestric et al., 2014; Worland et al., 2020); (5) Post-translational modifications closer to mammalian cell factories - up to 40% similarity with animal proteome (Swennen & Beckerich, 2007).

[0014] To obtain a proof of concept, we conducted a one-factor-at-a-time growth assay in which a growth factor or accessory protein in the Beefy-9 media composition is replaced by lysates of Yarrowia lipolytica expressing that growth factor or accessory protein. Furthermore, we combine the use of layered strain engineering, and foundrylevel multi-component combinatorial screening to obtain a high-performing serum-free media for cultivated meat solely based on lysates of engineered Y. lipolytica.

[0015] It is a main aspect of the present disclosure to provide a Yarrowia cell capable of producing one or more bovine growth-associated factors. In some embodiments, said one or more bovine growth-associated factors are fibroblast growth factor 2 (FGF2), transforming growth factor-beta (TGF-beta), transferrin, albumin, and / or fetuin.P7124PC00

[0016] It is a further main aspect to provide a Yarrowia lysate, wherein said lysate comprises one or more bovine growth-associated factors. Such factors may be, for example, FGF2, TGF-beta, transferrin, albumin, and / or fetuin. The Yarrowia lysate is preferably derived from the Yarrowia cell capable of producing one or more bovine growth-associated factors as described herein.

[0017] It is another main aspect to provide a fetal bovine serum (FBS)-replacement composition comprising a Yarrowia lysate, wherein said composition comprises one or more bovine growth-associated factors. Preferably, wherein said bovine growth-associated factors are, for example, FGF2, TGF-beta, transferrin, and / or albumin.

[0018] It is also an aspect of the present disclosure to provide a mammalian cell or fish cell culture medium composition, wherein said mammalian cell or fish cell culture medium composition comprises the FBS-replacement composition. Thus, said mammalian cell or fish cell culture medium composition comprises the Yarrowia lysate, which comprises one or more bovine growth-associated factor, such as one or more of FGF2, TGF-beta, transferrin, and / or albumin.

[0019] Provided herein is also a method of obtaining one or more bovine growth-associated factors, comprising:

[0020] i. providing a Yarrowia cell capable of producing one or more bovine growth- associated factors;

[0021] ii. incubating and optionally propagating said Yarrowia cell in a medium, thereby producing one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin albumin and / or fetuin; and

[0022] iii. optionally, recovering said one or more bovine growth-associated factors, whereby said one or more bovine growth-associated factors are obtained.

[0023] It is also an aspect to provide a method of producing a Yarrowia lysate, comprising: i. providing a Yarrowia cell capable of producing one or more bovine growth- associated factors;

[0024] ii. propagating said Yarrowia cell in a medium,

[0025] iii. thereby obtaining a Yarrowia cell biomass; and

[0026] iv. lysing said Yarrowia cell biomass to produce said Yarrowia lysate, whereby said Yarrowia lysate is produced.P7124PC00

[0027] Preferably, said Yarrowia lysate is as described herein, and comprises one or more bovine growth-associated factors, for example, one or more of FGF2, TGF-beta, transferrin albumin and / or fetuin.

[0028] It is also an aspect of the present disclosure to provide a method for producing an FBS-replacement composition comprising a Yarrowia lysate and one or more bovine growth-associated factors, comprising:

[0029] i. providing a Yarrowia cell capable of producing one or more bovine growth- associated factors;

[0030] ii. propagating said Yarrowia cell in a medium,

[0031] thereby obtaining Yarrowia cell biomass;

[0032] iii. recovering and / or preparing a lysate of said Yarrowia cell biomass,

[0033] thereby obtaining a Yarrowia lysate; and

[0034] iv. optionally, combining one or more Yarrowia lysates obtained in step iii.;

[0035] v. formulating the one or more Yarrowia lysates of step iii. and / or iv. into an FBS- replacement composition;

[0036] whereby said FBS-replacement composition is produced.

[0037] Also provided herein is a method for cultivation of mammalian cells using said FBS-replacement composition, as described herein, comprising:

[0038] i. providing a FBS-replacement composition as described herein;

[0039] ii. mixing said FBS-replacement composition with a culture medium suitable for cultivation of said mammalian cells, thereby obtaining a mixture; and

[0040] iii. using said mixture to cultivate mammalian cells.

[0041] Also provided herein is a method for producing a food composition, comprising:

[0042] i. providing a Yarrowia lysate;

[0043] ii. formulating said Yarrowia lysate into an FBS-replacement composition;

[0044] iii. cultivating mammalian cells in the presence of said FBS-replacement composition, optionally further comprising a culture medium,

[0045] thereby obtaining a biomass of mammalian cells; and

[0046] iv. formulating said biomass of mammalian cells into a food composition, thereby producing said food composition.

[0047] The present disclosure also provides for the use of the Yarrowia lysate as describedP7124PC00

[0048] herein, and / or the FBS-replacement composition as described herein for cultivating mammalian cells or fish cells, preferably in combination with a culture medium.

[0049] Also provided is the use of mammalian cells or fish cells obtainable by a method described herein, wherein said mammalian cells or fish cells are used as a food product, food ingredient, and / or food material.

[0050] Provided herein is also a food composition comprising mammalian cells or fish cells obtainable by a method described herein. Also provided herein is the use of said food composition as a food product, food material, and / or food ingredient. In some embodiments, said food composition is used as a cultivated meat and / or is used in a method for producing a meat alternative. Provided herein is also a cultivated meat and / or ingredient therefor comprising mammalian cells obtainable by a method as described herein. The mammalian cells of said food composition and / or cultivated meat may have been cultured in a mammalian cell culture medium comprising said Yarrowia lysate or FBS-replacement serum as described herein.

[0051] In preferred embodiments, the mammalian cells are primary bovine cells, such as primary satellite bovine cells.

[0052] Also provided herein is an expression system for expression in a Yarrowia cell, comprising one or more:

[0053] i. nucleic acid encoding a FGF2, optionally wherein the FGF2 is as set forth in SEQ ID NO: 1;

[0054] ii. nucleic acid encoding a TGF-beta, optionally wherein the TGF-beta is as set forth in SEQ ID NO: 2;

[0055] iii. nucleic acid encoding a transferrin, optionally wherein the transferrin is as set forth in SEQ ID NO: 3;

[0056] iv. nucleic acid encoding an albumin, optionally wherein the albumin is as set forth in SEQ ID NO: 4;

[0057] v. nucleic acid encoding a FGF2, optionally wherein the FGF2 is as set forth in SEQ ID NO: 5,

[0058] or functional variants thereof having at least 70% sequence identity thereto.

[0059] In another aspect, also provided herein is a kit of parts, comprising:P7124PC00

[0060] i. a Yarrowia cell;

[0061] ii. at least one nucleic acid as described herein; and / or

[0062] iii. the expression system as described herein;

[0063] iv. and optionally instructions for use.

[0064] Preferably the Yarrowia cell of the kit is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic, and / or wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

[0065] Description of Figures

[0066] Figure 1. The effect of replacing individual growth factors or accessory proteins, FGF2 and albumin in Beefy-9 media, with lysates of Y. lipolytica comprising FGF2-G3 (i.e. FGF2 lysate) and albumin (i.e. albumin lysate) respectively, on the growth of bovine satellite cells was investigated. Hoechst nuclei count (A) and high-contrast bright-field count (B) showed that 0.5% (v / v) of the respective yeast lysates can successfully replace these constituents in Beefy-9 media and support the growth of bovine satellite cells. (C) Overlayed Hoechst and brightfield images showing post-72 hours confluency of cells in (i) 10% FBS, compared to FGF2 dropout media supplemented with (ii) Y. lipolytica lysate, (iii) FGF2 lysates; and albumin dropout media supplemented with (iv) Y. lipolytica lysate and (v) albumin lysate.

[0067] Figure 2. Four of the protein constituents, FGF2 (A), albumin (B), transferrin (C) and TGF-beta (D) of the Beefy-9 were replaced with varying concentrations (between 0, 0.25%, 0.5%, and 1% v / v) of their respective lysates of recombinant yeasts in individual media formulation. Recombinant yeast lysates abolished the need to exogenously add these proteins into the Beefy-9 media formulation for the cultivation of satellite cells (A, B, C). Yeast lysate with TGF-beta3 (i.e. TGF-beta lysate) exhibited a trend different from other proteins (D) as tight concentration control (< 2 ng / mL) is required to maintain anti-differentiation potential over anti-proliferative effect.

[0068] Figure 3. The growth factors (FGF2-G3 and TGF-beta3) and accessory proteins (serotransferrin, serum albumin, and fetuin-A) were tagged with 11 amino acid Nanoluc Hibit tag sequence (SEQ ID NO: 11). Soluble lysates of Y. lipolytica W29 engineered to express these proteins were prepared by bead beating and subjected to protein electrophoresis and Hibit blotting. Hibit Blotting confirmed the expression of theP7124PC00

[0069] proteins of interest at the expected sizes: (A) FGF2-G3 (18kDa) (B) serum albumin (68kDa); (C) serotransferrin (77kDa); (D) TGF-beta3 (55kDa); (E) unglycosylated fetuin (37kDa) and glycosylated fetuin (55kDa).

[0070] Figure 4. Lysates of Y.lipolytica expressing individual growth factors (FGF2, Albumin, and Transferrin) were prepared as described herein. The three individual lysates were combined in all possible combinations of concentration between the range of 0-1% (v / v). Growth profiling of GFP-tagged satellite cells in these media formulations was performed in triplicates and monitored overtime using Incucyte S3. Each dot represents a unique combination of lysates: grey dots represent combinations with reduced cell viability compared to the initial seeding cell number; black dots represent combinations with increased (above the 1 fold threshold) cell viability compared to the initial seeding cell number. The Y-axis is the order of each combination in a 96 well plate (serial number).

[0071] Figure 5. Lysates from a strain co-expressing multiple growth factors and accessory protein offers numerous advantages over combining lysates from different single protein-expressing strains. The effects of increasing concentrations of Yarrowia lipolytica lysates engineered to co-express FGF2-G3, serum albumin, fetuin-A, and serrotransferrin (referred to as 4GF-coEx) were tested in DMEM (A) and DT (DMEM supplemented with 2 ng / mL TGF-beta3) (B). BSC proliferation assay showed that 4GF-coEx promoted cell growth at levels comparable to growth medium (GM, containing 10% FBS and 1 ng / mL FGF2) regardless of TGF-beta supplementation. Statistical significance was assessed using one-way ANOVA with multiple comparisons, comparing the effects of wild-type lysate (WT) and 4GF-coEx to GM. Statistical outcomes are labelled as ‘p<0.0001 (**“)’ for significant differences and ‘ns’ for nonsignificant differences.

[0072] Figure 6. Engineered Y. lipolytica lysates eliminate the requirement for fetal serum in satellite cell culture. (A) Six-day growth progression of satellite cells cultured in basal medium (DM EM Plus) supplemented with increasing concentrations of Y.

[0073] lipolytica 4GF-coEx lysates, compared with state-of-the-art serum-based growth medium (GM). GM: 20% FBS + 1 ng / mL FGF2; DMEMPIus: DMEM:F12 supplemented with 67 ng / mL sodium selenite, 200 pg / mL ascorbate, and 5 pg / mL insulin.

[0074] WT: lysates from non-engineered Y. lipolytica', 4GF-coEx: lysates from Y. lipolyticaP7124PC00

[0075] expressing FGF2-G3, serum albumin, fetuin-A, and serotransferrin. (B) Satellite cell growth in 4GF-coEx-based media compared with GM and Beefy9, quantified by endpoint fluorescent cytometry. (C) qPCR analysis of key myogenic markers (Pax3 and MyoD) in satellite cells cultured in lysate-based media relative to serum-based media.

[0076] (D) Lysate stability under different storage conditions: storage at -20 °C with or without glycerol, or lyophilization by freeze-drying followed by room-temperature storage.

[0077] Figure 7. Media optimization in a fluorescent satellite cell line is transferable to primary satellite cells. (A) Endpoint nuclei count of primary satellite cells cultured in serumcontaining (PGM) and serum-free (Tribasal 2.0+) media, compared with lysate-based media (DMEMPIus supplemented with WT or 4GF-coEx lysates). PGM: DMEM GlutaMAX supplemented with 10% FBS and 10% horse serum; Tribasal 2.0+: DMEM:F12 supplemented with 2 ng / mL FGF2, 600 pg / mL fetuin, 75 pg / mL BSA, and 1* ITS. Lysate-based media consisted of a basal medium (DMEMPIus): DMEM:F12 supplemented with 200 pg / mL 2-phospho-L-ascorbic acid, 67 ng / mL sodium selenite, and 5 pg / mL insulin. WT: lysates from non-engineered Y. lipolytica', 4GF-coEx: lysates from Y. lipolytica expressing FGF2-G3, serum albumin, fetuin-A, and serotransferrin.

[0078] (B)(i-ii) Phalloidin staining illustrating differences in myogenicity of cells cultured in PGM and 4GF-coEx-based media for 8 days. Hoechst (blue) and phalloidin (red) label nuclei and myotubes, respectively.

[0079] Detailed description

[0080] Definitions

[0081] The term growth factor herein refers to a biologically active polypeptides that are capable of stimulating and / or regulating cellular processes related to cell proliferation, differentiation and / or survival of mammalian and / or fish cells in culture. Non-limited examples of growth factors include FGF2 and TGF-beta.

[0082] The term accessory protein herein refers to biologically active polypeptides that contribute positively to the overall health and viability of mammalian and / or fish cells in culture. Accessory proteins may be involved in cellular functions such as nutrient uptake, maintenance of osmotic balance or cell adhesion / attachment, which are not directly related to cell proliferation and / or differentiation. Non-limited examples of accessory proteins include transferrin, albumin and fetuin.P7124PC00

[0083] The term growth-associated factor herein refers to both growth factors and accessory proteins. Thus, non-limited examples of growth-associated factors are FGF2, TGF-beta, transferring, albumin and fetuin.

[0084] The term bovine as used herein in the context of growth-associated factors refers to growth-associated factors from Bos taurus, as well as growth-associated factors derived therefrom.

[0085] The term lysate herein refers to the product obtained from the lysis of cells, wherein the lysis process disrupts the cell membrane and / or cell wall and leads to the release of intracellular components, including at least one growth-associated factor, as well as other biomolecules, such as minerals and fatty acids. Said lysates may be useful for supporting and / or promoting the maintenance, growth, proliferation and / or differentiation of mammalian and / or fish cells in culture.

[0086] The term FBS-replacement composition herein refers to a composition comprising or consisting of lysate, said lysate comprising one or more bovine growth-associated factors. In other words, said lysate comprises at least one bovine growth factor or at least one bovine accessory protein as defined herein. The FBS-replacement composition may be used in combination with FBS or serum in the cultivation of mammalian and / or fish cells, hence the term replacement composition should not be construed as always replacing all FBS in a mammalian and / or fish cell culture, as it may also be useful for as a partial-FBS-replacement composition. The FBS-replacement compositions of the present disclosure may be useful for supporting and / or promoting the maintenance, growth, proliferation and / or differentiation of mammalian and / or fish cells in culture, particularly when used in combination with media appropriate for the culture of said mammalian and / or fish cells. The skilled person knows what media is appropriate for the culture of their cells of interest.

[0087] The term cultivated meat herein refers to meat that is not derived directly from animal slaughter, but rather is derived from cell biomass that has been propagated in a lab. For example, said cultivated meat may be derived animal cell lines that have been incubated and propagated using in vitro cultivation techniques and process. Cultivated meat may also be referred to as lab-grown meat, cultured meat, slaughter-free meat, in vitro meat, cell-based meat or synthetic meat.P7124PC00

[0088] The terms protein, polypeptide, and amino acid sequence, as well as the plural version thereof, may be used interchangeably herein throughout.

[0089] The terms nucleic acid, nucleic acid sequence, nucleic acid construct, nucleic acid molecule oligonucleotide, and polynucleotide, as well as the plural versions thereof, may be used interchangeably herein.

[0090] The term identity with respect to a nucleic acid sequences (polynucleotide, DNA, RNA) or polypeptide sequences (protein), are defined herein as the percentage of nucleotides or amino acids, respectively, in the candidate sequence that are identicalhomologous or similar, respectively, to the residues of a corresponding native (may be codon-optimised) nucleotide or amino acid sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and considering any conservative substitutions according to the NCIIIB rules ([https: / / iubmb.qmul.ac.uk / misc / naseq.html; NC-llIB, Eur J Biochem 10 P7131 EP00 (1985)]) as part of the sequence identity. The percentage similarity of two or more nucleic acids, or two more polypeptide sequences can also be calculated. The percentage of similarity refers to the percentage of residues conserved with similar physiochemical properties. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity or similarity. Methods and computer programs for the alignments are well known in the art. Generally, a given identity between two sequences implies that the similarity between these sequences is at least equal to the identity; for example, if two sequences are 70% identical to one another, they cannot be less than 70% similar to one another - but could share 80% similarity. Thus, throughout the present disclosure, it will be understood that any variant, such as a functional variant, or homologue said to have at least 70% sequence identity to a specified sequence (nucleic acid sequence (polynucleotide) or polypeptide) refers to a sequence having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, suchP7124PC00

[0091] as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity or similarity thereto. The term homology may be used interchangeably with identity in this context.

[0092] The term functional variant herein refers to a polypeptide with at least 70% sequence identity to the polypeptide of interest, which retains at least some of the activity of the protein of interest. The term functional variant herein may also refers to a nucleic acid with at least 70% sequence identity to the nucleic acid of interest, which retains at least some of the activity of the nucleic acid of interest. In the context of the present disclosure, a functional variant of FGF2 is any polypeptide having at least at least 70% sequence identity to FGF2, wherein said polypeptide is capable of supporting mammalian and / or fish cell cultivation and / or propagation in place of FGF2 and wherein said polypeptide may be either more, less or equivalently as potent as FGF2. This definition of functional variant applies to all of the growth-associated factors disclosed herein, including TGF-beta, transferrin, albumin, and fetuin. The functions of each of growth-associated factors disclosed herein are described elsewhere herein, for example in the section “Yarrowia cells”.

[0093] The term homologue herein refers to a variant of a nucleic acid encoding the same polypeptide. The skilled person knows that different codons may encode the same amino acid, and it therefore follows that two different nucleic acid sequences may encode the same polypeptide. The skilled person knows how to derive one or more distinct DNA sequences from an amino acid sequence. The skilled person also knows how to derive an amino acid sequence from a DNA sequence. Software for performing reverse translations from an amino acid sequence to DNA sequences and translations from DNA sequences to amino acid sequences are available for performing this task.

[0094] The term heterologous, when referring to a polypeptide or to a polynucleotide (such as a gene, coding sequence of a gene or genetic element), shall herein be construed to refer to an amino acid sequence (polypeptide) or a nucleic acid sequence (polynucleotide), such as a gene, which is not naturally present in a wild-type cell.

[0095] Yarrowia cells

[0096] This disclosure relates to Yarrowia cells that are capable of producing bovine growth-associated factors. The Yarrowia cells of the present disclosure are modified YarrowiaP7124PC00

[0097] cells, which have been modified to make them capable of producing bovine growth-associated factors, such as growth factors and accessory proteins. This disclosure further relates to cell lysates, fetal bovine serum (FBS)-replacement compositions, methods and uses for all of the aforementioned, as well as methods of culturing mammalian and / or fish cells and their use as and in food compositions.

[0098] The Yarrowia cells disclosed herein producing or capable of producing bovine growth-associated factors might be referred to as production organisms, microbial factories, microbial production organisms, hosts, host cells, host organisms, production hosts, cell factories, and the like.

[0099] The growth-associated factors as described herein, may be referred to as recombinant growth-associated factors. Said growth-associated factors are recombinantly produced because the factors are heterologous to the Yarrowia cells that produce them. That is to say, the growth-associated factors produced by the Yarrowia cells of the present disclosure, are not naturally produced by said Yarrowia cells, rather the Yarrowia cells have be modified via the introduction of heterologous DNA comprising or consisting of one or more nucleic acids that encode said growth-associated factors. Thus, said bovine growth-associated factors can be said to be recombinant growth-associated factors and / or recombinantly produced bovine growth-associated factors.

[0100] The Yarrowia cells described herein may comprise additional modifications beyond comprising said heterologous nucleic acid encoding one or more growth-associated factors. Said additional modifications may include modifications such as deletion or inactivation of Yarrowia genes encoding proteases and the like, which may aid in increasing the resultant yields of growth-associated factors present in Yarrowia lysates produced from the lysis of Yarrowia cells comprising such modifications.

[0101] Provided herein is a Yarrowia cell capable of producing one or more bovine growth-associated factors. For example, in some embodiments, the cell is capable of producing or produces two, three, four, five or more bovine growth-associated factors. Preferably, the growth-associated factors are preferentially support bovine cell development, growth, proliferation and / or differentiation. Said growth-associated factors may comprise both growth factor and accessory proteins, wherein accessory proteins are non-growth factor proteins that may be useful in supporting theP7124PC00

[0102] development, growth, proliferation and / or differentiation of mammalian cells and / or fish cells in the context of in vitro cultivation.

[0103] In some embodiments of the present disclosure the one or more bovine growth-associated factors are fibroblast growth factor 2 (FGF2), transforming growth factorbeta (TGF-beta), transferrin, albumin, and / or fetuin. In some embodiments the bovine growth-associated factor is FGF2. In some embodiments the bovine growth-associated factor is transforming growth factor-beta (TGF-beta). In some embodiments the bovine growth-associated factor is transferrin. In some embodiments the bovine growth-associated factor is albumin. In some embodiments the bovine growth-associated factor is fetuin.

[0104] In some embodiments, the FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto. In some embodiments, the FGF2-G3 comprises or consists of SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0105] In some embodiments, the FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 1. In some embodiments, the FGF2-G3 comprises or consists of SEQ ID NO: 1 , or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 1.P7124PC00

[0106] Fibroblast growth factor 2 (FGF2 or FGF-2) might also be referred to as basic fibroblast growth factor (bFGF), FGF-p, Heparin-binding growth factor-2, or Endothelial cell growth factor-2 and these terms may be used interchangeably herein; when used herein these terms refer to bovine FGF2, in order words FGF2 from Bos taurus or derived therefrom, unless indicated otherwise. FGF2 is any tagged, conjugated, glycosylated, phosphorylated, acylated, methylated, acetylated, nitrosylated, ubiquitinated, SUMOylated etc. FGF2 protein. FGF2-G3 is a sub-type of FGF2.

[0107] The function of FGF2 is dependent on the combination of factors present in the cell culture media. In the context of in vitro cell culture, FGF2 plays a role in promoting cell survival, proliferation and differentiation. FGF2 is the primary growth factor that stimulates proliferation. FGF2 promotes cell survival by acting as an inhibitor of apoptosis, thus increasing cell viability and survival by reducing the number and frequency of cell death events. FGF2 acts as a mitogen, promoting cell division and thus proliferation in cell cultures. The FGF2 plays on cell differentiation differs based on the combination of other factors present in the media and the cell line that is being cultured; for example, FGF2 may inhibit spontaneous differentiation of embryonic stem cells, while contributing to the differentiation and / or maturation of other partially differentiated cell types.

[0108] In some embodiments, the TGF-beta comprises or consists of TGF-beta3 as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto. In some embodiments, the TGF-beta3 comprises or consists of SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0109] In some embodiments, the TGF-beta comprises or consists of TGF-beta3 as set forth in SEQ ID NO: 2, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptideP7124PC00

[0110] as set forth in SEQ ID NO: 2. In some embodiments, the TGF-beta3 comprises or consists of SEQ ID NO: 2, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 2.

[0111] Transforming growth factor-beta (TGF-beta) might also be referred to as TGF-p or TGFB and these terms may be used interchangeably herein; when used herein these terms refer to bovine TGF-beta, in order words TGF-beta from Bos taurus or derived therefrom, unless indicated otherwise. TGF-beta is any tagged, conjugated, glycosylated, phosphorylated, acylated, methylated, acetylated, nitrosylated, ubiquitinated, SUMOylated etc. TGF-beta protein. TGF-beta3 is a sub-type of TGF-beta.

[0112] The function of TGF-beta is dependent on its concentration, the cell type being cultured and the presence of additional factors in the culture environment. In the context of in vitro cell culture, TGF-beta plays a role in influencing cell proliferation, differentiation and survival. TGF-beta may induce or inhibit cell differentiation depending on the cell type being cultured and other factors present in the culture environment. TGF-beta plays a role in the regulation of apoptosis, thus affecting cell survival, while also having both proliferative properties and antiproliferative properties depending on the culture environment. Thus, careful regulation of TGF-beta concentrations is required in order to maintain optimal conditions for cell maintenance, growth, proliferation, survival and differentiation in the context of in vitro cultivations.

[0113] In some embodiments, the transferrin comprises or consists of serotransferrin as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto. In some embodiments, the serotransferrin comprises or consists of SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.P7124PC00

[0114] In some embodiments, the transferrin comprises or consists of serotransferrin as set forth in SEQ ID NO: 3, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 3. In some embodiments, the serotransferrin comprises or consists of SEQ ID NO: 3, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 3.

[0115] Transferrin might also be referred to TF and these terms may be used interchangeably herein; when used herein these terms refer to bovine transferrin, in order words transferrin from Bos taurus or derived therefrom, unless indicated otherwise.

[0116] Transferrin is any tagged, conjugated, glycosylated, phosphorylated, acylated, methylated, acetylated, nitrosylated, ubiquitinated, SUMOylated etc. transferrin protein. Serotransferrin is a subtype of transferrin.

[0117] Transferrin plays a role regulating cellular iron uptake, transport and utilisation. Iron availability is important for a range of cellular processes including cellular respiration and certain enzyme activity, making it important for the general well-being of cultured cells.

[0118] In some embodiments, the albumin comprises or consists of serum albumin as set forth in SEQ ID NO: 4, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto. In some embodiments, the serum albumin comprises or consists of SEQ ID NO: 4, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.P7124PC00

[0119] In some embodiments, the albumin comprises or consists of serum albumin as set forth in SEQ ID NO: 4, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 4. In some embodiments, the serum albumin comprises or consists of SEQ ID NO: 4, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 4.

[0120] Albumin as used herein refers to bovine albumin, in other words albumin from Bos taurus or derived therefrom, unless indicated otherwise. Albumin is any tagged, conjugated, glycosylated, phosphorylated, acylated, methylated, acetylated, nitrosylated, ubiquitinated, SUMOylated etc. albumin protein. Serum albumin is a subtype of albumin.

[0121] Albumin provides a source of nitrogen and essential amino acids to the cell, in the context of cell culture. It also functions as a carrier protein for many biomolecules, ions and vitamins etc., thus contributing to the regulation of nutrient availability in the media and helping to maintain osmotic balance in the cell culture environment.

[0122] In some embodiments, the fetuin comprises or consists of fetuin-A as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto. In some embodiments, the fetuin-A comprises or consists of SEQ ID NO: 5, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.P7124PC00

[0123] In some embodiments, the fetuin comprises or consists of fetuin-A as set forth in SEQ ID NO: 5, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 5. In some embodiments, the fetuin-A comprises or consists of SEQ ID NO: 5, or a functional variant thereof having a three dimensional structure with a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a polypeptide as set forth in SEQ ID NO: 5.

[0124] Fetuin might also be referred to Alpha-2-HS-glycoprotein or Asialofetuin and these terms may be used interchangeably herein; when used herein these terms refer to bovine fetuin, in order words fetuin from Bos taurus or derived therefrom, unless indicated otherwise. Fetuin is any tagged, conjugated, glycosylated, phosphorylated, acylated, methylated, acetylated, nitrosylated, ubiquitinated, SUMOylated etc. fetuin protein. Fetuin-A is a subtype of fetuin.

[0125] Fetuin is a glycoprotein that plays a role in cell attachment, facilitating cellular adhesion to culture surfaces. Thus, it is particularly useful in the context of in vitro cell culture, wherein the cells are grown attached to a surface. Fetuin is also able to bind molecules such as lipids, as well as binding to calcium and phosphate, thus also contributing to the general maintenance and stability of cell culture environment.

[0126] A Yarrowia cell may be modified in such a manner that it produces or is capable of producing one or more bovine growth-associated factors. Yarrowia cells that produce only one bovine growth associated factor may be useful for producing FBS-replacement compositions; lysates generated from populations of Yarrowia cells that each express only one bovine growth-associated factor may be combined to produce an FBS-replacement composition, wherein the ratio of each of the bovine growth-associated factors in the composition, may be adjusted as required by altering ratio of the corresponding Yarrowia lysate. Furthermore, this approach also allows FBS-P7124PC00

[0127] replacement compositions comprising different combinations of bovine growth-associated factors to be obtained using the same Yarrowia cell populations. Thus, in some embodiments one bovine growth associated factor is expressed by a Yarrowia cell.

[0128] Conversely, co-expression of multiple bovine growth-associated factors within the same Yarrowia cell may reduce the processing required to produce said FBS-replacement composition, by reducing the number of lysates required to produce an FBS-replacement composition comprising two or more bovine growth-associated factors. Thus, in some embodiments, one or more of said bovine growth-associated factors are co-expressed within the same Yarrowia cell.

[0129] In some embodiments, the Yarrowia cell comprises one or more of:

[0130] i. a nucleic acid encoding FGF2, such as a nucleic acid encoding a protein comprising or consisting of FGF2-G3 (SEQ ID NO: 1);

[0131] ii. a nucleic acid encoding TGF-beta, such as a nucleic acid encoding a protein comprising or consisting of a TGF-beta3 (SEQ ID NO: 2);

[0132] iii. a nucleic acid encoding transferrin, such as a nucleic acid encoding a protein comprising or consisting of serotransferrin (SEQ ID NO: 3);

[0133] iv. a nucleic acid encoding albumin, such as a nucleic acid encoding a protein comprising or consisting of serum albumin (SEQ ID NO: 4); and / or

[0134] v. a nucleic acid encoding fetuin, such as a nucleic acid encoding a protein comprising or consisting of fetuin-A (SEQ ID NO: 5);

[0135] or functional variants thereof having at least 70% sequence identity thereto.

[0136] In some embodiments, the Yarrowia cell comprises a nucleic acid encoding a protein comprising or consisting of FGF2-G3 (SEQ ID NO: 1), or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the Yarrowia cell comprises a nucleic acid encoding a protein comprising or consisting of a TGF-beta3 (SEQ ID NO: 2), or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the Yarrowia cell comprises a nucleic acid encoding a protein comprising or consisting of serotransferrin (SEQ ID NO: 3), or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the Yarrowia cell comprises a nucleic acid encoding a protein comprising or consisting of serum albumin (SEQ ID NO: 4), or functional variants thereof having at least 70%P7124PC00

[0137] sequence identity thereto. In some embodiments, the Yarrowia cell comprises a nucleic acid encoding a protein comprising or consisting of fetuin-A (SEQ ID NO: 5), or functional variants thereof having at least 70% sequence identity thereto.

[0138] In some embodiments, the Yarrowia cell expresses one or more of:

[0139] i. FGF2, optionally comprising or consisting of FGF2-G3 (SEQ ID NO: 1);

[0140] ii. TGF-beta, optionally comprising or consisting of TGF-beta3 (SEQ ID NO: 2); iii. transferrin, optionally comprising or consisting of serotransferrin (SEQ ID NO:

[0141] 3);

[0142] iv. albumin, optionally comprising or consisting of serum albumin (SEQ ID NO: 4);

[0143] and / or

[0144] v. fetuin, optionally comprising or consisting of fetuin-A (SEQ ID NO: 5);

[0145] or functional variants thereof having at least 70% sequence identity thereto.

[0146] In some embodiments, the Yarrowia cell produces or is capable of producing FGF2 and TGF-beta. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2 and transferrin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2 and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2 and fetuin. In some embodiments, the Yarrowia cell produces or is capable of producing TGF-beta and transferrin. In some embodiments, the Yarrowia cell produces or is capable of producing TGF-beta and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing TGF-beta and fetuin. In some embodiments, the Yarrowia cell produces or is capable of producing transferrin and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing transferrin and fetuin. In some embodiments, the Yarrowia cell produces or is capable of producing albumin and fetuin.

[0147] In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, and transferrin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, and fetuin. In some embodiments, the Yarrowia cell produces or is capable of producing TGF-beta, transferrin, and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing TGF-beta, transferrin, and fetuin. In some embodiments, the Yarrowia cell produces or is capable of producing transferrin, albumin, and fetuin.P7124PC00

[0148] In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, transferrin, fetuin and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, transferrin, and fetuin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, transferrin, and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, TGF-beta, fetuin and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing FGF2, transferrin, fetuin and albumin. In some embodiments, the Yarrowia cell produces or is capable of producing TGF-beta, transferrin, fetuin and albumin.

[0149] Various Yarrowia species may be useful according to the present disclosure. In some embodiments, the host cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic. The Yarrowia cells of the present disclosure may be used in FBS-replacement compositions, which may be used to culture and propagate cultivated meat and / or lab-grown meat. Hence, Yarrowia species with GRAS and / or QPS status are preferred.

[0150] The Yarrowia cell of the present disclosure may belong to species of Yarrowia lipolytica, Yarrowia porcina, Yarrowia bubula, Yarrowia deformans, Yarrowia yakushimensis, Yarrowia parophonii, Yarrowia galli, Yarrowia oslonensis, Yarrowia alimentaria, Yarrowia hollandica, or Yarrowia phangngaensis. In some embodiments, the Yarrowia cell belongs to the species of Yarrowia lipolytica.

[0151] Thus, in some embodiments, the Yarrowia cell expresses FGF2-G3 comprising or consisting of SEQ ID NO: 1, serotransferrin comprising or consisting of SEQ ID NO: 3, and serum albumin comprising or consisting of SEQ ID NO: 4, or functional variants thereof having at least 70% sequence identity thereto.

[0152] In other some embodiments, the Yarrowia cell expresses FGF2-G3 comprising or consisting of SEQ ID NO: 1, serotransferrin comprising or consisting of SEQ ID NO: 3, serum albumin comprising or consisting of SEQ ID NO: 4, and fetuin-A comprising or consisting of SEQ ID NO: 5, or functional variants thereof having at least 70% sequence identity thereto.P7124PC00

[0153] In the field of bioinformatics, the "template modelling score" (TM-score) is a recognized metric used to assess the similarity between two protein structures. The TM-score quantitatively measures this similarity on a scale from 0 to 1. Typically, scores below 0.20 indicate randomly chosen, unrelated proteins, whereas scores above 0.5 suggest that the structures share roughly the same fold. A detailed methodology for calculating the TM-score is provided in the publication "Zhang Y and Skolnick J (2004). Scoring function for automated assessment of protein structure template quality. Proteins. 57 (4): 702-710."

[0154] The TM-score can be calculated, for example, by uploading two three-dimensional structures in PDB format to the online resource available at https: / / zhanggroup.org / TM-score / . In situations where a pair of three-dimensional structures for comparison is not available, established methods for predicting the three-dimensional structure of a polypeptide are well known to those skilled in the art. For instance, a neural network trained for this specific task, such as AlphaFold, can be employed.

[0155] 3D structures of proteins and / or domains thereof are available through different sources. Thus, 3D structures to be used with the present invention may be available through various databases or can be predicted structures. AlphaFold3, referred to herein as AlphaFold, represents a state-of-the-art artificial intelligence (Al) system developed by DeepMind for predicting the three-dimensional (3D) structures of proteins from their amino acid sequences. This system is detailed in the publication: Jumper, J., Evans, R., Pritzel, A. et al. "Highly accurate protein structure prediction with AlphaFold." Nature 596, 583-589 (2021). https: / / doi.org / 10.1038 / s41586-021-03819-2.

[0156] AlphaFold DB is an online database which hosts over 200 million entries, encompassing the human proteome as well as the proteomes of 47 other key organisms relevant to research and global health. These entries are freely accessible at https: / / alphafold.ebi.ac.uk / . The (predicted) structure of FGF2 can be retrieved by inputting identifiers such as Fibroblast growth factor 2, FGF2, or P09038.

[0157] Structures of polypeptides not included in the AlphaFold DB may for example be predicted using the source code available at https: / / github.com / google-deepmind / alphafold, and / or a Colab notebook accessible at

[0158] https: / / colab.research.google.com / github / deepmind / alphafold / blob / main / notebooks / AlpP7124PC00

[0159] haFold.ipynb. To generate a 3D structure using the Colab notebook, the amino acid sequence of FGF2-G3 of SEQ ID NO: 1 can be inserted.

[0160] Yarrowia lysate

[0161] Herein described are lysates of the cells described in the section “Yarrowia cells”, wherein a lysate is prepared by lysing cells, for example by rupture, disintegration and / or other form of disruption of the cell wall and / or membrane. A Yarrowia lysate may comprise lysed cells from one or more strain, isolate or population of Yarrowia cells, each Yarrowia population expressing one or more bovine growth-associated factor. Hence provided herein are lysates of Yarrowia cells capable of producing one or more bovine growth-associated factors.

[0162] As used herein, a lysate of a Yarrowia cell, a Yarrowia lysate, a Yarrowia cell lysate, a lysate produced from a Yarrowia cell, and a lysed obtained from a Yarrowia cell, are all used as synonyms.

[0163] Lysates, as described herein, may be produced by physical methods, such as by sonication or mechanical homogenization, and / or by chemical methods, such as by contacting the cell with lysis buffers, detergents, enzymes and like, which are capable of disrupting the cell wall and / or membrane. Cell lysis may also be achieved through cellular processes, such as apoptosis, and these cellular processes may in turn be induced via the use of external factors, such as nutrient limitation, exposure to toxins and the like. The preparation of cell lysates, such as Yarrowia cell lysates as described herein, may involve the use of protease inhibitors, so that protease activity may be reduced during lysate preparation and thus less growth-associated factors are degraded during the lysis process.

[0164] The lysate as described herein may comprise a partial cell lysate, wherein said partial cell lysate may be composed of a mixture of intact and lysed cells, and / or wherein said partial cell lysate may comprise cells that are, on the level of individual cells, partially lysed, in other words the cells may be lysed incompletely.

[0165] Without being bound by theory, the one or more bovine growth-associated factors produced by Yarrowia cells, for example as described in the section “Yarrowia cells”, may not be secreted and may hence remain intracellularly, for example in the cellP7124PC00

[0166] cytoplasm. Thus, in order to produce a useful FBS-replacement composition comprising said bovine growth-associated factors it may be useful to lyse said Yarrowia cells to allow said bovine growth-associated factors to be released from the cell.

[0167] The use of cell lysate, as described herein, may confer additional advantages over the mere replacement of growth-associated factors in the context of mammalian and / or fish cell culture. Yarrowia cells comprise many components, for example salts, minerals, vitamins, fatty acids, carbon, nitrogen and the like, which may be useful in promoting and / or supporting mammalian and / or fish cell growth and / or proliferation in the context of in vitro cell culture. The use of cell lysate may be advantageous over intact cells, as said components are more readily available to said mammalian and / or fish cells when said Yarrowia cells are provided as a lysate.

[0168] Provided herein is a Yarrowia lysate comprising one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, albumin, and / or fetuin. Hence in some embodiments, the Yarrowia lysate comprises FGF2. In some embodiments, the Yarrowia lysate comprises TGF-beta. In some embodiments, the Yarrowia lysate comprises transferrin. In some embodiments, the Yarrowia lysate comprises albumin. In some embodiments, the Yarrowia lysate comprises fetuin. In some embodiments, the lysate comprises a combination of one or more of the aforementioned bovine growth-associated factors.

[0169] In some embodiments, the Yarrowia lysate comprises FGF2 and TGF-beta. In some embodiments, the Yarrowia lysate comprises FGF2 and transferrin. In some embodiments, the Yarrowia lysate comprises FGF2 and albumin. In some embodiments, the Yarrowia lysate comprises FGF2 and fetuin. In some embodiments, the Yarrowia lysate comprises TGF-beta and transferrin. In some embodiments, the Yarrowia lysate comprises TGF-beta and albumin. In some embodiments, the Yarrowia lysate comprises TGF-beta and fetuin. In some embodiments, the Yarrowia lysate comprises transferrin and albumin. In some embodiments, the Yarrowia lysate comprises transferrin and fetuin. In some embodiments, the Yarrowia lysate comprises albumin and fetuin.

[0170] In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta, and transferrin. In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta,P7124PC00

[0171] and albumin. In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta, and fetuin. In some embodiments, the Yarrowia lysate comprises TGF-beta, transferrin, and albumin. In some embodiments, the Yarrowia lysate comprises TGF-beta, transferrin, and fetuin. In some embodiments, the Yarrowia lysate comprises transferrin, albumin, and fetuin.

[0172] In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta, transferrin, fetuin and albumin. In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta, transferrin, and fetuin. In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta, transferrin, and albumin. In some embodiments, the Yarrowia lysate comprises FGF2, TGF-beta, fetuin and albumin. In some embodiments, the Yarrowia lysate comprises FGF2, transferrin, fetuin and albumin. In some embodiments, the Yarrowia lysate comprises TGF-beta, transferrin, fetuin and albumin.

[0173] In some embodiments the Yarrowia lysate comprises one or more bovine growth-associated factors. In some embodiments the Yarrowia lysate comprises two or more bovine growth-associated factors. In some embodiments the Yarrowia lysate comprises three or more bovine growth-associated factors. In some embodiments the Yarrowia lysate comprises four or more bovine growth-associated factors. In some embodiments the Yarrowia lysate comprises five or more bovine growth-associated factors.

[0174] In some embodiments, the Yarrowia lysate comprises FGF2, transferrin, albumin, and optionally fetuin.

[0175] In some embodiments, the Yarrowia lysate is as described above, wherein:

[0176] i. FGF2 comprises or consists of FGF2-G3 (SEQ ID NO: 1);

[0177] ii. TGF-beta comprises or consists of TGF-beta3 (SEQ ID NO: 2);

[0178] iii. transferrin comprises or consists of serotransferrin (SEQ ID NO: 3);

[0179] iv. albumin comprises or consists of serum albumin (SEQ ID NO: 4); and / or v. fetuin comprises or consists of fetuin-A (SEQ ID NO: 5);

[0180] or functional variants thereof having at least 70% sequence identity thereto.

[0181] Thus, in some embodiments, the Yarrowia lysate is as described above and comprises FGF2-G3 (SEQ ID NO: 1), TGF-beta3 (SEQ ID NO: 2), serotransferrin (SEQ ID NO: 3), serum albumin (SEQ ID NO: 4) and fetuin-A (SEQ ID NO: 5), or functional variantsP7124PC00

[0182] thereof having at least 70% sequence identity thereto. The lysate need not necessarily be a lysate of a single Yarrowia strain and / or isolate. The lysate may be a composite lysate, comprising lysates of two or more, such as two or more, such as three or more, such as four or more, such as a composite lysate of five Yarrowia strains, isolates and / or populations.

[0183] In some embodiments, the Yarrowia cell of the lysate is as described in the section “Yarrowia cells”.

[0184] In some embodiments, the Yarrowia lysate comprises or consists of a whole-cell lysate. Whole-cell lysates may differ from yeast extracts in that whole-cell lysates comprise material derived from every component of the cell, whereas yeast extracts comprise no or minimal cellular macromolecules such as, cell wall material, such as p-glucans and mannans, membrane structures, particularly lipids, and polynucleotide structures, such as DNA and RNA. Conversely, yeast extracts may comprise greater concentrations of free amino acids, free nucleotides, water-soluble vitamins, such as B-vitamins, inorganic salts, and / or ash, relative to a yeast lysate produced from the same yeast strain, and are depleted in cellular macromolecules, some of which may possess bioactive properties. Yeast extracts may be processed with acid, which may cause protein denaturation and / or protein breakdown, such as breakdown of proteins into peptides and free amino acids. Yeast extracts may also be processed to remove insoluble fractions, such as those fractions comprising cell wall material.

[0185] In some embodiments, the Yarrowia lysate comprises Yarrowia cell wall components, such as mannoproteins, glucans, mannan, chitin, glycoproteins, and / or DNA. In some embodiments, the Yarrowia lysate comprises a greater concentration of insoluble macromolecular components, such as cell wall components, polynucleotides, membrane structures and / or membrane lipids, relative to a Yarrowia extract produced from the same strain. In some embodiments, the Yarrowia lysate comprises a reduced concentration of free amino acids, free nucleotides, water soluble vitamins, such as thiamine or riboflavin, relative to a Yarrowia extract produced from the same strain.

[0186] In some embodiments, the Yarrowia lysate as described herein, comprises Yarrowia derived fatty acids, such as oleic acid and / or linoleic acid. Fatty acids may be useful in the cultivation, incubation and propagation of mammalian and / or fish cells, in particularP7124PC00

[0187] oleic acid and linoleic acid; hence said Yarrowia derived fatty acids may be useful components of FBS-replacement compositions comprising said Yarrowia lysate. Furthermore, Yarrowia species are oleaginous yeast, wherein the cells may have a fat content of 20% or more. Thus, the concentration of such fatty acids in the lysate may be used to differentiate the Yarrowia lysate from lysates of other yeasts, such as Saccharomyces lysates that would be expected to have a relatively lower fatty acid content. In some embodiments, the Yarrowia lysate comprises polyunsaturated fatty acids and / or omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0188] In some embodiments, the Yarrowia lysate may be a liquid composition. In some embodiments, the Yarrowia lysate may be a lyophilized powder.

[0189] Fetal bovine serum replacement composition

[0190] Mammalian and fish cells require factors in order to grow and proliferate that may be derived from serum, such as FBS; however, serum can be costly, inconsistent across batches, and raises issues with regards to animal welfare. With the increasing interest in lab-cultivated meat as an alternative to meat derived from animal slaughter, alternatives to animal derived serums, such as FBS are required to support the propagation and culture of said lab-cultivated meat, in a manner that is both more sustainable and prioritises animal welfare.

[0191] The FBS-replacement compositions described herein comprise at least one growth-associated factor, which is to say growth factor and / or accessory protein as defined herein, produced by the Yarrowia cell and thereby found in the Yarrowia lysate. The FBS-replacement compositions described herein may comprise both Yarrowia-produced bovine growth associated factors as well as exogenous growth-associated factors. Said exogenous growth-associated factors need not be bovine growth-associated factors.

[0192] Described herein is a fetal bovine serum (FBS)-replacement composition. Said FBS-replacement composition may be useful in the cultivation or culturing of mammalian and / or fish cells, for example when used as a replacement for serum, such as FBS, or when used to provide grow-associated factors that might otherwise be provided by serum.P7124PC00

[0193] Provided herein is a fetal bovine serum (FBS)-replacement composition comprising a Yarrowia lysate, wherein said composition comprises one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, and / or albumin. Said FBS-replacement composition may comprise one or more additional components such as for example water; aqueous solutions, for example comprising salts; buffer agents, such as HEPES, ascorbate and / or sodium bicarbonate; media; micronutrients, such as selenium; exogenous and / or non-Yarrow / a-derived fatty acids; exogenous and / or non-Yarrow / a-derived proteins, and the like. Said additional components may be used formulate the Yarrowia lysate into a FBS-replacement composition by incorporating additional mammalian and / or fish cell growth supporting and / or promoting components, alternatively they may be used to dilute the Yarrowia lysate to a obtain a reduced concentration of bovine growth-associated factors in the FBS-replacement composition.

[0194] In some embodiments, the FBS-replacement composition consists of a Yarrowia lysate and one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, and / or albumin. In some embodiments, the FBS-replacement composition consists of a Yarrowia lysate and two or more bovine growth-associated factors. In some embodiments, the FBS-replacement composition consists of a Yarrowia lysate and three or more bovine growth-associated factors. In some embodiments, the FBS-replacement composition consists of a Yarrowia lysate and four or more bovine growth-associated factors.

[0195] In some embodiments, the Yarrowia lysate of the FBS-replacement composition is as described in the section “Yarrowia lysate”.

[0196] In some embodiments, the Yarrowia lysate of the FBS-replacement composition comprises one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, and / or albumin. In some embodiments, the Yarrowia lysate of the FBS-replacement composition comprises FGF2. In some embodiments, the Yarrowia lysate of the FBS-replacement composition comprises TGF-beta. In some embodiments, the Yarrowia lysate of the FBS-replacement composition comprises transferrin. In some embodiments, the Yarrowia lysate of the FBS-replacement composition comprises albumin. In some embodiments, the Yarrowia lysate of the FBS-replacement composition comprises fetuin.P7124PC00

[0197] In some embodiments, the Yarrowia lysate of the FBS-replacement composition further comprises fetuin.

[0198] In some embodiments, the FBS-replacement composition is as described herein above, wherein:

[0199] i. FGF2 comprises or consists of FGF2-G3 (SEQ ID NO: 1);

[0200] ii. TGF-beta comprises or consists of TGF-beta3 (SEQ ID NO: 2);

[0201] iii. transferrin comprises or consists of serotransferrin (SEQ ID NO: 3);

[0202] iv. albumin comprises or consists of serum albumin (SEQ ID NO: 4); and / or v. fetuin comprises or consists of fetuin-A (SEQ ID NO: 5);

[0203] or functional variants thereof having at least 70% sequence identity thereto.

[0204] In some embodiments, the FBS-replacement composition comprises FGF2-G3 as set forth in SEQ ID NO: 1 , or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the FBS-replacement composition comprises TGF-beta3 as set forth in SEQ ID NO: 2, or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the FBS-replacement composition comprises serotransferrin as set forth in SEQ ID NO: 3, or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the FBS-replacement composition comprises serum albumin as set forth in SEQ ID NO: 4, or functional variants thereof having at least 70% sequence identity thereto. In some embodiments, the FBS-replacement composition comprises fetuin-A as set forth in SEQ ID NO: 5, or functional variants thereof having at least 70% sequence identity thereto.

[0205] In some embodiments, the FBS-replacement composition, comprises lysates of at least two different Yarrowia populations, wherein said at least two Yarrowia populations each produce at least one different bovine growth-associated factor and each Yarrowia population consists of Yarrowia cells as described herein, for example in the section “Yarrowia cells”. Yarrowia lysates comprising only one bovine growth associated factor may be useful for producing FBS-replacement compositions; lysates comprising only one bovine growth-associated factor may be combined to produce an FBS-replacement composition, wherein the ratio of each of the bovine growth-associated factors in the composition, can be adjusted as required by altering ratio of the corresponding Yarrowia lysate. This approach also allows FBS-replacement compositions comprising different combinations of bovine growth-associated factors toP7124PC00

[0206] be obtained using the same Yarrowia lysate, by including or omitting a particular lysate from the composition. Conversely, a Yarrowia lysate comprising two or more bovine growth-associated factors, such as three or more, such as four or more, such as five bovine growth-associated factors may simplify the processing required to produce said FBS-replacement composition, by reducing the number of lysates required to produce said FBS-replacement composition comprising two or more bovine growth-associated factors.

[0207] In some embodiments, the FBS-replacement composition comprises a Yarrowia lysate comprising FGF2, transferrin, albumin, and optionally fetuin. In some embodiments, the FBS replacement composition comprises at least one Yarrowia lysate comprising at least one bovine growth-associated factor, wherein said at least one bovine growth-associated factor is FGF2, transferrin, albumin and / or fetuin, and wherein the FBS replacement composition further comprises exogenous TGF-beta. In some embodiments, the FBS replacement composition comprises a lysate of a Yarrowia cell capable of producing FGF2, transferrin, albumin and optionally fetuin, and exogenous TGF-beta. Preferably wherein said exogenous TGF-beta is a non- Yarrowia derived source of TGF-beta. The term “exogenous” as used herein, particularly in the context of exogenous TGF-beta , refers to non- Yarrowia produced or derived factors and / or compounds, such as bovine growth-associated factors not produced by the Yarrowia cell, such as TGF-beta not produced or derived from the Yarrowia cell as described elsewhere herein.

[0208] Provided herein is a mammalian and / or fish cell culture medium composition, wherein said mammalian and / or fish cell culture medium composition comprises the FBS-replacement composition as described herein. Generally, mammalian and / or fish cell media comprises a source of carbon, a source of nitrogen, vitamins and salts. When said media is used for the cultivation and / or propagation of mammalian and / or fish cells it is generally combined with serum, such as FBS and / or recombinant or purified growth factors. Thus, the FBS- replacement composition of the present disclosure may be used in combination with mammalian and / or fish cell media for the cultivation and / or propagation of mammalian and / or fish cells. The FBS-replacement composition as described herein may be used to replace and / or in addition to serum, such as FBS, and / or recombinant or purified growth factors, when used for the cultivation and / or propagation of mammalian and / or fish cells.P7124PC00

[0209] Examples of suitable media, which may be combined with the FBS-replacement composition of the present disclosure for the cultivation and / or propagation of mammalian and / or fish cells include, but are not limited to: minimum essential media (MEM; also referred to as Eagle’s minimum essential media), Dulbecco's Modified Eagle Medium (DMEM), optimised minimum essential media (Opti-MEM), and Roswell Park Memorial Institute media (RPMI, also known as RPMI-1640 and RPMI 1640 medium). The skilled person can identify other such suitable media for use in the cultivation and / or propagation of mammalian and / or fish cells in combination with the FBS-replacement composition of the present disclosure. Furthermore, the skilled person knows that the aforementioned media may be available under alternative names and formulations, such as BenchStable formulations, and that such alternatives are functionally equivalent.

[0210] In some embodiments, said mammalian and / or fish cell culture medium composition comprising the FBS-replacement composition as described herein, comprises in the between 0.25%v / v and 1%v / v FBS-replacement composition, such as between 0.5%v / v and 1%v / v FBS-replacement composition, such as between 0.75%v / v and 1%v / v FBS-replacement composition.

[0211] Methods

[0212] Described herein are methods, which may be useful producing and / or obtaining one or more bovine growth-associated factors, a Yarrowia lysate, an FBS-replacement composition, mammalian and / or fish cell biomass and / or a food composition.

[0213] Provided herein is a method of obtaining one or more bovine growth-associated factors, comprising:

[0214] i. providing the Yarrowia cell defined herein;

[0215] ii. incubating and optionally propagating said Yarrowia cell in a medium, iii. thereby producing one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, albumin and / or fetuin; and

[0216] iv. optionally, recovering said one or more bovine growth-associated factors, whereby said one or more bovine growth-associated factors are obtained, preferably wherein said one or more bovine growth-associated factors are as described herein.P7124PC00

[0217] Provided herein is a method of obtaining one or more bovine growth-associated factors, comprising:

[0218] i. providing a Yarrowia cell capable of producing one or more bovine growth- associated factors;

[0219] ii. incubating and optionally propagating said Yarrowia cell in a medium, iii. thereby producing one or more bovine growth-associated factors, wherein said growth-associated factors are FGF2, TGF-beta, transferrin, albumin and / or fetuin; and

[0220] iv. optionally, recovering said one or more bovine growth-associated factors, whereby said one or more bovine growth-associated factors are obtained, preferably wherein said one or more bovine growth-associated factors are as described herein.

[0221] Provided herein is also a method of producing a Yarrowia lysate, comprising:

[0222] i. providing a Yarrowia cell as described herein, for example in the section “Yarrowia cells”;

[0223] ii. propagating said Yarrowia cell in a medium,

[0224] iii. thereby obtaining a Yarrowia cell biomass; and

[0225] iv. lysing said Yarrowia cell biomass to produce said Yarrowia lysate; whereby a Yarrowia lysate is produced, preferably wherein said Yarrowia lysate is as described herein, for example in the section “Yarrowia lysate”.

[0226] Provided herein is also a method for producing an FBS-replacement composition comprising a Yarrowia lysate and one or more bovine growth-associated factors, comprising:

[0227] i. providing a Yarrowia cell as described herein, for example in the section “Yarrowia cells”;

[0228] ii. propagating said Yarrowia cell in a medium, thereby obtaining Yarrowia cell biomass;

[0229] iii. recovering and / or preparing a lysate of said Yarrowia cell biomass, thereby obtaining a Yarrowia lysate; and

[0230] iv. optionally, combining one or more Yarrowia lysates obtained in step iii.; v. formulating the one or more Yarrowia lysates of step iii. and / or iv. into an FBS- replacement composition;

[0231] whereby said FBS-replacement composition is produced.P7124PC00

[0232] The skilled person knows that step v. of the aforementioned method, comprising formulating one or more Yarrowia lysates into an FBS-replacement composition, may comprise such steps as: dilution of the Yarrowia lysate, for example with water or an aqueous solution and / or incorporation of additional growth-factors, accessory proteins or growth-associated factors into the FBS-replacement composition.

[0233] In some embodiments, the methods are as described above, wherein the step of recovering said Yarrowia cell biomass comprises centrifugation.

[0234] The skilled person knows that there are many methods that may be used to disrupt the cell wall and / or cell membrane of a Yarrowia cell, thereby producing a Yarrowia lysate, wherein the majority of the Yarrowia produced bovine growth-associated factors are not destroyed, disrupted and / or denatured. The Yarrowia lysate may as described herein, for example in the section “Yarrowia lysate”. In some embodiments, the methods of obtaining bovine growth-associated factors, producing a Yarrowia lysate and producing an FBS-replacement composition are as described above, wherein the step of preparing a lysate of said Yarrowia cell comprises cell homogenisation, such as by bead beating and / or by cooling cycles, and optionally resuspension in an alkaline buffer, or such as by alternative mechanical means of cell lysis, such as by sonication or by hydraulic French press. Thus, the step of obtaining / producing a Yarrowia lysate from a Yarrowia cell may be mechanical lysis or chemical lysis means, or a combination.

[0235] Also provided herein is a method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition, as described herein, for example in the section “Fetal bovine serum replacement composition”, comprising:

[0236] i. providing a FBS-replacement composition as described herein, for example in the section “Fetal bovine serum replacement composition”;

[0237] ii. mixing said FBS-replacement composition with a culture medium suitable for cultivation of said mammalian and / or fish cells, thereby obtaining a mixture; and iii. using said mixture to cultivate mammalian and / or fish cells.

[0238] In some embodiments, the method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition is as described above, wherein the mammalian and / or fish cells are cultivated in vitro.P7124PC00

[0239] In some embodiments, the method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition is as described above, wherein the mammalian and / or fish cells are cultivated in suspension. Culturing mammalian and / or fish cells in suspension offers advantages, such as ease of scalability, ease of automation, and increased culture homogeneity. In some embodiments, adipose cells, such as adipocytes, may be cultivated in suspension.

[0240] In some embodiments, the method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition is as described above, wherein the mammalian and / or fish cells are cultivated attached to a surface. Culturing mammalian and / or fish cells attached to a surface offer advantages such as cell types that cannot be cultured in suspension may be grown. In some embodiments, satellite cells and / or mammary epithelial cells may be cultivated attached to a surface.

[0241] In the production of cultivated meat it may be desirable to combine several distinct cultivated mammalian and / or fish tissue cells into a composition, in order to more accurately mimic meat derived from animal slaughter. The FBS-replacement composition may be used for the cultivation and / or propagation of mammalian and / or fish cells derived from distinct mammalian and / or fish tissues. In some embodiments, the method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition is as described above, wherein the mammalian and / or fish cells comprise muscle cells, satellite cells, mammary epithelial cells, adipose cells and / or fibroblast cells.

[0242] In some embodiments, the method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition is as described above, wherein the mammalian and / or fish cells are any one of bovine cells, human cells, equine cells, murine cells, goat cells, sheep cells, chicken cells, fish cells, porcine cells, or lapine cells. The mammalian or fish cells may be satellite cells. The cell types may be primary cells, such as primary satellite cells, or immortalized cells, such as immortalized satellite cells. Thus, in some embodiments the bovine cells are primary bovine cells, such as primary bovine satellite cells. In some embodiments, the bovine cells are immortalized bovine satellite cells. The skilled person knows that the FBS-replacement composition as described herein may be useful for the cultivation and / or propagation ofP7124PC00

[0243] cells derived from mammals and / or fish. Such mammals may include cows, humans, horses, mice, goats, sheep, chicken, fish, pigs, or rabbits.

[0244] In preferred embodiments, the mammalian cells are primary bovine cells, such as primary satellite bovine cells.

[0245] Provided herein is a method for producing a food composition, comprising:

[0246] i. providing a Yarrowia lysate;

[0247] ii. formulating said Yarrowia lysate into an FBS-replacement composition; iii. cultivating mammalian and / or fish cells, wherein said mammalian cells are not human cells, in the presence of said FBS-replacement composition, optionally further comprising a culture medium;

[0248] thereby obtaining a biomass of mammalian and / or fish cells; and

[0249] iv. formulating said biomass of mammalian cells and / or fish into a food composition,

[0250] thereby producing said food composition. Said food composition may be, for example, cultured meat, or an ingredient therefor.

[0251] In some embodiments, the method for producing a food composition is as described above, wherein said Yarrowia lysate is as described herein, for example in the section “Yarrowia lysate”.

[0252] In some embodiments, the method for producing a food composition is as described above, wherein said FBS-replacement composition is as described herein, for example in the section “Fetal bovine serum replacement composition”.

[0253] In some embodiments, the method for producing a food composition is as described above, wherein step iii is performed as in the method for the cultivation of mammalian and / or fish cells using said FBS-replacement composition.

[0254] Any of the methods described herein, for example in the section “Methods”, may additionally comprise cleavage of any tag, such as a purification tag, covalently linked to said bovine growth-associated factor.

[0255] Product Uses

[0256] Described herein are uses for the products described elsewhere herein.P7124PC00

[0257] Disclosed herein is the use of the Yarrowia lysate as described herein, for example in the section “Yarrowia lysate” and / or the FBS-replacement composition as described herein, for example in the section “Fetal bovine serum replacement composition” for cultivating mammalian and / or fish cells, preferably in combination with a culture medium.

[0258] Disclosed herein is the use of mammalian and / or fish cells obtainable by a method described herein, for example in the section “Methods”, wherein the mammalian and / or fish cells are used as a food product, food ingredient, and / or food material.

[0259] Provided herein is also a food composition comprising mammalian and / or fish cells obtainable by a method described herein, for example in the section “Methods”.

[0260] In some embodiments, the food composition is a cultivated meat and / or an ingredient therefor.

[0261] Disclosed herein is also the use of the food composition as a food product, food material, and / or food ingredient.

[0262] Disclosed herein is also the use of the food composition in a cultivated meat and / or in a method for producing a meat alternative. Said cultivated meat may be cultivated meat and / or lab-grown meat. Said cultivated meat may also be an ingredient for cultivated meat and / or lab-grown meat. Said food composition may comprise mammalian and / or fish tissue of several types, for example muscle tissue, adipose tissue and / or fibroblast tissue.

[0263] Provided herein is also a cultivated meat and / or ingredient therefor comprising mammalian and / or fish cells obtainable by a method described herein, for example in the section “Methods”.

[0264] Nucleic acids

[0265] Provided herein are expression systems useful for obtaining a Yarrowia cell capable of producing one or more bovine growth-associated factors, as disclosed herein. Provided are also nucleic acids and / or Yarrowia cells comprising said nucleic acids useful for producing one or more bovine growth-associated factors. The present nucleic acidsP7124PC00

[0266] disclosed herein may be provided as one or more nucleic acid molecules, for example they may be comprised in one or more vectors and / or expression systems. Such nucleic acids may be introduced in the Yarrowia cell by methods known in the art. The terms nucleic acid, nucleic acid sequence, nucleic acid constructs, nucleic acid molecules, oligonucleotide, and polynucleotides may be used interchangeably herein. Said nucleic acids may be useful for expression in, engineering and / or modification of a Yarrowia cell.

[0267] Provided herein is a nucleic acid encoding a polypeptide comprising or consisting of FGF2-G3 as set forth in SEQ ID NO: 1, such as a nucleic acid comprising or consisting of SEQ ID NO: 6 (FGF2-G3), or a functional variant thereof having at least 70% sequence identity thereto.

[0268] Provided herein is also a nucleic acid encoding a polypeptide comprising or consisting of TGF-beta3 as set forth in SEQ ID NO: 2, such as a nucleic acid comprising or consisting of SEQ ID NO: 7 (TGF-beta3), or a functional variant thereof having at least 70% sequence identity thereto.

[0269] Also provided herein is a nucleic acid encoding a polypeptide comprising or consisting of serotransferrin as set forth in SEQ ID NO: 3, such as a nucleic acid comprising or consisting of SEQ ID NO: 8 (serotransferrin), or a functional variant thereof having at least 70% sequence identity thereto.

[0270] Provided herein is also a nucleic acid encoding a polypeptide comprising or consisting of serum albumin as set forth in SEQ ID NO: 4, such as a nucleic acid comprising or consisting of SEQ ID NO: 9 (serum albumin), or a functional variant thereof having at least 70% sequence identity thereto.

[0271] Provided herein is a nucleic acid encoding a polypeptide comprising or consisting of fetuin-A as set forth in SEQ ID NO: 5, such as a nucleic acid comprising or consisting of SEQ ID NO: 10 (fetuin-A), or a functional variant thereof having at least 70% sequence identity thereto.

[0272] In some embodiments, the nucleic acid encodes a bovine FGF2, preferably wherein said FGF2 is FGF2-G3 as set forth in SEQ ID NO: 1 , or a functional variant having atP7124PC00

[0273] least 70% sequence identity thereto. In some embodiments, the nucleic acid encodes a bovine TGF-p, preferably wherein said TGF-p is TGF-beta3 as set forth in SEQ ID NO: 2, or a functional variant having at least 70% sequence identity thereto. In some embodiments, the nucleic acid encodes a bovine transferrin, preferably wherein said transferrin is serotransferrin as set forth in SEQ ID NO: 3, or a functional variant having at least 70% sequence identity thereto. In some embodiments, the nucleic acid encodes a bovine albumin, preferably wherein said albumin is serum albumin as set forth in SEQ ID NO: 4, or a functional variant having at least 70% sequence identity thereto. In some embodiments, the nucleic acid encodes a bovine fetuin, preferably wherein said fetuin is fetuin-A as set forth in SEQ ID NO: 5, or a functional variant having at least 70% sequence identity thereto.

[0274] In some embodiments, the nucleic acid as described herein further comprises at least one promoter, such as at least one constitutive promoter or at least one inducible promoter. In some embodiments, the promoter is a strong promoter, for example pL41 (SEQ ID NO: 13) or pTEF-intron (SEQ ID NO: 14), or a weak promoter, for example pASN2 (SEQ ID NO: 15). In the context where two or more growth-associated factors are co-expressed in the same cell, expression of said two or more growth-associated factors may be individually expressed using the same promoter sequence, for example said two or more growth-associated factors may be expressed from pL41. Conversely, it may also be advantageous to select promoters for expression of said two or more promoters, to account for the desired level of each individual growth-associated in the cell lysate. For example, a medium or weak strength promoter, such as pASN2 may be particularly suitable for driving the expression of TGF-beta, as the expression of TGF-beta requires careful balancing to produce an FBS-replacement composition with optimal properties for the support and / or proliferation of mammalian and / or fish cells in culture.

[0275] The skilled person knows whether a given promoter is classified as a medium or strong promoter and / or how to characterise the promoter strength of an uncharacterised promoter. For example, RNA levels can be measured using RNA sequencing, or protein levels can be quantified with a reporter protein, such as green fluorescent protein (GFP), and used for determining the strength of the promoter. A medium or strong promoter may either be a native promoter or a non-native promoter of the hostP7124PC00

[0276] cell that it is comprised within. A non-native promoter may be a promoter that is native to another organism, or it may be a synthetic promoter.

[0277] In some embodiments, the nucleic acid is codon-optimised for expression in a Yarrowia cell as described herein, for example in the section “Yarrowia cells”.

[0278] Purifications tags may aid in the recovery of a protein from a host cell culture, whether the protein is secreted or maintained inside the host cell after being translated. Hence, in some embodiments, said nucleic acid further comprises a purification tag, preferably wherein said purification tag is operably linked to said bovine growth-associated factor, optionally wherein said purification tag is as set forth in SEQ ID NO: 12.

[0279] It may be desirable to cleave any tag covalently linked to the present analogues, in particular if said tags infer with the functionality of said bovine growth-associated factors that said tag is operably linked to. The skilled person will have no difficulty in designing such cleavable purification tags or cleavable secretion tags.

[0280] Expression system

[0281] Provided herein are expression systems useful for obtaining a Yarrowia cell capable of producing one or more bovine growth-associated factor, as disclosed herein. Such expression systems may be introduced in the Yarrowia cell by methods known in the art. Said expression systems may be useful for expression in, engineering and / or modification of a Yarrowia cell.

[0282] In one aspect the present disclosure provides an expression system for expression in a Yarrowia cell, comprising one or more of:

[0283] i. a nucleic acid comprising FGF2 as described herein, for example in the section “Nucleic acids”;

[0284] ii. a nucleic acid comprising TGF-beta as described herein, for example in the section “Nucleic acids”;

[0285] iii. a nucleic acid comprising transferrin as described herein, for example in the section “Nucleic acids”;

[0286] iv. a nucleic acid comprising albumin as described herein, for example in the section “Nucleic acids”;P7124PC00

[0287] v. a nucleic acid comprising fetuin as described herein, for example in the section “Nucleic acids”,

[0288] or functional variants thereof having at least 70% sequence identity thereto.

[0289] In some embodiments, the expression systems comprises a bovine FGF2, preferably wherein said FGF2 is FGF2-G3 as set forth in SEQ ID NO: 1 , or a functional variant having at least 70% sequence identity thereto. In some embodiments, the expression systems comprises a bovine TGF-p, preferably wherein said TGF-p is TGF-beta3 as set forth in SEQ ID NO: 2, or a functional variant having at least 70% sequence identity thereto. In some embodiments, the expression systems comprises a bovine transferrin, preferably wherein said transferrin is serotransferrin as set forth in SEQ ID NO: 3, or a functional variant having at least 70% sequence identity thereto. In some embodiments, the expression systems comprises a bovine albumin, preferably wherein said albumin is serum albumin as set forth in SEQ ID NO: 4, or a functional variant having at least 70% sequence identity thereto. In some embodiments, the expression systems comprises a bovine fetuin, preferably wherein said fetuin is fetuin-A as set forth in SEQ ID NO: 5, or a functional variant having at least 70% sequence identity thereto.

[0290] In some embodiments, the nucleic acid of the expression system comprises or consists of any of the nucleic acids as described herein, for example in the section “Nucleic acids”.

[0291] In some embodiments, the expression system is as described herein, for example in the section “Expression system”, wherein the Yarrowia cell is as described herein, for example in the section “Yarrowia cells”. Preferably wherein the Yarrowia cell belongs to the species Yarrowia lipolytica.

[0292] In some embodiments, the Yarrowia cell, the lysate, FBS-replacement composition, the method, and / or the use are as described herein, wherein said Yarrowia cell comprises the expression system as described herein, for example in the section “Expression system”, whereby said Yarrowia cell is capable of producing one or more bovine growth-associated factors.P7124PC00

[0293] Kit of parts

[0294] Herein disclosed is also a kit of parts, comprising:

[0295] i. a Yarrowia cell;

[0296] ii. at least one nucleic acid as described herein, for example in the section “Nucleic acids”; and / or

[0297] iii. the expression system as described herein, for example in the section “Expression system”;

[0298] iv. and optionally instructions for use;

[0299] preferably wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic, and / or wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

[0300] Such kits are useful for modifying a host cell so that it can produce one or more bovine growth-associated factors, as described herein, such as one or more of FGF2, TGF-beta, transferrin, albumin, and / or fetuin.

[0301] The nucleic acid may be SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5, or a nucleic acid encoding the polypeptide as set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and / or SEQ ID NO: 10 or functional variants thereof having at least 70% sequence identity thereto.

[0302] Examples

[0303] Example 1: Material and Methods

[0304] Microbial Strains, Media, and Growth Conditions

[0305] One Shot Top10 E.coli competent cells (Invitrogen # C404003) were used as cloning hosts. Transformants were selected on LB plates containing 100 ug / mL ampicillin at 37°C. Yarrowia lipolytica W29 (ku70::cas9) of the EasyClone Yali has been described in Holkenbrink et al. (2018), and was grown on YPD agar / broth at 30°C. Yeast transformant selection was performed on YPD plates containing 250 ug / mL nourseothricin or 300 ug / mL hygromycin.

[0306] Vector Construction and Transformation

[0307] The amino acid sequences of bovine FGF2-G3 (SEQ ID NO: 1), TGF-beta3 (SEQ ID NO: 2), serotransferrin (SEQ ID NO: 3), serum albumin (SEQ ID NO: 4), and fetuin-AP7124PC00

[0308] (SEQ ID NO: 5) were obtained from UniprotDB. Codon optimization based on Yarrowia lipolytica codon usage was performed using the IDT codon optimization tool to obtain the respective coding sequences (CDS). The growth factor or accessory protein CDS was cloned by USER assembly into Markerless CRISPR-based integration vectors as described by Holkenbrink et al. (2018). To allow for easy Ni-NTA purification, a subset of vectors (His-series) with the CDS preceded by 6X-His Tag (SEQ ID NO: 12), and followed by a cleavable TEV site, was generated by Gibson Assembly. Another subset of vectors (Hibit-series) was generated by introducing the codon-optimized sequence of 11 amino acid Hibit-Tag (SEQ ID NO: 11) to allow for quick quantification and blotting. Plasmid sequences were verified using Nanopore sequencing and transformed into One Shot Top10 E.coli competent cells by electroporation. Linear integration cassettes were generated by Notl digestion of the circular plasmids, and integrated into Yarrowia lipolytica using LiAC method as described by (Holkenbrink et al., 2018).

[0309] Protein Expression and Lysate Preparation

[0310] To assess growth factor protein and / or accessory protein expression, a seed culture was prepared by inoculating a single loop of transformant Yarrowia lipolytica strains into 10 mL YPD broth and cultivated overnight at 30°C and 225 rpm agitation. The seed culture was then transferred into a 250 mL baffled flask containing 40 mL YPD broth and allowed to grow overnight at 30°C and 225rpm agitation. All transformant (recombinant) strains were normalized to an optical density (OD) of 15 to allow for uniform lysate preparation and quantification of growth factor or accessory protein expression. Cells were then harvested by centrifugation at 4500 g, and the pellets were resuspended in 100 mM Tris-HCI pH 8.0. To prepare yeast lysates, 3 mL of the harvested biomass was transferred into 7 mL tubes Precellys tubes containing 0.5mm glass beads and homogenized using Precellys® 24 Tissue Homogeniser (Bertin Technologies) at 6500 rpm for 25 seconds and rapidly cooled on dry ice. This lyse-cool cycle was repeated 7 times, and cell lysis was confirmed under a light microscope (Zeiss Primostar 3). Soluble yeast lysate was obtained by centrifugation at 16,000 xg for 10 minutes, followed by filtration using a 0.2 pm syringe filter. For easy nomenclature, we named lysates of engineered yeast according to the growth factor or accessory protein the lysate is replacing, i.e. FGF2 lysate, albumin lysate, TGF-beta lysate, transferrin lysate and fetuin lysate.P7124PC00

[0311] Hibit Quantification, and Blotting

[0312] Growth factor expression or accessory protein expression was quantified using Nano-Glo Hibit Extracellular Detection System (Promega N2420) with a slight modification. Hibit Extracellular Reagent was prepared by diluting the LgBit protein and NanoGio Extracellular substrate in Nano-Gio Extracellular buffer at a ratio of 1:100 and 1:50 respectively. To quantify growth factor expression or accessory protein expression, a 1:1 mixture of Hibit Extracellular reagent and yeast lysate was prepared in a black clear bottom 96-well plate and incubated for 5 minutes on an orbital shaker. Luminescence was measured using a microplate reader (Synergy H1).

[0313] SDS-PAGE was performed on yeast lysate samples using 4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels. Separated protein gels were transferred to a nitrocellulose membrane (Thermofisher# IB23001) using Invitrogen™ iBIot™ 2 Gel Transfer Device at 20V for 7 minutes. Subsequently, the membrane was washed briefly with 1XTBST (20 mM Tris-HCI pH 7.6, 150 mM, 0.1% v / v Tween-20) and incubated in 10 mL Hibit Blotting Reagent consisting of 1:200 LgBit protein and 1X Nano-Gio blotting buffer respectively, for 1 hour under orbital shaking. 20 pL of Nano-Gio Luciferase assay substrate was introduced to the mixture and allowed to incubate for 2 minutes. CCD Chemiluminescent imaging of the membrane was performed using Amersham imager 680.

[0314] Bovine Satellite Cell Culture and Cell Counting

[0315] Satellite cells isolated from Holstein dairy cows (Danish Crown, Aalborg, Denmark) (Skrivergaard et al., 2021), immortalized satellite cells of Simmental calf origin (Tufts Cummings School of Veterinary Medicine) (Stout, Arnett, et al., 2023) as well as GFP-tagged satellite cells (Stout et al., 2020) were used in this work. All bovine satellite cells were maintained in a growth medium (Dulbecco's Modified Eagle Medium (Thermofisher# 10569010) supplemented with 10% FBS, 100 units / mL penicillin, and 0.1 mg / mL streptomycin). Growth medium for immortalized satellite cells was supplemented with 2.5 ug / mL puromycin. Routinely, 1 mL cryovials of bovine satellite cells were quickly thawed and resuspended in 9 mL growth medium in a 15 mL falcon tube. The cell suspension was centrifugated at 500 g for 5 minutes to remove cryoprotectant DMSO. The pellet was resuspended in 1 mL growth medium and seeded onto matrigel-coated T-25 / 75 plates and incubated at 37°C and 5% CO2.

[0316] Routine trypsinization was carried out using 0.25% Trypsin-EDTA (ThermofisherP7124PC00

[0317] #25200056) and cells were counted using NucleoCounter® NC-200™ automated cell counter.

[0318] Nuclei stain-based Cell Counting and Fluorescence-based Live Assay

[0319] Bovine satellite cells grown in 96-well format for 3-5 days in serum-containing and serum-free media were washed twice with phosphate-buffered saline (PBS without calcium or magnesium) and stained with 20 pM Hoechst 33342 solution (Thermofisher #62249) for 5-10 minutes. After incubation, the cells were washed with 1X PBS and imaged using Cytation 5 DAPI filter cube at 10x magnification. Whole well image analysis was performed on the stitched image using the Gen5 image analysis software.

[0320] For fluorescence-based cell counting, the growth of GFP-tagged satellite cells seeded into 96 well plates was monitored over time in the Incucyte S3 imager (Sartorius, UK). Eccentric fluorescent objects above 80 pm were counted as satellite cells and changes in morphology and cell number were monitored for 3-5 days.

[0321] Cell characterization

[0322] RNA was isolated and the expression of the proliferation-associated

[0323] markers Pax3 and MyoD was quantified using TaqMan assays, following the methodology described by Stout et al. (2022). Lipid accumulation in satellite cells was evaluated by Nile Red staining, with Nile Red (Thermo Fisher Scientific, #N1142) prepared in acetone according to the protocol of Greenspan et al. (1985). Myotube morphology was assessed through visualization of actin filaments, using Actin-555 ReadyProbes (Thermo Fisher Scientific, #R37112) for iBSC and Phalloidin-AF647 (1:400; Thermo Fisher Scientific, #A30107) for primary satellite cells. Continuous, label-free quantification of myotube formation was carried out using the Cytation 5 imaging system.

[0324] Example 2: Results 1

[0325] Lysates of Engineered Yeast Supports Satellite Cell Proliferation

[0326] To establish a growth profiling protocol for bovine satellite cells grown on lysates of Y. lipolytica cells (yeast lysates), we conducted a mock cell-counting experiment using satellite cells from Holstein dairy cow. The cells were seeded onto 96 well plates at a density of 2500 cells / cm2onto matrigel-coated plates in growth medium containing 10%P7124PC00

[0327] FBS and allowed to attach to the plate surface overnight. Subsequently, the media was removed and replaced with a preparation of Beefy-9 media in which FGF2 and albumin were each replaced with varying concentrations (0, 0.5%, 2%, and 5%) of their respective recombinant yeast lysates.

[0328] High contrast brightfield (HCBF) imaging and DNA-stain-based cell counting were impossible when the yeast lysate concentration in the media was 2% and 5% (data not shown). This is because at high concentrations, less soluble cellular constituents from yeast lysates precipitate and obscure the satellite cells at the bottom of plates from clear microscopic visualization. Yeast lysates also contain nucleic acids that are receptive to Hoechst DNA stain, making it difficult to differentiate between satellite cells and clumps of fragmented yeast nucleic acids, especially at high lysate concentrations. However, we observed relatively low interference from yeast cell constituents at a lower lysate concentration of 0.5% (Fig. 10). Therefore, we focused on elucidating the pro-proliferative effect of recombinant yeast lysates at this concentration.

[0329] Using HCBF-guided counting of Hoechst-stained satellite cells, we observed that lysates of Y. lipolytica engineered to express FGF2-G3 (FGF2-lysate) and serum albumin (albumin Lysate) abolished the need to add commercial bovine FGF2 or human albumin to the Beefy-9 media supplement without any obvious detriment to the bovine satellite cells - on the contrary, satellite cells count in recombinant lysates containing media exceed the level observed when the satellite cells were grown in 10% FBS (Fig. 1A, 1B).

[0330] Fluorescence Cytometry Enables Accurate Real-Time Growth Assay of Satellite Cells Cell counting based on nuclei staining and contrast-object counts are subject to interference from particulates in lysates of Y. lipolytica cells (yeast lysates). Fluorescent tagging of bovine satellite cells ensures that only bovine satellite cells are counted during growth assays in complex media formulations containing yeast lysates.

[0331] Moreover, fluorescent tagging allowed us to monitor the cell counts over time and eliminated bias in cell count resulting from non-uniform cell seeding.

[0332] Using GFP-tagged satellite cells, we confirmed that within the range of 0.25 to 1% (v / v) FGF2 lysate (comprising recombinantly produced FGF2-G3) and albumin lysate (comprising recombinantly produced serum albumin) can effectively replace FGF2 orP7124PC00

[0333] albumin in the Beefy-9 media formulation (Fig. 2A, 2B). Subsequently, we prepared lysates of Y. lipolytica strains expressing other protein components of the Beefy-9 media (i.e. TGF-beta and transferrin). All tested lysates, except for TGF-beta lysate (comprising recombinantly produced TGF-beta3), could substitute for their respective exogenous growth factor or accessory protein in supporting bovine satellite cell proliferation (Fig. 20, 2D). We attribute the difference seen with TGF-beta3 to its innate dual opposing effects - at low concentrations (< 5ng / mL), TGF-beta is known to be useful in media preparations to prevent myoblast differentiation, however, at higher concentrations, TGF-beta is known to exhibit a potent anti-proliferative effect.

[0334] Therefore, an intricate balancing of the level of TGF-beta3 expression level in Y. lipolytica by the use of a weaker and / or an inducible promoter may be advantageous to support bovine satellite cell proliferation.

[0335] Proteins of mammalian origin are notoriously rich in post-translational modifications (PTMs) such as disulfide bonds, phosphorylation, and glycosylation which are critical for protein folding and functionality. When expressed in non-native hosts such as lesser eukaryotes, these proteins may misfold and form insoluble inclusion bodies. The target proteins in this work are no exception, comprising multiple disulfide bonds, and glycosylation sites per protein. We set out to confirm the expression of the growth factors and accessory proteins in Y. lipolytica. For easy detection and quantification by luciferase assay and Hibit Blotting, we added the 11 amino acids Hibit-tag (SEQ ID NO: 11) to the N-terminal of the growth factors and accessory proteins. Fig. 3A-E shows that Y. lipolytica successfully expressed FGF2-G3, serum albumin, serotransferrin, TGF-beta3, and fetuin-A in a soluble form confirming the observed bioactive effects of the recombinant lysates on bovine satellite cells. Taken together, our findings show that lysates of growth factor-expressing Y. lipolytica can substitute for FBS in cultivating bovine satellite cells for cultivated meat production and other applications where crude mammalian biomass is required.

[0336] Complete Lysate-based Supplement Formulation by Automation-assisted Combinatorial Screening

[0337] We have shown that lysate of Y. lipolytica engineered to produce growth factor or accessory protein components of serum-free media can be used at low concentrations (0-1% v / v) in cell culture media formulations without the need for a protein purification step. However, this was done in a single-component (one factor at a time) dropoutP7124PC00

[0338] manner in which only one growth factor or accessory protein is supplied through the lysate and other constituents are exogenously added. To obtain a serum-free media supplement completely based on yeast lysate, we used automated liquid handling platforms (ECHO 525 Acoustic Liquid Handler and Mantiss Liquid Handler) to combine the lysates of all 5 growth factors or accessory proteins of interest (FGF2-G3, TGF-beta3, serotransferrin, serum albumin, and fetuin-A (introduced for cell attachment)), to reach a media formulation requiring zero, or minimal, amount of exogenously added growth factors or accessory proteins. Our initial results have shown some wellperforming combinations (Fig. 4).

[0339] Lysates from a Strain Engineered to Express Multiple Growth Factor and / or Accessory Proteins

[0340] Creating a media supplement by combining lysates of different growth factor or accessory protein producing strains poses the need for a separate bioprocess (glycerol stock maintenance, seed train, and bioreactor cultivation) for each strain - a strategy bound to be costly in the long term. However, a single strain producing all desired growth factors and accessory proteins will eliminate this challenge and make the use of engineered lysates as culture media supplements more cost-effective. Consequently, we constructed a single Y. lipolytica strain capable of expressing FGF2-G3, serotransferrin, serum albumin, and fetuin-A (hereon referred to as 4GF-coEX).

[0341] To evaluate the effect of lysates of the new strain on satellite cells, we performed a 6-day fluorescent cell counting-based growth assay of satellite cells propagated in basal media (DMEM / F12) containing 0-1% (v / v) concentration of 4GF-coEX lysates in the presence (Fig. 5A) and absence of TGF-beta3 supplementation (Fig 5B; 6A; 6B). We observed that 4GF-coEX lysates-based media preparations resulted in a remarkable growth of bovine satellite in both media, far exceeding the level observed for cells grown in 10% FBS and reaching similar level as growth medium (DMEM / F12 containing 10% FBS and 1ng / mL FGF2).

[0342] Example 3: Results 2

[0343] Further characterization of lysates of 4GF-coEX

[0344] To further assess cell proliferation of Y. lipolytica 4GF-coEX, we analyzed the expression of key myogenic markers, including the myogenic differentiation factor MyoD (Uniprot ID: Q7YS82) and the paired box transcription factor Pax3 (Uniprot ID:P7124PC00

[0345] F1MTX0). Elevated MyoD expression in bovine satellite cells cultured in 4GF-coEx-based media indicated active proliferation, while Pax3 expression confirmed maintenance of the satellite cell stem state under these conditions (Fig. 60, i-ii).

[0346] Preservation strategies for engineered lysates

[0347] Engineered yeast lysates function as bioactive supplements, and maintaining the integrity of their recombinant protein components is essential for sustained activity. Common protein preservation approaches include storage in polyols, such as glycerol, to mitigate damage from freeze-thaw cycles, or lyophilization to enable long-term storage at ambient temperatures. We evaluated the applicability of these strategies to 4GF-coEx lysates. Freshly prepared lysates were aliguoted and stored either in 50% glycerol at -20 °C or freeze-dried and maintained at room temperature. Lysate stability was subseguently assessed based on functional activity after four weeks of storage. Cell proliferation assays indicated that 4GF-coEx lysates retained full bioactivity following low-temperature storage, independent of glycerol addition (ANOVA, F = 3.33, p = 0.31) (Fig. 6D). Nonetheless, glycerol-based storage may be operationally advantageous, as it avoids repeated freeze-thaw cycles that can lead to ice crystal formation and protein denaturation during routine handling. Lyophilized lysates similarly preserved more than 85% of their initial bioactivity after storage (ANOVA, F = 3.33, p = 0.11), supporting the feasibility of low-energy, room-temperature storage for the engineered lysate.

[0348] Primary cells can grow on engineered yeast lysates

[0349] Although immortalized fluorescent satellite cells were valuable for robust cell guantification during optimization of media composition and storage conditions, a cultivated meat product is more likely to rely on primary satellite cells.

[0350] To assess the applicability of 4GF-coEx lysates for primary cell culture, we employed primary bovine satellite cells isolated from Holstein dairy cows. Cell proliferation in lysatebased media was compared with standard serum-containing (PGM) and serum-free (Tribasal 2.0+) formulations previously optimized for these cells.

[0351] Primary satellite cell growth was monitored over five days using time-course high-contrast brightfield (HCBF) imaging, followed by end-point Hoechst nuclear staining and guantification. Both analyses showed that lysate-based media supported significantlyP7124PC00

[0352] higher proliferation than all conventional media tested (ANOVA, F = 308.1, p < 0.0001) (Fig. 7A), indicating its potential as an alternative to serum-based formulations.

[0353] An extended culture of primary satellite cells in 4GF-coEx lysate-based media was subsequently performed to evaluate myogenic capacity. Differentiation was tracked over eight days using real-time myotube area quantification (MyoQuant) derived from established HCBF imaging and image analysis workflows. Morphological analysis and quantification of fluorescence intensity following phalloidin staining indicated that cells cultured in lysate-based media retained myogenic capacity and formed extensive myotubes.

[0354] Collectively, these results support the functional capacity of the engineered lysates to sustain satellite cell proliferation.

[0355] Sequence overview

[0356]

[0357] P7124PC00

[0358]

[0359] P7124PC00

[0360]

[0361] P7124PC00

[0362]

[0363] P7124PC00

[0364]

[0365] P7124PC00

[0366]

[0367] P7124PC00

[0368]

[0369] References

[0370] Holkenbrink et al. (2018). EasyCloneYALI: CRISPR / Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. Biotechnology Journal, 13(9).

[0371] Sinke et al. (2023). Ex-ante life cycle assessment of commercial-scale cultivated meat production in 2030. International Journal of Life Cycle Assessment, 28(3), 234-254. Skrivergaard et al. (2023). A simple and robust serum-free media for the proliferation of muscle cells. Food Research International, 172.

[0372] Stout et al. (2022). Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat. Communications Biology, 5(1).

[0373] Stout et al. (2023). A Beefy-R culture medium: Replacing albumin with rapeseed protein isolates. Biomaterials, 296.

[0374] Tuomisto et al. (2022). Prospective life cycle assessment of a bioprocess design for cultured meat production in hollow fiber bioreactors. Science of the Total Environment, 851.P7124PC00

[0375] Items

[0376] 1. A Yarrowia cell capable of producing one or more bovine growth-associated factors.

[0377] 2. The Yarrowia cell according to item 1 , wherein said one or more bovine growth- associated factors are fibroblast growth factor 2 (FGF2), transforming growth factorbeta (TGF-beta), transferrin, albumin, and / or fetuin.

[0378] 3. The Yarrowia cell according to any one of the preceding items, wherein said FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0379] 4. The Yarrowia cell according to any one of the preceding items, wherein said TGF- beta comprises or consists of TGF-beta3 as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0380] 5. The Yarrowia cell according to any one of the preceding items, wherein said transferrin comprises or consists of serotransferrin as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0381] 6. The Yarrowia cell according to any one of the preceding items, wherein said albumin comprises or consists of serum albumin as set forth in SEQ ID NO: 4, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0382] 7. The Yarrowia cell according to any one of the preceding items, wherein said fetuin comprises or consists of fetuin-A as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 70% sequence identity thereto, such as at least 75%,P7124PC00

[0383] such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity thereto.

[0384] 8. The Yarrowia cell according to any one of the preceding items, wherein said Yarrowia cell expresses:

[0385] i. FGF2-G3 comprising or consisting of SEQ ID NO: 1 , serotransferrin comprising or consisting of SEQ ID NO: 3, and serum albumin comprising or consisting of SEQ ID NO: 4; or

[0386] ii. FGF2-G3 comprising or consisting of SEQ ID NO: 1 , serotransferrin comprising or consisting of SEQ ID NO: 3, serum albumin comprising or consisting of SEQ ID NO: 4, and fetuin-A comprising or consisting of SEQ ID NO: 5;

[0387] or functional variants thereof having at least 70% sequence identity thereto.

[0388] 9. The Yarrowia cell according to any of the preceding items, wherein said Yarrowia cell expresses FGF2-G3 (SEQ ID NO: 1), serotransferrin (SEQ ID NO: 3), and serum albumin (SEQ ID NO: 4), or functional variants thereof having at least 70% sequence identity thereto.

[0389] 10. The Yarrowia cell according to any of the preceding items, wherein said Yarrowia cell expresses FGF2-G3 (SEQ ID NO: 1), serotransferrin (SEQ ID NO: 3), serum albumin (SEQ ID NO: 4), and fetuin-A (SEQ ID NO: 5), or functional variants thereof having at least 70% sequence identity thereto.

[0390] 11. The Yarrowia cell according to any one of the preceding items, wherein one or more of said bovine growth-associated factors are co-expressed within the same Yarrowia cell.

[0391] 12. The Yarrowia cell according to any one of the preceding items, wherein said Yarrowia cell comprises one or more of:

[0392] i. a nucleic acid encoding FGF2, such as a nucleic acid encoding a protein comprising or consisting of FGF2-G3 (SEQ ID NO: 1);

[0393] ii. a nucleic acid encoding TGF-beta, such as a nucleic acid encoding a protein comprising or consisting of a TGF-beta3 (SEQ ID NO: 2);P7124PC00

[0394] iii. a nucleic acid encoding transferrin, such as a nucleic acid encoding a protein comprising or consisting of serotransferrin (SEQ ID NO: 3);

[0395] iv. a nucleic acid encoding albumin, such as a nucleic acid encoding a protein comprising or consisting of serum albumin (SEQ ID NO: 4); and / or v. a nucleic acid encoding fetuin, such as a nucleic acid encoding a protein comprising or consisting of fetuin-A (SEQ ID NO: 5);

[0396] or functional variants thereof having at least 70% sequence identity thereto.

[0397] 13. The Yarrowia cell according to any one of the preceding items, wherein said Yarrowia cell expresses:

[0398] i. FGF2, optionally comprising or consisting of FGF2-G3 (SEQ ID NO: 1); ii. TGF-beta, optionally comprising or consisting of TGF-beta3 (SEQ ID NO:

[0399] 2);

[0400] iii. transferrin, optionally comprising or consisting of serotransferrin (SEQ ID NO: 3);

[0401] iv. albumin, optionally comprising or consisting of serum albumin (SEQ ID NO:

[0402] 4); and / or

[0403] v. fetuin, optionally comprising or consisting of fetuin-A (SEQ ID NO: 5); or functional variants thereof having at least 70% sequence identity thereto.

[0404] 14. The Yarrowia cell according to any of the preceding items, wherein said Yarrowia cell expresses:

[0405] i. FGF2, optionally comprising or consisting of FGF2-G3 (SEQ ID NO: 1); ii. transferrin, optionally comprising or consisting of serotransferrin (SEQ ID NO: 3); and

[0406] iii. albumin, optionally comprising or consisting of serum albumin (SEQ ID NO:

[0407] 4);

[0408] iv. and optionally fetuin, optionally comprising or consisting of fetuin-A (SEQ ID NO: 5);

[0409] or functional variants thereof having at least 70% sequence identity thereto.

[0410] 15. The Yarrowia cell according to any one of the preceding items, wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic.P7124PC00

[0411] 16. The Yarrowia cell according to any one of the preceding items, wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

[0412] 17. A Yarrowia lysate, wherein said lysate comprises one or more bovine growth- associated factors, such as FGF2, TGF-beta, transferrin, albumin, and / or fetuin.

[0413] 18. The Yarrowia lysate according to item 17, wherein:

[0414] i. FGF2 comprises or consists of FGF2-G3 (SEQ ID NO: 1);

[0415] ii. TGF-beta comprises or consists of TGF-beta3 (SEQ ID NO: 2);

[0416] iii. transferrin comprises or consists of serotransferrin (SEQ ID NO: 3);

[0417] iv. albumin comprises or consists of serum albumin (SEQ ID NO: 4); and / or v. fetuin comprises or consists of fetuin-A (SEQ ID NO: 5);

[0418] or functional variants thereof having at least 70% sequence identity thereto.

[0419] 19. The Yarrowia lysate according to any one of items 17 to 18, wherein the Yarrowia lysate is obtained from a Yarrowia cell, preferably the Yarrowia cell is as defined in any one of items 1 to 16.

[0420] 20. The Yarrowia lysate according to any one of items 17 to 19, wherein the lysate comprises or consists of a whole-cell lysate.

[0421] 21. The Yarrowia lysate according to any one of items 17 to 20, wherein the lysate comprises Yarrowia cell wall components, such as mannoproteins, glucans, mannan, chitin, glycoproteins, and / or DNA.

[0422] 22. The Yarrowia lysate according to any one of items 17 to 21 , wherein the Yarrowia lysate comprises Yarrowia derived fatty acids, such as oleic acid, and / or linoleic acid.

[0423] 23. A fetal bovine serum (FBS)-replacement composition comprising a Yarrowia lysate, wherein said composition comprises one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, and / or albumin.P7124PC00

[0424] 24. The FBS-replacement composition according to item 23, wherein said composition consists of a Yarrowia lysate and one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, and / or albumin.

[0425] 25. The FBS-replacement composition according to any one of items 23 to 24, wherein the Yarrowia lysate is as defined in any one of items 17 to 22.

[0426] 26. The FBS-replacement composition of item 23 to 25, wherein the Yarrowia lysate comprises one or more bovine growth-associated factors, such as FGF2, TGF- beta, transferrin, and / or albumin.

[0427] 27. The FBS-replacement composition according to item 23 to 26, wherein the Yarrowia lysate further comprises fetuin.

[0428] 28. The FBS-replacement composition according to any one of items 23 to 27, wherein:

[0429] i. FGF2 comprises or consists of FGF2-G3 (SEQ ID NO: 1);

[0430] ii. TGF-beta comprises or consists of TGF-beta3 (SEQ ID NO: 2);

[0431] iii. transferrin comprises or consists of serotransferrin (SEQ ID NO: 3);

[0432] iv. albumin comprises or consists of serum albumin (SEQ ID NO: 4); and / or v. fetuin comprises or consists of fetuin-A (SEQ ID NO: 5);

[0433] or functional variants thereof having at least 70% sequence identity thereto.

[0434] 29. The FBS-replacement composition according to any one of items 23 to 28, comprising lysates of at least two different Yarrowia populations, wherein said at least two Yarrowia populations each produce at least one different bovine growth- associated factor and each Yarrowia population consists of Yarrowia cells as defined in any one of items 1 to 16.

[0435] 30. The FBS-replacement composition according to any one of items 23 to 29, wherein the Yarrowia lysate comprises at least three bovine growth associated factors, wherein said at least three bovine growth associated factors comprise:

[0436] i. FGF2, optionally wherein said FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1;

[0437] ii. Transferrin, optionally wherein said transferrin comprises or consists of serotransferrin, as set forth in SEQ ID NO: 3;P7124PC00

[0438] iii. Albumin, optionally wherein said albumin comprises or consists of serum albumin, as set forth in SEQ ID NO: 4;

[0439] or functional variants thereof having at least 70% sequence identity thereto, and optionally wherein said FBS-replacement composition further comprises fetuin, optionally wherein said fetuin comprises or consists of fetuin-A, as set forth in SEQ ID NO: 5, or a functional variant thereof having at least 70% sequence identity thereto.

[0440] 31. A mammalian cell or fish cell culture medium composition, wherein said mammalian cell or fish cell culture medium composition comprises the FBS-replacement composition as defined in any one of items 23 to 30.

[0441] 32. The mammalian cell and / or fish cell culture medium composition according to item 31, comprising the FBS-replacement composition according to items 23 to 30, comprises in the between 0.25%v / v and 1%v / v FBS-replacement composition, such as between 0.5%v / v and 1%v / v FBS-replacement composition, such as between 0.75%v / v and 1%v / v FBS-replacement composition.

[0442] 33. A method of obtaining one or more bovine growth-associated factors, comprising:

[0443] i. providing the Yarrowia cell as defined in any one of items 1 to 16;

[0444] ii. incubating and optionally propagating said Yarrowia cell in a medium, thereby producing one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, albumin and / or fetuin; and

[0445] iii. optionally, recovering said one or more bovine growth-associated factors, whereby said one or more bovine growth-associated factors are obtained, preferably wherein said one or more bovine growth-associated factors are as defined in any one of items 2 to 7.

[0446] 34. A method of producing a Yarrowia lysate, comprising:

[0447] i. providing a Yarrowia cell as defined in any one of items 1 to 16;

[0448] ii. propagating said Yarrowia cell in a medium,

[0449] thereby obtaining a Yarrowia cell biomass; and

[0450] iii. lysing said Yarrowia cell biomass to produce said Yarrowia lysate; whereby a Yarrowia lysate is produced,

[0451] preferably wherein said Yarrowia lysate is as defined in any one of items 17 to 22.P7124PC00

[0452] 35. A method for producing an FBS-replacement composition comprising a Yarrowia lysate and one or more bovine growth-associated factors, comprising:

[0453] i. providing a Yarrowia cell as defined in any one of items 1 to 16;

[0454] ii. propagating said Yarrowia cell in a medium,

[0455] thereby obtaining Yarrowia cell biomass;

[0456] iii. recovering and / or preparing a lysate of said Yarrowia cell biomass,

[0457] thereby obtaining a Yarrowia lysate; and

[0458] iv. optionally, combining one or more Yarrowia lysates obtained in step iii.; v. formulating the one or more Yarrowia lysates of step iii. and / or iv. into an FBS-replacement composition;

[0459] whereby said FBS-replacement composition is produced.

[0460] 36. The method according to any one of items 34 to 35, wherein the step of recovering said Yarrowia cell biomass comprises centrifugation.

[0461] 37. The method according to any one of items 34 to 36, wherein the step of preparing a lysate of said Yarrowia cell comprises cell homogenisation, such as by bead beating and / or by cooling cycles, and optionally resuspension in an alkaline buffer, or such as by alternative mechanical means of cell lysis, such as by sonication or by hydraulic French press.

[0462] 38. A method for cultivation of mammalian cells and / or fish cells using said FBS- replacement composition, as defined in any one of items 23 to 30, comprising: i. providing a FBS-replacement composition as defined in any one of items 23 to 30;

[0463] ii. mixing said FBS-replacement composition with a culture medium suitable for cultivation of said mammalian cells and / or said fish cells, thereby obtaining a mixture; and

[0464] iii. using said mixture to cultivate mammalian cells and / or fish cells.

[0465] 39. The method according to item 38, wherein the mammalian cells and / or fish cells are cultivated in vitro.

[0466] 40. The method according to any one of items 38 to 39, wherein the mammalian cells and / or fish cells are cultivated in suspension.P7124PC00

[0467] 41. The method according to any one of items 38 to 40, wherein the mammalian cells and / or fish cells are cultivated attached to a surface.

[0468] 42. The method according to any one of items 38 to 41 , wherein the mammalian cells comprise muscle cells, satellite cells, mammary epithelial cells, adipose cells and / or fibroblast cells.

[0469] 43. The method according to any one of items 38 to 42, wherein the mammalian cells are any one of bovine cells, human cells, equine cells, murine cells, goat cells, sheep cells, chicken cells, porcine cells, or lapine cells, preferably the mammalian cells are primary bovine cells, such as primary satellite bovine cells.

[0470] 44. A method for producing a food composition, comprising:

[0471] i. providing a Yarrowia lysate;

[0472] ii. formulating said Yarrowia lysate into an FBS-replacement composition; iii. cultivating mammalian cells and / or fish cells in the presence of said FBS- replacement composition, optionally further comprising a culture medium, thereby obtaining a biomass of mammalian cells and / or fish cells; and iv. formulating said biomass of mammalian cells and / or fish cells into a food composition,

[0473] thereby producing said food composition.

[0474] 45. The method according to item 44, wherein said Yarrowia lysate is as defined in any one of items 17 to 22.

[0475] 46. The method according to any one of items 44 to 45, wherein said FBS-replacement composition is as defined in any one of items 23 to 30.

[0476] 47. The method according to item 44, wherein step iii is performed according to any one of items 38 to 43.

[0477] 48. Use of the Yarrowia lysate as defined in any one of items 17 to 22 and / or the FBS- replacement composition as defined in any one of items 23 to 30 for cultivating mammalian cells and / or fish cells, preferably in combination with a culture medium.P7124PC00

[0478] 49. Use of mammalian cells and / or fish cells obtainable by a method according to any one of items 38 to 43, wherein the mammalian cells and / or fish cells are used as a food product, food ingredient, and / or food material.

[0479] 50. A food composition comprising mammalian cells and / or fish cells obtainable by a method according to any one of items 38 to 43.

[0480] 51. The food composition as according to item 50, wherein said food composition is a cultivated meat and / or ingredient therefor.

[0481] 52. Use of the food composition as defined in item 50, as a food product, food material, and / or food ingredient.

[0482] 53. Use of the food composition as defined in item 50 in a cultivated meat and / or in a method for producing a meat alternative.

[0483] 54. A cultivated meat and / or ingredient therefor comprising mammalian cells and / or fish cells obtainable by a method according to any one of items 38 to 43.

[0484] 55. A nucleic acid encoding a polypeptide comprising or consisting of FGF2-G3 as set forth in SEQ I D NO: 1 , such as a nucleic acid comprising or consisting of SEQ I D NO: 6 (FGF2-G3), or a functional variant thereof having at least 70% sequence identity thereto.

[0485] 56. A nucleic acid encoding a polypeptide comprising or consisting of TGF-beta3 as set forth in SEQ ID NO: 2, such as a nucleic acid comprising or consisting of SEQ ID NO: 7 (TGF-beta3), or a functional variant thereof having at least 70% sequence identity thereto.

[0486] 57. A nucleic acid encoding a polypeptide comprising or consisting of serotransferrin as set forth in SEQ ID NO: 3, such as a nucleic acid comprising or consisting of SEQ ID NO: 8 (serotransferrin), or a functional variant thereof having at least 70% sequence identity thereto.P7124PC00

[0487] 58. A nucleic acid encoding a polypeptide comprising or consisting of serum albumin as set forth in SEQ ID NO: 4, such as a nucleic acid comprising or consisting of SEQ ID NO: 9 (serum albumin), or a functional variant thereof having at least 70% sequence identity thereto.

[0488] 59. A nucleic acid encoding a polypeptide comprising or consisting of fetuin-A as set forth in SEQ ID NO: 5, such as a nucleic acid comprising or consisting of SEQ ID NO: 10 (fetuin-A), or a functional variant thereof having at least 70% sequence identity thereto.

[0489] 60. The nucleic acid according to any one of items 55 to 59, wherein the nucleic acid further comprises at least one promoter, such as at least one constitutive promoter or at least one inducible promoter.

[0490] 61. The nucleic acid according to item 60, wherein the promoter is a strong promoter, for example pL41 (SEQ ID NO: 13) or pTEF-intron (SEQ ID NO: 14), or a weak promoter, for example pASN2 (SEQ ID NO: 15).

[0491] 62. The nucleic acid according to any one of items 55 to 61 , wherein the nucleic acid is codon-optimised for expression in a Yarrowia cell as defined in any one of items 1 to 16.

[0492] 63. The nucleic acid according to any one of items 55 to 62, wherein said nucleic acid further comprises a purification tag, preferably wherein said purification tag is operably linked to said bovine growth-associated factor, optionally wherein said purification tag is as set forth in SEQ ID NO: 12.

[0493] 64. An expression system for expression in a Yarrowia cell, comprising one or more:

[0494] i. nucleic acid as defined in any one of items 55 or 60 to 63;

[0495] ii. nucleic acid as defined in any one of items 56 or 60 to 63;

[0496] iii. nucleic acid as defined in any one of items 57 or 60 to 63;

[0497] iv. nucleic acid as defined in any one of items 58 or 60 to 63;

[0498] v. nucleic acid as defined in any one of items 59 to 63,

[0499] or functional variants thereof having at least 70% sequence identity thereto.P7124PC00

[0500] 65. The expression system of item 64, wherein the nucleic acid comprises or consists of any of the nucleic acids as defined in any one of items 55 to 63.

[0501] 66. The expression system according to any one of the items 64 to 65, wherein the Yarrowia cell is as defined in any one of items 1 to 16.

[0502] 67. The Yarrowia cell, the lysate, FBS-replacement composition, the method, and / or the use according to any one of items 1 to 53, wherein said Yarrowia cell comprises the expression system as defined in any one of items 64 to 66, whereby said Yarrowia cell is capable of producing one or more bovine growth-associated factors.

[0503] 68. A kit of parts, comprising:

[0504] i. a Yarrowia cell;

[0505] ii. at least one nucleic acid as defined in any one of the items 55 to 63; and / or iii. the expression system as defined in any one of items 64 to 66; and

[0506] iv. optionally instructions for use;

[0507] preferably wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic, and / or wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

[0508] 3. A kit of parts, comprising:

[0509] i. a Yarrowia cell;

[0510] ii. at least one nucleic acid encoding at least one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5, or functional variants thereof having at least 70% sequence identity thereto; and

[0511] iii. optionally instructions for use;

[0512] preferably wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic, and / or wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

Claims

67P7124PC00Claims1. A Yarrowia lysate comprising one or more bovine growth-associated factors, such as fibroblast growth factor 2 (FGF2), transforming growth factor-beta (TGF-beta), transferrin, albumin, and / or fetuin.

2. The Yarrowia lysate according to claim 1 , wherein the one or more bovine growth- associated factors are FGF2, TGF-beta, transferrin, albumin, and / or fetuin.

3. The Yarrowia lysate according to any one of the preceding claims, wherein the Yarrowia lysate is obtained from a Yarrowia cell capable of producing the one or more bovine growth-associated factors, preferably wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

4. The Yarrowia lysate according to any one of the preceding claims, wherein:i. said FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1; ii. said TGF-beta comprises or consists of TGF-beta3 as set forth in SEQ ID NO: 2;iii. said transferrin comprises or consists of serotransferrin as set forth in SEQ ID NO: 3;iv. said albumin comprises or consists of serum albumin as set forth in SEQ ID NO: 4; and / orv. said fetuin comprises or consists of fetuin-A as set forth in SEQ ID NO: 5; or functional variants thereof having at least 70% sequence identity thereto.

5. The Yarrowia lysate according to any of the preceding claims, wherein said Yarrowia cell expresses:i. FGF2, optionally comprising or consisting of FGF2-G3 (SEQ ID NO: 1); ii. transferrin, optionally comprising or consisting of serotransferrin (SEQ ID NO: 3); andiii. albumin, optionally comprising or consisting of serum albumin (SEQ ID NO:4);or functional variants thereof having at least 70% sequence identity thereto.68P7124PC006. The Yarrowia lysate according to any of the preceding claims, wherein said Yarrowia cell expresses:i. FGF2, optionally comprising or consisting of FGF2-G3 (SEQ ID NO: 1); ii. transferrin, optionally comprising or consisting of serotransferrin (SEQ ID NO: 3);iii. albumin, optionally comprising or consisting of serum albumin (SEQ ID NO:4); andiv. fetuin, optionally comprising or consisting of fetuin-A (SEQ ID NO: 5); or functional variants thereof having at least 70% sequence identity thereto.

7. The Yarrowia lysate according to any of the preceding claims, wherein said Yarrowia cell expresses FGF2-G3 (SEQ ID NO: 1), serotransferrin (SEQ ID NO: 3), and serum albumin (SEQ ID NO: 4), or functional variants thereof having at least 70% sequence identity thereto.

8. The Yarrowia lysate according to any of the preceding claims, wherein said Yarrowia cell expresses FGF2-G3 (SEQ ID NO: 1), serotransferrin (SEQ ID NO: 3), serum albumin (SEQ ID NO: 4), and fetuin-A (SEQ ID NO: 5), or functional variants thereof having at least 70% sequence identity thereto.

9. The Yarrowia lysate according to any of the preceding claims, wherein said Yarrowia cell expresses:i. FGF2-G3 comprising or consisting of SEQ ID NO: 1 , serotransferrin comprising or consisting of SEQ ID NO: 3, and serum albumin comprising or consisting of SEQ ID NO: 4; orii. FGF2-G3 comprising or consisting of SEQ ID NO: 1 , serotransferrin comprising or consisting of SEQ ID NO: 3, serum albumin comprising or consisting of SEQ ID NO: 4, and fetuin-A comprising or consisting of SEQ ID NO: 5;or functional variants thereof having at least 70% sequence identity thereto.

10. The Yarrowia lysate according to any one of the preceding claims, wherein the lysate comprises or consists of a whole-cell lysate, preferably wherein the lysate comprises Yarrowia DNA, cell wall components, such as mannoproteins, glucans,69P7124PC00mannan, chitin, glycoproteins, and / or Yarrowia derived fatty acids, such as oleic acid, and / or linoleic acid.

11. A fetal bovine serum (FBS)-replacement composition comprising a Yarrowia lysate, wherein said composition comprises one or more bovine growth-associated factors, such as FGF2, TGF-beta, transferrin, and / or albumin,preferably wherein the Yarrowia lysate is as defined in any one of claims 1 to 10.

12. The FBS-replacement composition according to claim 11, wherein:i. FGF2 comprises or consists of FGF2-G3 (SEQ ID NO: 1);ii. TGF-beta comprises or consists of TGF-beta3 (SEQ ID NO: 2);iii. transferrin comprises or consists of serotransferrin (SEQ ID NO: 3);iv. albumin comprises or consists of serum albumin (SEQ ID NO: 4); and / or v. fetuin comprises or consists of fetuin-A (SEQ ID NO: 5);or functional variants thereof having at least 70% sequence identity thereto.

13. The FBS-replacement composition according to any one of claims 11 to 12, wherein the Yarrowia lysate comprises at least three bovine growth associated factors, wherein said at least three bovine growth associated factors comprise: i. FGF2, optionally wherein said FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1;ii. transferrin, optionally wherein said transferrin comprises or consists of serotransferrin, as set forth in SEQ ID NO: 3; andiii. albumin, optionally wherein said albumin comprises or consists of serum albumin, as set forth in SEQ ID NO: 4;or functional variants thereof having at least 70% sequence identity thereto.

14. The FBS-replacement composition according to any one of claims 11 to 13, wherein the Yarrowia lysate comprises at least four bovine growth associated factors, wherein said at least four bovine growth associated factors comprise: i. FGF2, optionally wherein said FGF2 comprises or consists of FGF2-G3 as set forth in SEQ ID NO: 1;ii. transferrin, optionally wherein said transferrin comprises or consists of serotransferrin, as set forth in SEQ ID NO: 3;70P7124PC00iii. albumin, optionally wherein said albumin comprises or consists of serum albumin, as set forth in SEQ ID NO: 4; andiv. fetuin, optionally wherein said fetuin comprises or consists of fetuin-A, as set forth in SEQ ID NO: 5,or functional variants thereof having at least 70% sequence identity thereto.

15. The FBS-replacement composition according to any one of claims 11 to 14, comprising lysates of at least two different Yarrowia populations, wherein said at least two Yarrowia populations each produce at least one different bovine growth- associated factor and each Yarrowia population consists of Yarrowia cells as defined in any one of claims 2 to 9.

16. The FBS-replacement composition according to any one of claims 11 to 15, wherein the FBS-replacement composition further comprises exogenous TGF-beta.

17. A Yarrowia cell as defined in any one of claims 2 to 9.

18. A method of producing a Yarrowia lysate, comprising:i. providing a Yarrowia cell as defined in any one of claims 2 to 9;ii. propagating said Yarrowia cell in a medium,thereby obtaining a Yarrowia cell biomass; andiii. lysing said Yarrowia cell biomass to produce said Yarrowia lysate, whereby said Yarrowia lysate is produced;preferably wherein said Yarrowia lysate is as defined in any one of claims 1 to 10.

19. A method for producing an FBS-replacement composition comprising a Yarrowia lysate and one or more bovine growth-associated factors, comprising:i. providing a Yarrowia cell as defined in any one of claims 2 to 9;ii. propagating said Yarrowia cell in a medium, thereby obtaining Yarrowia cell biomass;iii. recovering and / or preparing a lysate of said Yarrowia cell biomass, thereby obtaining a Yarrowia lysate; andiv. optionally, combining one or more Yarrowia lysates obtained in step iii.; v. formulating the one or more Yarrowia lysates of step iii. and / or iv. into an FBS-replacement composition;71P7124PC00whereby said FBS-replacement composition is produced, optionally wherein the step of recovering said Yarrowia cell biomass comprises centrifugation and / or wherein the step of preparing a lysate of said Yarrowia cell comprises cell homogenisation, such as by bead beating and / or by cooling cycles, and optionally resuspension in an alkaline buffer, or such as by alternative mechanical means of cell lysis, such as by sonication or by hydraulic French press.

20. An expression system for expression in a Yarrowia cell, comprising two or more of:i. a nucleic acid encoding a polypeptide comprising or consisting of FGF2-G3 as set forth in SEQ ID NO: 1, such as a nucleic acid comprising or consisting of SEQ ID NO: 6 (FGF2-G3);ii. a nucleic acid encoding a polypeptide comprising or consisting of TGF- beta3 as set forth in SEQ ID NO: 2, such as a nucleic acid comprising or consisting of SEQ ID NO: 7 (TGF-beta3yiii. a nucleic acid encoding a polypeptide comprising or consisting of serotransferrin as set forth in SEQ ID NO: 3, such as a nucleic acid comprising or consisting of SEQ ID NO: 8 (serotransferrinyiv. a nucleic acid encoding a polypeptide comprising or consisting of serum albumin as set forth in SEQ ID NO: 4, such as a nucleic acid comprising or consisting of SEQ ID NO: 9 (serum albumin andv. a nucleic acid encoding a polypeptide comprising or consisting of fetuin-A as set forth in SEQ ID NO: 5, such as a nucleic acid comprising or consisting of SEQ ID NO: 10 (fetuin-Ayor functional variants thereof having at least 70% sequence identity thereto.

21. A kit of parts, comprising:i. a Yarrowia cell;ii. at least one nucleic acid encoding at least one of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5, or functional variants thereof having at least 70% sequence identity thereto; andiii. optionally instructions for use;preferably wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic, and / or wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.72P7124PC0022. A kit of parts, comprising:i. a Yarrowia cell;ii. at least one nucleic acid as defined in any one of the claim 20; and / or iii. the expression system as defined in claim 20; andiv. optionally instructions for use;preferably wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, has qualified presumption of safety (QPS) status, and / or is non-pathogenic, and / or wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica.

23. A method of obtaining one or more bovine growth-associated factors, comprising: i. providing the Yarrowia cell as defined in any one of claims 2 to 9;ii. incubating and optionally propagating said Yarrowia cell in a medium, thereby producing one or more bovine growth-associated factors, such as FGF2, TGF- beta, transferrin, albumin and / or fetuin; andiii. optionally, recovering said one or more bovine growth-associated factors, whereby said one or more bovine growth-associated factors are obtained, preferably wherein said one or more bovine growth-associated factors are as defined in any one of claims 2 to 16.

24. A mammalian cell and / or fish cell culture medium composition, wherein said mammalian cell and / or fish cell culture medium composition comprises the FBS- replacement composition as defined in any one of claims 11 to 16.

25. A method for cultivation of mammalian cells and / or fish cells using said FBS- replacement composition, as defined in any one of claims 11 to 16, comprising: i. providing a FBS-replacement composition as defined in any one of claims 11 to 16;ii. mixing said FBS-replacement composition with a culture medium suitable for cultivation of said mammalian cells and / or said fish cells, thereby obtaining a mixture; andiii. using said mixture to cultivate mammalian cells and / or fish cells.

26. A method for producing a food composition, comprising:P7124PC00i. providing a Yarrowia lysate, preferably a Yarrowia lysate as defined in any one of claims 1 to 10;ii. formulating said Yarrowia lysate into an FBS-replacement composition, preferably an FBS-replacement composition as defined in any one of claims 11 to 16;iii. cultivating mammalian cells and / or fish cells in the presence of said FBS- replacement composition, optionally further comprising a culture medium, thereby obtaining a biomass of mammalian cells and / or fish cells; and iv. formulating said biomass of mammalian cells and / or fish cells into a food composition,thereby producing said food composition.

27. Use of the Yarrowia lysate as defined in any one of claims 1 to 10 and / or the FBS- replacement composition as defined in any one of claims 11 to 16 for cultivating mammalian cells and / or fish cells, preferably in combination with a culture medium.

28. Use of mammalian cells and / or fish cells obtainable by a method according to claim 25, wherein the mammalian cells and / or fish cells are used as a food product, food ingredient, and / or food material.

29. A food composition comprising mammalian cells and / or fish cells obtainable by a method according to claim 25.

30. Use of the food composition as defined in claim 29, as a food product, food material, and / or food ingredient.

31. Use of the food composition as defined in claim 29 in a cultivated meat and / or in a method for producing a meat alternative.

32. A cultivated meat and / or ingredient therefor comprising mammalian cells and / or fish cells obtainable by a method according to claim 25.

33. The method, use, food composition and / or cultivated meat according to any one of the preceding claims, wherein the mammalian cell are any one of bovine cells, human cells, equine cells, murine cells, goat cells, sheep cells, chicken cells, ,P7124PC00porcine cells, or lapine cells, preferably the mammalian cells are primary bovine cells, such as primary satellite bovine cells.

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