FOR PROTEIN EXPRESSION

RELATED APPLICATION

[001] This application claims the benefit of Australian provisional patent application number 2022902508, filed on 1 September 2022, and of US provisional patent application number 63/535043, filed on 28 August 2023, which are hereby incorporated by reference herein in their entirety.

FIELD

[002] The present disclosure relates generally to microbial strains and their preparation for growth and protein expression. More specifically, the present disclosure relates to expression systems for microbial strains, various growth media and methodology for achieving expression from these strains, as well as expressed proteins obtained from these strains and products obtained therefrom.

BACKGROUND

[003] It is estimated that more than 35 million tonnes of food in the United States ends up in landfill each year (EPA: Facts and figures about materials, waste and recycling; see https://www.epa.gov). At least 7.6 tonnes of food in Australia is lost or wasted each year (Foodbank AU: Food waste facts; see https://www.foodbank.org.au). Food ends up in landfills worldwide despite some attempts to utilise food waste in animal feed, biofuels, and fertilisers.

[004] In addition to economic losses, food in landfills create methane gas, which contributes to climate change. According to the World Wildlife Federation, 6-8% of all human- caused greenhouse gas emissions could be reduced if food wastage were addressed (WWF: Fight climate change by preventing food waste; see https://www.worldwildlife.org/stories/).

[005] Food production is also a significant contributor to climate change. As a key example, global demand for dairy continues to increase in large part due to population growth, rising incomes, urbanisation and westernisation of diets in countries such as China and India. Currently, there are at least 270 million dairy cows worldwide, and with growing demand, there are increasing greenhouse gas production and increasing threats to natural resources, including freshwater and soil (WWF: Sustainable agriculture - Dairy; see https://www.worldwildlife. org/industries/dairy) . [006] Dairy cows produce methane gas, nitrous oxide, and carbon dioxide, all of which promote climate change. Globally, dairy farming is said to be responsible for the equivalent of about 2 billion metric tons of carbon dioxide per year (Food and Agriculture Organization of the United Nations, 2013, Tackling climate change through livestock: A global assessment of emissions and mitigation opportunities). Unsustainable dairy farming and feed production can lead to the loss of ecologically important areas, such as prairies, wetlands, and forests. Dairy operations can also contribute to water pollution and soil deterioration when manure, fertilisers, and feed crops are badly managed (WWF: Sustainable agriculture — Dairy; see https://www.worldwildlife.org/industries/dairy).

[007] Certain microorganisms have been tested in relation to waste mitigation (e.g., bacteria; see https://techcrunch.com/2020/03/16/yc-graduate-genecis-bioind ustries-turns- food-waste-into-compostable-plastics/), and specific microorganisms have been tested in relation to the production of recombinant proteins (e.g., yeast; see Vandermies and Fickers, 2019, Bioreactor-scale strategies for the production of recombinant protein in the yeast Yarrowia lipolytica Microorganisms 7:40; doi.org/10.3390/microorganisms7020040). However, to our knowledge, no research groups have connected these pathways.

[008] Agricultural and food waste has been used for the production of ethanol, polysaccharides, fatty-acids, pigments, hydrolytic enzymes and other value-added products, but not for the production of high-quality food proteins. For example, in relation to microbial species such as Trichoderma, Kluyveromyces, and Pichia, there is no available information as to how waste media influences recombinant protein expression, particularly media obtained from food waste. Yet, this is an important area to explore, as it provides a locus where waste mitigation and protein production intersect. The ability to produce useful food constituents from these microbes, using a substrate considered a waste product from another industry, provides a circular method of operation having sustainability and greenhouse gas mitigation at its core.

[009] The present disclosure seeks to address these needs or at least to provide the public with a useful alternative.

SUMMARY

[0010] As described herein, the present inventors have identified waste substrates that have surprising efficacy for the growth of microbial strains. Also identified are highly effective compositions and methods for obtaining expression from these organisms. Notably, this is the first research performed on the creation of high-value consumer products and ingredients from food waste. This disclosure thereby provides compositions, systems, methods that enable food waste to be converted into beneficial dairy proteins, including whey proteins and casein proteins, which can then be used for producing animal-free dairy products, such as yoghurts, ice creams and cheeses, amongst others.

[0011] In one particular aspect, the present disclosure encompasses a polypeptide expression system comprising (i) at least one microbial cell which is capable of expressing one or more dairy proteins, and (ii) a food waste substrate for growing the at least one microbial cell.

[0012] In various aspects:

[0013] The food waste substrate consists essentially of an aqueous extract obtained from food waste.

[0014] The food waste is waste from plant material.

[0015] The food waste is selected from the group consisting of fruit waste material, vegetable waste material, nut or grain waste material, and any combination thereof.

[0016] The fruit waste material includes one or more of flesh, pulp, and skin of a fruit.

[0017] The vegetable waste material includes one or more of flesh, leaf, root, and skin of a vegetable.

[0018] The food waste is selected from cruciferous vegetable waste material, tuber vegetable waste material, root vegetable waste material, berry fruit waste material, and any combination thereof.

[0019] The food waste is selected from the group consisting of broccoli waste material, brussels sprouts waste material, cabbage waste material, cauliflower waste material, eggplant waste material, leafy greens waste material, carrot waste material, parsnips waste material, kumara waste material, yam waste material, oca waste material, and any combination thereof.

[0020] The food waste is selected from the group consisting of apple waste material, grape waste material, olive waste material, kiwifruit waste material, and any combination thereof.

[0021] The food waste is beverage processing waste material. [0022] The beverage processing waste material is selected from the group consisting of fruit pomace, grape marc, spent grain, and any combination thereof.

[0023] The at least one microbial cell is a yeast cell, a fungal cell, a bacterial cell, or any combination thereof.

[0024] The at least one microbial cell is at least one of a Trichoderma cell, a Pichia cell, a Kluyveromyces cell, or any combination thereof.

[0025] The at least one Trichoderma cell is selected from the group consisting of Trichoderma reesei, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma atroviride, Trichoderma virens, Trichoderma citrinoviride, Trichoderma viride, and any combination thereof.

[0026] The at least one Pichia cell is selected from the group consisting of Pichia pastoris, Pichia finlandic a, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, and any combination thereof.

[0027] The at least one Kluyveromyces cell is a cell selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, and Kluyveromyces thermotolerans, and any combination thereof.

[0028] The at least one microbial cell contains at least one polynucleotide capable of expressing the dairy protein(s).

[0029] The at least one polynucleotide is codon optimised for expression in the cell.

[0030] The at least one polynucleotide encodes a fusion protein.

[0031] The at least one polynucleotide encodes a protein fragment.

[0032] The one or more dairy proteins comprise one or more cow milk proteins.

[0033] The one or more dairy proteins are selected from the group consisting of whey proteins and casein proteins, and any combination thereof.

[0034] The whey proteins are selected from the group consisting of α-lactalbumin,β- lactoglobulin, lactotransferrin/lactoferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, glycomacropeptide, and any combination thereof. [0035] The casein proteins are selected from the group consisting ofβ- casein, K-casein, α-S1-casein, α-S2-casein, and any combination thereof.

[0036] In still one other aspect, the present disclosure encompasses a method for producing a dairy protein comprising: growing at least one microbial cell capable of expressing one or more dairy proteins under conditions to obtain expression of the one or more dairy proteins, wherein the at least one microbial cell is in and/or on a food waste substrate for growing the at least one microbial cell.

[0037] In various aspects:

[0038] The food waste substrate consists essentially of an aqueous extract obtained from food waste.

[0039] The food waste is waste from plant material.

[0040] The food waste is selected from the group consisting of fruit waste material, vegetable waste material, nut or grain waste material, and any combination thereof.

[0041] The fruit waste material includes one or more of flesh, pulp, and skin of a fruit.

[0042] The vegetable waste material includes one or more of flesh, leaf, root, and skin of a vegetable.

[0043] The food waste is selected from cruciferous vegetable waste material, tuber vegetable waste material, root vegetable waste material, berry fruit waste material, and any combination thereof.

[0044] The food waste is selected from the group consisting of broccoli waste material, brussels sprouts waste material, cabbage waste material, cauliflower waste material, eggplant waste material, leafy greens waste material, carrot waste material, parsnips waste material, kumara waste material, yam waste material, oca waste material, and any combination thereof.

[0045] The food waste is selected from the group consisting of apple waste material, grape waste material, olive waste material, kiwifruit waste material, and any combination thereof.

[0046] The food waste is beverage processing waste material.

[0047] The beverage processing waste material is selected from the group consisting of fruit pomace, grape marc, spent grain, and any combination thereof. [0048] The at least one microbial cell is a yeast cell, a fungal cell, a bacterial cell, or any combination thereof.

[0049] The at least one microbial cell is at least one of a Trichoderma cell, a Pichia cell, a Kluyveromyces cell, or any combination thereof.

[0050] The at least one Trichoderma cell is selected from the group consisting of Trichoderma reesei, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma atroviride, Trichoderma virens, Trichoderma citrinoviride, Trichoderma viride, and any combination thereof.

[0051] The at least one Pichia cell is selected from the group consisting of Pichia pastoris, Pichia finlandic a, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, and any combination thereof.

[0052] The at least one Kluyveromyces cell is a cell selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, and Kluyveromyces thermotolerans, and any combination thereof.

[0053] The at least one microbial cell contains at least one polynucleotide capable of expressing the dairy protein(s).

[0054] The at least one polynucleotide is codon optimised for expression in the cell.

[0055] The at least one polynucleotide encodes a fusion protein.

[0056] The at least one polynucleotide encodes a protein fragment.

[0057] The one or more dairy proteins comprise one or more cow milk proteins.

[0058] The one or more dairy proteins are selected from the group consisting of whey proteins and casein proteins, and any combination thereof.

[0059] The whey proteins are selected from the group consisting of α-lactalbumin,β- lactoglobulin, lactotransferrin/lactoferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, glycomacropeptide, and any combination thereof.

[0060] The casein proteins are selected from the group consisting of β-casein, K-casein, α-Sl-casein, α-S2-casein, and any combination thereof. [0061] In one further aspect, the present disclosure encompasses at least one microbial cell capable of expressing one or more dairy proteins, the at least one microbial cell being in and/or on a food waste substrate for growing the at least one microbial cell.

[0062] In various aspects:

[0063] The food waste substrate consists essentially of an aqueous extract obtained from food waste.

[0064] The food waste is waste from plant material.

[0065] The food waste is selected from the group consisting of fruit waste material, vegetable waste material, nut or grain waste material, and any combination thereof.

[0066] The food waste is fruit waste material and/or vegetable waste material.

[0067] The fruit waste material includes one or more of flesh, pulp, and skin of the fruit.

[0068] The vegetable waste material includes one or more of flesh, leaf, root, and skin of the vegetable.

[0069] The food waste is selected from cruciferous vegetable waste material, tuber vegetable waste material, root vegetable waste material, berry fruit waste material, and any combination thereof.

[0070] The food waste is selected from the group consisting of broccoli waste material, brussels sprouts waste material, cabbage waste material, cauliflower waste material, eggplant waste material, leafy greens waste material, carrot waste material, parsnips waste material, kumara waste material, yam waste material, oca waste material, and any combination thereof.

[0071] The food waste is selected from the group consisting of apple waste material, grape waste material, olive waste material, kiwifruit waste material, and any combination thereof.

[0072] The food waste is beverage processing waste material.

[0073] The beverage processing waste material is selected from the group consisting of fruit pomace, grape marc, spent grain, and any combination thereof.

[0074] The at least one microbial cell is a yeast cell, a fungal cell, a bacterial cell, or any combination thereof. [0075] The at least one microbial cell is at least one of a Trichoderma cell, a Pichia cell, a Kluyveromyces cell, or any combination thereof.

[0076] The at least one Trichoderma cell is selected from the group consisting of Trichoderma reesei, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma atroviride, Trichoderma virens, Trichoderma citrinoviride, Trichoderma viride, and any combination thereof.

[0077] The at least one Pichia cell is selected from the group consisting of Pichia pastoris, Pichia finlandic a, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, and any combination thereof.

[0078] The at least one Kluyveromyces cell is a cell selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, and Kluyveromyces thermotolerans, and any combination thereof.

[0079] The at least one microbial cell contains at least one polynucleotide capable of expressing the dairy protein(s).

[0080] The at least one polynucleotide is codon optimised for expression in the cell.

[0081] The at least one polynucleotide encodes a fusion protein.

[0082] The at least one polynucleotide encodes a protein fragment.

[0083] The one or more dairy proteins comprise one or more cow milk proteins.

[0084] The one or more dairy proteins are selected from the group consisting of whey proteins and casein proteins, and any combination thereof.

[0085] The whey proteins are selected from the group consisting of α-lactalbumin,β- lactoglobulin, lactotransferrin/lactoferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, glycomacropeptide, and any combination thereof.

[0086] The casein proteins are selected from the group consisting of β-casein, K-casein, α-Sl-casein, α-S2-casein, and any combination thereof.

[0087] In yet one further aspect, the present disclosure encompasses a dairy protein produced by an expression system, method, or cell according to any preceding aspect. [0088] The dairy protein is a cow milk protein.

[0089] The dairy protein is a sheep milk protein or a goat milk protein.

[0090] The dairy protein is an alpaca milk protein, a buffalo milk protein, a camel milk protein, a human milk protein, a llama milk protein, or a yak milk protein.

[0091] The dairy protein is selected from the group consisting of whey proteins and casein proteins.

[0092] The whey proteins are selected from the group consisting of α-lactalbumin, β- lactoglobulin, lactotransferrin/lactoferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, and glycomacropeptide.

[0093] The casein proteins are selected from the group consisting of β-casein, K-casein, α-Sl-casein, and α-S2-casein.

[0094] The dairy protein is isolated.

[0095] The dairy protein is isolated to about 80% purity or greater, about 90% purity or greater, about 95% purity or greater, or about 99% purity or greater.

[0096] The dairy protein is concentrated and/or dried.

[0097] The dairy protein is powdered.

[0098] In still one further aspect, the present disclosure encompasses a dairy product or dairy ingredient obtained using the dairy protein produced by an expression system, method, or cell according to any preceding aspect.

[0099] In various aspects:

[00100] The dairy product is selected from the group consisting of milk, evaporated milk, and milk powder.

[00101] The dairy product is selected from the group consisting of milk substitute, evaporated milk substitute, and milk substitute powder.

[00102] The dairy product is selected from the group consisting of cream, creamer, cocktail mixer, custard, cheese, dessert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink, and frozen yoghurt. [00103] The dairy product is selected from the group consisting of infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement, weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food.

[00104] The dairy ingredient is an ingredient for one or more of baked goods, beverages, cooked goods, frozen goods, cooking mixes, baking mixes, beverage mixes, and sauce mixes.

[00105] The dairy ingredient is selected from the group consisting of milk protein concentrate and milk protein isolate.

[00106] The milk protein concentrate is selected from the group consisting of whey protein concentrate, α-lactalbumin concentrate,β- lactoglobulin concentrate, glycomacropeptide concentrate, and casein concentrate.

[00107] The milk protein isolate is selected from the group consisting of whey protein isolate, α-lactalbumin protein isolate,β- lactoglobulin protein isolate, glycomacropeptide protein isolate, and casein protein isolate

[00108] Novel features that are believed to be characteristic will be better understood from the detailed description when considered in connection with any accompanying figures and examples. However, the figures and examples provided herein are intended to help illustrate or assist with developing an understanding of this disclosure; these are not intended to limit the scope of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[00109] Figure 1 : Schematic showing a research design in accordance with one embodiment of the present disclosure.

[00110] Figure 2: Graph showing the growth rates of 7. reesei in different food waste substrates. Growth was measured as optical density at 600 nm. Growth was monitored over 48 hours. Readings were taken every 5 minutes. Control media = PDB (potato dextrose broth). Abbreviations: apple (A); apple + dextrose (AD); broccoli (B); broccoli + dextrose (BD); carrot (C); carrot + dextrose (CD); cabbage (Cb); cabbage + dextrose (CbD); potato (P); potato + dextrose (PD).

[00111] Figure 3A: Photograph depicting agar squares containing hyphal tips from nine colonies of T. reesei that were transformed with expression construct pEM06T. [00112] Figure 3B: Agarose gel electrophoresis of PCR analysis of two T. reesei transformants. Genomic DNA isolated from T. reesei transformants was use as PCR template to amplify the hygromycin selectable marker using gene specific primers. Lane A = T. reesei transformed with pUE08. Lane J = T. reesei transformed with pEM06T. Lane M = Thermo Scientific™ GeneRuler 1 kb DNA Ladder. The top band indicates the presence of the hygromycin selectable marker.

[00113] Figure 3C: Agarose gel electrophoresis of PCR analysis of two T. reesei transformants. Genomic DNA isolated from T. reesei transformants was use as PCR template to amplify the K-casein gene using gene specific primers. Lane 1 = T. reesei transformed with pUE08. Lane 2 = T. reesei transformed with pEM06T. Lane M = Thermo Scientific™ GeneRuler 1 kb DNA Ladder. The band in lane 2 indicate the presence of the k-casein gene in the transformant with the pEM06T plasmid.

[00114] Figure 4: Graph showing the growth rates of K. lactis in different food waste substrates. Growth was measured as optical density at 600 nm. Growth was monitored over 48 hours. Readings were taken every 5 minutes. Control media = no inoculant. Abbreviations: apple (A); apple + dextrose (AD); broccoli (B); broccoli + dextrose (BD); carrot (C); carrot + dextrose (CD); cabbage (Cb); cabbage + dextrose (CbD); potato (P); potato + dextrose (PD).

[00115] Figure 5: SDS-PAGE analysis showing expression of BLG in K. lactis. Lane M = pre-stained protein ladder; mid-range molecular weight (10 - 180 kDa) (abl 16027); Lane 1 = K. lactis NEB kit positive control expressing the E. coli maltose binding protein; Lane 2 = K. lactis NEB kit negative control expressing the empty pKLAC2 vector; Lane 3 = K. lactis expressing BLG on plasmid pDL3O3.

[00116] Figure 6A: Bovine β-lactoglobulin (BLG) variant B amino acid sequence (SEQ ID NO:1). NCBI reference sequence: NP_776354.2. Bovine secretion signal noted in amino acid sequence (underlined). Without secretion signal = SEQ ID NO:4.

[00117] Figure 6B:β- lactoglobulin (BLG) variant B nucleotide sequence, which has been codon optimised for expression in K. lactis (SEQ ID NO:7). Bovine secretion signal replaced with mating factor signal (underlined). Without signal = SEQ ID NO: 10.

[00118] Figure 6C:β- lactoglobulin (BLG) variant B nucleotide sequence (SEQ ID NO: 13), which has been codon optimised for expression in K. lactis and includes a stop codon (final triplet) and HA tag (capitalised). Bovine secretion signal replaced with mating factor signal (underlined).

[00119] Figure 7A: Bovineβ- casein (CSN2) amino acid sequence (SEQ ID NO:2. GenBank accession number: M16645.1. Bovine secretion signal noted in amino acid sequence (underlined). Without secretion signal = SEQ ID NO:5.

[00120] Figure 7B:β- casein (CSN2) nucleotide sequence, which has been codon optimised for expression in K. lactis (SEQ ID NO:8). Start (atg) codon and mating factor signal is on vector. Bovine secretion signal replaced with mating factor signal (underlined). Without signal = SEQ ID NO: 11.

[00121] Figure 7C: β- casein (CSN2) nucleotide sequence (SEQ ID NO: 14), which has been codon optimised for expression in K. lactis and includes a stop codon (final triplet) and HA tag (capitalised). Start (atg) codon and mating factor signal is on vector. Bovine secretion signal replaced with mating factor signal (underlined).

[00122] Figure 8A: Bovine K-casein (CSN3) amino acid sequence (SEQ ID NOG). GenBank accession number: A Y380229.1. Bovine secretion signal noted in amino acid sequence (underlined). Without secretion signal = SEQ ID NO:6.

[00123] Figure 8B: K-casein (CSN3) nucleotide sequence, which has been codon optimised for expression in K. lactis (SEQ ID NO:9). Start (atg) codon and the mating factor signal is on vector. Bovine secretion signal has been removed. Without signal = SEQ ID NO:12.

[00124] Figure 8C: K-casein (CSN3) nucleotide sequence (SEQ ID NO: 15), which has been codon optimised for expression in K. lactis and includes a stop codon (final triplet) and HA tag (capitalised). Start (atg) codon and the mating factor signal is on vector. Bovine secretion signal has been removed.

[00125] Figure 9: Graph showing the growth rates of Pichia pastoris pKC15P in different food waste substrates. 96 well plate assay. Growth was measured as optical density at 600 nm. Growth was monitored over 48 hours. Measurements were taken every 5 minutes for 48 hours. Samples were shaken continuously. Abbreviations: apple (A); apple + dextrose (AD); broccoli (B); broccoli + dextrose (BD); carrot (C); carrot + dextrose (CD); cabbage (Cb); cabbage + dextrose (CbD); grape (G); grape + dextrose (GD); kiwifruit (K); kiwifruit + dextrose (KD); potato (P); potato + dextrose (PD). [00126] Figure 10: Graph showing the growth rates of Pichia pastoris pKC23P-6 in different food waste substrates. Deep well plate growth assay. Growth was measured as optical density at 600 nm. Growth was monitored over 72 hours. Abbreviations: apple (A); apple + dextrose (AD); broccoli (B); broccoli + dextrose (BD); carrot (C); carrot + dextrose (CD); cabbage (Cb); cabbage + dextrose (CbD); grape (G); grape + dextrose (GD); kiwifruit (K); kiwifruit + dextrose (KD); potato (P); potato + dextrose (PD).

[00127] Figure 11 A: SDS-PAGE results showing expression rates for bovineβ- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from deep well studies.

[00128] Figure 11B: SDS-PAGE results showing expression rates for bovineβ- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from deep well studies.

[00129] Figure 12: Graph showing the growth rates of Pichia pastoris pKC23P-6 in different food waste substrates. Test tube growth assay. Growth was measured as optical density at 600 nm. Growth was monitored over 72 hours. Abbreviations: apple (A); apple + dextrose (AD); broccoli (B); broccoli + dextrose (BD); carrot (C); carrot + dextrose (CD); cabbage (Cb); cabbage + dextrose (CbD); grape (G); grape + dextrose (GD); kiwifruit (K); kiwifruit + dextrose (KD); potato (P); potato + dextrose (PD).

[00130] Figure 13A: SDS-PAGE results showing expression rates for bovineβ- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from test tube studies.

[00131] Figure 13B: SDS-PAGE results showing expression rates for bovineβ- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from test tube studies.

Figure 14: Graph showing the growth rates of Pichia pastoris pKC23P-6 in different food waste substrates. Test tube growth assay. Growth was measured as optical density at 600 nm. Growth was monitored over 72 hours. Abbreviations: kumara waste extract (M); kumara waste extract + dextrose (KM); kale waste extract (L); kale waste extract + dextrose (KL); yam waste extract (Y); yam waste extract + dextrose (YD); cauliflower waste extract (F); cauliflower waste extract + dextrose (FD); parsnip waste extract (R); parsnip waste extract + dextrose (RD); Brussels sprouts waste extract (T); Brussels sprouts waste extract + dextrose (TD).

[00132] Figure 15 A: SDS-PAGE results showing expression rates for Bovine [3- lactogloBulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste suBstrates. Results shown from test tube studies. Samples were taken at 48 hours except forthose specified as 72 hour samples.

[00133] Figure 15B: SDS-PAGE results showing expression rates for bovine [3- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from test tube studies. Samples were taken at 48 hours except forthose specified as 72 hour samples.

[00133a] Figure 15C: SDS-PAGE results showing expression rates for bovine [3- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from test tube studies. Samples were taken at 48 hours except forthose specified as 72 hour samples.

[00133b] Figure 15D: SDS-PAGE results showing expression rates for bovine [3- lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates. Results shown from test tube studies. Samples were taken at 48 hours except forthose specified as 72 hour samples.

[00134] Figure 16: Expression rates for bovine [3-lactoglobulin (BLG) from Pichia pastoris pKC23P-6 grown in different food waste substrates and normalised by growth rates ( OD ₆₀₀ ). Results shown from test tube studies. Samples were taken at 48 hours except for those specified as 72 hour samples.

DETAILED DESCRIPTION

[00135] The following description sets forth numerous exemplary configurations, parameters, and the like. It should be recognised, however, that such description is not intended as a limitation on the scope of this disclosure; it is instead provided as exemplary embodiments.

Definitions

[00136] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise.

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RECTIFIED SHEET (RULE 91) [00137] The various examples, embodiments, and aspects as set out herein may be readily combined, without departing from the scope or spirit of this disclosure. Thus, the phrase “in one example”, “in one embodiment”, or “in one aspect” is not necessarily exclusive of other examples, embodiments, or aspects that are also described. In the same way, the phrase “in another example”, “in another embodiment”, or “in another aspect” is not necessarily exclusive of other examples, embodiments, or aspects that are described.

[00138] In each instance herein, in descriptions, embodiments, and examples of the present disclosure, the terms “comprising”, “including”, etc, are to be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as to opposed to an exclusive sense, that is to say in the sense of “including but not limited to”.

[00139] Where a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Thus, each range that is specified (e.g., 1 to 10) includes all possible combinations ofnumerical values between the lowest value and the highest value enumerated (e.g., 1, 1.1, 2, 3, 3.3, 4, 5.5, 6, 7, 8.9, 9 and 10) and also any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.9), and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. The numeric values provided in parentheses here are only examples of what is specifically intended and all possible combinations of numerical value between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure in a similar manner.

[00140] The term “consisting essentially of’, as used herein, refers to the proportion of waste substance present in a substrate. For example, for solid or semi-solid substrates, the waste substance may be at least 50% by weight of the substrate, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or approximately 100.0% by weight of the substrate (% w/w). For liquid substrates, the waste substance may be at least 50% by volume of the substrate, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or

15

RECTIFIED SHEET (RULE 91) at least 80%, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or approximately 100.0% by volume of the substrate (% v/v).

[00141] As used herein “and/or” means additionally or alternatively.

[00142] As used herein “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. The meaning of “in” includes “in” and “on.”

[00143] Any use of a term in the singular also encompasses plural forms. Thus, throughout the specification, the meaning of “a”, “an”, and “the” include plural references.

[00144] The term “about” or “approximately” means up to 10% greater than or up to 10% lesser than a particular value.

[00145] As used herein, an “isolated” component (e.g., isolated polynucleotide, polypeptide, or enzyme) refers to a component that has been purified from (e.g., separated from) other components. An isolated component may be removed from its originating environment, e.g., natural cellular environment or synthetic environment. The isolated component of this disclosure may be prepared by at least one purification step. An isolated component may have: about 70% purity or greater, about 80% purity or greater, about 90% purity or greater, about 95% purity or greater, or, in particular aspects, about 99% purity or greater. An isolated component may be obtained by any method or combination of methods as known and used in the field, including biochemical, recombinant, and synthetic techniques.

[00146] ‘Isolated” when used herein in reference to a cell or host cell describes a cell or host cell that has been obtained or removed from an organism or from its natural environment or from an artificial environment. The term encompasses single cells, per se, as well as cells or host cells comprised in a cell culture and can include a single cell or single host cell.

[00147] The term “construct”, e.g., “expression construct”, refers to a polynucleotide molecule, usually double-stranded DNA, which may have cloned or inserted into it another polynucleotide molecule. For example, a construct may have an unidentified polynucleotide insert that is prepared from an environmental sample or as a cDNA, but not limited thereto. A construct may contain the necessary elements that permit transcription of a cloned or inserted polynucleotide molecule, and, optionally, for translating the transcript into a peptide or

16

RECTIFIED SHEET (RULE 91) polypeptide. The inserted polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism. Once inside the host cell the construct may become integrated in the host chromosomal DNA. The construct may be linked to a vector.

[00148] The term “vector” as used herein refers to a polynucleotide molecule, usually double stranded DNA, which is used to replicate or express a construct. The vector may be used to transport a construct into a given host cell.

[00149] The term “polynucleotide(s),” as used herein, means a single or double -stranded deoxyribonucleotide or ribonucleotide polymer of any length, and include as non-limiting examples, coding and non-coding sequences of a gene, genomic DNA, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, fragments, constructs, and vectors. Reference to nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences is to be similarly understood.

[00150] The term “polypeptide”, as used herein, encompasses amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds. “Polypeptide” may refer to a polypeptide that is a purified natural product, or that has been produced partially or wholly using recombinant or synthetic techniques. Useful as polypeptides include: aggregates of one or more polypeptides such as dimers or other multimers, fusion polypeptides, polypeptide fragments, polypeptide variants, as well as aggregates, fusions, or fragments of such variants. The term “polypeptide” is used interchangeably herein with the terms “protein” and “enzyme”.

[00151] A “fragment” of a polypeptide is a subsequence of a particular polypeptide, i.e., truncation. In certain aspects, the fragment is a functional fragment. A functional fragment performs a function that is required for a biological activity and/or provides three dimensional structure of the polypeptide. The term may refer to a polypeptide fragment, an aggregate of a polypeptide fragment, a fusion polypeptide fragment, a fragment of a polypeptide variant, or a fragment of a polypeptide derivative thereof that is capable of performing the polypeptide activity.

[00152] The term “full length” as used herein with reference to a sequence means a peptide or polypeptide that comprises a contiguous sequence of amino acid residues where each amino acid residue has been expressed from each of its corresponding codons in the polynucleotide over the entire length of the coding region and resulting in a fully functional

17

RECTIFIED SHEET (RULE 91) polypeptide, peptide, or protein. As will be appreciated by a person of ordinary skill in the field, a “full length” sequence contains the amino acid sequence that corresponds to and has been expressed from each and every codon encoded by the polynucleotide comprising the entire coding region of the polypeptide, wherein each of said codons is located between the start codon and the termination codon normally associated with that coding region.

[00153] The term “expressing” refers to the expression of a nucleic acid transcript from a nucleic acid template and/or the translation of that transcript into a peptide or polypeptide, and is used herein as commonly used in the field.

[00154] The term “incubating” refers to the placing together of elements so they may interact and is used herein as commonly used in the field.

[00155] The term “endogenous” as used herein refers to a constituent of a cell, tissue or organism that originates or is produced naturally within that cell, tissue or organism.

[00156] The term “exogenous” as used herein refers to any constituent of a cell, tissue or organism that does not originate or is not produced naturally within that cell, tissue or organism. An exogenous constituent may be, for example, a polynucleotide sequence that has been introduced into a cell, tissue or organism, or a peptide or polypeptide expressed in that cell, tissue or organism from that polynucleotide sequence.

[00157] “Naturally occurring” as used herein with reference to a polynucleotide or polypeptide sequence refers to a sequence that is found in nature. A synthetic sequence that is identical to a wild type sequence is, for the purposes of this disclosure, considered a naturally occurring sequence. What is important for a naturally occurring sequence is that the actual sequence (e.g., nucleotide or amino acid sequence) is found or known from nature.

[00158] “Non-naturally occurring” as used herein with reference to a polynucleotide or polypeptide refers to a molecule that is not found in nature, i.e., non-wild type. Examples of non-naturally occurring sequences include artificially produced and variant sequences, made for example by recombination, domain swapping, point mutation, insertion, deletion, or other methods, or combinations of these methods. Non-naturally occurring sequences also include chemically evolved sequences as well as sequences that are altered to include modifications, e.g., post-translational modifications. What is important for a non-naturally occurring sequence is that the actual sequence (e.g., nucleotide or amino acid sequence) is not found or known from nature.

18

RECTIFIED SHEET (RULE 91) [00159] The term, “wild type” when used herein with reference to a polynucleotide refers to a naturally occurring, non-mutant form of a polynucleotide, peptide, polypeptide, or organism. A wild type peptide or polypeptide is capable of being expressed from a wild type polynucleotide. In one embodiment, a wild type polypeptide is a wild type amino acid sequence that is expressed from a wild type polynucleotide.

[00160] “Homologous” as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is a native and naturally occurring polynucleotide regulatory element. A homologous polynucleotide regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a, vector, construct, or expression cassette according to this disclosure.

[00161] “Heterologous” as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is not a native and naturally occurring polynucleotide regulatory element. A heterologous polynucleotide regulatory element is not normally associated with the coding sequence to which it is operably linked. A heterologous regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a vector, construct, or expression cassette according to this disclosure.

[00162] The term “recombinant” refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context. A “recombinant” peptide or polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.

[00163] The term “modified” refers to a molecule modification that is not a naturally occurring, i.e., non-wild type. In the same way, a “modified polypeptide” or “modified enzyme” refers to a polypeptide that is not a naturally occurring. Modification may be carried out in accordance with known methods, e.g., post-translational modifications. Modified enzymes and polypeptides useful in this disclosure may have biological activities, stability, and/or production levels that are the same or similar to those of a corresponding wild type molecule i.e., functional modifications. Alternatively, modified enzymes and polypeptides may have biological activities that are improved when compared to corresponding wild type molecules. In certain embodiments, the differences can include one or more of increased activity, stability, interactions, and production levels.

19

RECTIFIED SHEET (RULE 91) [00164] As used herein, the term “variant” refers to an nucleotide or amino acid sequence that is not naturally occurring, i.e., non-wild type. A polynucleotide, peptide, or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, transposed, substituted, or added. Variants may be fusion sequences, e.g., fusion polynucleotide sequences or fusion polypeptide sequences. In certain embodiments, the variants useful in this disclosure have biological activities that are the same or similar to those of a corresponding wild type molecule; i.e., functional variants of the parent polypeptide or polynucleotide. In certain embodiments, the variants have biological activities that are improved compared to corresponding wild type molecules. In certain embodiments, the differences can include one or more of improved activity, stability, interactions, and production levels.

[00165] The term “variant” with reference to polynucleotides, polypeptides, and enzymes encompasses all forms of polynucleotides, peptides, and polypeptides as defined herein.

[00166] As used herein, the term in vitro refers to a reaction performed outside of the confines of a living cell or a host organism. The term in vivo refers to a reaction performed within a living cell and/or within a host organism.

[00167] A “dairy protein” as used herein includes any milk protein, including, for example, whey proteins and casein proteins. Non-limiting examples include cow milk proteins, sheep milk proteins, goat milk proteins, alpaca milk proteins, buffalo milk proteins, camel milk proteins, human milk proteins, llama milk proteins, and yak milk proteins. A dairy protein may be produced partly or wholly by recombinant means, and may be produced as a wild type or non-wild type molecules, as described in detail herein.

[00168] The term “food waste” as used herein includes but is not limited to fruit waste material, vegetable waste material, nut or grain waste material, dairy processing waste material, beverage (e.g., beer, wine, juice) processing waste material, food (e.g., canned or frozen fruits, canned or frozen vegetables, breads) processing waste material, dairy processing wastewater, beverage (e.g., beer, wine, cider, juice) processing wastewater, food (e.g., canned or frozen fruits, canned or frozen vegetables, breads) processing wastewater, and any combination of these. Waste material may include a whole food component (e.g., whole fruit waste, whole vegetable waste, whole grain waste, etc), or any portion thereof (e.g., one or more of peels,

20

RECTIFIED SHEET (RULE 91) shells, seeds, hearts, juice, pulp, oil, and flesh), or any combination of whole food components and portions.

[00169] The term “growth substrate” includes any media useful for supporting the growth of microbial strains. Such substrate can include one or more solid components obtained from food waste and/or one or more liquid components obtained from food waste. Extracts obtained from food waste are specifically noted, including aqueous extracts as described in detail herein.

[00170] It is understood that, for any DNA molecule disclosed herein, the corresponding RNA molecule and peptide/polypeptide molecules are also encompassed and disclosed. Likewise, for any peptide/polypeptide molecule disclosed herein, the corresponding RNA and DNA sequences are also considered to be encompassed and disclosed. In addition, where there are multiple sequence identifiers, e.g., “SEQ ID NO: 1-18” or “SEQ ID NO: 1 to SEQ ID NO: 18”, this format may be understood as referring to each sequence individually, or any combination thereof.

Polynucleotides and polypeptides

[00171] In one embodiment of the present disclosure, a microbial strain such as a Trichoderma, Kluyveromyces, or Pichia sp. is adapted to enable expression of at least one dairy protein (e.g., a wild type or non-wild type molecule). This can be achieved using one or more appropriate polynucleotide sequence, for example, at least one expression construct, and growth can be achieved using a suitable substrate, such as those described in detail herein.

[00172] In accordance with this disclosure, dairy proteins (e.g., a wild type or variant molecules) can include whey proteins and casein proteins. As non-limiting examples, whey proteins can include a-lactalbumin, [3-lactoglobuhn, lactotransferrin/lactoferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, and glycomacropeptide. As non-limiting examples, casein proteins can include [3-casein, K-casein, α-Sl -casein, and α-S2-casein. Specifically noted are cow milk proteins (bovine proteins); however, any milk proteins may be utilised.

[00173] Specific exemplifications include polynucleotides that encode an amino acid sequence of any one of SEQ ID NO: 1-6, or encode any variants of these; as well as

21

RECTIFIED SHEET (RULE 91) polynucleotides that comprise a nucleotide sequence of any one of SEQ ID NO:7-15, or any variants as these; along with polynucleotides that consist of a nucleotide sequence of any one of SEQ ID NO:7-15.

[00174] In addition the particular nucleotide sequences set out herein, other variant sequences may also be utilised. In various embodiments, polynucleotide variants encompass naturally occurring or non-naturally occurring (e.g., recombinantly or synthetically produced) polynucleotides. As exemplifications, variant polynucleotide sequences exhibit at least 50%, at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence of the present disclosure. In the same way, the coding sequence for a polypeptide domain or other fragment may be varied to include the above noted levels of sequence identity.

[00175] As a variant polynucleotide sequence, a fragment of a polynucleotide sequence includes a subsequence of contiguous nucleotides. In one embodiment, the polynucleotide fragment allows expression of at least a portion of a polypeptide, e.g., expression of one or more functional domains of the polypeptide.

[00176] Variant polynucleotides include polynucleotides that differ from the disclosed sequences but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a disclosed polynucleotide. A sequence alteration that does not change the amino acid sequence of the polypeptide is termed a silent variation. Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by recognised techniques in the field, e.g., to optimise codon expression in a particular host organism.

[00177] For polynucleotides, sequence identity may be found over a comparison window of at least 1500 nucleotide positions, at least 2000 nucleotide positions, at least 2500 nucleotide positions, at least 3000 nucleotide positions, at least 3500 nucleotide positions, at least 3800 nucleotide positions, or over the entire length of a polynucleotide used according to a method of this disclosure. Alternatively, shorter regions may be compared, for example, at least 50 nucleotide positions, at least 100 nucleotide positions, at least 200 nucleotide positions,

22

RECTIFIED SHEET (RULE 91) at least 300 nucleotide positions, at least 400 nucleotide positions, at least 500 nucleotide positions, at least 600 nucleotide positions, at least 700 nucleotide positions, at least 800 nucleotide positions, at least 900 nucleotide positions, or at least 1000 nucleotide positions.

[00178] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in this disclosure. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al. 1990).

[00179] Polynucleotide sequence identity and similarity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using sequence alignment algorithms and sequence similarity search tools such as in GenBank, EMBL, Swiss-PROT and other databases. Nucleic Acids Res 29: 1 - 10 and 11-16, 2001 provides examples of online resources.

[00180] Specific exemplifications for dairy proteins include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences; as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6. It will be understood that, where wild type and variant polypeptides are encompassed and disclosed, the correlating wild type and variant polynucleotides are considered to be encompassed and disclosed.

[00181] In addition the particular amino acid sequences set out herein, variant sequences may also be utilised. In various embodiments, polypeptide variants encompass naturally occurring or non-naturally occurring (e.g., recombinantly or synthetically produced) polypeptides. As exemplifications, variant polypeptide sequences exhibit at least 50%, at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least

76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

97%, at least 98%, or at least 99% identity to a sequence of the present disclosure. In the same way, a polypeptide domain or other fragment may be varied to include the above noted levels of sequence identity. .

23

RECTIFIED SHEET (RULE 91) [00182] As to polypeptide variants, an amino acid sequence may differ from a polypeptide disclosed herein by one or more conservative amino acid substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[00183] Other variants include peptides with modifications which influence peptide stability. Such analogues may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are analogues that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta or gamma amino acids and cyclic analogues.

[00184] Substitutions, deletions, additions, or insertions may be made by mutagenesis methods known in the field. A skilled worker will be aware of methods for making phenotypically silent amino acid substitutions. See, for example, Bowie et al. 1990. A polypeptide may be modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, phosphorylation, amidation, by derivatisation using blocking/protecting groups and the like. Such modifications may increase stability or activity of the polypeptide.

[00185] For polypeptides, sequence identity may be found over a comparison window of at least 600 amino acid positions, at least 700 amino acid positions, at least 800 amino acid positions, at least 900 amino acid positions, at least 1000 amino acid positions, at least 1100 amino acid positions, at least 1200 amino acid positions, or over the entire length of a polypeptide used in or identified according to a method of this disclosure. Alternatively, shorter regions may be compared, for example, at least 8 amino acid positions, at least 10 amino acid positions, at least 20 amino acid positions, at least 30 amino acid positions, at least 40 amino acid positions, at least 50 amino acid positions, at least 60 amino acid positions, at least 70 amino acid positions, at least 80 amino acid positions, at least 90 amino acid positions, or at least 100 amino acid positions.

[00186] Polypeptide variants also encompass those that exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional

24

RECTIFIED SHEET (RULE 91) equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. For polynucleotides and polypeptides, exemplary sequence alignment platforms include but are not limited to: homology alignment algorithms (Needleman and Wunsch (1970) J Mol Biol 48: 443); local homology algorithms (Smith and Waterman (1981) Adv Appl Math 2: 482); searches for similarity (Pearson and Lipman (1988) PNAS USA 85: 2444).

[00187] In specific embodiments, the BLAST algorithm may be used (Altschul et al. (1990) J Mol Biol 215: 403-410; Henikoffand Henikoff (1989) PNAS USA 89: 10915; Karlin and Altschul (1993) PNAS USA 90: 5873-5787). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Other examples of alignment software include GAP, BESTFIT, FASTA, PILEUP, and TFASTA provided by Wisconsin Genetics Software Package (Genetics Computer Group), and CLUSTAL programs such as ClustalW, ClustalX, and Clustal Omega (see, e.g., Thompson et al. (1994) Nuc Acids Res 22: 4673-4680).

Polypeptide expression

[00188] In this disclosure, a microbial strain such as a Trichoderma, Kluyveromyces, or Pichia sp. is adapted to express at least one dairy protein, such as a whey protein and/or casein protein (e.g., a wild type or non-wild type molecule).

[00189] Specific exemplifications include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences; as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6.

[00190] As examples, one or more expression constructs may be used, i.e., a nucleic acid expression construct that comprises a polynucleotide sequence that encodes the desired polypeptide. This polynucleotide sequence can be operatively linked to a promoter that allows expression of the polynucleotide sequence to produce the polypeptide. In specific aspects, this process produces a wild type or non-wild type molecule, for example, a functional variant of this polypeptide may be produced.

[00191] An expression cassette may be used to include the necessary elements that permit the transcription of a polynucleotide molecule that has been cloned or inserted into the construct. Optionally, the expression cassette may comprise some or all of the necessary

25

RECTIFIED SHEET (RULE 91) elements for translating the transcript produced from the expression cassette into a polypeptide . An expression cassette may include necessary coding regions. It may also include any necessary noncoding regions. Particular exemplifications include the constructs, polynucleotides, and polypeptides set out in US provisional patent application number 63/535043, filed on 28 August 2023, which is hereby incorporated by reference herein.

[00192] A polynucleotide sequence encoding the polypeptide may be suitable for expression in any organism. The organism may be a microbial strain, such as a yeast strain or fungal strain or bacterial strain. Exemplifications include filamentous fungus strains, such as Trichoderma, Aspergillus, and Myceliophthora strains, and also yeast strains, such as Pichia, Kluyveromyces, Arxula, Candida, Hansenula, Saccharomyces, and Yarrowia strains.

[00193] Specifically noted are Trichoderma reesei, Aspergillus niger, Aspergillus oryzae, and Myceliophthora thermophila strains. Further noted are Pichia pastoris, Kluyveromyces lactis, Arxula adeninivoran, Candidia hiodini, Hansenula polymorpha, Saccharomyces cerevisiae, Schizosaccharomyces pomhe, and Yarrowia lipolytica strains. Other useful strains are set out in detail herein.

[00194] The polynucleotide sequence encoding the polypeptide may be a naturally occurring (i.e., wild type) or non-naturally occurring (i.e., non-wild type) polynucleotide sequence. For example, a wild type or a non-wild type polynucleotide sequence for one or more dairy proteins may be used. In particular, the polynucleotide sequence encoding the polypeptide may be a variant sequence, modified sequence, fusion sequence, or fragment sequence, as described herein.

[00195] Specific exemplifications include polynucleotides that encode an amino acid sequence of any one of SEQ ID NO: 1-6, or encode any variants of these; as well as polynucleotides that comprise a nucleotide sequence of any one of SEQ ID NO:7-15, or any variants as these; along with polynucleotides that consist of a nucleotide sequence of any one of SEQ ID NO:7-15.

[00196] In one embodiment, a construct is made by cloning a polynucleotide sequence encoding at least one polypeptide into an appropriate vector. An appropriate vector is any vector that comprises a promoter operatively linked to the cloned, inserted polynucleotide sequence that allows expression of the polypeptide from the vector. A skilled worker appreciates that different vectors may be employed in the methods of this disclosure. In

26

RECTIFIED SHEET (RULE 91) addition methods for constructing vectors, including the choice of an appropriate vector, and the cloning and expression of a polynucleotide sequence inserted into an appropriate vector as described above is believed to be within the capabilities of a person of skill in the field (Sambrook et al. 2003).

[00197] In certain embodiments, the expressed polypeptide comprises a functional amino acid sequence. Similar approaches may be used for the polypeptides disclosed herein, and any functional variants thereof. The person of skill in the field recognises that there are also many suitable alternative expression systems available that may be used in the methods of this disclosure to express a polypeptide.

[00198] In various embodiments, expression is obtained in a suitable host cell or strain or cell free expression system. In one embodiment, the host cell or strain may be a yeast or fungus cell or strain. The expression vector may be chosen to allow inducible or constitutive expression in a host cell or strain. Expression may be inducible, for example, with IPTG. Expression may also be obtained using in vitro expression systems; such systems are well known in the field. The polynucleotide may be adapted for use in such strains or systems. For example, the nucleotide sequence may be varied to include appropriate codon usage. Codon usage for particular microbial strains, including yeast and fungal strains, will be known by the skilled practitioner.

[00199] In one embodiment, multiple polypeptides are co-expressed in the same host cell or strain or expression system. To achieve expression within the same host cell or strain, the nucleotide sequences encoding the polypeptides may be cloned into one or more suitable expression vectors. Suitable vectors may have the same or compatible origins of replication in order to be stably maintained in the same host cell or strain. It is also possible to express multiple polypeptides as one or more fusion proteins. In specific embodiments, an expression vector can encode at least one polypeptide (e.g., one or more of SEQ ID NO: 1-6) and/or at least one functional variant thereof.

[00200] In another embodiment, one or more polynucleotide sequences encoding a polypeptide may be integrated into the chromosome of an appropriate host organism as described herein, to produce a strain useful in accordance with the present disclosure. In one embodiment, a construct comprises at least one nucleotide sequence encoding a dairy protein and suitable regulatory promoter sequences that are integrated into the chromosome of a

27

RECTIFIED SHEET (RULE 91) Trichoderma, Kluyveromyces, or Pichia strain, or other host organism, in an appropriate orientation to allow expression of the polypeptide or polypeptides in the cell.

[00201] In one particular embodiment, a construct encoding one or more polypeptide is integrated into a host cell. For example, a construct may be integrated and then expressed in vivo. The constructs may allow co-expression of wild type polypeptides or non-wild type polypeptides. Thus, in a specific embodiment, a construct that encodes a single or multiple polypeptides is expressed in a host cell or strain, or cell free expression system.

[00202] In particular embodiments, the present disclosure provides polynucleotide libraries that include nucleic acids encoding dairy proteins. For example, a polynucleotide library may include at least 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleic acids. Libraries of polynucleotides, and specifically variant polynucleotides, may be generated using standard methods.

[00203] As exemplifications, nucleic acid libraries may be generated to include a plurality of polynucleotides with variant sequences. For example, a nucleic acid library may include polynucleotides with substitutions or swapped domains. In addition, libraries of polynucleotides may be generated using random mutagenesis of one or more domains (e.g., conserved sequence or regions). For example, error prone PCR may be utilised (see, e.g., Beaudry and Joyce (1992) Science 257: 635 and Bartel and Szostak (1993) Science 261: 1411). Alternative means for mutagenesis may be used, for example, chemical mutagens, radiation, amongst others. Commercial kits are also available, e.g., GeneMorph® II EZClone domain mutagenesis kit (Agilent) and Diversify™ PCR random mutagenesis kit (Clontech Laboratories, Inc). The library may be provided as a mixture of polynucleotides, or may be provided via a host cell or strain.

[00204] As one embodiment of the present disclosure, a kit is provided which includes one or more polynucleotides or polypeptides. The one or more polynucleotides or polypeptides may be non-wild type molecules as described herein. The one or more polynucleotide or polypeptide may be provided in one or more containers in the kit. Additional components may also be provided with the kit, for example, one or more components to obtain expression, or one or more components to measure activity, which are intended for use with the polynucleotide(s) or polypeptide(s). Optionally, instructions may be provided with the kit, as

28

RECTIFIED SHEET (RULE 91) well as any other item, such as any number of containers, labels, or measurement tools. The one or more polynucleotide or polypeptide of the kit may be provided as isolated components, or as mixtures, or may be provided via a host cell or strain.

Host cells, strains, and substrates

[00205] According to this disclosure, expression of one or more dairy proteins (e.g., a wild type or non-wild type molecules) may be achieved using an appropriate expression construct and growth on a suitable substrate. Methods of production for polypeptides (e.g., wild type or non-wild type molecules) are provided, and methods of use of these polypeptides are also provided.

[00206] Specific exemplifications include polypeptides that comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences; as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6.

[00207] Host cells and their use for such production are set out in detail in this description. By use of a host cell comprising one or more dairy proteins, this allows production of dairy products. The expression of a polypeptide (e.g., wild type or non-wild type molecule) may be carried out in vitro or in vivo. In vivo expression may be carried out in a suitable host cell or strain. In one embodiment, the host cell or strain is a yeast, fungal, or bacterial host cell or strain.

[00208] For larger scale applications, the host strain may be a filamentous fungal strain, such as Trichoderma, Aspergillus, or Myceliophthora strain; or a yeast strain, such as aPichia, Kluyveromyces, Arxula, Candida, Hansenula, Saccharomyces, or Yarrowia strain; or a bacterial strain such as Bacillus, Corynebacterium, Escherichia, Lactococcus, Mycobacterium, Pseudomonas, Ralstonia, Streptomyces, and Vibrio strains.

[00209] Specifically noted are Trichoderma reesei, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Myceliophthora thermophila strains. Also noted are Pichia pastoris, Kluyveromyces lactis, Arxula adeninivoran, Candidia biodini, Hansenula polymorpha, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Yarrowia lipolytica strains. Further noted are Bacillus subtilis, Bacillus megaterium, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Escherichia coli, Lactococcus lactis, Lactobacillus plantarum, Mycobacterium smegmatis, Pseudomonas fluorescens, Ralstonia eutropha, Streptomyces lividans, and Vibrio natriegens, strains.

29

RECTIFIED SHEET (RULE 91) [00210] Trichoderma reesei is of particular interest. Other Trichoderma strains may also be used, for example, Trichoderma harzianum, Trichoderma koningii, Trichoderma longihrachiatum, Trichoderma atroviride, Trichoderma virens, Trichoderma citrinoviride, Trichoderma viride, and any combination of these.

[00211] Pichia pastoris is of particular interest. Other Pichia strains may also be used, for example, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia memhranaefaciens, Pichia minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans , Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, and any combination of these.

[00212] Kluyveromyces lactis is of particular interest. Other Kluyveromyces strains may also be used, for example, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, and any combination of these.

[00213] Introduction of a polynucleotide or expression construct into an appropriate host cell or strain may be achieved using any of a number of available standard protocols and/or as described herein as known and used in the field (Sambrook et al. 2003). The construct may be a construct for protein production as set out herein. The construct may be introduced via transformation, transduction, transfection, or other techniques (see, e.g., Sambrook et al. 2003).

[00214] In one embodiment, the expressed polypeptide is an exogenous polypeptide in the host cell, strain, or cell free expression system, which is expressed from a construct according to this disclosure. Alternatively, the polypeptide is expressed from the genome of the host cell or strain. The polypeptide may be exogenous and non-naturally occurring with respect of the host cell or strain. The polypeptide so expressed may be a dairy protein as set out herein, or a functional variant thereof. In one specific embodiment, a single host organism or cell free expression system could be utilised to allow expression of multiple polypeptides (e.g., two or more variant polypeptides), to maximise production.

[00215] The production of a polypeptide may be carried out in vitro or in vivo. The choice host cell or strain may be made based on the expression componentry or other factors. For example, host cells or strains may be chosen based on promoter activity, codon bias, protein solubility, or other factors. Therefore, the use of different host strains and systems provides alternative means suitable for use in production of any polypeptides of interest.

30

RECTIFIED SHEET (RULE 91) [00216] In specific embodiments, the polypeptide (expressed or otherwise produced) may be isolated using various biochemical techniques. These techniques include but are not limited to filtration, centrifugation, and various types of chromatography, such as ionexchange, affinity, hydrophobic interaction, size exclusion, and reverse-phase chromatography. In one particular embodiment, Ni-affmity chromatography is used. As exemplifications, the polypeptides may be linked to a solid format such as beads, filters, fibres, paper, membranes, chips, and plates such as multiwell plates.

[00217] The polypeptides may also be prepared as a polypeptide conjugates in accordance with known methods. Protein affinity tags may be used to assist with purification, for example, albumin-binding protein (ABP), biotin-carboxy carrier protein (BCCP), calmodulin binding peptide (CBP), cellulose binding domain (CBP), chitin binding domain (CBD), galactose-binding protein (GBP), glutathione S-transferase (GST), HaloTag®, LacZ, polyhistidine (His-tag), polyphenylalanine (Phe-tag), S-tag, small ubiquitin-like modifier (SUMO), Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep- tag, streptavidin, thioredoxin (Trx), tandem affinity purification (TAP), and ubiquitin protein tags. In one particular embodiment, a His-tag and a standard linker sequence are added to the N-terminus of the polypeptide.

[00218] The production of the polypeptide may be carried out in and/or on various substrates. For example, the substrate may comprise food waste (e.g., solids and/or liquids obtained from food waste) or may consist essentially of food waste (e.g., solids and/or liquids obtained from food waste). Examples of food waste include but are not limited to plant waste material, fruit waste material, vegetable waste material, nut or grain waste material, dairy processing waste material, beverage processing waste material, food processing waste material, dairy processing wastewater, beverage processing wastewater, food processing wastewater, and any combination of these.

[00219] In specific aspects, the food waste may be waste from plant material, such as plants or plant parts used for food. This includes, for example, fruit waste material, vegetable waste, and nut and grain waste material. Thus, food processing waste material and food processing waste water can relate, specifically, to foods comprising or consisting essentially of plant material, e.g., frozen fruits, frozen vegetables, canned fruits, canned vegetables, breads, etc. In the same way, beverage processing waste material and beverage processing waste water

31

RECTIFIED SHEET (RULE 91) can relate, specifically, to beverages produced from plant material, e.g., beers, wines, ciders, juices, etc.

[00220] As exemplifications, fruit waste material may include one or more of the flesh, pulp, and skin of one or more fruits. Similarly, vegetable waste material may include one or more of flesh, leaf, root, and skin of one or more vegetables. Waste material may include a whole food component (e.g., whole fruit waste, whole vegetable waste, whole grain waste, etc), or any portion thereof. For example, it will be possible to utilise one or more of peels, shells, seeds, hearts, juice, pulp, oil, and flesh (e.g., from fruit waste, vegetable waste, grain waste, etc). Any combination of whole food components and their portions may be used.

[00221] Cruciferous vegetable waste material is particularly noted, for example, arugula, bok choy, broccoli (including Asian broccoli, broccoli rabe, broccoli Romanesco), brussels sprouts, cabbage (including Asian cabbage), cauliflower, collard greens, cress (including land cress and watercress) daikon, garden cress, horseradish, kale, kohlrabi, komatsuna, mizuna, mustard, radish, rutabaga, tatsoi, turnips, and wasabi waste material, and any combination of these. As exemplifications, broccoli waste material may include one or more of the broccoli heads, florets, stems, leaves, and roots; cabbage waste material may include one or more of the cabbage leaves, buds, and roots; brussels sprouts waste material may include one or more of the brussels sprouts buds, leaves, stalks, and roots.

[00222] Tuber and root vegetable waste material is particularly noted, for example, American groundnut, arrowhead, beet, carrot, cassava, chufa sedge, elephant ear, Jerusalem artichoke, jicama, mashua, oca, parsnip, potato, sweet potato, taro, yacon, and yam (including air potatoes, elephant foot yam) waste material, and any combination of these. As exemplifications, carrot waste material may include one or more of the carrot tops, skin, and taproot; parsnip waste material may include one or more of the parsnip tops, skin, and taproot; potato waste material may include one or more of the potato skin, tuber, and roots; yam waste material may include one or more of the yam greens, skin, tuber, and roots; oca waste material may include one or more of the oca greens, skin, tuber, flower, and roots.

[00223] Berry fruit waste material is particularly noted, for example, barberry, buckthome berry, currant (including black currant and red currant), grapes (including Oregon grapes), elderberry, gooseberry, mayapple, nannyberry, and rose hips waste material, and as further examples, blueberry, chokeberry, cranberry, huckleberry, and lingonberry waste

32

RECTIFIED SHEET (RULE 91) material, and as still further examples, bayberry, blackberry, boysenberry, dewberry, juneberry, loganberry, mulberry, raspberry, strawberry, tayberry, and wineberry waste material, and any combination of these. As exemplifications, berry fruit waste material (e.g., grapes, gooseberry, currant, etc) can include one or more of the flesh, skin, and seeds.

[00224] Specifically noted are broccoli waste material, brussels sprouts waste material, cabbage waste material, cauliflower waste material, and any combination thereof. Also specifically noted are eggplant waste material, leafy greens waste material (e.g., lettuce waste material, mesclun waste material, kale waste material, spinach waste material, etc). Also specifically noted are kumara waste material, potato waste material, carrot waste material, parsnip waste material, yam waste material, oca waste material, and any combination thereof. Also specifically noted are apple waste material, feijoa waste material, grape waste material, olive waste material, kiwifruit waste material, and any combination thereof.

[00225] Additionally noted are beverage processing waste materials, for example, fruit pomace, grape marc, spent grain, and any combination of these. Beverage processing can include alcoholic beverages prepared from plant materials and non-alcoholic beverages prepared from plant materials. Alcoholic beverages can include brewed or fermented beverages. Non-alcoholic beverages can include pressed, steeped, or fermented beverages. Examples include but are not limited to beer, bourbon, cider, gin, sake, vodka, wine, whiskey as alcoholic beverages; as well as juice, kombucha, smoothies, and tea as non-alcoholic beverages. Any combination of waste materials of this disclosure may be utilised.

[00226] To be considered as a vegetable waste material or a fruit waste material, the material may be, for example, showing one or more indications of disposable material, e.g., wilting, bruising, softening, spotting, decaying, over ripening, going to seed, going to flower, being past a use by date, or being past a best by date. Alternatively, to be considered as a vegetable waste material, a fruit waste material, or a grain waste material, the material may be, for example, a remainder that is left over from one or more processing steps, e.g., peeling, pressing, grinding, shredding, squeezing, slicing, steeping, or fermenting.

[00227] In certain aspects of this disclosure, particular plant materials can be excluded as food and can also be excluded as food waste. For example, in particular embodiments of the present disclosure, materials that are not normally defined as food for humans (i.e., generally accepted as edible/digestible for humans) can be ruled out as food and can also be

33

RECTIFIED SHEET (RULE 91) ruled out as food waste. As specific exemplifications, this can include woody materials such as tree wood and tree bark, grass or straw materials such as wheat straw and rice straw, stover materials such as shucked com leaves, com stalks, com silk, and stripped com cobs. In this way, lignocellulosic materials may be generally excluded as food and food waste, according to specified aspects of the present disclosure.

[00228] As disclosed herein, a food waste substrate may include one or more solid components and/or one or more liquid components obtained from food waste. For example, extracts including aqueous extracts may be utilised, these being obtained from one or more types of food waste as set out herein. The solid(s) and/or liquid(s) obtained from food waste may be formulated to produce a liquid media (e.g., broth) or solid media (e.g., agar plate), or semi-solid media (e.g., agar slurry). As such, it will be understood that various gelling agents such as agar may be utilised in conjunction with the food waste substrate.

[00229] As specific exemplifications, the food waste substrate may take the form of culture media, minimal media, selective media, differential media, transport media, and indicator media. In certain aspects, the food waste substrate may be enriched to include one or more additional ingredients to enhance growth. For example, one or more additional energy sources (e.g., added carbohydrates) and/or nutrients (e.g., added minerals or metals) and/or buffering agents may be included.

[00230] In particular aspects, one or more sugars, sugar alcohols, starches, minerals, metal compounds, and/or buffering compounds may be added to a food waste substrate. Nonlimiting examples of these include dextrose, fructose, sucrose, sorbitol, glycerol, soluble starch; as well as calcium, magnesium, iron, trace metals, phosphates, acetates and sulphates. As particular exemplifications, the sugar (e.g., one or more of dextrose, sucrose, glycerol, etc) may be utilised in the food waste substrate at about 0.5% to about 3.5%, or at about 1% to about 3%, or at about 1.5% to about 2% w/w or w/v. In certain circumstances, the addition of one or more exogenous sugars (e.g., dextrose and/or glycerol) can be omitted.

[00231] For example, broccoli waste material, brussels sprouts waste material, cabbage waste material, cauliflower waste material, eggplant waste material, leafy greens waste material (e.g., lettuce waste material, mesclun waste material, kale waste material, spinach waste material, etc), kumara waste material, potato waste material, carrot waste material, parsnip waste material, yam waste material, oca waste material, apple waste material, feijoa

34

RECTIFIED SHEET (RULE 91) waste material, grape waste material, olive waste material, kiwifruit waste material, and any combination thereof, may be utilised with or without the addition of one or more sugars (e.g., dextrose, glycerol, etc).

Products and production methods

[00232] According to this disclosure, expression of one or more dairy proteins (e.g., wild type or non-wild type molecules) may be achieved using appropriate expression constructs and substrates. The resulting polypeptides can be isolated and used to produce various products, including dairy products and dairy ingredients.

[00233] In particular exemplifications, the polypeptides may comprise an amino acid sequence of any one of SEQ ID NO: 1-6, and any variants of these sequences; as well as polypeptides that consist of an amino acid sequence of any one of SEQ ID NO: 1-6.

[00234] As provided in this disclosure, polypeptide(s) may be produced by growing at least one microbial cell capable of expressing one or more dairy proteins, under conditions to obtain expression of the one or more dairy proteins, wherein the at least one microbial cell is in and/or on a food waste substrate for growing the at least one microbial cell.

[00235] As also provided in this disclosure, a polypeptide expression system may be utilised, this system comprising (i) at least one microbial cell which is capable of expressing one or more dairy proteins, and (ii) a food waste substrate for growing the at least one microbial cell.

[00236] As additionally further provided in this disclosure, at least one microbial cell capable of expressing one or more dairy proteins may be utilised, the at least one microbial cell being in and/or on a food waste substrate for growing this at least one microbial cell.

[00237] In non-limiting examples, the cell may be a filamentous fungal cell, such as

Trichoderma, Aspergillus, or Myceliophthora cell; or a yeast cell, such as a Pichia, Kluyveromyces, Arxula, Candida, Hansenula, Saccharomyces, or Yarrowia cell; or a bacterial cell, such as Bacillus, Corynebacterium, Escherichia, Lactococcus, Mycobacterium, Pseudomonas, Ralstonia, Streptomyces, or Vibrio cell.

[00238] Specifically noted are Trichoderma reesei, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Myceliophthora thermophila cells. Also noted are Pichia

35

RECTIFIED SHEET (RULE 91) pastoris, Kluyveromyces lactis, Arxula adeninivoran, Candidia biodini, Hansenula polymorphci, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Yarrowici lipolyticci cells. Further noted are Bacillus subtilis, Bacillus megaterium, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Escherichia coli, Lactococcus lactis, Lactobacillus plantarum, Mycobacterium smegmatis, Pseudomonas fluorescens, Ralstonia eutropha, Streptomyces lividans, and Vibrio natriegens cells. Other suitable host cells may be utilised as described in detail herein. Any combination of host cells may be utilised.

[00239] As set out herein, dairy proteins (e.g., a wild type or variant molecules) can include whey proteins and casein proteins, for example, a-lactalbumin, [3-lactoglobulin, lactotransferrin/lactoferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, glycomacropeptide, [3-casein, K-casein, α-Sl-casein, and α-S2-casein, and any combinations of these. Specifically noted are cow milk proteins, as well as sheep milk proteins, goat milk proteins. Other suitable proteins may be utilised as described in detail herein.

[00240] The host cell may include at least one polynucleotide capable of expressing the dairy protein(s). This polynucleotide may be codon optimised for expression in the cell. This polynucleotide may encode a fusion protein. The polynucleotide may encode a protein fragment, e.g., a functional fragment. The polynucleotide may encode protein variant, e.g., a functional variant.

[00241] The substrate may comprise food waste or may consist essentially of food waste, for example, the substrate may comprise or may consist essentially of solid matter or liquid matter obtained from food waste. The substrate may comprise an extract of food waste or may consist essentially of an extract of food waste, for example, the substrate may comprise or may consist essentially of an aqueous extract obtained from food waste. As particular exemplifications, the food waste may include plant materials that are designated as waste, for example, the food waste may be fruit waste material and/or vegetable waste material, including one or more of the flesh, pulp, and skin of the fruit(s), and/or one or more of flesh, leaf, root, and skin of the vegetable(s). Other types of food waste are set out herein.

[00242] Once expressed by the microbial cell, a dairy protein may be isolated, concentrated, and/or dried. Methods involving air drying or heat-assisted drying (e.g., oven drying) may be used. Drying may be obtained, for example, by one or more of: sun or solar drying, hot air drying, batch drying, rotary drying, tunnel drying, belt drying, fluidised bed

36

RECTIFIED SHEET (RULE 91) drying, impingement drying, puff drying, drum drying, spray drying, vacuum drying, freeze drying, or osmotic drying. Lyophilisation (freeze drying) is specifically noted as a suitable drying method. Concentration may be achieved by precipitation or evaporation. Evaporation may be obtained, for example, by one or more of: pan evaporation, batch evaporation, tube evaporation, rising film evaporation, falling film evaporation, rising-falling film evaporation, or agitated film evaporation. In some circumstances, evaporation may be used to obtain product that is dry or essentially dry, and in specific circumstances, it may be useful to combine evaporation methods and drying methods, for example, an initial evaporation step followed by heat-assisted drying.

[00243] The resulting product (e.g., evaporated or dried protein) may then be milled into a powder, which can then be utilised as appropriate. Milling methods are well known and widely used by skilled persons in the field. Standard mesh sizes may be used to produce the powder, for example, US 20, US 23, US 30, US 35, US 40, US 45, or US 50 mesh sizes may be used. The sieve size for the powder may range from 1.0 to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or may be about 1.0 mm, about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47 mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm. Other meshes and/or sieves may be employed in accordance with established methods.

[00244] The dairy protein (e.g., concentrated protein, dried protein, or powder) may then be used to produce a dairy product or dairy ingredient. As non-limiting examples, the dairy product may be milk, evaporated milk, milk powder, milk substitute, evaporated milk substitute, or milk substitute powder; or the dairy product may be cream, creamer, cocktail mixer, custard, cheese, desert topping, gelato, ghee, ice cream, milkshake, pudding, butter, spread, sauce, sorbet, smoothies, snack food, yoghurt, yoghurt drink, and frozen yoghurt; or the diary product may be infant formula, toddler formula, nutritional supplement, energy supplement, sports supplement, weight gain supplement, weight loss supplement, energy drink, sports drink, weight gain drink, weight loss drink, energy food, sports food, weight gain food, and weight loss food.

[00245] As other non-limiting examples, a dairy ingredient may be produced for use in one or more of a baked good, beverage, cooked good, frozen good, cooking mix, baking mix, beverage mix, or sauce mix; or the diary ingredient may be milk protein concentrate or milk protein isolate; or the dairy ingredient may be whey protein concentrate, a-lactalbumin

37

RECTIFIED SHEET (RULE 91) concentrate, β- lactoglobulin concentrate, glycomacropeptide concentrate, and casein concentrate; or the dairy ingredient may be whey protein isolate, a-lactalbumin protein isolate, P-lactoglobulin protein isolate, glycomacropeptide protein isolate, and casein protein isolate.

[00246] Specific dairy products include protein bars, protein drinks, protein gels, and protein powders, as well as fermented dairy products, and long-life dairy products. Specific dairy ingredients include demineralised whey, demineralised whey protein, demineralised whey powder, as well as dairy ingredients to provide one or more of emulsifying, stabilising, texturising, viscosity, colour, flavour, and flavour masking in foods and/or beverages.

[00247] The examples provided herein are provided for the purpose of illustrating specific embodiments and aspects and are not intended to limit this disclosure in any way. Persons of ordinary skill can utilise the disclosures and teachings herein to produce other embodiments, aspects, and variations without undue experimentation. All such embodiments, aspects, and variations are considered to be part of this disclosure.

EXAMPLES

Example 1: Growth studies for fungal strains

(1) Strains and standard media

[00248] Trichoderma reesei (T. reesei) QM6a was obtained from Artemio Mendoza at Lincoln University, New Zealand. Standard growth substrates for Trichoderma reesei (T. reesei) were prepared as follows.

(1) Potato dextrose broth (PDB)

4 g Potato Infusion Powder (Sigma-Aldrich) and 20 g glucose (Sigma-Aldrich) was mixed with 1 L ddH ₂ O.

(2) Potato dextrose agar (PDA)

4 g Potato Infusion Powder (Sigma- Aldrich), 20 g glucose (Sigma- Aldrich), and 15 g Agar (Sigma- Aldrich) was mixed with 1 L ddkhO . The mixture was autoclaved at 121°C for 15 minutes to sterilise, cooled, and poured into plates.

(2) Food waste broth

[00249] Growth substrates for Trichoderma reesei (T. reesei) were prepared as follows.

38

RECTIFIED SHEET (RULE 91) For each of the fruit or vegetable growth media: The starting fruit and vegetables were originally obtained from Countdown and stored in a refrigerator. Their condition was deteriorated beyond best eating, but still edible. To begin, 50 g of cut fruit or vegetable (whole, unpeeled) was weighed. The weighed material was boiled for approximately 30 minutes in 300 mL ddH ₂ O. until soft. The boiled material was strained in a muslin cloth to collect extract. The extract was topped up ddH ₂ O to 500 mL. For media with dextrose, 5 g dextrose was added and dissolved. The media were autoclaved to sterilise. Tested media: apple waste extract (A), apple waste extract with dextrose (AD), broccoli waste extract (B), broccoli waste extract with dextrose (BD), cabbage waste extract (Cb), cabbage waste extract with dextrose (CbD), carrot waste extract (C), carrot waste extract with dextrose (CD), potato waste extract (P), potato waste extract with dextrose (PD). Control was potato dextrose broth (PDB) prepared from potato infusion powder (Millipore®).

(3) Growth assays

[00250] The growth of T. reesei was measured as follows. Standard and experimental growth media were prepared as described above. T. reesei was grown on a PDA plate for 5 days. Next, 3 mL Tween 80 with NaCl was added to the plate and a cotton bud was used to scrape the spores from the plate. Using a pipette, the mixture was passed through a glass wool tube (see further below). This fdtered out any mycelia. The spore solution passed through and was collected in a plastic test tube. Then, 3 μL of spore solution was loaded onto a haemocytometer and spores were counted. Dilutions were performed to achieve 10 ⁶ - 10 ⁷ spores per mL with the respective growth media. Following this, 150 μL inoculated growth media was added to each well with 50 μL of mineral oil. Each plate contained 5 replicates of inoculated media and 3 replicates of the negative control (media with no organism). Plates were read for 48 hours at 27°C. Readings were taken every 5 minutes with 20 seconds of shaking prior to each reading.

(4) Results for growth assays

[00251] T. reesei (without transformation) was grown on 11 different media as described above. Optical density measurements were taken every 5 minutes at 600 nm for 48 hours. Replicate wells were averaged and OD ₆₀₀ readings from negative controls were subtracted to account for any influence that the media colour had on OD ₆₀₀ readings.

[00252] The results are shown in Figure 2. See also Tables 1A and IB, below. For all

39

RECTIFIED SHEET (RULE 91) media tested, the growth curves replicated the pattern expected and were similar to those observed for the PDB control (potato dextrose broth). While growth on PDB media reached the highest OD ₆₀₀ levels at around 12 hours, this did not significantly out-perform growth on the other organic waste media. Growth on carrot waste media showed comparable performance as growth on the PDB control, even surpassing growth on PDB media until about 21 hours. Growth on apple waste media and apple waste media with dextrose also showed consistent and impressive levels. Surprisingly, potato waste media proved to be the worst performing media, and potato waste media with dextrose did not perform much better. Similarly broccoli waste media and broccoli waste media with dextrose did not stand out significantly when compared to other media. Despite certain media being comparably better than other media, all of the tested waste media demonstrated potential as a growth source. See Figure 2.

40

RECTIFIED SHEET (RULE 91) Table 1A: Averaged growth levels for Trichoderma reesei QM6a in different substrates

Table IB: Averaged growth levels for Trichoderma reesei QM6a in different substrates

Example 2: Transformation of fungal strains

(1) Transformation solutions and substrates

[00253] Growth substrates and protocol solutions were prepared for T. reesei as follows. Strain used is as described in Example 1, above.

(1) PDB (see above)

(2) Sorbitol osmoticum solution

Added were: CaCl ₂ 50 mM, sorbitol 700 mM, MES 50 mM, and NaOH to adjust to pH 5.5. The mixture was filter sterilised using 0.2 pm cellulose acetate membrane.

(3) Regeneration medium

Added were: Potato infusion powder 4 g (Millipore®), glucose 20 g, sucrose 171 g, ddH ₂ O 1 L, agar 8 g (0.8%). This mixture was autoclaved at 121°C for 15 minutes to sterilise.

(4) PEG 40% solution

Added were: PEG-3350400 g (40%) (Sigma- Aldrich) and sorbitol osmoticum 1 L. This was heated to dissolve if needed. To sterilise, autoclaving was carried out at 121°C for 15 minutes.

(5) Enzyme solution

Added were: 50 mg Glucanex (Artemio Mendoza of Lincoln University; Sigma-Aldrich product) and 5 mL sorbitol osmoticum solution. This was vortexed to dissolve then filter sterilised with a 0.2 pm cellulose acetate membrane syringe filter.

(6) Hygromycin B

Hygromycin B solution (Sigma-Aldrich) was filter sterilised using 0.2 pm cellulose acetate membrane.

(7) PDA plates with hygromycin

Added were: PDA 39 g and ddH ₂ O 1 L. This was autoclaved at 121°C for 15 minutes to sterilise, then cooled to less than 50°C to allow addition of Hygromycin B at 50 μg/mL.

(8) PDA plates with Hygromycin plus Triton X1000.1% Added were: Triton X100 1 mL and water 1 L. This was mixed and added with PDA 39 g. The mixture was then autoclaved at 121°C for 15 minutes to sterilise, then cooled to less than 50°C to allow addition of add Hygromycin B at 50 |ag/mL.

(9) Freezing protoplasts

To 1 mL protoplasts was added: 0.5 ml 40% PEG solution and 15 μL DMSO. This was mixed gently. Aliquots of 100 pl were frozen.

(10) Glass wool tubes

Eppendorf tubes were pierced at the bottom with a hot needle. Glass wool was placed inside tube with tweezers. This was autoclaved at 121°C for 15 minutes to sterilise.

(2) Plasmids

[00254] Plasmids were constructed for transformation into T. reesei. See Table 2, below.

Table 2: Plasmids to be used * Aspergillus awamori alpha-amylase secretion signal

(3) Preparation for transformation

[00255] In preparation for transformation, pUE08 and pEM06T constructs were digested with restriction enzyme. See Table 3, below. The digested DNA was purified using a OMEGA EZNA Cycle Pure Kit following the instruction in the kit. The quantity of DNA was determined using Qubit set at double strand DNA broad range setting. 20 μl of each mixture was used for transformation. pUE08 = 1.4 μg DNA; pEM06T = 2 μg DNA. The restriction digests were incubated at room temperature overnight.

Table 3: Plasmid digestion mixtures

(4) Preparation of protoplasts and spores

[00256] For preparation of protoplasts: The preparations were carried out to transform the pUE08 and pEM06T constructs as indicated directly above. T. reesei spores were plated on a PDA plate. This was incubated at 25°C for 5-7 days.

[00257] For washing spores: 5 ml water was added to a PDA plate containing T. reesei spores. A bacterial spreader was used to dislodge spores from the plate. The suspended spores were pipetted into a sterile glass wool Eppendorf tube. The spores were allowed to run through. This was done to remove the mycelia via filtration. A haemocytometer (Neubauer chamber) was used to count spores under a microscope. The spores were diluted to 1 x 10 ⁸ concentration with 100 ml PDB in a 500 ml flask. This was incubated for 15 hours at 28°C with shaking. A microscope was used to check the Trichoderma for germ tubes. Ideally, cells were just germinated, without branching. Multiple branches include multiple nuclei and present issues later for identifying single spores with the desired construct(s).

(5) Preparation of germ tubes

[00258] The germ tubes were centrifuged at room temperature at 10,000 rpm for 10 minutes. The supernatant was removed to a 50 mL falcon tube. This was centrifuged again to remove as much media as possible. The pellet was washed with 20 mL sorbitol osmoticum. This was centrifuged at 10,000 rpm for 10 minutes at room temperature. The supernatant was carefully removed, noting that cells may not have been well compacted and shaking could potentially disturb the pellet. Next, 5 ml enzyme solution (Glucanex; see above) was added. This was vortexed briefly to mix the germ tubes and enzyme solution.

[00259] Incubation was then carried out for 2-3 hours at 28°C with 100 rpm shaking. The tube was placed horizontally and fixed with tape to allow the cells good contact with the enzymatic solution. The protoplasts were counted using a haemocytometer counting chamber. For each transformation, 10 ⁸ protoplasts were used; these were handled gently. Suspension was carried out in 30 mL sorbitol osmoticum. This was centrifuged at 7,400 rpm for 10 minutes. The supernatant was carefully removed and discarded. Another 10 mL sorbitol osmoticum was added, and then centrifuged at 7,400 rpm for 10 minutes. The supernatant was carefully removed and discarded.

[00260] Gently, protoplasts were resuspended with 500 μL to 2 mL sorbitol osmoticum solution. The amount used was dependent on the concentration of the protoplasts. 10 ⁸ protoplasts were utilised for each transformation. The protoplasts were kept on ice. The regeneration medium was melted and kept at 50°C until use. The 40% PEG was checked for precipitation. If present, this was microwaved and kept at 42°C. The solution was used at 42°C or room temperature.

(6) Transformation of protoplasts

[00261] The protoplasts were prepared for pUE08 and pEM06T transformations as indicated directly above. For transformation, at least 1 μg linearised plasmid DNA was added into a maximum of 20 μL of water. A higher water volume could cause osmotic stress on protoplasts. 4 tubes were labelled for each of the transformations: (1) Control - protoplasts with water added; (2) Control - protoplasts with water added; (3) Protoplasts with DNA; (4) Protoplasts with a plasmid containing hygromycin resistance cassette (positive control).

[00262] For each tube, 240 μL protoplasts was added, along with: Tube 1 - 20 μL of sterile water (this sample was plated on regeneration agar without hygromycin B); Tube 2 - 20 μL of sterile water; Tube 3 - 20 μL of target gene; Tube 4 - 20 μL pUOE8 (around 1.4 ug). The tubes were incubated on ice for 20 minutes and then returned to room temperature. 260 μL PEG solution was added. This was gently mixed with a pipette tip. The protoplasts in PEG solution were incubated at room temperature for 30 minutes.

[00263] Four 15 ml sterile tubes were placed in a rack. The regeneration media was prepared. In 50 mL tubes, 35 mL regeneration media was added and 1.75 mg hygromycin B. The tube was inverted gently to mix. Shaking was avoided. 10 mL of the solution was poured immediately into three 10 cm petri dishes. 10 mL of regeneration media was poured into another 10 cm petri dish without hygromycin. The plates were allowed to solidify for 5-10 minutes.

[00264] In the Tube 1, 8-10 mL regeneration media was placed without hygromycin using a 1 mL pipette tip that had been cut to increase the hole size. A sterile heated scalpel was used to do this. Protoplasts were taken from Tube 1 and added to the first 15 mL tube containing regeneration media without hygromycin. The tube was closed and gentle mixing was carried out by inverting 3-4 times. Immediately, the protoplasts were poured over the plate with regeneration media. The plates were moved in circles on the benchtop to allow even spreading and mixing with regeneration media. The plate was left open to solidify for 10 minutes.

[00265] For Tubes 2-4, the same steps were repeated except that regeneration media supplemented with hygromycin B was used. Once all plates were poured, plates were sealed Parafilm M. Plates were incubated at 25°C for 5-7 days depending on the resistance and phenotype of the mutant. After 7 days of incubation, agar block was cut from several colonies and transferred to a new selective media plate (PBA with 50 μg/mL hygromycin). See Figure 3 A.

(7) Isolation of transformants

[00266] Transformation was carried out for pUE08 and pEM06T constructs as indicated directly above. Colonies that grew were transferred to plates of 25 ml PDA with hygromycin B. These were incubated for 1 to 2 days at 25°C. The hyphae tips were cut with a sterile or flamed scalpel. The cut material was transferred to new PDA-hygromycin B plates. These were incubated for 1 to 2 days at 25°C. The hypha tips from a single colony were transferred to a PDA plate without hygromycin. This was incubated for 5 days at 25-28°C under light dark cycles (12 h:12 h). After this period, the spores were generated.

[00267] For this, 40 pl of spores were taken using sterile water and placed on the border of a PDA plate with hygromycin and Triton X100 (0.1 %). The spores were separated with the help of sterile loop. These were incubated for 3 to 5 days at 25°C until single colonies appeared. Single colonies were then transferred to a PDA-hygromycin plate. These were grown for 2 days at 25°C and the hyphal tips were cut. The cut material was transferred to new PDA- hygromycin B plates as noted in the preceding paragraph, and the subsequent steps were repeated 2-3 times. The insert was then confirmed by PCR. See Figures 3B-3C.

Example 3: Growth studies for yeast strains

(1) Growth assay

[00268] The growth of K. lactis GG799 (New England Biolabs) was measured as follows. Standard and experimental growth media were prepared as described above. Experimental media: apple waste extract (A), apple waste extract with dextrose (AD), broccoli waste extract (B), broccoli waste extract with dextrose (BD), cabbage waste extract (Cb), cabbage waste extract with dextrose (CbD), carrot waste extract (C), carrot waste extract with dextrose (CD), potato waste extract (P), potato waste extract with dextrose (PD). A single colony of each strain was resuspended in 500 μL of ddH ₂ O . Next, 3 μL of the suspension was inoculated into 3 mL media and incubated at 28°C for 48 hours. Growth was observed of both strains with none in the negative controls (media with no inoculant).

[00269] To ensure the growth was not as a result of any residual media taken from the plate during the colony suspension, a further 1:100 dilution as performed. For this, 30 μL of the overnight culture was diluted with 2970 μL media. Cultures were left 84 hours at 28°C with shaking. For plate reading, 1 mL of overnight culture grown in YPD was spun down and pelleted then resuspended in PBS. This was repeated 3 times to ensure removal of any residual YPD growth media. 2 μL of the final resuspension in PBS was added to 148 μL of each media and pipetted into a well of a in a 96 well plate. Five replicates for each media were done with three negative control replicates (media with no inoculant). Then, 50 μL of mineral oil was placed on top to reduce evaporation on the lid which could impact on OD ₆₀₀ readings. The negative controls were averaged and subtracted from the growth assay data to account for an influence the media colour has on measurements. Yeast peptone dextrose (YPD) was used as the positive control. There were five replicates for each experiment with two negative control replicates for each media. (2) Results for growth assay

[00270] The results are shown in Figure 4. See also Tables 4A and 4B, below. Growth on media supplemented with dextrose was better than growth on media without dextrose. The best growth was observed in broccoli waste media with dextrose which outperformed the positive control, and standard K. lactis growth media of YPD (yeast peptone dextrose). Growth in apple waste media with dextrose reached the stationary phase much earlier than all other media tested, including apple alone. However, the initial OD ₆₀₀ measurement was much higher than the rest of the media. Growth in broccoli waste media also followed a similar pattern with a very short exponential growth phase, entering stationary phase quickly. Growth in both potato waste media and potato waste media with dextrose gave interesting results. K. lactis GG799 in potato waste media with dextrose showed a shallow extended exponential growth phase, but eventually reached an OD higher than seen for all other media without dextrose, except for carrot. Growth on broccoli waste media with dextrose, cabbage waste media with dextrose, YPD, and carrot waste media with dextrose all follow a very similar curve with a similar gradient during the exponential growth phase and all reaching a plateau around the 24 -hour mark. Growth on carrot waste media without dextrose followed this same initial growth phase gradient but reached a plateau earlier at about 12 hours. As in the earlier studies, certain media were comparably better than others, but all of the waste media demonstrated potential as a growth source.

Table 4A: Averaged growth levels for K. lactis GG799 in different substrates

Table 4B: Averaged growth levels for K. lactis GG799 in different substrates

Example 4: Transformation of yeast strains

(1) pDL303 vector for p-lactoglobulin expression

[00271] The native bovine nucleotide sequence for β-lactoglobulin variant B was codon optimised to be better suited for Kluyveromyces lactis expression by reference to codon usage tables (see Figure 6B). Codon usage tables were found on publicly available websites (seewww.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=289 85&aa=l&style=N). Lastly, three codon optimised HA-tags were inserted just before the stop codon. HA-tag = YPYDVPDYA (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala; SEQ ID NO: 16) (see Figure 6C).

[00272] DNA for the codon optimised nucleotide sequence including HA-tag was obtained from Twist Bioscience. This was cloned into the Xhol restriction site in the multiple cloning site of the pKLAC2 expression vector (NEB #E1000S K. lactis Protein Expression Kit), producing the pDL3O3 vector. The sequence leading up to the KEX2 cleavage site between the a-factor secretion signal and the β-lactoglobulin gene was kept intact. The construction method was as follows. The pKLAC2 vector was linearised with a Xhol digest. The codon optimised gene was amplified by PCR using gene specific primers. See Table 5, below. In Table 5, the lowercase lettering shows sequence homologous to pKLAC2 upstream of the Xhol site. The uppercase lettering shows sequence homologous to pKLAC2 downstream of the Xhol site.

Table 5: PCR primer sequences

[00273] The primers were synthesised by Integrated DNA Technologies to introduce flanking regions homologous to the pKLAC2 vector on either side of the Xhol site. The new construct was assembled using the NEB Gibson Assembly® Master Mix. The pDL3O3 vector was transformed into Top 10 E. coli using divalent cation-mediated transformation and plated on LB agar with 100 μg/mL ampicillin for selection. A single colony was selected from the plate and the presence of the desired gene was confirmed by colony PCR. The colony was used to inoculate 50 mL of LB media, which was incubated at 37°C with shaking overnight and then used for a plasmid midi extraction (using the Qiagen® Plasmid Midi Kit). The extracted plasmid was linearised with a Sacll-HF digest and transformed into K. lactis GG799 (NEB #E1000S K. lactis Protein Expression Kit) following the instructions in the kit. The negative control and positive control plasmid were transformed in the same way. After 5 days ten colonies were patched on a fresh YCB with 5 mM acetamide plate.

(2) Expression of β -lactoglobulin

[00274] Standard growth substrates for Kluyveromyces lactis were prepared as follows.

(1) YPGal medium

10 g yeast extract and 20 g peptone were dissolved in 950 ml deionized water. This was autoclaved for 20 min at 121 °C; and allowed to cool to room temperature. Aseptically, 50 ml of sterile 40% galactose was added to this.

(2) YCB plates

YCB agar medium was prepared with 5 mM acetamide (500 ml): For this, 15 ml 1 M Tris-HCl buffer stock solution (pH 7.5) was mixed with 5.85 g YCB medium powder, and 10 g bacto agar. The volume was brought up to 495 ml with deionized water. This was autoclaved for 20 minutes at 121°C and allowed to cool to ~60°C. Aseptically, 5 ml of 100X acetamide stock solution was added to this.

[00275] After incubation for 2 days at 30°C all of the colonies on the patch plate were inoculated into 2 mL of YPGal in test tubes and incubated at 30°C and 250 rpm for 48 hours. Cells (400 pl) were collected from each tube (including the negative and positive controls) and tested for integration of multiple copies of the BLG expression cassette at the correct locus using the PCR primers and instruction in the kit. A 200 μl sample was taken from each of the above cultures and centrifuged at 3000 rpm for 3 minutes to remove the cells. From the liquid fraction, 15 μl was taken and analysed via SDS-PAGE to assess the expression ofβ- lactoglobulin.

(3) Results for expression studies

[00276] The results are shown in Figure 5. The results showed that K. lactis was able to express BLG from plasmid pDL3O3. Example 5: Experimental evolution studies

(1) Overview

[00277] An experimental evolution approach will be used to develop and select the microbial colonies that are best suited to digesting each particular substrate. Particular focus will be placed on food waste substrates obtained from apples, grapes, kiwifruit, olives, and spent grain. For these studies, different substrates will be aliquoted into multiwell plates, and then inoculated with a microbial strain. A sample will be taken from a well that demonstrates the best growth for a particular substrate. This will be used to inoculate several replicates of fresh media in new multiwell plates. The plate reader assay will be repeated and improved growth is detected again. These steps are repeated several times to evolve strains for improved growth on the substrate of interest. Improved protein expression on each substrate will also be evaluated with SDS-PAGE. Strains exhibiting increased protein expression will be selected for and used in subsequent expression trials.

(2) SDS-PAGE solutions

[00278] Solutions are prepared as follows.

(1) Sample buffer (SDS reducing buffer)

Added are: 3.55 mL deionised water, 1.25 mL 0.5 M Tris-HCL. pH 6.8, 2.5 mL glycerol, 2.0 mL 10% (w/v) SDS, 0.2 mL 0.5% (w/v) bromophenol blue. The total volume is 9.5 mL. Prior to use, 50 μL β-mercaptoethanol is added to 950 μL sample buffer.

(2) 10X Electrode (running) buffer, pH 8.3

Added are: 30.3 g Tris base, 144.0 g glycine, 10.0 g SDS. This mixture is dissolved and brought to a total volume of 1000 ml with deionised water. For each electrophoresis run, 50 mL stock solution is diluted with 450 mL deionised water. This is mixed thoroughly before use.

(3) Coomassie blue stain

Added are: 1.25 g Coomassie brilliant blue R250 (0.125%), 500 mL methanol (50%), 100 mL acetic acid (10%), and 400 mL deionised water. The total volume is 1 L.

(4) Destain solution

Added are 300 mL methanol (30%), 100 mL acetic acid (10%), and 600 mL deionised water (60%). The total volume is 1 L. (4) SDS-PAGE protocol

[00279] For SDS-PAGE analysis: Pellets are resuspended in 100 μL sample buffer and heated at 95°C for 10 minutes. The samples are spun down at 13,000 rpm for 1 minute. Into a pre-cast stacking gel, 5 μL protein marker and 10 μL of sample are loaded. The gel is run for 30 minutes at 200 volts. The gel is removed from the chamber and extracted from the plastic plates. The gel is stained with Coomassie blue and incubated for an hour at room temperature with gentle shaking (about 100 rpm). The Coomassie blue stain is poured out and the gel is washed with tap water twice to remove excess stain. The gel is covered with destain solution and incubated at room temperature with gentle shaking for 15 minutes. The destain solution is poured out and replaced with fresh destain solution. This is left overnight with shaking or for several hours until all background stain has been washed away.

Example 6: Extended plate growth studies for yeast strains in waste media

(1) Media preparation

[00280] To prepare waste media, 100 g of the selected waste fruit or waste vegetable was boiled in 500 mL of ddH ₂ O as per the standard potato dextrose agar (PDA) recipes. Substrates were boiled for approximately 30 minutes or until the substrate was soft and mushy. The boiled substrate was strained through a muslin cloth into a 250 mL bottle. The media was autoclaved at 121°C at 15 minutes. For dextrose media, a 40% dextrose solution was made an autoclaved. 750 μL of 40% dextrose was added to 14.25 mL of waste media to give a 2% final concentration of dextrose.

[00281] The following waste media was prepared:

(1) Apple waste extract (A)

(2) Apple waste extract + dextrose (AD)

(3) Broccoli waste extract (B)

(4) Broccoli waste extract + dextrose (BD)

(5) Carrot waste extract (C)

(6) Carrot waste extract + dextrose (CD)

(7) Cabbage waste extract (Cb) (8) Cabbage waste extract + dextrose (CbD)

(9) Grape waste extract (G)

(10) Grape waste extract + dextrose (GD)

(11) Kiwifruit waste extract (K)

(12) Kiwifruit waste extract + dextrose (KD)

(13) Potato waste extract (P)

(14) Potato waste extract + dextrose (PD)

(2) Strains and growth assays

[00282] The following strains were utilised:

(1) pKC15P - Pichia pastoris BG11 comprising a inducible promoter controlling the expression of the bovine β-lactoglobulin gene. The parent strain, BG11 and parent plasmid were obtained from BioGrammatics (California, USA).

[00283] P. Pastoris pKC15P strain construction for β-lactoglobulin expression: An open reading frame (ORF) sequence comprising the S. cerevisiae α-mating factor pre-pro peptide (si) purchased from by BioGrammatics, Inc. and the native bovine nucleotide sequence forβ- lactoglobulin variant B was edited to replace the stop codon with the stop codon preferred by P. pastoris (TAA) and remove any Pmel, Bsal and Bsu36I recognition sites. The resulting ORF was ordered from Twist Bioscience (San Francisco, CA) as a clonal gene in the Bsal insertion site of the pJAG vector-(BioGrammatics, Inc.). The vector was digested with Pmel from New England Biolabs (Ipswich, MA). The linearized vector was then transformed into electrocompetent P. pastoris cells using electroporation. Transformed cells were incubated in 1:1 YPD: 1 M sorbitol for ~3 hours and then plated on selective YPD G418 media. Distinct transformant colonies were streaked on selective media for single colonies. One of these single colonies was selected to move forward with and is hereto referred to as pKC15P.

(2) pKC23P-6 - Pichia pastoris BG11 comprising a constitutive promoter (UPP) controlling the expression of the bovine β-lactoglobulin gene. The parent strain, BG11 and parent plasmid were obtained from BioGrammatics (California, USA).

[00284] P. Pastoris pKC23P-6 strain construction for β-lactoglobulin expression: The nativeβ- lactoglobulin variant B amino acid sequence was back-translated for Pichia pastoris using Benchling Biology Software (2023. https://benchling.com) to create a codon optimized (DL encoding) version of the BLG gene. An open reading frame (ORF) sequence comprising the S. cerevisiae a-mating factor pre-pro peptide purchased from by BioGrammatics, Inc. with a deletion of the Glu-Ala repeats after the Kex2 cleavage site (si.5) followed by the DL encoding for β-lactoglobulin variant B was edited to replace the stop codon with the stop codon preferred by P. pastoris (TAA) and remove any Pmel, Bsal and Bsu36I recognition sites. The resulting ORF was ordered from Twist Bioscience (San Francisco, CA) as a clonal gene in the Bsal insertion site of the pJUG vector (BioGrammatics, Inc.). The vector was digested with Bsu36I from New England Biolabs (Ipswich, MA). The linearized vector was then transformed into electrocompetent P. pastoris cells using electroporation. Transformed cells were incubated in 1 : 1 YPD: IM Sorbitol for ~3 hours and then plated on selective YPD G418 media. Distinct transformant colonies were streaked on selective media for single colonies. One of these single colonies was selected to move forward with and is hereto referred to as pKC23P-6.

[00285] A 96 well plate assay was performed as follows. For two days, a culture of Pichia pastoris pKC15P was grown with shaking at 28°C. This culture was used to make the inoculum. To do this, 1 mL of the culture was spun down and washed 3 times with 1 mL PBS to remove any residual growth. From this washed pellet, 2 μL was used to inoculate each well that contained 148 μL of waste media. The plate reader was left for 48 hours with fast shaking at 28°C. The plate set up is shown in Table 6, below.

Table 6: Plate set up for growth assays [00286] Three replicates of each media was performed with three negative control wells which consisted of the media without any culture inoculum. The negative controls are indicated by ‘-ve’ on the 96 well plate schematic above. The OD ₆₀₀ of replicates was averaged, and the negative control was subtracted from the respective cultured media.

[00287] The results are shown in Figure 9. See also Tables 7A and 7B, below. Growth of Pichia pastoris pKC15P was seen on all waste media trialled. Growth on grape waste media with dextrose media gave the highest final OD ₆₀₀ reading. This was followed by growth in cabbage waste media with dextrose and broccoli waste media with dextrose. Growth on potato waste media with dextrose and kiwifruit waste media resulted in the lowest OD ₆₀₀ measurements. Growth on several of the media resulted in noisy trend lines which was particularly noted for growth on grape waste media. Typically media supplemented with dextrose resulted in Pichia pastoris pKC15P reaching a higher OD ₆₀₀ reading than the same media without dextrose.

Table 7A: Averaged growth levels for Pichia pastoris pKC15P in 96 well plate studies

Table 7B: Averaged growth levels for Pichia pastoris pKC15P in 96 well plate studies

[00288] Next, a 96 deep well plate trial was conducted. Plate 1: 592 μL of each waste media was pipetted into the wells as per the deep well plate set up. See Table 8, below. Following this, 1 mL of Pichia pastoris pKC23P-6 overnight culture was spun down. The pellet was washed 3 times in PBS and resuspended in 1 mL PBS.

[00289] At the zero hour mark, each well, except for the negative controls, was inoculated with 8 μL of resuspended overnight culture. A sterile breathable seal film was placed over the top of the plate. The plate was left in the 28°C room in the floor plate shaking incubator which was also set to 28°C and set to 1000 RMP. Twelve hours later, 1.5% glycerol (GY) was added to each of the wells labelled GY, including the GY negative controls. This was done by adding 20 μL of 50% GY to the appropriate wells. A sterile breathable film was placed over the top of the plate.

Table 8: Deep well plate set up for growth assays

[00290] Two replicates of each well was performed with two negative controls for each waste media which contained no inoculum. These wells are indicated by a ‘-ve’ on the 96 well schematic above.

[00291] At the 24 hours mark, 100 μL of culture from each well was removed from the deep well plate (Plate 1) and placed in a new 96 well plate (Plate 2). To each GY well (Plate 1), 1.5% glycerol was added. This was done by adding 17 μL of 50% glycerol to the appropriate wells. A sterile breathable film was placed over the deep well plate (Plate 1) and it was placed back in the shaking incubator at 1000 RPM at 28°C.

[00292] Next, 10 μL was taken from the 100 μL 24 hour sample (Plate 2) and placed into another 96 well plate (Plate 3) containing 90 μL of each media to produce a 1:10 dilution. An end point assay was performed in the plate reader at 600 nm. The plate with the remaining 90 μL sample (Plate 2) was spun down and the supernatant was removed from the pellet and transferred to a new plate (Plate 2S). Both plates (Plates 2 and 2S) were transferred to -20°C.

[00293] At the 36 hour mark, another 17 μL of glycerol was added (Plate 1). At the 48 hour mark, 100 μL of sample was taken from the deep well plate (Plate 1) and placed in a new 96 well plate (Plate 2). To each GY well (Plate 1), 1.5% glycerol was added. This was done by adding 13 μL of 50% glycerol to the appropriate wells. A sterile breathable film was placed over the top of the deep well plate (Plate 1), and this was placed back in the shaking incubator at 1000 RPM at 28°C.

[00294] Next, 4 μL was taken out of the 100 μL sample plate (Plate 2) and placed in another plate (Plate 3) that already had 96 μL of each media to result in a 1:25 dilution. An end point assay reading was taken at 600 nm. The plate that had the remaining 96 μL sample (Plate 2) was and spun down for several minutes to pellet the cultures. The supernatant was removed carefully and placed in another 96 well plate (Plate 2S). Both plates (Plates 2 and 2S) were transferred to -20°C.

[00295] At the 72 hour mark, 100 μL of sample from each well out of the deep well plate (Plate 1) was placed in a new 96 well plate (Plate 2). Next, 1 μL was taken from the 100 μL plate (Plate 2) and placed in another plate (Plate 3) that already had 99 μL of each media to result in a 1:100 dilution. An end point assay reading was done at 600 nm. The plate with the 99 μL sample (Plate 62 was spun down for several minutes. The supernatant was carefully removed and placed in another 96 well plate (Plate 2S). Both plates (Plates 2 and 2S) were transferred to -20°C.

[00296] The OD ₆₀₀ readings demonstrating the growth of Pichia pastoris pKC23P-6 on waste media are shown in Figure 10. See also Table 8A, below. Growth of Pichia pastoris pKC23P-6 was seen on all waste media trialled, with the exception of kiwifruit waste media with dextrose (KD) which resulted in zero or negative values upon subtraction of the negative control OD ₆₀₀ value from the experimental value. As expected, growth in the standard growth media of BMGY with additional glycerol feeds (BMGYGY), resulted in the highest OD ₆₀₀ values. This was followed by growth in broccoli waste media with glycerol feeds. Growth on BMGY and broccoli waste media with dextrose followed closely. Generally, growth on all forms of apple waste media and kiwifruit waste media results in the lowest OD ₆₀₀ level seen. For some media, supplementation with glycerol resulted in a higher OD ₆₀₀ level than those with either dextrose or no supplementation but this was not a pattern seen consistently across all waste media substrates. In fact, Pichia pastoris pKC23P-6 growth in grape waste media and carrot waste media with no supplementation resulted in a higher OD ₆₀₀ level than their respective supplemented medias.

Table 8A: Averaged growth levels for Pichia pastoris pKC23P-6 in deep well plate studies

Example 7: Protein expression analysis for plate growth studies

[00297] SDS-PAGE was used to assess protein expression from Pichia pastoris pKC23P-6 grown in waste media as described in Example 5. SDS-PAGE solutions were prepared as described in Example 5. For gel analysis, 15 μL of sample supernatant was added to 3 pl of loading dye. Samples were boiled at 95°C for 10 minutes. 3 μL of a molecular weight protein ladder was loaded and 10 μL of each sample into a pre-cast stacking gel.

[00298] The gel was removed from the chamber and extracted from the plastic plates. The gel was stained with Coomassie blue and incubated for an hour at room temperature with gentle shaking (about 100 rpm). The Coomassie blue stain was poured out and the gel is washed with tap water twice to remove excess stain. The gel was covered with destain solution and incubated at room temperature with gentle shaking for 15 minutes. The destain solution was poured out and replaced with fresh destain solution. This was left overnight with shaking or for several hours until all background stain has been washed away.

[00299] The SDS-PAGE gels demonstrating bovine β -lactoglobulin protein expression levels are shown in Figures 11A and 1 IB. Surprisingly, the highest expression levels were seen with Pichia pastoris pKC23P-6 grown in broccoli waste media, without addition of dextrose or glycerol. The inclusion of dextrose in the broccoli waste media produced a notable decrease in expression, which was unexpected. Substantial expression levels were also seen for Pichia pastoris pKC23P-6 grown in cabbage waste media. Substantial expression was also noted for Pichia pastoris pKC23P-6 grown in the potato waste media, including with and without addition of dextrose or glycerol. Some degradation of the bovine β -lactoglobulin protein was observed in the samples. Additional experiments were implemented to measure protein expression levels.

Example 8: Test tube growth studies for yeast strains in waste media

[00300] Test tube growth studies were conducted to obtain improved aeration as compared to the deep well plate assays. A 3 mL culture of YPD was inoculated with Pichia pastoris pKC23P-6 and left with shaking for 48 hours at 28°C. The pKC23P-6 culture was spun down. The pellet was washed 3 times with PBD solution to remove all residual YPD media. The pellet was resuspended in 1 mL PBS.

[00301] The waste media was prepared as described in Example 6. Once prepared, 2 mL of each different media was inoculated with 30 μL of resuspended Pichia pastoris pKC23P-6. To the GY tubes, 1.5% glycerol was added. To achieve this, 61 μL of 50% glycerol was introduced to give a final concentration of 1.5%. The cultures were left shaking for 24 hours at 28°C.

[00302] After 24 hours, a 200 μL sample was taken from each test tube and placed in a 96 well plate (Plate 1). From these samples, 15 μL was taken and added to 135 μL PBS solution to make a final dilution of 1 : 10 in another 96 well plate (Plate 2). An end point assay was done and samples read at 600 nm. To the GY tubes, 1.5% glycerol was added. To achieve this, 57 μL of 50% glycerol was introduced to give a final concentration of 1.5%. The cultures were left shaking for another 24 hours at 28°C.

[00303] After 48 hours, a 200 μL sample was taken from each test tube and placed in a 96 well plate (Plate 2). From these samples, 6 μL was taken and added to 144 μL PBS solution to make a final dilution of 1:25 in another 96 well plate (Plate 3). An end point assay was done and samples read at 600 nm. To the GY tubes, 1.5% glycerol was added. To achieve this, 53 μL of 50% glycerol was introduced to give a final concentration of 1.5%. The cultures were left shaking for another 24 hours at 28°C.

[00304] After 72 hours, a 200 μL sample was taken from each test tube and placed in a 96 well plate (Plate 2). From these samples, 6 μL was taken and added to 144 μL PBS solution to make a final dilution of 1:25 in another 96 well plate (Plate 3). An end point assay was done and samples read at 600 nm.

[00305] The results are shown in Figure 12. See also Table 9A, below. BMGY supplemented with glycerol produced the highest final OD ₆₀₀ reading. This was followed by grape waste media, grape waste media with dextrose, and grape waste media with glycerol. The OD ₆₀₀ readings appear to have been affected by the change in dilution ratios between the 24 hour and 48 hour time point. As noted above, the first reading at 24 hours included a 1:10 dilution, while the second and third reading at 48 and 72 hours respectively, included a 1:25 dilution to account for the increased biomass and to ensure the reading would remain within the plate readers limits. While the dilution factor was accounted for in the calculations, the higher dilution rate may have reduced reading accuracy. Very low to low growth levels for Pichia pastoris pKC23P-6 were observed in cabbage waste media with dextrose, parsnip waste media, and all of the kiwifruit waste media.

Example 9: Protein expression analysis for test tube growth studies

[00306] SDS-PAGE was utilised as indicated in Example 8, except that gels were stained with Sypro Ruby stain (Thermofisher) overnight then destained with 10% methanol and 7% acetic acid for 30 minutes. The gels were imaged then stained with Coomassie blue stain. The relative quantity of protein was calculated for each lane as compared to the size ladder lane. The size ladder was loaded at 3 μL and 10 μL of each sample was loaded. The gel was imaged using a BioRad gel imager, and BioRad Image Lab 6.01 software was used to determine band intensity and thus relative protein concentrations.

[00307] The SDS-PAGE gels demonstrating bovineβ- lactoglobulin (BLG) protein expression levels are shown in Figures 13A and 13B. The quantification results for these gels are shown in Tables 10 and 11, respectively.

Table 9A: Averaged growth levels for Pichia pastoris pKC23P-6 in test tube studies

Table 10: Relative quantity of protein in gel lanes Table 11: Relative quantity of protein in gel lanes

[00308] Consistent with the results from Example 7, the highest expression levels were seen with Pichia pastoris pKC23P-6 grown in broccoli waste media. This was significantly higher than expression shown by the positive control BMGY. The inclusion of dextrose in the broccoli waste media produced a notable decrease in expression levels. This was a repeat of the surprising results found in Example 7.

[00309] Very high expression levels were also seen with Pichia pastoris pKC23P-6 grown in cabbage waste media. This was significantly higher than the positive control BMGY. In addition, reasonable expression levels were seen with Pichia pastoris pKC23P-6 grown in grape waste media, without added dextrose or glycerol.

[00310] For most of the waste-grown samples, glycerol additions did not produce substantial increases in expression levels. This was unexpected considering the significant effect of glycerol addition on the positive control, noted as BMGY glycerol. For example, in broccoli waste media, the addition of glycerol every 24 hours improves expression by ~ 13%. For BMGY, the addition of glycerol every 24 hours improves expression by -350%. Noting that these relative concentration calculations were prepared using the BioRad Image Fab 6.01 software.

[00311] One other important observation is that Pichia pastoris pKC23P-6 grown on waste media shows cleaner expression of bovine β-lactoglobulin. In particular, in waste media, there are reduced levels of endogenous proteins as compared to standard media. This results is also unexpected and highly advantageous.

Example 10: Extended test tube growth studies for yeast strains in waste media

[00312] Media was prepared using the methods described in Example 6.

[00313] The following waste media was made:

(1) Kumara waste extract (M)

(2) Kumara waste extract + dextrose (KM)

(3) Kale waste extract (L)

(4) Kale waste extract + dextrose (KL)

(5) Yam waste extract (Y) (6) Yam waste extract + dextrose (YD)

(7) Cauliflower waste extract (F)

(8) Cauliflower waste extract + dextrose (FD)

(9) Parsnip waste extract (R)

(10) Parsnip waste extract + dextrose (RD)

(11) Brussels sprouts waste extract (T)

(12) Brussels sprouts waste extract + dextrose (TD)

[00314] Test tube studies were carried using the methods described in Example 8. The results are shown in Figure 14. See also Table 12A, below. Growth on BMGY (standard Pichia pastoris growth media) resulted in the highest final OD ₆₀₀ reading, followed by Brussel sprouts waste media supplemented with dextrose, followed by parsnip waste media supplemented with dextrose. Interestingly, there were several media that decreased in OD ₆₀₀ from 48 hours to 72 hours including parsnip waste media with glycerol, yam waste media with glycerol, and yam waste media with dextrose. Both readings were done with a 1:25 dilution of inoculated media. The majority of the OD ₆₀₀ measurements remained stable across the 72 hour experiment. Noting that the tuber indicated as “yam” in these experiments is not a true yam. It is more correctly identified as oca.

Table 12A: Averaged growth levels for Pichia pastoris pKC23P-6 in extended test tube studies Example 11: Protein expression analysis for extended test tube growth studies

[00315] SDS-PAGE analysis was performed as described in Example 9. Growth samples from Example 10 were run alongside a BSA standard to allow quantification. Selected growth samples from Example 8 were run in parallel. For each gel, 20 μL of each sample was mixed with 5 μL 5X loading dye and boiled for 10 minutes at 95°C. Next, 10 μL of each sample was loaded into a precast gel along with 3 μL of molecular weight ladder and 2.5 μL of a 100 μg/mL BSA standard and 2.5 μL of a 200 μg/mL BSA standard. Each gel was run for 35 minutes at 200 volts and then stained overnight with Sypro Ruby stain. Gels were de- stained for 30 minutes with a 10% methanol, 7% acetic acid solution. The gels were imaged and bands were quantified using the BioRad Lab Imager 6.01 software. The 100 μg/mL standard was used as a reference lane to compare the protein in each well. Noting that the sample tested was the 48 hour sample unless stated otherwise.

[00316] The results are shown in Figures 15A-15D. The positive controls of BMGY and BMGY with glycerol produced very good expression levels for bovine β -lactoglobulin (BLG), as expected. Consistent with our previous observations, very good expression levels were seen for Pichia pastoris pKC23P-6 grown in broccoli waste media. Very good expression levels were also seen for Pichia pastoris pKC23P-6 grown in brussels sprout waste media, kumara waste media, and parsnip waste media. Growth in kumara waste media with dextrose appeared to reduce BLG expression levels. Growth in parsnip waste media with dextrose also reduced BLG expression. Moderate expression levels were seen for Pichia pastoris pKC23P- 6 grown in grape waste media. Expression was observed for kale, cauliflower, and yam waste media but only with added glycerol. Interestingly, parsnip waste media supplemented with glycerol seemed to have some degradation of BLG. In addition, for the parsnip waste media, expression appeared to be higher in the 48 hour samples, as opposed to the 72 hour sample. The smaller band (likely degraded BLG) looked to be the brightest, indicating the most abundant protein levels.

[00317] To account for the different levels of growth for Pichia pastoris pKC23P-6 in different media, the expression quantification data was normalised by the OD ₆₀₀ value of the sample. For each media sample, the negative control value was subtracted from the experimental value. The estimated yield in mg/L was then divided by the OD ₆₀₀ value. For samples with degradation, the band intensities/yields were combined together to calculate the total amount of BLG present. [00318] The results are shown in Figure 16. See also Tables 13-16, below. After normalising based on OD ₆₀₀ values, the highest expression levels of bovine β -lactoglobulin (BLG) were seen for Pichia pastoris pKC23P-6 grown in kumara waste media with glycerol. Very high expression levels were also seen for Pichia pastoris pKC23P-6 grown in broccoli waste media, brussels sprouts waste media, cabbage waste media, and parsnip waste media. The expression levels observed for Pichia pastoris pKC23P-6 grown in this waste media (kumara waste media with glycerol, and broccoli waste media, brussels sprouts waste media, cabbage waste media, and parsnip waste media) were substantially higher than expression levels for Pichia pastoris pKC23P-6 grown in standard media (BMGY positive control). These results are very surprising and point to significant technical advantages for the waste substrates.

[00319] In sum, the above studies have identified novel compositions and methods which find significant utility in waste mitigation and protein expression, amongst other uses.

Table 13: Expression levels normalised to OD ₆₀₀

Table 14: Expression levels normalised to OD ₆₀₀

Table 15: Expression levels normalised to OD ₆₀₀

Table 16: Expression levels normalised to OD ₆₀₀

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[00349] SEQ ID NO: 1 to SEQ ID NO: 18 correspond to the various polypeptide and polynucleotide sequences set out herein. The database sequence information (including sequences and accession numbers) provided with this description corresponds to the information accessed online as of 1 September 2022. [00350] Any references cited in this specification are hereby incorporated by reference. All amino acid and nucleotide sequences in the references cited in this specification are hereby incorporated into this disclosure. No admission is made that any reference constitutes prior art. Nor does discussion of any reference constitute an admission that such reference forms part of the common general knowledge in the art, in Australia or in any other country

[00351] Persons of ordinary skill can utilise the disclosures and teachings herein to produce other embodiments and variations without undue experimentation. All such embodiments and variations are considered to be encompassed herein.

[00352] Accordingly, one of ordinary skill in the field will readily appreciate from the disclosure that later modifications, substitutions, and/or variations performing substantially the same function or achieving substantially the same result as embodiments described herein may be utilised according to such related embodiments. Thus, the present disclosure is intended to encompass, within its scope, the modifications, substitutions, and variations to processes, manufactures, compositions of matter, compounds, means, methods, and/or steps set out herein

[00353] The description herein may contain subject matter that falls outside of the scope of the claimed invention. This subject matter is included to aid understanding of the invention.