Recombinant components and compositions for use in food products

Abstract

To provide methods for use in producing alternatives to animal-derived food products from recombinant components, as well as compositions used in and obtained from such methods.SOLUTION: Provided are methods for producing food products comprising recombinant components, and compositions used in and food products produced by such methods.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates generally to replacements for animal-derived food products. In particular, the present invention relates to recombinant ingredients and compositions for use in food products, methods for producing these ingredients and compositions, and methods for producing food products containing these ingredients and compositions. [Background technology]

[0002] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 746,918, filed October 17, 2018, which is incorporated herein by reference in its entirety.

[0003] Animal-derived food products (e.g., meat, milk, eggs) are popular sources of nutrition. They contain high-quality protein, essential minerals (e.g., calcium, phosphorus, zinc, magnesium), and vitamins (e.g., riboflavin, vitamin A, vitamin B12). In addition, many such food products have advantageous functional properties that allow for the production of a wide variety of derivative food products (e.g., yogurt, cheese, cream, ice cream, butter, mayonnaise).

[0004] However, animal-derived food products contain ingredients (e.g., lactose, allergens, saturated fat, cholesterol) that can cause unhealthy reactions in humans. Furthermore, the production of these food products involves livestock farming, which has significant impacts on animal welfare and the environment and the potential for contamination with pathogens, pesticide residues, heavy metals, and aflatoxin M1.

[0005] These concerns have encouraged the development of alternatives to animal-derived food products. Some such alternatives contain plant-derived ingredients (e.g., proteins, lipids, vitamins). However, increasingly, alternatives to animal-derived food products are produced from ingredients (e.g., proteins, lipids) that are recombinantly (e.g., using microbial host cells).

[0006] The use of recombinant components in food products raises new problems. One such problem is the fact that food products produced from recombinant components often contain significant amounts of such recombinant components (more than was typical in previous products in which recombinant components were utilized), and the use of large amounts of recombinant components can be affected by other, potentially undesirable, components that are co-produced by the microbial host cell that produces the recombinant component.

[0007] One such other component is esterase activity. Esterases can hydrolyze ester bonds in diglycerides, triglycerides, phospholipids, and / or lipoproteins to release free fatty acids. Compositions including diglycerides, triglycerides, phospholipids, and / or lipoproteins are used in a variety of food products in which recombinant components may be used. In some such food products, the esterase-catalyzed release of free fatty acids can have detrimental effects on properties such as texture, emulsification, aroma, flavor, and nutrient content. In other such food products, the esterase-catalyzed release of free fatty acids can have beneficial effects on properties such as flavor profile (e.g., flavor profile of aged cheese).

[0008] As a result, challenges must be overcome in the production, processing, and use of recombinant components for the production of alternatives to animal-derived food products, particularly with regard to esterase activity.

[0009] Example embodiments will be described and explained with additional specificity and detail through the use of the accompanying drawings. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] PCT filing PCT / US2015 / 046428 [Patent Document 2] PCT filing PCT / US2017 / 48730 [Patent Document 3] International Publication No. 96 / 29391 Brochure [Non-patent literature]

[0011] [Non-Patent Document 1] Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK. [Non-Patent Document 2] Pearson.1990.Methods Enzymol.183:63 [Non-Patent Document 3] Altschul et al.1990.J.Mol.Biol.215:403 [Non-Patent Document 4] Malyshev et al.2014.Nature 509:385 [Non-Patent Document 5] Li et al.2014.J.Am.Chem.Soc.136:826 [Non-Patent Document 6] Deshpande et al.2008.Glycobiology 18(8):626 [Non-Patent Document 7] Kabir & Kazi.2011.Genet Mol Biol.34(4):587 Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, a need exists for methods that can be used to produce alternatives to animal-derived food products from recombinant ingredients, as well as compositions used in and resulting from such methods. [Means for solving the problem]

[0013] In one aspect, provided herein is a method for producing a food product comprising a recombinant component produced by a recombinant microbial host cell capable of producing the recombinant component, the method comprising at least one step in which the activity of an esterase is essentially eliminated or modulated.

[0014] In another aspect, provided herein are preparations comprising recombinant components and an esterase activity that is essentially eliminated or modulated compared to the esterase activity contained in a corresponding preparation.

[0015] In another aspect, provided herein are recombinant microbial host cells capable of producing recombinant components and comprising an esterase activity that is essentially eliminated or modulated relative to the esterase activity contained in the corresponding recombinant microbial host cell.

[0016] In another aspect, provided herein is a food product comprising a recombinant component produced by a recombinant microbial host cell, the food product further comprising an esterase activity that is essentially eliminated or modulated compared to the activity of the esterase in the corresponding food product. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a chart showing the results of a free fatty acid (FFA) assay of recombinant β-lactoglobulin preparations resulting from purification removal of esterase activity based on charge (i.e., ionic complexation) and heat lability / thermostability, where the "final material" is the preparation after buffer exchange to remove high salts and concentrate the recombinant β-lactoglobulin, according to various representative embodiments of the present invention. [Diagram 2]FIG. 1 shows a stained native PAGE gel of a recombinant β-lactoglobulin preparation resulting from purifying away esterase activity based on charge (i.e., ion complexation) and heat lability / thermostability, where the "final material" is the preparation after buffer exchange to remove high salts and concentrate the recombinant β-lactoglobulin according to various representative embodiments of the present invention. [Diagram 3] FIG. 1 is a chart showing the results of an FFA assay of a recombinant β-lactoglobulin preparation resulting from purification of esterase activity based on charge (i.e., ion complexation) and hydrophobicity, where the "final material" is the preparation after buffer exchange to remove high concentrations of salt and concentrate the recombinant β-lactoglobulin, according to various representative embodiments of the present invention. [Figure 4] FIG. 1 shows a stained native PAGE gel of a recombinant β-lactoglobulin preparation resulting from purifying away esterase activity based on charge (i.e., ion complexation) and hydrophobicity, where the "final material" is the preparation after buffer exchange to remove high salts and concentrate the recombinant β-lactoglobulin, according to various representative embodiments of the present invention. [Diagram 5] 1 is a diagram showing the results of a pNPL assay using Tukey Kramer analysis of clarified fermentation broth obtained from cultures containing the indicated antifoam agents, according to various exemplary embodiments of the present invention. [Figure 6] 1 is a map of a targeting vector used to generate a recombinant host cell containing ablated cutinase activity according to an exemplary embodiment of the present invention. [Figure 7] 1 is a chart showing the results of an FFA assay for recombinant β-lactoglobulin preparations obtained from recombinant host cells that contain removed cutinase activity compared to recombinant β-lactoglobulin preparations obtained from corresponding recombinant host cells according to various exemplary embodiments of the present invention. [Figure 8]FIG. 1 shows a stained native PAGE gel of a recombinant β-lactoglobulin preparation obtained from a recombinant host cell containing removed cutinase activity compared to a recombinant β-lactoglobulin preparation obtained from a corresponding recombinant host cell according to various representative embodiments of the present invention. [Figure 9A] 1 shows photographs of gel compositions of frozen confections containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various exemplary embodiments of the present invention. [Figure 9B] 1 shows photographs of gel compositions of frozen confections containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various exemplary embodiments of the present invention. [Figure 9C] 1 shows photographs of gel compositions of frozen confections containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various exemplary embodiments of the present invention. [Figure 9D] 1 shows photographs of gel compositions of frozen confections containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various exemplary embodiments of the present invention. [Figure 9E] 1 shows photographs of gel compositions of frozen confections containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various exemplary embodiments of the present invention. [Figure 9F] 1 shows photographs of gel compositions of frozen confections containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various exemplary embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0019] definition As used herein, the terms "a," "an," and "the" and similar references refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0020] As used herein, the term "about" refers to being within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined or the limitations of the measurement system.

[0021] As used herein, the term "and / or" refers to multiple components in combination with or to the exclusion of one another. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," or "x or y or z."

[0022] As used herein, the term "corresponding preparation" refers to a preparation that is identical to the preparation to which it is being compared, except that the esterase activity contained in the "corresponding preparation" has not been essentially removed or modulated as provided herein (e.g., because the preparation is not derived from a recombinant microbial host cell that contains esterase activity that has been essentially removed or modulated, and / or from a recombinant microbial host cell that has been cultured under conditions suitable to essentially remove or modulate esterase activity, and / or from a fermentation broth that contains esterase inhibitors, or because the recombinant components have not been purified to remove esterase activity).

[0023] As used herein, the term "corresponding food product" refers to a food product produced by a process that is identical to the process used to produce the food product to which the "corresponding food product" is being compared, except that the process used to produce the "corresponding food product" does not include at least one step in which esterase activity is essentially eliminated or modulated as provided in the methods provided herein.

[0024] The term "corresponding recombinant microbial host cell" refers to a recombinant microbial host cell that is identical to the recombinant microbial host cell to which it is compared, except that the esterase activity of the "corresponding recombinant microbial host cell" has not been essentially eliminated or modulated as provided in the recombinant microbial host cell provided herein.

[0025] As used herein, the terms "essentially free" and "essentially removed" mean that the indicated component is not detectable in the indicated composition by common analytical methods, or that the indicated component is present in such small amounts that it is not functional. As used in this context, the term "functional" means that the indicated component does not contribute to the properties of the composition containing the indicated component in a trace amount, or that the indicated component has no activity (e.g., enzymatic activity) in the indicated composition containing the indicated component in a trace amount, or that the indicated component has no adverse health effects upon consumption of the composition containing the indicated component in a trace amount.

[0026] As used herein, the term "esterase activity" or "activity of an esterase" refers to the activity of an enzyme capable of hydrolyzing an ester bond (i.e., a hydrolase acting on an ester bond). Esterases are designated by the Enzyme Code (EC) number 3.1. The terms are used interchangeably herein.

[0027] As used herein, the term "fermentation broth" refers to a culture containing recombinant microbial host cells capable of producing a recombinant component.

[0028] As used herein, the term "filamentous fungal cell" refers to a cell derived from any filamentous form of the subdivision Fungi and Oomycota (as defined by E. Schneider, "Filamentous Fungal Cells," vol. 133, no. 1, 1999). Filamentous fungal cells are distinguished from yeasts by the elongation of their hyphae during vegetative growth.

[0029] As used herein, the term "fungi" refers to organisms of the phyla Ascomycota, Basidiomycota, Zygomycota, and Chytridiomycota, Oomycota, and Glomeromycota, however, it is understood that fungal taxonomy is constantly evolving and thus this particular definition of the fungal kingdom may be adjusted in the future.

[0030] The term "heterologous" as used herein refers to something not normally present in the context in which it is described. In other words, an entity so characterized is foreign to the context in which it is described. When used in reference to a protein produced by a filamentous fungal cell, the term implies that the protein is not naturally produced by the filamentous fungal cell.

[0031] The term "host cell" as used herein refers to a cell into which a recombinant nucleic acid is introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications due to mutation or environmental influences may occur in successive generations, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term "host cell" as used herein.

[0032] The term "identical" as used herein in the context of polynucleotide or polypeptide sequences refers to the residues in the reference window of two sequences that are the same when aligned for maximum matching.There are several different algorithms known in the art that can be used to measure the identity of nucleotide or protein sequences.For example, sequences can be compared using FASTA (e.g., provided in Wisconsin Genetics Software Package Version 10.0, Genetics Computer Group, Madison, WI), GAP (e.g., provided in Wisconsin Genetics Software Package Version 10.0, Genetics Computer Group, Madison, WI), BESTFIT (e.g., provided in Wisconsin Genetics Software Package Version 10.0, Genetics Computer Group, Madison, WI), ClustalW (e.g., using the default parameters of version 1.83), or BLAST (e.g., using reciprocal BLAST, PSI-BLAST, BLASTP, BLASTN; provided through the National Center for Biotechnology Information) (see, e.g., Non-Patent Document 2; Non-Patent Document 3). Alignments by these methods may use default parameters (e.g., for BLASTN the default parameters are Gap opening penalty=5 and Gap extension penalty=2, and for BLASTP the default parameters are Gap opening penalty=11 and Gap extension penalty=1). The reference window may range from the entire length of the two sequences to only a region of each of the two sequences (e.g., a region that is at least 50 nucleotides or at least 10 amino acids in length).

[0033] As used herein, the terms "including," "includes," "having," "has," "with," or variations thereof, are intended to be inclusive similar to the term "comprising."

[0034] The term "microbe" as used herein is a contraction of microorganism and refers to a single-celled organism. As used herein, the term includes all bacteria, archaea, single-celled protists, single-celled animals, single-celled plants, single-celled fungi, single-celled algae, protozoa, and chromista organisms.

[0035] The term "modulated" as used herein in relation to esterase activity refers to any alteration in the activity of an esterase (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein), including an increase or decrease in the activity of the esterase. Such modulated esterase activity is typically due to an increase or decrease in the concentration of the esterase or an increase or decrease in the enzymatic activity of the esterase.

[0036] As used herein, the term "naturally occurring" refers to something found in nature.

[0037] As used herein, the term "optionally" or "as needed" refers to the presence or absence of a feature or structure, or the occurrence or non-occurrence of an event or circumstance. The description includes instances when the particular feature or structure is present and when the feature or structure is not present, or when the event or circumstance occurs and when the event or circumstance does not occur.

[0038] The term "polynucleotide" disclosed herein refers to both the sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the above. Polynucleotides may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more analogs of naturally occurring nucleotides, uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Examples of modified nucleotides are known in the art (see, for example, Non-Patent Document 4; Non-Patent Document 5). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to designated sequences via hydrogen bonds and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages replace the phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains bridging moieties or other structures such as those found in "locked" polynucleotides.

[0039] The term "preparation" as used herein refers to a preparation obtained once a recombinant component is separated from one or more other components of the fermentation broth.

[0040] As used herein, the term "protein" refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, naturally occurring and non-naturally occurring amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0041] As used herein, the term "purified" refers to a recombinant protein that has been substantially separated from cellular components (e.g., membrane lipids, chromosomes, proteins) of a recombinant microbial host cell. The term does not require (even though it is recognized) that the recombinant protein has been separated from all other chemicals.

[0042] The term "recombinant microbial host cell" as used herein refers to a microbial cell that contains a recombinant polynucleotide. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications due to mutation or environmental influences may occur in successive generations, such progeny may not be identical to the parent cell, but are still included within the scope of the term "recombinant microbial host cell" as used herein.

[0043] The term "recombinant polynucleotide" as used herein refers to a polynucleotide that has been removed from its naturally occurring environment, that is not associated with all or a portion of the polynucleotide that borders or is adjacent to the polynucleotide as it is found in nature, that is operably linked to a polynucleotide to which it is not linked in nature, or that is not naturally occurring. The term can be used to describe, for example, a cloned DNA isolate, or a polynucleotide that includes a chemically synthesized nucleotide analog. A polynucleotide is also considered "recombinant" if it contains any genetic modification that does not occur in nature. For example, an endogenous polynucleotide is considered a "recombinant polynucleotide" if it contains an insertion, deletion, or substitution of one or more nucleotides that are artificially introduced, for example, by human intervention. Such modifications can introduce point mutations, substitution mutations, deletion mutations, insertion mutations, missense mutations, frameshift mutations, duplication mutations, amplification mutations, translocation mutations, or inversion mutations into the polynucleotide. The term includes polynucleotides in the chromosomes of a host cell as well as polynucleotides that are not in the chromosomes of a host cell (e.g., polynucleotides contained in episomes).

[0044] As used herein, the term "recombinant protein" refers to a protein that is produced in a cell of a different species or type compared to the species or type of cell that naturally produces the protein, or that is produced in a cell at a level in which it is not produced in nature.

[0045] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome.

[0046] As used herein, the term "yeast" refers to organisms in the order Saccharomycetales, such as Saccharomyces cerevisiae and Pichia pastoris. Vegetative growth of yeasts occurs by budding / blebbing of a unicellular thallus and carbon catabolism can be fermentative.

[0047] Methods for Producing Food Products Containing Recombinant Ingredients In one aspect, a method is provided for producing a food product comprising a recombinant component (e.g., any of the recombinant components disclosed herein) produced by a recombinant microbial host cell capable of producing the recombinant component (e.g., any of the recombinant microbial host cells provided herein), the method comprising at least one step in which the activity of an esterase (i.e., esterase activity; e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially eliminated or modulated.

[0048] In some embodiments, the present invention provides one or more methods for producing a food product comprising a recombinant component (e.g., any of the recombinant components disclosed herein), based on the discovery that recombinant microbial host cells that produce the recombinant component typically also produce esterase activity. Such esterase activity can be co-purified with the recombinant component. When the recombinant component is then used to produce a food product, the esterase activity can hydrolyze ester bonds in diglycerides, triglycerides, phospholipids, and / or lipoproteins contained in the food product, thus catalyzing the production of free fatty acids in the food product.

[0049] As discussed above, the production of free fatty acids in food products can have deleterious effects, for example, by producing unpleasant odors and / or tastes, preventing emulsion formation, having undesirable effects on texture, and interacting with essential nutrients (e.g., vitamins) thereby reducing nutrient content and reducing shelf life. The production of free fatty acids in food products can also have beneficial effects, for example, by producing a desired flavor profile (e.g., the flavor profile of aged cheese) or by making enzyme-treated cheese for use in processed cheese.

[0050] In some embodiments, the present invention provides one or more methods for producing food products that include recombinant components (e.g., any of the recombinant components disclosed herein) based on the discovery that esterase activity produced by a recombinant host cell is distinct from the esterase activity contained in milk produced from a mammal.

[0051] Thus, various embodiments of the present invention include specific methods for essentially eliminating or modulating esterase activity produced by recombinant microbial host cells. By essentially eliminating or modulating esterase activity in food products containing recombinant components produced by recombinant microbial host cells, the present invention provides a method for preventing or delaying the production of off-odors and tastes, forming emulsions, increasing nutrient content, modulating texture, extending shelf life, and / or more quickly producing a desired flavor profile in a food product.

[0052] In some embodiments, the methods provided herein for producing a food product include at least one step in which esterase activity is essentially eliminated.

[0053] In some embodiments, the methods provided herein for producing a food product include at least one step of reducing esterase activity. In some such embodiments, the esterase activity is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0054] In some embodiments, the methods provided herein for producing a food product include at least one step of increasing esterase activity, hi some embodiments, the esterase activity is increased by at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, 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 1,000%.

[0055] In some embodiments, the methods provided herein for producing a food product include at least one step of essentially eliminating a first esterase activity and decreasing a second esterase activity. In some embodiments, the methods provided herein for producing a food product include at least one step of essentially eliminating a first esterase activity and increasing a second esterase activity. In some embodiments, the methods provided herein for producing a food product include at least one step of increasing a first esterase activity and decreasing a second esterase activity.

[0056] In some embodiments, the methods provided herein for producing a food product include a step in which the activity of an esterase (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially eliminated or modulated.

[0057] In some embodiments, the methods provided herein for producing a food product include two or more steps (e.g., two steps, three steps, four steps, five steps) in which the activity of an esterase (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially eliminated or modulated (e.g., two or more steps selected from the steps disclosed herein that provide essentially eliminated or modulated esterase activity, e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein).

[0058] Essentially eliminates or modulates esterase activity The at least one step in the methods provided herein for producing a food product in which esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially eliminated or modulated can include any step that provides essentially eliminated or modulated esterase activity.

[0059] In some embodiments, at least one step in the methods provided herein for producing a food product in which esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially eliminated or modulated comprises culturing a recombinant microbial host cell capable of producing the recombinant component under conditions suitable to essentially eliminate or modulate the esterase activity.

[0060] Thus, in some such embodiments, the methods provided herein comprise the steps of: a) obtaining a recombinant microbial host cell capable of producing a recombinant component (e.g., any of the recombinant components disclosed herein); b) culturing the recombinant microbial host cell in a culture medium under conditions suitable for essentially removing or modulating esterase activity, and a culture medium under conditions suitable for production and / or secretion of the recombinant component to obtain a fermentation broth comprising the recombinant component and the essentially removed or modulated esterase activity; c) optionally purifying the recombinant component from the fermentation broth to obtain a preparation comprising the recombinant component and the essentially removed or modulated esterase activity; and d) preparing a food product from the fermentation broth or preparation comprising the recombinant component and the essentially removed or modulated esterase activity.

[0061] Suitable conditions for culturing recombinant microbial host cells in a culture medium under suitable conditions to essentially eliminate or modulate esterase activity are, for example, conditions under which the recombinant microbial host cells essentially eliminate or modulate their production of esterase activity. Non-limiting examples of such conditions include suitable pH, suitable temperature, suitable feed rate, suitable pressure, suitable nutrient content (e.g., suitable carbon content, suitable nitrogen content, suitable phosphorus content), suitable nutritional supplement content, suitable trace metal content, and / or suitable oxygenation level. In some embodiments, suitable pH at which the recombinant microbial host cells essentially eliminate or modulate their production of esterase activity is between 2 and 7.5, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, or 2.5; between 2.5 and 7.5, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3; between 3 and 7.5, 6.5, 6, 5.5, 5, 4.5, 4, or between 3.5; between 3.5 and 7.5, 6.5, 6, 5.5, 5, 4.5, 4; between 4 and 7.5, 6.5, 6, 5.5, 5, 4.5; between 4.5 and 7.5, 6.5, 6, 5.5, 5; between 5 and 7.5, 6.5, 6, 5.5; between 5.5 and 7.5, 6.5, 6; between 6 and 7.5, 6.5; between 6.5 and 7.5, or 7.5. In some embodiments, a suitable supplement content in the culture medium in which the recombinant microbial host cell essentially eliminates or modulates its production of esterase activity is an antifoam agent.Non-limiting examples of suitable antifoam agents include Struktol J 673 A (Schill & Seilacher GmbH, Hamburg, Germany), Industrol DF204 (BASF Canada, Inc., Mississauga, Canada), polyglycol P-2000 (Dow, Midland, MI), Hodag K-60K (Hodag Chemical Corp., Chicago, IL), and Erol DF6000K (PMC Ouvrie, Carvin, France), ACP 1500 (Dow Chemical Company, Midland, MI), Antifoam 204 (Sigma-Aldrich, St Louis, MO), SAG 471 (Momentive Performance Materials Inc., Waterford, NY), SAG 5693 (Momentive Performance Materials Inc., Waterford, NY), SAG 710 (Momentive Performance Materials Inc., Waterford, NY), SAG 810 (Momentive Performance Materials Inc., Waterford, NY), SAG 820 (Momentive Performance Materials Inc., Waterford, NY), SAG 830 (Momentive Performance Materials Inc., Waterford, NY), SAG 840 (Momentive Performance Materials Inc., Waterford, NY), SAG 850 (Momentive Performance Materials Inc., Waterford, NY), SAG 860 (Momentive Performance Materials Inc., Waterford, NY), SAG 870 (Momentive Performance Materials Inc., Waterford, NY), SAG 880 (Momentive Performance Materials Inc., Waterford, NY), SAG 89 ... 730 (Momentive Performance Materials Inc., Waterford, NY), a silicone defoamer, Struktol J647 (Schill & Seilacher, Hamburg, Germany), and sunflower oil.

[0062] In some embodiments, at least one step in which esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially removed or modulated in the methods provided herein for producing a food product comprises essentially removing or modulating esterase activity in a fermentation broth or preparation obtained from a culture of a recombinant microbial host cell capable of producing the recombinant component.

[0063] Thus, in some such embodiments, the methods provided herein comprise the steps of: a) obtaining a recombinant microbial host cell capable of producing a recombinant component (e.g., any of the recombinant components disclosed herein); b) culturing the recombinant microbial host cell in a culture medium under conditions suitable for production and / or secretion of the recombinant component to obtain a fermentation broth comprising the recombinant component; c) optionally purifying the recombinant component from the fermentation broth to obtain a preparation comprising the recombinant component; d) essentially removing or modulating esterase activity in the fermentation broth or preparation to obtain a fermentation broth or preparation comprising the recombinant component and essentially removed or modulated esterase activity; and e) preparing a food product from the fermentation broth or preparation comprising the recombinant component and essentially removed or modulated esterase activity.

[0064] Esterase activity in a fermentation broth or preparation can be essentially eliminated or modulated, for example, by adding an esterase inhibitor to the fermentation broth or preparation. Non-limiting examples of suitable esterase inhibitors include synthetic inhibitors (e.g., phosphonates, boronic acids, lipid analogs) as well as compounds derived from plants such as, for example, Juniperus communis, Illicium religiosum, Panax japonicus rhizomes, Panax ginseng, Panax quinquefolius, Acanthopanax senticosus, Camellia sinensis var. sinensis, Camellia sinensis var. assamica, Kochia scoparia, Platycodi radix, Salacia reticulate, Nelumbo nucifera, Salix salicina, and others. Natural inhibitors isolated from sources such as Eriochea villosa, Eriochea matsudana, Eriochea villosa, Eriochea tschiliensis Grun, and Eriochea sessiliflorus (e.g., β-lactones (e.g., valilactone, ebelactone A & B, and vibralactone), mannooligosaccharides, acetylcholinesterase inhibitors, cholinesterase inhibitors, polyphenols, saponins, panclicin, hesperidin, caulerpenin, proanthocyanidins, orlistat (Xenical), carnosic acid, escin, crocin, crocetin, chlorogenic acid, neochlorogenic acid, feruloyl quinic acid, e-polylysine, chitosan, chitin). In some such embodiments, the esterase inhibitors require the addition of pH and / or ionic strength adjusters for activity.

[0065] Esterase activity in a fermentation broth or preparation can also be essentially removed or modulated, for example, by removing and / or adding to the fermentation broth or preparation a cofactor required for the activity of the esterase and / or a cofactor required for the activity of an esterase inhibitor. Non-limiting examples of such cofactors include metals (e.g., divalent cations such as calcium). In some embodiments, divalent cations (e.g., calcium) are removed by binding the divalent cation to a chelating agent. A non-limiting example of a suitable chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0066] Esterase activity in a fermentation broth or preparation can be essentially eliminated or modulated, for example, by heat or non-heat treatment. Non-limiting examples of heat treatment include pasteurization and sterilization. Non-limiting examples of non-heat treatment include high pressure pasteurization (i.e., high pressure processing, HPP), ultrasonic treatment, pulsed electric field treatment, and irradiation. In some embodiments, esterase activity in a fermentation broth or preparation is essentially eliminated or reduced by incubating the fermentation broth or preparation at high temperature for a relatively short time (e.g., at a temperature of 85°C to 90°C for 5 to 10 minutes).

[0067] In some embodiments, at least one step in the methods provided herein for producing a food product in which esterase activity (e.g., the activity of any one esterase disclosed herein or any combination of at least two esterases disclosed herein) is essentially eliminated or modulated comprises obtaining a recombinant microbial host cell capable of producing a recombinant component, the recombinant microbial host cell comprising the essentially eliminated or modulated esterase activity.

[0068] Thus, in some such embodiments, the methods provided herein comprise the steps of: a) obtaining a recombinant microbial host cell capable of producing a recombinant component (e.g., any of the recombinant components disclosed herein), the recombinant microbial host cell comprising an esterase activity that is essentially removed or modulated compared to an esterase activity contained in a corresponding recombinant microbial host cell; b) culturing the recombinant microbial host cell in a culture medium under conditions suitable for the production and / or secretion of the recombinant component to obtain a fermentation broth comprising the recombinant component and the essentially removed or modulated esterase activity; c) optionally purifying the recombinant component from the fermentation broth to obtain a preparation comprising the recombinant component; and d) preparing a food product from the fermentation broth or preparation comprising the recombinant component and the essentially removed or modulated esterase activity.

[0069] Esterase activity can be essentially eliminated or modulated in a recombinant microbial host cell by essentially eliminating or modulating the expression (i.e., production of active protein) of an esterase (e.g., any one of the esterases disclosed herein or any combination of at least two esterases disclosed herein) or by essentially eliminating or modulating the activity of an esterase. Essentially eliminating or modulating the expression of an esterase in a recombinant microbial host cell can be achieved, for example, by genetically modifying a control sequence (e.g., a promoter sequence, an enhancer sequence, a signal peptide, a transcription terminator, or any other sequence that controls the expression (i.e., transcription and / or translation) of a gene) that drives the expression of the esterase, or a functional portion thereof (i.e., a portion sufficient for the function of the control sequence), such that expression of the esterase is essentially eliminated or modulated in the recombinant microbial host cell.

[0070] Substantially eliminating or modulating expression of an esterase in a recombinant microbial host cell can, for example, be achieved by omitting a regulatory sequence, or a functional portion thereof (e.g., a transcription factor (e.g., a sequence at least 50% identical to a sequence selected from UniProt sequences G0RX49, G0RHG1, A0A2T4B416, A0A2T4BJU6, A2R2J1, A2R903, G2Q2Z5, and G2Q8I6) that drives expression of a protein required for expression of the esterase, such that expression of the esterase is essentially eliminated or modulated in the recombinant microbial host cell. The expression of an esterase can also be achieved by genetically modifying a protein that contains an amino acid sequence that is identical to or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of the amino acid sequence of the esterase, or a post-translational modification enzyme required for production of an active form of the esterase, or by genetically modifying a coding sequence that encodes a protein or a functional portion thereof required for expression of the esterase (e.g., a DNA binding domain of a transcription factor, a catalytic domain of a post-translational modification enzyme).

[0071] Essentially eliminating or modulating expression of an esterase in a recombinant microbial host cell can also be accomplished, for example, by introducing into the recombinant microbial host cell a recombinant polynucleotide comprising a nucleotide sequence encoding an RNAi construct that is complementary to a coding sequence encoding an esterase or is specific for an esterase.

[0072] Essentially eliminating or modulating the expression or activity of an esterase in a recombinant microbial host cell can also be accomplished, for example, by genetically modifying a coding sequence encoding the esterase or a functional portion thereof (e.g., a catalytic domain) in the recombinant microbial host cell.

[0073] Essentially eliminating or modulating the activity of an esterase in a recombinant microbial host cell can also be achieved, for example, by genetically modifying a control sequence driving the expression of an endogenous inhibitor of an esterase in the recombinant microbial host cell and / or genetically modifying a coding sequence encoding an endogenous inhibitor of an esterase, such that expression and / or activity of the endogenous inhibitor of an esterase is essentially eliminated or modulated in the recombinant microbial host cell, or by introducing a recombinant polynucleotide encoding a heterologous inhibitor of an esterase into the recombinant microbial host cell.

[0074] Genetic modification can be, for example, by introducing, substituting or removing one or more nucleotides in a nucleotide sequence.For example, one or more nucleotides can be inserted or removed to introduce a stop codon, remove a start codon, insert a frameshift in an open reading frame, or create a point mutation, a missense mutation, a substitution mutation, a deletion mutation, a frameshift mutation, an insertion mutation, a duplication mutation, an amplification mutation, a translocation mutation, or an inversion mutation.

[0075] Methods for genetically modifying microbial host cells are well known in the art and include, but are not limited to, random mutagenesis and screening, site-directed mutagenesis, PCR mutagenesis, insertional mutagenesis, chemical mutagenesis (e.g., using hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, nucleotide analogs), irradiation (e.g., ultraviolet (UV) irradiation), deletion of coding or non-coding nucleotide sequences, homologous recombination, FLP / FRT recombination, gene disruption, CRISPR gene editing, and gene conversion.

[0076] In some embodiments, at least one step in the methods provided herein for producing a food product in which esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially removed or modulated comprises purifying the recombinant component away from the esterase activity produced by the recombinant microbial host cell.

[0077] Thus, in some such embodiments, the methods provided herein include the steps of: a) obtaining a recombinant microbial host cell capable of producing a recombinant component (e.g., any of the recombinant components disclosed herein); b) culturing the recombinant microbial host cell in a culture medium under conditions suitable for production and / or secretion of the recombinant component to obtain a fermentation broth comprising the recombinant component; c) purifying the recombinant component from the fermentation broth to obtain a preparation comprising the recombinant component, such that the preparation comprises essentially removed or modulated esterase activity; and d) preparing a food product from the preparation comprising the recombinant component and the essentially removed or modulated esterase activity.

[0078] Purification of the recombinant component from the fermentation broth can be accomplished based on any one or combination of properties that distinguish the recombinant component from esterases or that are shared between the recombinant component and esterases. Non-limiting examples of such properties include size, molecular weight, degradation by specific proteases or combinations of proteases, isoelectric point (pI), charge, thermolability / thermostability, affinity for specific molecules, solubility, pH stability, hydrophobicity, and hydrophilicity.

[0079] In some embodiments, the recombinant components are purified based on their thermolability / thermostability. In some such embodiments, a fermentation broth (e.g., a clarified fermentation broth (i.e., a fermentation broth from which cells and cell debris have been removed)) or preparation containing the recombinant components is heated to a temperature at which the esterases are denatured and precipitate out of solution, but the recombinant components remain structurally intact and soluble. The precipitated esterases can then be separated from the soluble recombinant components by any suitable method, including various filtration techniques, including, but not limited to, 1-g sedimentation, accelerated sedimentation by centrifugation, and / or depth filtration or tangential flow filtration using filter pads, sheets, or membranes.

[0080] In some embodiments, the recombinant components are purified based on hydrophobicity. In some such embodiments, a suitable chromatographic support is added to a fermentation broth (e.g., clarified fermentation broth) or preparation containing the recombinant components, and the pH and / or ionic strength of the fermentation broth or preparation is adjusted such that the esterase or recombinant components (but not both) bind to the chromatographic support (e.g., based on hydrophobic interactions), leaving the soluble recombinant components or esterase, respectively, in the unbound portion. Non-limiting examples of suitable chromatographic supports include phenyl sepharose, butyl sepharose, and octyl sepharose. The chromatographic support with bound esterase or recombinant components can subsequently be separated from the soluble recombinant components or esterase, respectively, by any suitable method known in the art, including, but not limited to, 1-g sedimentation, centrifugation, or filtration. Alternatively, the chromatographic support can be a stationary support (e.g., an adsorbent in a column) onto which the fermentation broth or preparation containing the recombinant components is transferred, with the recombinant components being obtained in the unbound portion and the esterase being bound to the chromatographic support, or the recombinant components are bound to the chromatographic support, with the esterase remaining in the unbound portion, and the bound recombinant components being subsequently released from the chromatographic support, e.g., by adjusting the pH and / or ionic strength.

[0081] In some embodiments, the recombinant components are purified based on charge. In some such embodiments, a counterion or ion exchange resin or a sodium acid salt is added to a fermentation broth (e.g., clarified fermentation broth) or preparation containing the recombinant components, and the pH and / or ionic strength of the fermentation broth or preparation is adjusted such that the counterion or ion exchange resin or the sodium acid salt forms a complex with the recombinant components or esterase, leaving behind the soluble esterase or recombinant components, respectively. The complex can then be isolated by any suitable method known in the art, including, but not limited to, 1-g sedimentation, accelerated sedimentation by centrifugation, and / or various filtration techniques, including depth filtration or tangential flow filtration using filter pads, sheets, or membranes. In embodiments in which the recombinant components are complexed with a counterion or ion exchange resin or a sodium acid salt, the recombinant components can be extracted from the complex by adjusting the pH and / or ionic strength such that the recombinant components and the counterion or ion exchange resin or the sodium acid salt are repelled from each other.

[0082] In some embodiments, the recombinant components are purified based on pH stability. In some such embodiments, the pH of the fermentation broth (e.g., clarified fermentation broth) or preparation containing the recombinant components is adjusted so that the esterase is denatured and precipitates out of solution, leaving behind the soluble recombinant components. The precipitated esterase can then be separated from the soluble recombinant components by any suitable method, including, but not limited to, 1-g sedimentation, accelerated sedimentation by centrifugation, and / or various filtration techniques, including, but not limited to, depth filtration or tangential flow filtration using filter pads, sheets, or membranes.

[0083] In some embodiments, recombinant components are purified based on two or more of charge, thermolability / thermostability, hydrophobicity, size, molecular weight, pH stability, isoelectric point (pI), affinity for a particular molecule, and solubility, and various strategies for separation are performed sequentially (e.g., separation based on charge followed by separation based on hydrophobicity, separation based on hydrophobicity followed by separation based on pH stability) or simultaneously (e.g., separation based on pH stability and thermolability / thermostability, separation based on pH stability and affinity for a particular molecule, separation based on solubility and pH stability and / or thermolability / thermostability and / or pI).

[0084] Esterase activity The essentially eliminated or modulated esterase activity provided herein (e.g., contained in a fermentation broth or preparation comprising a recombinant component, or contained in a recombinant microbial host cell provided herein capable of producing a recombinant component) can be essentially eliminated or modulated activity of any one esterase or any combination of at least two (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more) esterases.

[0085] Non-limiting examples of such esterases include carboxyester hydrolases (EC number 3.1.1), phosphodiester hydrolases (EC number 3.1.4), exoribonucleases (EC number 3.1.13), carboxylesterases (EC number 3.1.1.1), arylesterases (EC number 3.1.1.2), triacylglycerol lipases (EC number 3.1.1.3), phospholipase A2 (EC number 3.1.1.4), lysophospholipases (EC number 3.1.1.5), acetylesterases (EC number 3.1.1.6), acetylcholinesterases (EC number 3.1.1.7), phospholipase C (EC number 3.1.4.3), phospholipase D (EC number 3.1.4.4), phosphoinositide phospholipase C (EC number 3.1.4.11), pectinesterases (EC number 3.1.1. 11), gluconolactonase (EC number 3.1.1.17), acylglycerol lipase (EC number 3.1.1.23), 3-oxoadipate enol lactonase (EC number 3.1.1.24), 1,4-lactonase (EC number 3.1.1.25), galactolipase (EC number 3.1.1.26), phospholipase A1 (EC number 3.1.1.32), and lipoprotein lipase (EC number 3.1.1.33). 1.34), cephalosporin-C deacetylase (EC number 3.1.1.41), carboxymethylenebutenolidase (EC number 3.1.1.45), 2-pyrone-4,6-dicarboxylic acid lactonase (EC number 3.1.1.57), feruloyl esterase (EC number 3.1.1.73), cutinase (EC number 3.1.1.74), and hormone-sensitive lipase (EC number 3.1.1.79).

[0086] Non-limiting examples of suitable carboxylester hydrolases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the carboxylester hydrolase-encoding sequences listed in Table 1.

[0087] [Table 1]

[0088] In some embodiments, the carboxylester hydrolase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from UniProt sequences G0RIU1, A2R1N7, G0RBM4, A0A2T4BBP9, G2Q379, A0A2T4AZ21, A2R8Z3, G0R9X3, A2QPC2, G0RCG3, A0A2T4BCL7, A0A2T4B416, and A0A2T4BNI9.

[0089] Non-limiting examples of suitable phosphodiester hydrolases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the phosphodiester hydrolase-encoding sequences listed in Table 1.

[0090] Non-limiting examples of suitable exoribonucleases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the exoribonuclease-encoding sequences listed in Table 1.

[0091] Non-limiting examples of suitable carboxylesterases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the carboxylesterase-encoding sequences listed in Table 1.

[0092] Non-limiting examples of suitable aryl esterases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the aryl esterase-encoding sequences listed in Table 1.

[0093] Non-limiting examples of suitable triacylglycerol lipases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the triacylglycerol lipase-encoding sequences listed in Table 1. In some embodiments, the triacylglycerol lipase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from UniProt sequences A2QM14 and A2R709.

[0094] Non-limiting examples of suitable phospholipase A2s include esterases comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from any of the phospholipase A2-encoding sequences listed in Table 1.

[0095] Non-limiting examples of suitable lysophospholipases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the lysophospholipase-encoding sequences listed in Table 1. In some embodiments, the lysophospholipase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from UniProt sequences G0RX90, G2Q0K1, A0A2T4B235, A2QC75, and A2QF42.

[0096] Non-limiting examples of suitable acetyl esterases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the acetyl esterase-encoding sequences listed in Table 1. In some embodiments, the acetyl esterase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from UniProt sequences G0RHJ4, A0A2T4BJD9, and G2QL32.

[0097] Non-limiting examples of suitable acetylcholinesterases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the acetylcholinesterase-encoding sequences listed in Table 1.

[0098] Non-limiting examples of suitable phospholipase C enzymes include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the phospholipase C enzyme-encoding sequences listed in Table 1. In some embodiments, the phospholipase C comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from UniProt sequences G0REM9, A0A2T4BFY3, and A2QAD7.

[0099] Non-limiting examples of suitable phospholipase D include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the phospholipase D-encoding sequences listed in Table 1.

[0100] Non-limiting examples of suitable phosphoinositide phospholipase C include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the phosphoinositide phospholipase C-encoding sequences listed in Table 1.

[0101] Non-limiting examples of suitable pectin esterases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the pectin esterase-encoding sequences listed in Table 1.

[0102] Non-limiting examples of suitable gluconolactonase include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the gluconolactonase-encoding sequences listed in Table 1.

[0103] Non-limiting examples of suitable acylglycerol lipases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the acylglycerol lipase-encoding sequences listed in Table 1.

[0104] Non-limiting examples of suitable 3-oxoadipate enol lactonase include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the 3-oxoadipate enol lactonase-encoding sequences listed in Table 1.

[0105] Non-limiting examples of suitable 1,4-lactonases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the 1,4-lactonase-encoding sequences listed in Table 1.

[0106] Non-limiting examples of suitable galactolipases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the galactolipase-encoding sequences listed in Table 1.

[0107] Non-limiting examples of suitable phospholipase A1s include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the phospholipase A1-encoding sequences listed in Table 1.

[0108] Non-limiting examples of suitable lipoprotein lipases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the lipoprotein lipase-encoding sequences listed in Table 1.

[0109] Non-limiting examples of suitable cephalosporin-C deacetylases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the cephalosporin-C deacetylase-encoding sequences listed in Table 1.

[0110] Non-limiting examples of suitable carboxymethylenebutenolidases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the carboxymethylenebutenolidase-encoding sequences listed in Table 1.

[0111] Non-limiting examples of suitable 2-pyrone-4,6-dicarboxylic acid lactonases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the 2-pyrone-4,6-dicarboxylic acid lactonase-encoding sequences listed in Table 1.

[0112] Non-limiting examples of suitable feruloyl esterases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the feruloyl esterase-encoding sequences listed in Table 1.

[0113] Non-limiting examples of suitable cutinases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the cutinase-encoding sequences listed in Table 1. In some embodiments, the cutinase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from UniProt sequences G0RH85, A2QBP1, A0A2T4BJB5, A5ABE6, A2R2W3, and G2QH51.

[0114] Non-limiting examples of suitable hormone-sensitive lipases include esterases comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence selected from any of the hormone-sensitive lipase-encoding sequences listed in Table 1.

[0115] Non-limiting examples of additional suitable esterases include esterases that comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence listed in Table 1, and esterases that comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% identical to a sequence listed in Table 2. The invention includes esterases comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to any one of the listed InterPro domains.

[0116] [Table 2]

[0117] The esterases disclosed herein include esterases that are conservatively modified variants of the esterases disclosed herein. As used herein, the term "conservatively modified variants" refers to discrete substitutions, deletions, or additions to the encoded amino acid sequence, whereby an amino acid is replaced with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. The esterases disclosed herein further include polymorphic variants, interspecies homologs, and alleles of the esterases disclosed herein.

[0118] In embodiments where the essentially eliminated or modulated esterase activity (e.g., contained in a fermentation broth or preparation comprising a recombinant component, or contained in a recombinant microbial host cell provided herein capable of producing a recombinant component) comprises essentially eliminated or modulated activity of at least two esterases, such at least two esterases can comprise one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, etc.) of any esterases (e.g., any esterases provided herein).

[0119] Non-limiting examples of such esterases include carboxylester hydrolases (EC number 3.1.1), phosphodiester hydrolases (EC number 3.1.4), exoribonucleases (EC number 3.1.13), carboxylesterases (EC number 3.1.1.1), arylesterases (EC number 3.1.1.2), triacylglycerol lipases (EC number 3.1.1.3), phospholipase A2 ( EC number 3.1.1.4), lysophospholipase (EC number 3.1.1.5), acetyl esterase (EC number 3.1.1.6), acetylcholinesterase (EC number 3.1.1.7), phospholipase C (EC number 3.1.4.3), phospholipase D (EC number 3.1.4.4), phosphoinositide phospholipase C (EC number 3.1.4.11), pectin esterase (EC number 3.1.1.11), Gluconolactonase (EC number 3.1.1.17), acylglycerol lipase (EC number 3.1.1.23), 3-oxoadipate enol lactonase (EC number 3.1.1.24), 1,4-lactonase (EC number 3.1.1.25), galactolipase (EC number 3.1.1.26), phospholipase A1 (EC number 3.1.1.32), lipoprotein lipase (EC number 3.1.1.34), Cephalosporin-C deacetylase (EC number 3.1.1.41), carboxymethylenebutenolidase (EC number 3.1.1.45), 2-pyrone-4,6-dicarboxylic acid lactonase (EC number 3.1.1.57), feruloyl esterase (EC number 3.1.1.73), cutinase (EC number 3.1.1.74), hormone sensitive lipase (EC number 3.1.1.79), and combinations thereof.

[0120] Non-limiting examples of such combinations include: one or more cutinases and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more other carboxyl ester hydrolases; one or more triacylglycerol lipases and one or more other carboxyl ester hydrolases; one or more phospholipase A2s and one or more other carboxyl ester hydrolases; one or more phospholipase Cs and one or more other carboxyl ester hydrolases; one or more acetyl xylan esterases and one or more other carboxyl ester hydrolases; one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases; one or more acetyl esterases and one or more other carboxyl ester hydrolases; one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more alpha / beta hydrolases and one or more other carboxylester hydrolases;one or more transcription factors regulating expression of esterases and one or more other carboxylester hydrolases;one or more cutinases and one or more lysophospholipases;one or more cutinases and one or more triacylglycerol lipases;one or more cutinases and one or more phospholipase A2s;one or more cutinases and one or more phospholipase Cs;one or more cutinases and one or more acetyl xylan esterases;one or more cutinases and one or more extracellular lipase-like proteins;one or more cutinases and one or more acetyl esterases;one or more cutinases and one or more GDSL lipases;one or more cutinases and one or more alpha / beta hydrolases;one or more cutinases and one or more transcription factors regulating expression of esterases;one or more lysophospholipases and one or more triacylglycerol lipases;one or more lysophospholipases and one or more phospholipase A2; one or more lysophospholipases and one or more phospholipase C; one or more lysophospholipases and one or more acetyl xylan esterases; one or more lysophospholipases and one or more extracellular lipase-like proteins; one or more lysophospholipases and one or more acetyl esterases; one or more lysophospholipases and one or more GDSL lipases; one or more lysophospholipases and one or more alpha / beta hydrolases; one or more transcription factors regulating the expression of one or more lysophospholipases and esterases; one or more triacylglycerol lipases and one or more phospholipase A2; one or more triacylglycerol lipases and one or more phospholipase C; one or more triacylglycerol lipases and one or more acetyl xylan esterases; one or more triacylglycerol lipases and one or more a number of extracellular lipase-like proteins; one or more triacylglycerol lipases and one or more acetyl esterases; one or more triacylglycerol lipases and one or more GDSL lipases; one or more triacylglycerol lipases and one or more alpha / beta hydrolases; one or more transcription factors regulating the expression of one or more triacylglycerol lipases and esterases; one or more phospholipase A2s and one or more phospholipase Cs; one or more phospholipase A2s and one or more acetyl xylan esterases; one or more phospholipase A2s and one or more extracellular lipase-like proteins; one or more phospholipase A2s and one or more acetyl esterases; one or more phospholipase A2s and one or more GDSL lipases; one or more phospholipase A2s and one or more alpha / beta hydrolases; one or more phospholipase A2s and one or more transcription factors regulating the expression of the esterases;one or more phospholipases C and one or more acetyl xylan esterases;one or more phospholipases C and one or more extracellular lipase-like proteins;one or more phospholipases C and one or more acetyl esterases;one or more phospholipases C and one or more GDSL lipases;one or more phospholipases C and one or more alpha / beta hydrolases;one or more phospholipases C and one or more transcription factors regulating the expression of the esterases;one or more acetyl xylan esterases and one or more extracellular lipase-like proteins;one or more acetyl xylan esterases and one or more acetyl esterases;one or more acetyl xylan esterases and one or more GDSL lipases;one or more acetyl xylan esterases and one or more alpha / beta hydrolases;one or more acetyl xylan esterases and one or more transcription factors regulating the expression of the esterases;one or more acetyl esterases and one or more GDSL lipases;one; or multiple acetyl esterases and one or more alpha / beta hydrolases; one or more acetyl esterases and one or more transcription factors regulating the expression of the esterases; one or more GDSL lipases and one or more alpha / beta hydrolases; one or more GDSL lipases and one or more transcription factors regulating the expression of the esterases; one or more alpha / beta hydrolases and one or more transcription factors regulating the expression of the esterases; one or more cutinases and one or more lysophospholipases and one or more other carboxyl ester hydrolases; one or more cutinases and one or more triacylglycerol lipases and one or more other carboxyl ester hydrolases; one or more cutinases and one or more phospholipase A2 and one or more other carboxyl ester hydrolases; one or more cutinases and one or more phospholipase C and one or more other carboxyl ester hydrolases; one or more cutinases and one or more acetyl xylan esterases and one or more other carboxyl ester hydrolases; one or more cutinases and one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases; one or more cutinases and one or more acetyl esterases and one or more other carboxyl ester hydrolases; one or more cutinases and one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more cutinases and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; one or more transcription factors regulating expression of one or more cutinases and esterases and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more triacylglycerol lipases and one or more other carboxyl ester hydrolases;one or more lysophospholipases and one or more phospholipase A2 and one or more other carboxylester hydrolases; one or more lysophospholipases and one or more phospholipase C and one or more other carboxylester hydrolases; one or more lysophospholipases and one or more acetyl xylan esterases and one or more other carboxylester hydrolases; one or more lysophospholipases and one or more extracellular lipase-like proteins; one or more lysophospholipases and one or more acetyl esterases and one or more other carboxylester hydrolases; one or more lysophospholipases and one or more acetyl esterases and one or more other carboxylester hydrolases a plurality of GDSL lipases and one or more other carboxylester hydrolases; a plurality of lysophospholipases and one or more alpha / beta hydrolases and one or more other carboxylester hydrolases; a plurality of transcription factors regulating expression of the one or more lysophospholipases and esterases and one or more other carboxylester hydrolases; a plurality of triacylglycerol lipases and one or more phospholipase A2 and one or more other carboxylester hydrolases; a plurality of triacylglycerol lipases and one or more phospholipase C and one or more other carboxylester hydrolases; or a plurality of triacylglycerol lipases and one or more acetyl xylan esterases and one or more other carboxyl ester hydrolases; a plurality of triacylglycerol lipases and one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases; a plurality of triacylglycerol lipases and one or more acetyl esterases and one or more other carboxyl ester hydrolases; a plurality of triacylglycerol lipases and one or more GDSL lipases and one or more other carboxyl ester hydrolases; a plurality of triacylglycerol lipases and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; a plurality of transcription factors that regulate the expression of a plurality of triacylglycerol lipases and an esterase and one or more other carboxyl ester hydrolases; a plurality of phospholipases A2 and one or more phospholipase C and one or more other carboxyl ester hydrolases; one or more phospholipase A2 and one or more acetyl xylan esterases and one or more other carboxyl ester hydrolases; one or more phospholipase A2 and one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases; one or more phospholipase A2 and one or more acetyl esterases and one or more other carboxyl ester hydrolases; one or more phospholipase A2 and one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more phospholipase A2 and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; one or more phospholipase A2 and one or more transcription factors that regulate the expression of the esterases and one or more other carboxyl ester hydrolases;one or more phospholipase C and one or more acetyl xylan esterases and one or more other carboxyl ester hydrolases;one or more phospholipase C and one or more extracellular lipase-like proteins;one or more phospholipase C and one or more acetyl esterases and one or more other carboxyl ester hydrolases;one or more phospholipase C and one or more GDSL lipases and one or more other carboxyl ester hydrolases;one or more phospholipase C and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases;one or more phospholipase C and one or more transcription factors that regulate the expression of the esterases and one or more other carboxyl ester hydrolases;one or more acetyl xylan esterases and one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases;one or more acetyl xylan esterases and one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases one or more acetyl esterases and one or more other carboxyl ester hydrolases;one or more acetyl xylan esterases and one or more GDSL lipases and one or more other carboxyl ester hydrolases;one or more acetyl xylan esterases and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases;one or more acetyl xylan esterases and one or more transcription factors that regulate the expression of the esterases and one or more other carboxyl ester hydrolases;one or more acetyl esterases and one or more GDSL lipases and one or more other carboxyl ester hydrolases;one or more acetyl esterases and one or more alpha / beta hydrolases ... transcription factors that regulate the expression of the esterases and one or more other carboxyl ester hydrolases;one or more GDSL lipases and one or more alpha / beta hydrolases and one or more other carboxylester hydrolases; one or more transcription factors that regulate the expression of one or more GDSL lipases and esterases and one or more other carboxylester hydrolases; one or more transcription factors that regulate the expression of one or more alpha / beta hydrolases and esterases and one or more other carboxylester hydrolases; one or more cutinases and one or more lysophospholipases and one or more triacylglycerol lipases; one or more cutinases and one or; one or more cutinases and one or more lysophospholipases and one or more phospholipases; one or more cutinases and one or more lysophospholipases and one or more acetyl xylan esterases; one or more cutinases and one or more lysophospholipases and one or more extracellular lipase-like proteins; one or more cutinases and one or more lysophospholipases and one or more acetyl esterases; one or more cutinases and one or more lysophospholipases and one or more GDSL lipases; one or more cutinases and one or more lysophospholipases and one or more alpha / beta hydrolases; one or more cutinases and one or more lysophospholipases and one or more transcription factors regulating the expression of the esterases; one or more cutinases and one or more triacylglycerol lipases and one or more phospholipase A2; one or more cutinases and one or more triacylglycerol lipases and one or more phospholipase C; one or more cutinases and one or more triacylglycerol lipases and one or more acetyl xylan esterases; one or more cutinases and one or more triacylglycerol lipases and one or more extracellular lipase-like proteins; one or more cutinases and one or more triacylglycerol lipases and one or more acetyl esterases; one or more cutinases and one or more triacylglycerol lipases and one or more GDSL lipases; one or more cutinases and one or more triacylglycerol lipases and one or more alpha / beta hydrolases; one or more transcription factors regulating the expression of one or more cutinases and one or more triacylglycerol lipases and esterases; one or more cutinases and one or more phospholipase A2 and one or more phospholipase C;one or more cutinases and one or more phospholipase A2 and one or more acetyl xylan esterases;one or more cutinases and one or more phospholipase A2 and one or more extracellular lipase-like proteins;one or more cutinases and one or more phospholipase A2 and one or more acetyl esterases;one or more cutinases and one or more phospholipase A2 and one or more GDSL lipases;one or more cutinases and one or more phospholipase A2 and one or more alpha / beta hydrolases;one or more cutinases and one or more phospholipase A2 and one or more transcription factors regulating the expression of the esterases;one or more cutinases and one or more phospholipase C and one or more acetyl xylan esterases;one or more cutinases and one or more phospholipase C and one or more extracellular lipase-like proteins;one or more cutinases and one or more phospholipase C and one or more extracellular lipase-like proteins;one or more cutinases and one or more phospholipase C and one or more or more acetyl esterases; one or more cutinases and one or more phospholipase C and one or more GDSL lipases; one or more cutinases and one or more phospholipase C and one or more alpha / beta hydrolases; one or more cutinases and one or more phospholipase C and one or more transcription factors regulating the expression of the esterases; one or more cutinases and one or more acetyl xylan esterases and one or more extracellular lipase-like proteins; one or more cutinases and one or more acetyl xylan esterases and one or more acetyl esterases; one or more cutinases and one or more acetyl xylan esterases and one or more GDSL lipases; one or more cutinases and one or more acetyl xylan esterases and one or more alpha / beta hydrolases; one or more cutinases and one or more acetyl xylan esterases and one or more transcription factors regulating the expression of the esterases;one or more cutinases and one or more extracellular lipase-like proteins and one or more acetyl esterases; one or more cutinases and one or more extracellular lipase-like proteins and one or more GDSL lipases; one or more cutinases and one or more extracellular lipase-like proteins and one or more alpha / beta hydrolases; one or more transcription factors regulating the expression of one or more cutinases and one or more extracellular lipase-like proteins and esterases; one or more cutinases and one or more acetyl esterases and one or more GDSL lipases; one or more cutinases and one or more acetyl esterases and one or more alpha / beta hydrolases; one or more transcription factors regulating the expression of one or more cutinases and one or more acetyl esterases and esterases; one or more cutinases and one or more GDSL lipases and one or more alpha / beta hydrolases; one or more transcription factors regulating the expression of one or more cutinases and one or more GDSL lipases and esterases; and one or more transcription factors regulating the expression of one or more cutinases and one or more alpha / beta hydrolase fold domain containing proteins and esterases.

[0121] The appropriate number and / or combination of esterases whose activity must be essentially eliminated or modulated in at least one step of the methods provided herein for producing a food product, or in the fermentation broth or preparation provided herein containing recombinant components, or in the recombinant microbial host cells provided herein capable of producing the recombinant components, can be identified by methods known in the art. For example, the esterases can be isolated by methods known in the art (e.g., affinity chromatography, zymogram assay, gel electrophoresis) and tested in vitro to determine which one or which combination of at least two esterases provides a significant amount of degradation of a particular diglyceride, triglyceride, phospholipid, or lipoprotein. Moreover, recombinant microbial host cells capable of producing a recombinant component (e.g., any of the recombinant components disclosed herein) can be obtained that contain essentially eliminated or modulated activity in any one or any combination of at least two esterases, and the degree of reduction or elimination of lipid degradation in food products containing recombinant components produced by each such recombinant microbial host cell can be measured by methods known in the art.

[0122] In some embodiments, the fermentation broths or preparations provided herein that contain recombinant components, or the recombinant microbial host cells provided herein that are capable of producing the recombinant components, contain a combination and / or level of esterase activity that provides a particular profile of esterase activity (i.e., esterase activity profile) that is useful for the production of a particular food product. In some such embodiments, the esterase activity profile is optimized to provide a desired flavor, aroma, texture, emulsification, nutritional content, and / or shelf life of the food product.

[0123] Esterase activity can be measured using enzyme assays. For example, enzyme activity can be determined by the production of a colorimetric reaction product or a product (e.g., free fatty acids and / or glycerol produced from the esterase-catalyzed hydrolysis of triglycerides) that can be detected using, for example, PAGE gels, spectrophotometers, imaging, UV / Vis, light, and HPLC.

[0124] Recombinant Microbial Host Cells Producing Recombinant Components and Containing Essentially Eliminated or Modulated Esterase Activity In another aspect, provided herein are recombinant microbial host cells capable of producing recombinant components and that contain an esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) that is essentially eliminated or modulated relative to the esterase activity contained in the corresponding recombinant microbial host cell.

[0125] The esterase activity contained in the recombinant microbial host cells provided herein can be essentially eliminated or modulated by any means. In some embodiments, the esterase activity is essentially eliminated or modulated by essentially eliminating or modulating the expression of the esterase (i.e., production of an active protein) or by essentially eliminating or modulating the activity of the esterase. Thus, in various embodiments, the recombinant microbial host cells provided herein include a) a genetic modification of a control sequence driving the expression of the esterase, or a functional portion thereof (i.e., a portion sufficient for the function of the control sequence) (e.g., a promoter sequence, an enhancer sequence, a signal peptide, a transcription terminator, or any other sequence that controls the expression (i.e., transcription and / or translation) of a gene), where the genetic modification essentially eliminates or modulates the expression of the esterase; b) a genetic modification of a coding sequence encoding an esterase or a functional portion thereof (e.g., a catalytic domain), where the genetic modification essentially eliminates or modulates the activity of the esterase; c) a protein required for the expression of the esterase (e.g., a transcription factor, , a post-translational modification enzyme required for the production of an active form of the esterase), or a functional portion thereof (i.e., a portion sufficient for the function of the control sequence) driving the expression of the protein required for the expression of the esterase, where the genetic modification essentially eliminates or modulates the expression of the protein, thereby essentially eliminating or modulating the expression of the esterase; d) a genetic modification of a coding sequence encoding a protein or a functional portion thereof required for the expression of the esterase (e.g., a DNA binding domain of a transcription factor, a catalytic domain of a post-translational modification enzyme), where the genetic modification essentially eliminates or modulates the activity of the protein required for the expression of the esterase, thereby essentially eliminating or modulating the expression of the esterase;e) a genetic modification of a control sequence, or a functional portion thereof (i.e., a portion sufficient for the function of the control sequence), that drives expression of an endogenous inhibitor of the esterase, where the genetic modification essentially eliminates or modulates expression of the endogenous inhibitor, thereby essentially eliminating or modulating expression of the esterase; f) a genetic modification of a coding sequence that encodes an endogenous inhibitor of the esterase, where the genetic modification essentially eliminates or modulates the activity of the endogenous inhibitor, thereby essentially eliminating or modulating expression of the esterase; and / or g) a genetic modification that introduces a coding sequence that encodes a heterologous (i.e., non-naturally occurring) inhibitor of the esterase, where the genetic modification provides for production of the heterologous inhibitor, thereby essentially eliminating or modulating expression of the esterase;

[0126] Genetic modification can be, for example, the insertion, substitution or deletion of one or more nucleotides in polynucleotide sequence.Genetic modification can be, for example, the introduction of stop codon; the movement of start codon; the insertion of open reading frame frame shift; or the creation of point mutation, missense mutation, substitution mutation, deletion mutation, frameshift mutation, insertion mutation, duplication mutation, multiplication mutation, translocation mutation or inversion mutation.

[0127] In some embodiments, the recombinant microbial host cells provided herein comprise a recombinant polynucleotide that comprises a nucleotide sequence that is complementary to a coding sequence encoding an esterase (e.g., any one of the esterases disclosed herein or any combination of at least two of the esterases disclosed herein), encodes an RNAi construct specific for an esterase, or encodes a heterologous inhibitor of esterase activity.

[0128] In some embodiments, where the recombinant microbial host cells provided herein comprise genetic modifications that essentially eliminate or modulate the expression or activity of a protein required for expression of an esterase, such a protein required for expression of an esterase is a transcription factor that regulates expression of an esterase. In some such embodiments, the transcription factor that regulates expression of an esterase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from any of the sequences encoding transcription factors that regulate expression of an esterase listed in Table 1. In some such embodiments, the transcription factor that regulates expression of the esterase comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or 100% amino acid sequence identity to a sequence selected from the UniProt sequences G0RX49, G0RHG1, A0A2T4B416, A0A2T4BJU6, A2R2J1, A2R903, G2Q2Z5, and G2Q8I6. In some such embodiments, the recombinant microbial host cells provided herein comprise one or more genetic modifications that essentially eliminate or modulate the expression or activity of a combination of at least two transcription factors that regulate expression of one or more esterases.

[0129] The recombinant microbial host cells provided herein are capable of producing recombinant components and comprise an esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) that is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the esterase activity contained in the corresponding recombinant microbial host cell. In some such embodiments, the recombinant microbial host cells provided herein comprise essentially eliminated esterase activity. In other embodiments, the recombinant microbial host cells provided herein are capable of producing recombinant components and comprise an esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) that is increased by at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, 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 1,000% compared to the esterase activity contained in a corresponding recombinant microbial host cell.

[0130] microbial host cell The recombinant microbial host cells provided herein can be derived from any microbial cell. In some embodiments, the recombinant microbial host cells provided herein are recombinant bacterial host cells. The recombinant bacterial host cells can be derived from any bacterial strain known in the art. Non-limiting examples of bacterial strains include those from the phylum Firmicutes, Cyanobacteria (Blue-green algae), Oscillatoriophcideae, Bacillales, Lactobacillales, Oscillales, Bacillaceae, Lactobacillaceae, Acetobacter suboxydans, Acetobacter xylinum, Actinoplanes missouriensis, Arthrospira platensis, Arthrospira maxima, Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactococcus lactis, Lactococcus lactis Lancefield Group N, Lactobacillus reuteri, Leuconostoc citrovorum, Leuconostoc dextrinicumdextranicum, Leuconostoc mesenteroides strain NRRL B-512(F), Micrococcus lysodeikticus, Spirulina, Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus, Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus, Tetrahymena thermophile, Tetrahymena hegewischi, Tetrahymena hyperangularis, Tetrahymena malaccensis, Tetrahymena pigmentosa, Tetrahymena pyriformis, and Tetrahymena vorax, Xanthomonas campestris. campestris), and their derivatives and hybrids.

[0131] In some embodiments, the recombinant microbial host cells provided herein are recombinant yeast host cells. Recombinant yeast host cells can be derived from any yeast strain known in the art. Non-limiting examples of yeast strains include Candida albicans, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Candida versatilis, Debaryomyces hansenii, Eremothecium ashbyii, Hansenula polymorpha, Kluyveromyces spp. sp., Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Pichia sp.), Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia piedperi pijperi, Pichia stiptis, Pichia methanolica, Rhodotorula sp., Saccharomyces sp.), Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Sporobolomyces roseus, Yarrowia lipolytica, Zygosaccharomyces rouxii, and their derivatives and hybrids.

[0132] In some embodiments, the recombinant microbial host cells provided herein are recombinant filamentous fungal host cells. The recombinant filamentous fungal host cells can be derived from any filamentous fungal strain known in the art, including any holomorphic, teleomorphic, or anamorphic forms thereof.Non-limiting examples of filamentous fungal strains include those of the genera Acremonium, Aspergillus, Aureobasidium, CAnariomyces, Chaetonium, Chaetomidium, Corynascus, Cryptococcus, Chrysosporium, Conemeria, and the like. Coonemeria, Dactylomyces, Emericella, Filibasidium, Fusarium, Gibberella, Humicola, Lentinula, Magnaporthe, Malbranchium, Melanocarpus, Mucor Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Rhizopus , Schizophyllum, Scytalidium, Sporotrichum, Stereum, Talaromyces, Thermoascus, Thermomyces, Thielavia, Tolypocladium, and Trichoderma strains.

[0133] Non-limiting examples of Acremonium strains include Acremonium alabamense. Non-limiting examples of Aspergillus strains include Aspergillus aculeatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus niger var.These include Aspergillus awamori, Aspergillus oryzae, Aspergillus sojae, and Aspergillus terreus, as well as Emericella, Neosartorya, and Petromyces species. Non-limiting examples of Chrysosporium species include Chrysosporium botryoides, Chrysosporium carmichaeli, Chrysosporium crassitunicatum, Chrysosporium europae, Chrysosporium evolceannui, Chrysosporium farinicola, Chrysosporium fastidium, Chrysosporium filiforme, Chrysosporium georgiae, Chrysosporium globiferum, Chrysosporium globiferum), Chrysosporium globiferum var. articulatum, Chrysosporium globiferum var. niveumniveum, Chrysosporium hirundo, Chrysosporium hispanicum, Chrysosporium holmii, Chrysosporium indicum, Chrysosporium iops, Chrysosporium keratinophilum, Chrysosporium kreiselii, Chrysosporium kuzurovianum, Chrysosporium lignorum, Chrysosporium ovatum obatum, Chrysosporium lucknowense, Chrysosporium lucknowense Garg 27K, Chrysosporium medium, Chrysosporium medium var. spissescens, Chrysosporium mephiticum, Chrysosporium merdarium, Chrysosporium merdarium var. roseum, Chrysosporium minor, Chrysosporium pannicola, Chrysosporium parvum parvum), Chrysosporium parvum var. cressence (Chrysosporium parvum var.crescens, Chrysosporium pilosum, Chrysosporium pseudomerdarium, Chrysosporium pyriformis, Chrysosporium queenslandicum, Chrysosporium sigleri, Chrysosporium sulfureum, Chrysosporium synchronum, Chrysosporium tropicum, Chrysosporium undulatum, Chrysosporium barrenalens vallenarense, Chrysosporium vespertilium, and Chrysosporium zonatum.

[0134] Non-limiting examples of Fusarium species include Fusarium moniliforme, Fusarium venenatum, Fusarium oxysporum, Fusarium graminearum, Fusarium proliferatum, Fusarium verticiollioides, Fusarium culmorum, Fusarium crookwellense, Fusarium poae, Fusarium sporotrichioides, Fusarium sambusinum, ... moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliform Examples of species of the genus include Myceliophthora sambuccinum, Fusarium torulosum, and related Gibberella teleomorphic forms. Non-limiting examples of Myceliophthora strains include Myceliophthora thermophila. Non-limiting examples of Mucor strains include Mucor miehei var. Cooney et Emerson (Rhizomucor miehei (Cooney & R. Emerson)) Schipper, and Mucor pusillus Lindt. Non-limiting examples of Neurospora strains include Neurospora crassa.

[0135] Non-limiting examples of Penicillium strains include Penicillium chrysogenum and Penicillium roquefortii. Non-limiting examples of Rhizopus strains include Rhizopus niveus. Non-limiting examples of Sporotrichum strains include Sporotrichum cellulophilum. Non-limiting examples of Thielavia strains include Thielavia terrestris. Non-limiting examples of Trichoderma strains include Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichoderma virens, Trichoderma citrinoviride, and Trichoderma viride, as well as alternative sexual / telemorphic forms thereof (i.e., Hypocrea spp.).

[0136] The recombinant microbial host cells provided herein can be sporulation competent or sporulation deficient.

[0137] The recombinant microbial host cells provided herein can be derived from wild-type microbial cells or genetic variants (eg, mutants) thereof.

[0138] The recombinant microbial host cells provided herein can be derived from strains that are Food and Drug Administration recognized as acceptable for sale (GRAS).

[0139] The recombinant microbial host cells provided herein can have a high exogenous secreted protein / biomass ratio, in some such embodiments, greater than about 1:1, greater than about 2:1, greater than about 3:1, greater than about 4:1, greater than about 5:1, greater than about 6:1, greater than about 7:1, or greater than about 8:1.

[0140] In some embodiments, a recombinant microbial host cell provided herein that comprises essentially eliminated or modulated activity of an esterase or a combination of at least two (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more) esterases can be a recombinant Trichoderma reesei host cell (i.e., a recombinant host cell derived from a Trichoderma reesei strain) that comprises essentially eliminated or modulated activity of an esterase or a combination of at least two (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more) esterases. recombinant Aspergillus niger host cells (i.e., recombinant host cells derived from an Aspergillus niger strain) that contain essentially eliminated or modulated activity of a combination of esterases or at least two (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more) of the esterases; recombinant Trichoderma citrinoviride host cells (i.e., recombinant host cells derived from a Trichoderma citrinoviride strain) that contain essentially eliminated or modulated activity of a combination of esterases or at least two (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or more) of the esterases;or more),

[0141] Recombinant ingredients The recombinant microbial host cells provided herein are capable of producing recombinant components, non-limiting examples of which include proteins, lipids, carbohydrates, small molecules, food additives, dietary supplements (e.g., vitamins), functional foods, and probiotics.

[0142] In some embodiments, the recombinant component is a recombinant protein. In some embodiments, the recombinant protein is a recombinant plant protein. As used herein, the term "plant protein" refers to a polypeptide that comprises a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to a sequence of amino acids in a protein naturally found in a plant (i.e., a protein native to a plant cell). Non-limiting examples of plant proteins include cycads, ginkgo, conifers, cypress, juniper, arborvitae, cedarwood, pine, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, cornflower, marigold, mugwort, safflower, chamomile, lettuce, wormwood, calendula, citronella, sage, thyme, chaseed, mustard, olive, coffee, chili pepper, eggplant, paprika, cranberry, kiwi, vegetable plants (e.g., carrot, celery), Tagetes, tansy, tarragon, sunflower, burdock, basil, hyssop, lavender, lemon verbena, marjoram, mentha, patchouli, mentha piperita, mentha piperita, Rosemary, sesame, spearmint, anemone, samara, chili pepper, pimento, potato, sweet potato, tomato, blueberry, day cherries, petunia, morning glory, lilac, jasmine, honeysuckle, snapdragon, psyllium, sage, buckwheat, amaranth, chard, quinoa, spinach, rhubarb, jojoba, Cypsera, chlorella, marula, hazelnut, canola, kale, bok choy, rutabaga, frankincense, myrrh, elemi, thyme, pumpkin, squash, urticaria, manioc, Dalbergia, legumes (e.g. alfalfa, lentil, bean, clover, pea, umbellifera, frijole bola roya)roja), black beans, bush clover, licorice, lupin, mesquite, carob, soybean, peanut, tamarind, wisteria, cassia, chickpea / garbanzo, fenugreek, green pea, yellow pea, snow pea, lima bean, broad bean), geranium, flax, pomegranate tree, cotton, okra, neem, fig, mulberry, clove, eucalyptus, tea tree, niaouli, fruit plants (e.g. apple, apricot, peach, plum, pear, safflower), strawberry, black Cupberry, raspberry, cherry, prune, rose, tangerine, citrus fruits (e.g. grapefruit, lemon, lime, orange, bitter orange, mandarin, tangerine), mango, citrus bergamot, boucou, grape, broccoli, Brussels sprouts, cauliflower, rapeseed, rapeseed (canola), turnip, cabbage, cucumber, watermelon, honeydew melon, zucchini, birch, walnut, cassava, baobab, allspice, almond , breadfruit, sandalwood, macadamia, taro, moonflower, aloe vera, garlic, onion, shallot, vanilla, yucca, vetiver, alpinia, barley, cereals, yellow turmeric, ginger, lemongrass, oats, palm, pineapple, rice, rye, sorghum, triticale, turmeric, yam, bamboo, barley, cajeput, canna, cardamom, corn, oats, wheat, cinnamon, sassafras, lindera benzoin, bay leaf, avocado, ylang-ylang Includes proteins from orchids, mace, nutmeg, moringa, jute, oregano, coriander leaves, chervil, chives, aggregate fruits, cereal plants, herbal plants, leafy vegetables, non-cereal legume plants, nut plants, succulents, terrestrial plants, aquatic plants, Dalbergia, millet, drupes, schisandra, flowering plants, non-flowering plants, cultivated plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, brushes, lianas, cacti, tropical plants, subtropical plants, temperate plants, and derivatives and hybrids thereof.

[0143] In some embodiments, the plant protein is a pea protein (e.g., legumin, vicilin, covicilin), in other embodiments, the plant protein is a potato protein (e.g., tuberin, protease inhibitor Noutyt II).

[0144] In some embodiments, the recombinant protein is a recombinant animal protein. The term "animal protein" as used herein refers to a polypeptide that comprises a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to a sequence of amino acids in a protein naturally found in an animal (i.e., a protein native to an animal cell). Non-limiting examples of animal proteins include proteins from insects (e.g., flies), mammals (e.g., cows, sheep, goats, rabbits, pigs, humans), or birds (e.g., chickens). In some embodiments, the animal protein is a structural protein (e.g., collagen, tropoelastin, elastin). In other embodiments, the animal protein is an egg protein (eg, ovalbumin).

[0145] In yet other embodiments, the recombinant protein is a recombinant milk protein. As used herein, the term "milk protein" refers to a polypeptide that includes a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to a sequence of amino acids in a protein naturally found in milk produced by a mammal (i.e., a protein native to the milk produced by a mammal).

[0146] The recombinant milk protein can be a recombinant whey protein or recombinant casein. As used herein, the term "whey protein" or "casein" refers to a polypeptide that includes a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to the sequence of amino acids in naturally occurring whey protein or casein, respectively. Non-limiting examples of whey proteins include α-lactalbumin, β-lactalbumin, lactoferrin, transferrin, serum albumin, lactoperoxidase, and glycomacropeptide. Non-limiting examples of caseins include β-casein, γ-casein, κ-casein, α-S1 casein, and α-S2 casein. Non-limiting examples of nucleic acid sequences encoding whey proteins and caseins are disclosed in U.S. Patent No. 5,993,433, filed Aug. 21, 2015, and U.S. Patent No. 5,993,433, filed Aug. 25, 2017, which are hereby incorporated by reference in their entireties.

[0147] Recombinant milk proteins can be derived from any mammalian species, including, but not limited to, cow, human, sheep, goat, water buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, and echidna.

[0148] Recombinant milk proteins can lack epitopes capable of eliciting an immune response in humans or animals, making them particularly suitable for use in food products.

[0149] Recombinant milk proteins can have post-translational modifications. As used herein, the term "post-translational modification" or its acronym "PTM" refers to the covalent attachment of a chemical group to a protein after protein biosynthesis. PTMs can occur at amino acid side chains or at the C- or N-terminus of the protein. Non-limiting examples of PTMs include glycosylation (i.e., the covalent attachment of a glucan group (i.e., monosaccharide, disaccharide, polysaccharide, linear glycan, branched glycan, glycan with galf residues, glycan with sulfate and / or phosphate residues, D-glucose, D-galactose, D-mannose, L-fucose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, N-acetyl-D-neuraminic acid, galactofuranose, phosphodiester, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and combinations thereof; see, e.g., Non-Patent Document 6) to a protein via C-, N-, or O-linkage, or via glypiation (i.e., addition of a glycosylphosphatidylinositol anchor) or phosphoglycosylation (i.e., linked through the phosphate of phosphoserine), phosphorylation (i.e., the attachment of a phosphate group to a protein). These include covalent attachment to proteins), alkylation (i.e., the covalent attachment of an alkane group (e.g., a methane group in methylation) to a protein), and lipidation (i.e., a lipid group (e.g., an isoprenide group in prenylation and isoprenylation (e.g., a farnesol group in farnesylation, a geraniol group in geranylation, and geranylgeraniol in geranylgeranylation), a fatty acid group in fatty acid acylation (e.g., myristic acid in myristoylation, palmitic acid in palmitoylation, and a glycosylphosphatidylinositol anchor in glypiation)), hydroxylation (i.e., the covalent attachment of a hydroxide group), SUMOylation (i.e., the attachment of a small ubiquitin-like modifier (or SUMO) protein to a protein), nitrosylation (i.e., the attachment of an NO group to a protein), and tyrosine nitration (i.e., the attachment of a nitrate group to a tyrosine residue in a protein). The PTMs of the recombinant milk proteins can be naturally occurring PTMs, non-naturally occurring PTMs, or a mixture of at least one naturally occurring PTM and at least one non-naturally occurring PTM.As used herein, a "non-native PTM" refers to a difference in one or more positions of one or more PTMs (e.g., glycosylation, phosphorylation) in a protein and / or a difference in the type of one or more PTMs at one or more positions in a protein compared to a naturally occurring protein (i.e., a protein having a "native PTM").

[0150] The recombinant milk protein can have milk protein repeats. As used herein, the term "milk protein repeat" refers to an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95% identical, at least 99% identical) to an amino acid sequence in a protein found in mammalian produced milk (e.g., whey protein, casein) and occurs more than once (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 75 times, at least 100 times, at least 150 times, or at least 200 times) in the recombinant milk protein. The milk protein repeat may include at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, or at least 150, and usually no more than 200 amino acids. The milk protein repeats in a recombinant milk protein can be contiguous (i.e., with no intervening amino acid sequences) or non-contiguous (i.e., with intervening amino acid sequences). If non-contiguous, the intervening amino acid sequences may play a passive role in providing molecular weight without introducing undesirable properties, or may play an active role in providing certain properties (e.g., solubility, biodegradability, binding to other molecules).

[0151] In some embodiments, the recombinant protein is a recombinant algal protein. As used herein, the term "algal protein" refers to a polypeptide that includes a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to a sequence of amino acids in a protein naturally found in algae (i.e., a protein native to an algal cell).

[0152] In some embodiments, the recombinant protein is a recombinant fungal protein. As used herein, the term "fungal protein" refers to a polypeptide that comprises a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to the sequence of amino acids in a protein that is naturally found in a fungus (i.e., a protein native to a fungal cell).

[0153] In some embodiments, the recombinant protein is a recombinant microbial protein. As used herein, the term "microbial protein" refers to a polypeptide that comprises a sequence of at least 20 amino acids (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually no more than 250 amino acids) that is at least 80% identical (e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) to the sequence of amino acids in a protein naturally found in a microorganism (i.e., a protein native to a microbial cell).

[0154] General method Recombinant microbial host cells capable of producing the recombinant components can be obtained using methods known in the art. Recombinant microbial host cells typically contain a recombinant polynucleotide (e.g., a recombinant vector) that encodes the recombinant components. Recombinant polynucleotides can be prepared by any suitable method known in the art, including, but not limited to, direct chemical synthesis and cloning.

[0155] A recombinant polynucleotide typically comprises one or more expression cassettes, each comprising: a promoter (e.g., a fungal promoter), an optional signal sequence (i.e., a sequence encoding a peptide that mediates delivery of the nascent protein linked to a peptide to the exterior of the cell in which the nascent protein is synthesized), a sequence encoding a recombinant protein, and a termination sequence (or sequences), wherein the promoter is operably linked to the optional signal sequence in a sense orientation (i.e., the promoter and the optional signal sequence and the subsequent sequence encoding the protein are positioned such that the promoter is effective to regulate transcription of the optional signal sequence and the sequence encoding the protein), the optional signal sequence is operably linked to the sequence encoding the recombinant protein in a sense orientation (i.e., the signal sequence and the sequence encoding the recombinant protein are positioned such that transcription and translation produces a recombinant protein comprising a functional signal sequence), and a termination sequence is operably linked to the sequence encoding the recombinant protein (i.e., the sequence encoding the recombinant protein and the termination sequence are positioned such that the terminator is effective to terminate transcription of the optional signal sequence and the sequence encoding the recombinant protein).

[0156] The promoter may be any suitable promoter that is functional in a recombinant microbial host cell. In some embodiments, the promoter is a constitutive promoter. In other embodiments, the promoter is an inducible or repressible promoter (e.g., a promoter that is induced or repressed in the presence of glucose, galactose, lactose, sucrose, cellulose, sophorose, gentiobiose, sorbose, a disaccharide inducing cellulase promoter, starch, tryptophan, or phosphate). Non-limiting examples of promoters suitable for use in recombinant filamentous fungal host cells include those encoding the following proteins: glucoamylase (e.g., Aspergillus niger, Aspergillus awamori, Aspergillus japonicus, Aspergillus tubingensis, Aspergillus foetidus, or Aspergillus carbonarius). carbonarius glaA), amylases (e.g., Aspergillus TAKA amylase, Aspergillus niger neutral alpha amylase, Aspergillus niger acid-stable alpha amylase, fungal α-amylase (amy), bacterial alpha amylase), proteases (e.g., Rhizomucor miehei aspartic protease, Aspergillus oryzae alkaline protease, Fusarium oxysporum trypsin-like protease, Trichoderma reesei protease), lipases (e.g., Rhizomucor miehei lipase), isomerases (e.g., Aspergillus oryzae triosephosphate isomerase, fungal triosephosphate isomerase (tpi), yeast triosephosphate isomerase), acetamidase (e.g., Aspergillus oryzae or other fungal acetamidase (amdS)), dehydrogenase (e.g., fungal alcohol dehydrogenase (adhA), fungal glyceraldehyde-3-phosphate dehydrogenase (gpd), yeast alcohol dehydrogenase), xylanase (e.g., fungal xylanase (xlnA), Trichoderma xylanase (Trichodermaxylanases (xyn1, xyn2, bxl1), kinases (e.g., yeast 3-phosphoglycerate kinase), hydrolases (e.g., fungal cellobiohydrolase I (cbh1), Trichoderma hydrolases (cbh2, egl1, egl2)), phosphatases (e.g., Fusarium acid phosphatase), and other fungal proteins (e.g., fungal endo-α-L-arabinase (abnA), fungal α-L-arabinofuranosidase A (abfA), fungal α-L-arabinofuranosidase B (abfB), fungal phytase, fungal ATP synthetase, fungal subunit 9 (oliC), fungal sporulation-specific protein (Spo 2), fungal SSO, yeast alcohol oxidase, yeast lactase, Neurospora crassa CPC1, Aspergillus oryzae trpC, fungal chitinolytic enzymes (e.g., endo- and exo-chitinase, beta-glucanase), fungal VAZMP-associated protein (VAP), fungal translation elongation factor (TEF1), fungal DNA damage response protein (DDRP), fungal (e.g., Fusarium or Neurospora crassa) hexagonal peroxisome (Hex1), fungal (e.g., Neurospora crassa) catalase), and any other protein that is produced at high levels in a recombinant filamentous fungal host cell, and functional portions thereof.

[0157] Non-limiting examples of promoters suitable for use in recombinant bacterial or yeast host cells include promoters of genes encoding any of the following proteins: LAC4, T7 polymerase, TAC, GAL1, lambda PL, lambda PR, beta-lactamase, spa, CYC1, TDH3, GPD, TEF1, ENO2, PGL1, GAP, SUC2, ADH1, ADH2, HXT7, PHO5, CLB1, AOX1, cellulase, amylase, protease, xylinase, and any other protein that is produced at high levels in a recombinant filamentous fungal host cell, and functional portions thereof.

[0158] In some embodiments, the promoter is a promoter of a stress (eg, heat shock) responsive gene (eg, hac1, BIP).

[0159] The signal sequence may be any suitable signal sequence which is functional in the recombinant microbial host cell.

[0160] The recombinant protein typically encoded by the recombinant polynucleotide can be any recombinant protein (e.g., a recombinant component disclosed herein), including a recombinant milk protein (e.g., any of the recombinant milk proteins disclosed herein (e.g., recombinant beta-lactoglobulin, recombinant alpha-lactalbumin, recombinant kappa-casein, recombinant beta-casein)), recombinant egg proteins (e.g., ovotransferrin (conalbumin), ovomucin, ovalbumin, ovotransferrin, lysozyme), recombinant proteins required for the production of vitamins, lipids, small molecules that create flavor, polymers that create texture and emulsions, and combinations thereof.

[0161] The termination sequence may be any suitable termination sequence that is functional in a recombinant microbial host cell. Non-limiting examples of termination sequences suitable for use in recombinant filamentous fungal host cells include those from Aspergillus oryzae (e.g., termination sequences of the TAKA amylase gene), Aspergillus niger (e.g., termination sequences of the glaA, gpdA, aamA, trpC, pdc1, adh1, amdS, or tef1 genes), Fusarium oxysporum (e.g., termination sequences of serine protease (trypsin) genes), Trichoderma reesei (e.g., termination sequences of the cbh1, p Termination sequences include, but are not limited to, termination sequences from yeast, Pichia pastoris (e.g., termination sequences from aox1, gap1, adh1, tef1, tps1, or pgk1 genes), Saccharomyces cerevisiae (e.g., termination sequences from adh1, cyc1, gal1, tef1, pdc1, pgk1, or tps1 genes), synthetic termination sequences, and any combination of the sequences listed above. Non-limiting examples of termination sequences suitable for use in recombinant yeast host cells include, but are not limited to, PGK1 and TPS1 termination sequences.

[0162] The recombinant polynucleotide may further comprise additional elements. Non-limiting examples of such additional elements include enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions, polyadenylation sequences, introns, replication origins, operators (i.e., sequences of nucleic acids adjacent to a promoter and containing a protein binding domain to which a repressor protein can bind to reduce or eliminate the activity of the promoter), and selection markers (i.e., genes encoding proteins that can complement auxotrophy of filamentous fungal cells, confer antibiotic resistance, or cause color change). Such elements are known in the art. Non-limiting examples of replication origins include AMA1 and ANSI. Non-limiting examples of suitable selection markers include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG / pyr4 (orotidine 5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and derivatives thereof. In some embodiments, the selection marker comprises an alteration that reduces production of the selection marker, thus increasing the copy number required for a filamentous fungal cell containing the polynucleotide to survive under selection.

[0163] In embodiments in which the recombinant polynucleotide comprises two or more expression cassettes, the operably linked promoters, optional signal sequences, sequences encoding polypeptide termination sequences, and optional additional elements can be the same or different between the two or more expression cassettes.

[0164] Methods for introducing recombinant polynucleotides into microbial cells to obtain recombinant microbial host cells are well known in the art. Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (e.g., cationic liposome transfection), cationic polymer transfection, cell squeezing, sonoporation, phototransfection, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transduction, electroporation, and chemical transformation (e.g., using PEG).

[0165] In some embodiments, the recombinant polynucleotide is maintained extrachromosomally in the recombinant microbial host cell on an expression vector (i.e., a nucleic acid that transduces, transforms, or infects a host cell and causes the host cell to express nucleic acids and / or proteins other than those native to the host cell in a manner that is not native to the host cell). In other embodiments, the recombinant polynucleotide is stably integrated into the genome (e.g., chromosome) of the recombinant microbial host cell. For integration into the genome, the recombinant polynucleotide can include sequences for integration into the genome by homologous or non-homologous recombination. In some embodiments, such sequences allow integration at a precise location into the host genome. The recombinant polynucleotide may include at least 100, at least 250, at least 500, at least 750, at least 1000, or at least 10,000 base pairs that are highly homologous to a target sequence in the genome of the recombinant microbial host cell to enhance the likelihood of homologous recombination. Such highly homologous sequences may be non-coding sequences. More than one copy of a recombinant polynucleotide may be inserted into a recombinant microbial host cell to increase production of the recombinant protein.

[0166] Recombinant microbial host cells can be cultured in any suitable fermentation vessel known in the art (e.g., culture plates, shake flasks, fermentors (e.g., stirred tank fermentors, airlift fermentors, bubble column fermentors, fixed bed bioreactors, laboratory fermentors, industrial fermentors, or any combination thereof)) and at any scale (e.g., small-scale, large-scale) and process (e.g., continuous, batch, fed-batch, or solid state) known in the art.

[0167] Suitable culture media include any culture medium in which recombinant microbial host cells can grow and / or remain viable. In some embodiments, the culture medium is an aqueous medium that includes carbon, nitrogen (e.g., anhydrous ammonia, ammonium sulfate, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, sodium nitrate, urea, peptone, protein hydrolysate, yeast extract), and phosphate sources. The culture medium can further include inorganic salts, minerals, metals, transition metals, vitamins, emulsifiable oils, surfactants, and any other nutrients. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, acetate, ethanol, methanol, glycerol, methane, and combinations thereof. Non-limiting examples of monosaccharides include dextrose (glucose), fructose, galactose, xylose, arabinose, and combinations thereof. Non-limiting examples of disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, amylose, hemicellulose, maltodextrin, and combinations thereof. Suitable culture media are available from commercial suppliers or may be prepared according to published compositions (e.g., in the American Type Culture Collection (ATCC) Catalog).

[0168] Suitable conditions for the production of recombinant components are those under which recombinant microbial host cells can grow and / or remain viable. Non-limiting examples of such conditions include suitable pH, suitable temperature, suitable feed rate, suitable pressure, suitable nutrient content (e.g., suitable carbon content, suitable nitrogen content, suitable phosphorus content), suitable nutritional supplement content, suitable trace metal content, and suitable oxidation levels.

[0169] In some embodiments, the culture medium further comprises a protease (e.g., a plant-derived protease) that can prevent degradation of the recombinant protein, a protease inhibitor that reduces the activity of a protease that can degrade the recombinant protein, and / or a sacrificial protein that can deflect protease activity. In some embodiments, the culture medium further comprises an unnatural amino acid for incorporation into the produced recombinant protein.

[0170] Other Genetic Modifications The recombinant microbial host cells provided herein can further comprise genetic modifications that improve the production of recombinant components. Non-limiting examples of such genetic modifications include altered promoters, altered kinase activity, altered phosphatase activity, altered protein folding activity, altered protein secretion activity, and altered gene expression induction pathways.

[0171] The recombinant microbial host cells provided herein can further have reduced or essentially eliminated protease activity to minimize degradation of recombinant proteins (see, for example, U.S. Patent No. 5,339,933). Filamentous fungal cells having reduced or essentially eliminated protease activity can be obtained by screening mutants or by specific genetic modification by methods known in the art.

[0172] The recombinant microbial host cells provided herein can further comprise native or heterologous glycosyltransferases. Non-limiting examples of such endogenous or heterologous glycosyltransferases include fucosyltransferases, galactosyltransferases, glucosyltransferases, xylosyltransferases, acetylases, glucoronyltransferases, glucoronylepimerases, sialyltransferases, mannosyltransferases, sulfotransferases, β-acetylgalactosaminyltransferases, and N-acetylglucosaminyltransferases.

[0173] The recombinant microbial host cells provided herein can further comprise native or heterologous kinases or phosphatases. Non-limiting examples of such native or heterologous kinases or phosphatases include protein kinase A, protein kinase B, protein kinase C, creatine kinase B, protein kinase C beta, protein kinase G, TmkA, Fam20 kinase (e.g., Fam20C), ATM, CaM-II, cdc2, cdk5, CK1, CKII, DNAPK, EGFR, GSK3, INSR, p38MAPK, RSK, SRC, phosphotransferase, alkaline phosphatase (e.g., UniProtKB-O77578), acid phosphatase, and others (see, e.g., Non-Patent Document 7).

[0174] Preparations Comprising Recombinant Components and Substantially Removed or Modulated Esterase Activity In another aspect, provided herein are preparations that contain recombinant components (e.g., any of the recombinant components disclosed herein) and esterase activity that is substantially eliminated or modulated compared to the esterase activity contained in a corresponding preparation (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein).

[0175] In some embodiments, the preparations provided herein comprise recombinant components and substantially removed or modulated esterase activity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% reduced compared to the esterase activity contained in a corresponding preparation. In some such embodiments, the preparations provided herein comprise recombinant components and substantially removed esterase activity. In other embodiments, the preparations provided herein contain recombinant components and an esterase activity that is reduced by at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, 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 1,000% compared to the esterase activity contained in a corresponding preparation.

[0176] The preparations provided herein may be obtained by purifying recombinant components produced by recombinant microbial host cells provided herein from a fermentation broth comprising the recombinant microbial host cells.

[0177] Methods for purifying recombinant components from fermentation broths are well known in the art (see, e.g., Protein Purification, JC Janson and L Ryden, Eds., VCH Publishers, New York, 1989; Protein Purification Methods: A Practical Approach, ELV Harris and S Angel, Eds., IRL Press, Oxford, England, 1989).

[0178] Recombinant components may be purified based on their molecular weight, for example, by size exclusion / exchange chromatography, ultrafiltration through a membrane, gel permeation chromatography (eg, preparative disc gel electrophoresis), or density centrifugation.

[0179] Recombinant components may also be purified on the basis of their surface charge or hydrophobicity / hydrophilicity, for example, by isoelectric precipitation, anion / cation exchange chromatography, isoelectric focusing (IEF), or reverse-phase chromatography.

[0180] Recombinant components may also be purified based on their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, detergent, or solvent extraction.

[0181] A recombinant component may also be purified based on its affinity for another molecule, for example, by affinity chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography may include the use of an antibody with specific binding affinity for the recombinant component, or nickel-NTA for His-tagged recombinant proteins, or lectins to bind to sugar moieties of recombinant proteins, or any other molecule that specifically binds to the recombinant component. In some embodiments, the recombinant component has a tag that facilitates purification. Non-limiting examples of such tags include epitope tags and protein tags. Non-limiting examples of epitope tags include c-myc, hemagglutinin (HA), polyhistidine (6x-HIS), GLU-GLU, and DYKDDDDK (FLAG) epitope tags. Non-limiting examples of protein tags include glutathione-S-transferase (GST), green fluorescent protein (GFP), and maltose binding protein (MBP). The epitope or protein tag may be removed (e.g., via protease cleavage) following isolation of the recombinant component.

[0182] In embodiments where the recombinant components are secreted by the recombinant microbial host cells, the recombinant components may be purified directly from the fermentation broth, hi other embodiments, the recombinant components may be purified from a cell lysate.

[0183] The identity of the recombinant components may be confirmed and / or quantified by high performance liquid chromatography (HPLC), Western blot analysis, Eastern blot analysis, polyacrylamide gel electrophoresis, capillary electrophoresis, enzyme product formation, disappearance of enzyme substrate, and two-dimensional mass spectrometry (2D-MS / MS) sequence identification.

[0184] In some embodiments, the recombinant components are purified to greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% purity relative to other components contained in the fermentation broth. In some embodiments, the recombinant components are purified to be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more abundant relative to other components in the preparation than in the fermentation broth. In some embodiments, the recombinant components are purified to a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% by weight.

[0185] In some embodiments, the preparation is a liquid. In other embodiments, the preparation is a solid. In some such embodiments, the preparation is a powder. In some such embodiments, the powder comprises a moisture content of less than 10% by weight, less than 7% by weight, less than 5% by weight, less than 3% by weight, or less than 1% by weight. The powder may be obtained, for example, by concentration via spray drying or evaporation.

[0186] In some embodiments, the preparation comprises a post-processed recombinant component. Post-processing of the recombinant component may include fragmentation (e.g., by chemical means or by exposure to a protease enzyme (e.g., trypsin, pepsin, carmapepsin)), removal of reactive sites (e.g., removal of reactive sites on methionine and / or tryptophan residues by oxidation), modulating (e.g., via chemical, photochemical, and / or enzymatic strategies), cyclizing, biotinylating (i.e., attaching biotin), and conjugating to other elements (e.g., polyethylene glycol, antibodies, liposomes, phospholipids, DNA, RNA, nucleic acids, sugars, disaccharides, polysaccharides, starch, cellulose, detergents, cell walls).

[0187] Post-treatment may be performed in a random or site-specific manner (e.g., sulfhydryl groups of cysteine ​​residues (e.g., for aminoethylation, formation of iodoacetamide, formation of maleimide, formation of Dha, covalent attachment via disulfide bonds, and desulfurization); primary amine groups of lysine residues (e.g., for attachment of activated esters, sulfonyl chlorides, isothiocyanates, unsaturated aldehyde esters, and aldehydes); phenolic hydroxyl groups of tyrosine residues; removal of specific epitopes (e.g., glycan groups) capable of eliciting an immune response in humans; azo electrocyclization).

[0188] Post-treatment may alter certain chemical and / or physical properties of the recombinant components, including, but not limited to, size, charge, hydrophobicity, hydrophilicity, solvation, protein folding, and chemical reactivity.

[0189] Food products containing recombinant ingredients In another aspect, provided herein are food products that contain recombinant components (e.g., any of the recombinant components disclosed herein) produced by a recombinant microbial host cell provided herein, and that contain esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) that is substantially removed or modulated compared to the esterase activity in the corresponding food product. As used herein, the term "food product" refers to a product that can be consumed by humans or animals, including domestic animals (e.g., dogs, cats), livestock (e.g., cows, pigs, horses), and wild animals (e.g., non-domestic predatory animals). The term includes products that can be combined with or added to one or more other ingredients to create a food product that can be consumed by humans or animals. In various embodiments, the food products provided herein meet the standards of food safety required by the U.S. Food and Drug Administration (FDA), the U.S. Department of Agriculture, the European Food Safety Authority, and / or other state or local food regulatory authorities.

[0190] In some embodiments, the food products provided herein comprise at least 0.000075% by weight, at least 0.0001% by weight, 0.00025% by weight, at least 0.0005% by weight, at least 0.00075% by weight, at least 0.001% by weight, at least 0.0025% by weight, at least 0.005% by weight, at least 0.0075% by weight, at least 0.01% by weight, at least 0.025% by weight, at least 0.05% by weight, at least 0.0 75% by weight, at least 0.1% by weight, at least 0.25% by weight, at least 0.5% by weight, at least 0.75% by weight, at least 1% by weight, at least 2.5% by weight, at least 5% by weight, at least 7.5% by weight, at least 10% by weight, at least 12.5% ​​by weight, at least 15% by weight, at least 17.5% by weight, at least 20% by weight, at least 22.5% by weight, at least 25% by weight, at least 27.5% by weight, at least 30% by weight, At least 32.5%, at least 35%, at least 37.5%, at least 40%, at least 42.5%, at least 45%, at least 47.5%, at least 50%, at least 52.5%, at least 55%, at least 57.5%, at least 60%, at least 62.5%, at least 65%, at least 67.5%, at least 70%, at least 72.5%, at least 75%, at least 77.5%, at least 80%, at least 82.5%, at least 85%, at least 87.5%, at least 90%, at least 92.5%, at least 95%, at least 97.5%, or 100% by weight of one or more recombinant components produced by one or more recombinant microbial host cells provided herein (e.g., any of the recombinant components disclosed herein).

[0191] In some embodiments, the food products provided herein are substantially free of esterase activity (e.g., the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein).

[0192] In some embodiments, the food product is primarily or completely composed of ingredients derived from non-animal sources. In alternative embodiments, the food product is composed of ingredients derived in part from animal sources, but with the addition of ingredients derived from non-animal sources. In some such embodiments, the food product is composed of between 5% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% by weight; between 10% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% by weight; between 20% and 100%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% by weight; between 30% and 100%, 90%, 80%, between 70%, 60%, 50%, or 40% by weight; between 40% and 100%, 90%, 80%, 70%, 60%, or 50% by weight; between 50% and 100%, 90%, 80%, 70%, or 60% by weight; between 60% and 100%, 90%, 80%, or 70% by weight; between 70% and 100%, 90%, or 80% by weight; between 80% and 100% or 90% by weight; or between 90% and 100% by weight.

[0193] In various embodiments, the food product is a food product selected from any of the food product categories defined by the National Health and Nutrition Examination Survey or is similar to such a food product (i.e., a food product substitute).

[0194] Non-limiting examples of such food product categories (and non-limiting examples of such food products) include snack foods and gums (e.g., snack bars, crackers, salty snacks from cereal products, chewing gum); breads, cereals, and pastas (e.g., oat breads and rolls, corn breads, corn muffins, tortillas, flour and dry mixes, biscuits, multigrain breads and rolls, whole wheat breads and rolls, pastas, rye breads and rolls, coarse wheat breads and rolls, white breads and rolls); beverages (e.g., beers and ales, concentrated beverages, beverages, energy drinks, sports drinks, fluids, soft drinks, carbonated drinks, juices, wines, cocktails, energy drinks, energy powders; confectionery and desserts (e.g., cakes, candies, chips, cookies, cobblers, pastries, ice cream or popsicles, muffins, pies, sugar substitutes or substitutes, syrups, honey, jellies, jams, preserves, salads, crepes, danishes, breakfast pastries, donuts); breakfast foods (e.g., cereals, rice, French toast, pancakes, waffles, coffee cake); eggs (e.g., egg dishes, egg soup, mixtures made with egg whites, egg substitutes, mixtures made with egg substitutes); dairy products (dairy product); salad dressings, oils, sauces, condiments (e.g., cooking fats, vegetable oils, salad dressings, tomato sauce, gravies); potatoes (e.g., potato salad, potato soup, chips and sticks, french fries, mashed potatoes, stuffed potatoes, puffs); and soups (e.g., vegetable soups, vegetable broths), meals, main dishes, proteins (e.g., meat substitutes), and seafood.

[0195] In some such embodiments, the food products provided herein are dairy products or similar to dairy products (i.e., dairy product substitutes). The term "dairy product", as used herein, includes milk (e.g., whole milk (at least 3.25% milk fat), partially skimmed milk (1% to 2% milk fat), skim milk (less than 0.2% milk fat), cooking milk, condensed milk, flavored milk, goat milk, sheep milk, milk powder, evaporated milk, milk foam), and, but not limited to, yogurt (e.g., whole milk yogurt (at least 6 grams of fat per 170 g), low-fat yogurt (between 2 and 5 grams of fat per 170 g), nonfat yogurt (0.5 grams of fat or less per 170 g), Greek yogurt (strained yogurt from which the whey has been removed), and other types of yogurt, including, but not limited to, yogurt, such as milk from which the whey has been removed, ... yogurt), whipped yogurt, goat's milk yogurt, labneh (labne), sheep's milk yogurt, yogurt drinks (e.g. whole milk kefir, low fat milk kefir), lassi), cheeses (e.g. whey cheeses, e.g. ricotta; pasta filata cheeses, e.g. mozzarella; semi-soft cheeses, e.g. Havarti and Muenster; semi-hard cheeses, e.g. Swiss and Jarlsberg; hard cheeses, e.g. Cheddar and Parmesan; washed curd cheeses, e.g. Colby and Monterey Jack; soft ripened cheeses, e.g. Brie and Camembert; fresh cheeses, e.g. cottage cheese, feta cheese, cream cheese, and curd; processed cheese; processed cheese foods; processed cheese products; processed cheese spreads; cheeses modulated with enzymes;cold-pack cheeses), dairy-based sauces (e.g., fresh, frozen, refrigerated, or shelf-stable), dairy spreads (e.g., low-fat spreads, low-fat butter), cream (e.g., dry cream, heavy cream, light cream, whipping cream, half-and-half, coffee whites, coffee cream, sour cream, crème fraîche, fraiche), frozen confectioneries (e.g. ice cream, smoothies, milkshakes, frozen yogurt, sundaes, gelato, custard), dairy desserts (e.g. fresh, refrigerated or frozen), butter (e.g. whipped butter, cultured butter), dairy powders (e.g. whole milk powder, skimmed milk powder, fat-filled milk powder (i.e. milk powder which contains vegetable fat instead of all or part of the animal fat), infant formula, milk protein concentrates (i.e. at least 80% protein content by weight), milk protein isolates (i.e. at least 90% protein content by weight), whey protein concentrates, whey protein isolates, demineralized refers to milk-derived products including whey protein concentrates, demineralized whey protein concentrates, β-lactoglobulin concentrates, β-lactoglobulin isolates, α-lactalbumin concentrates, α-lactalbumin isolates, glycomacropeptide concentrates, glycomacropeptide isolates, casein concentrates, casein isolates, nutritional supplements, texturizing blends, flavor blends, color blends), prepared beverages or prepared mix products (e.g., fresh, refrigerated, or shelf-stable dairy protein beverages, weight loss beverages, nutritional beverages, sports recovery beverages, and energy drinks), puddings, gels, chewables, crisps, and bars;

[0196] The term "food product substitute" (e.g., "dairy product substitute"), as used herein, refers to a food product that is similar to (e.g., can be used in place of) a conventional food product. Such similarity may be due to any physical, chemical, or functional attribute. In some embodiments, the similarity between the food products provided herein and conventional food products is due to physical attributes. Non-limiting examples of physical attributes include color, shape, mechanical properties (e.g., hardness, G' storage modulus value, shape retention, cohesiveness, texture (i.e., mechanical properties that correlate with perception (e.g., mouthfeel, fattiness, creaminess, homogeneity, richness, smoothness, thickness)), viscosity, and crystallinity. In some embodiments, the similarity between the food products provided herein and conventional food products is due to chemical / biological attributes. Non-limiting examples of chemical attributes include nutrient content (e.g., type and / or amount of amino acids (e.g., PDCAAS score), type and / or amount of lipids, type and / or amount of carbohydrates, type and / or amount of minerals, type and / or amount of vitamins), pH, digestibility, shelf life, hunger and / or satiety regulation, taste, and aroma. In some embodiments, the similarity between the food products provided herein and conventional food products is due to functional attributes.Non-limiting examples of functional attributes include gelation / aggregation behavior (e.g., gelation ability (i.e., the time required to form a maximum strength gel (i.e., a protein network having solvent-filled spaces bound to the protein molecules by hydrogen bonds) in response to physical and / or chemical conditions (e.g., agitation, temperature, pH, ionic strength, protein concentration, sugar concentration, ionic strength)), aggregation ability (i.e., the ability to form a precipitate (i.e., a strong protein network based on strong interactions between protein molecules and the exclusion of solvent) in response to physical and / or chemical conditions), gel strength (i.e., force / unit area (e.g., Pascal (Pa)) ), water retention capacity upon gelation, syneresis upon gelation (i.e., water seepage over time), foaming behavior (e.g., foaming capacity (i.e., amount of air retained in response to physical and / or chemical conditions), foam stability (i.e., half-life of foam formed in response to physical and / or chemical conditions), foam spread), thickening ability, versatility of use (i.e., ability to use food products in different ways and / or to obtain other diverse compositions from food products; e.g., ability to produce food products that are similar to dairy derivatives such as yogurt, cheese, cream, and butter), and ability to form protein dimers.

[0197] In various embodiments, the food products provided herein are milk substitutes (i.e., similar to milk), yogurt substitutes (i.e., similar to yogurt), cheese substitutes (i.e., similar to cheese), frozen dessert substitutes (i.e., similar to frozen desserts (e.g., ice cream)), or any other dairy product substitute (i.e., similar to a dairy product (e.g., any dairy product disclosed herein)).

[0198] Milk Protein Ingredients In some embodiments, the food product (e.g., dairy product, egg product) comprises a milk protein component, the milk protein component comprises recombinant milk protein, and the activity of an esterase has been substantially removed or modulated compared to the esterase activity of a milk protein component not produced according to the methods provided herein (i.e., not produced by a method including at least one step in which the activity of the esterase has been substantially removed or modulated). The term "dairy protein component" as used herein refers to a component consisting of a subset of whey proteins or a subset of caseins or a mixture of a subset of whey proteins and a subset of caseins (i.e., from a whey protein concentrate or micellar casein concentrate, or sodium caseinate, or acid casein, or some but not all of the proteins present in a milk protein concentrate or milk protein isolate). The term further implies that the milk proteins in the milk protein component are the only milk proteins contained in the food product (i.e., the food product does not contain any other milk proteins other than the milk proteins that make up the milk protein component).

[0199] In some embodiments, the food products provided herein comprise from 0.1% to 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6% by weight of the food products provided herein. , 0.5%, 0.4%, 0.3%, or 0.2% by weight; between 0.2% by weight and 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, Between 0.6%, 0.5%, 0.4% or 0.3% by weight; 0.3% to 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0. between 6%, 0.5% or 0.4% by weight; between 0.4% by weight and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6% or 0.Between 5% by weight; 0.5% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, double Amount %, between 1%, 0.9%, 0.8%, 0.7% or 0.6% by weight; between 0.6% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9% by weight , 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8% or 0.7% by weight; between 0.7% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13% %, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9% or 0.8% by weight; between 0.8% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% by weight. between 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.9% by weight; between 0.9% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% %, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% by weight; between 1% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12% %, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% by weight; between 2% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, %, between 7%, 6%, 5%, 4% or 3% by weight; between 3% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or 4% by weight;Between 4% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or 5% by weight; 5% to 100% by weight , 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight or between 6% by weight and 100% by weight, 95% by weight, 90% by weight, 85% by weight , 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight or between 7% by weight and 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, between 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9% or 8% by weight; between 8% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45% by weight; between 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10% or 9% by weight; between 9% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11% or 10% by weight %; between 10% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12% or 11% by weight; between 11% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, between 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13% or 12% by weight; between 12% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14% or 13% by weight; between 13% and 100%, 95%, 90% by weight , 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 14% by weight; between 14% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% by weight;between 15% by weight and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% by weight; between 20% by weight and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% or 25% by weight; between 25% by weight and 100%, 95%, 90%, %, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight or between 30% by weight and 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight or between 35% by weight and 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, between 50%, 45% or 40% by weight; between 40% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or 45% by weight; between 45% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% by weight; between 50% and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60% by weight, between 55% by weight and 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65% or 60% by weight; between 60% by weight and 100%, 95%, 90%, 85%, 80%, 75%, 70% or 65% by weight; between 65% by weight and 100%, 95%, 90%, 85%, 80%, 75% or 70% by weight; between 70% by weight and 100%, 95%, 90%, 85%, 80% or 75% by weight;between 75% and 100%, 95%, 90%, 85% or 80% by weight; between 80% and 100%, 95%, 90% or 85% by weight; or between 85% and 100%, 95%, 90% by weight; between 90% and 100% or 95% by weight; or between 95% and 100% by weight;

[0200] The dairy protein component included in the food products provided herein can be comprised of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) dairy proteins (e.g., one or more whey proteins, one or more caseins, or a mixture of one or more whey proteins and one or more caseins), where at least one of the dairy proteins is a recombinant dairy protein produced by a recombinant microbial host cell (e.g., any of the recombinant dairy proteins disclosed herein).

[0201] In some embodiments, the dairy protein component included in the food products provided herein is one or more (e.g., two, three, four or more) whey proteins (e.g., β-lactoglobulin, α-lactalbumin, a mixture of β-lactoglobulin and α-lactalbumin, a mixture of two or more β-lactoglobulins with different post-translational modifications (PTMs), a mixture of two or more α-lactalbumins with different PTMs, a mixture of two or more β-lactoglobulin ...α-lactalbumins with different PTMs, a mixture of two or more β-lactoglobulins with different PTMs, a mixture of two or more α-lactalbumins with different PTMs, a mixture of two or more β-lactoglobulins with different PTMs, a mixture of two or more α-lactalbumins with different PTMs, a mixture of two or more β-lactoglobulins with different PTMs, a mixture of two or more α a mixture of α-lactalbumin and β-lactoglobulin with different PTMs, a mixture of two or more α-lactalbumin and β-lactoglobulin with different PTMs, a mixture of two or more β-lactoglobulin with different PTMs, and a mixture of two or more α-lactalbumin with different PTMs; one or more (e.g., 2, 3, 4 or more) caseins (e.g., κ-casein, β-casein, γ-casein, a mixture of κ-casein and β-casein, κ-casein and γ-casein a mixture of β-casein and γ-casein, a mixture of two or more κ-caseins with different PTMs, a mixture of two or more β-caseins with different PTMs, a mixture of two or more γ-caseins with different PTMs, a mixture of two or more κ-caseins and β-caseins with different PTMs, a mixture of two or more κ-caseins and γ-caseins with different PTMs, a mixture of two or more β-caseins and κ-caseins with different PTMs a mixture of two or more β-caseins and γ-caseins with different PTMs, a mixture of two or more γ-caseins and κ-caseins with different PTMs, a mixture of two or more γ-caseins and β-caseins with different PTMs, a mixture of two or more κ-caseins with different PTMs and / or two or more β-caseins with different PTMs and / or two or more γ-caseins with different PTMs); or any combination thereof.

[0202] The at least one recombinant milk protein may be a single recombinant milk protein. The single recombinant milk protein may be a single recombinant whey protein (e.g., recombinant β-lactoglobulin or recombinant α-lactalbumin) or a single recombinant casein (e.g., recombinant κ-casein or recombinant β-casein or recombinant γ-casein). Alternatively, the at least one recombinant milk protein may be two or more recombinant milk proteins. The two or more recombinant milk proteins may be two or more recombinant whey proteins (e.g. recombinant β-lactoglobulin and recombinant α-lactalbumin), two or more recombinant caseins (e.g. recombinant κ-casein and recombinant β-casein, recombinant κ-casein and recombinant γ-casein, recombinant κ-casein and recombinant β-casein and recombinant γ-casein), or a mixture of at least one recombinant whey protein and at least one recombinant casein (e.g. a combination of one or both of recombinant β-lactoglobulin and recombinant α-lactalbumin with one or two or all of recombinant κ-casein and recombinant β-casein and recombinant γ-casein).

[0203] In some embodiments, the at least one recombinant milk protein comprises at least one recombinant milk protein having a PTM. In various such embodiments, the PTM may be a native PTM, a non-native PTM, or a mixture thereof. In some embodiments, the type and / or number of PTMs of the recombinant milk protein imparts desirable attributes to the food products provided herein.

[0204] In some embodiments, the dairy protein component included in the food products provided herein comprises one or more (e.g., two, three, four or more) recombinant whey proteins (e.g., recombinant β-lactoglobulin, recombinant α-lactalbumin, a mixture of recombinant β-lactoglobulin and recombinant α-lactalbumin, a mixture of two or more recombinant β-lactoglobulins with different PTMs, a mixture of two or more recombinant α-lactalbumins with different PTMs, a mixture of two or more recombinant β-lactoglobulins and recombinant α-lactalbumins with different PTMs, a mixture of two or more recombinant α-lactoglobulins and β-lactoglobulins with different PTMs, a mixture of two or more recombinant β-lactoglobulins with different PTMs, and a mixture of two or more recombinant α-lactalbumins with different PTMs);One or more (e.g., two, three, four, or more) recombinant caseins (e.g., recombinant κ-casein, recombinant β-casein, recombinant γ-casein, a mixture of recombinant κ-casein and recombinant β-casein, a mixture of recombinant κ-casein and recombinant γ-casein, a mixture of recombinant β-casein and recombinant γ-casein, a mixture of two or more recombinant κ-caseins with different PTMs, a mixture of two or more recombinant β-caseins with different PTMs, a mixture of two or more recombinant γ-caseins with different PTMs, a mixture of two or more recombinant κ-caseins and recombinant β-caseins with different PTMs, a mixture of two or more recombinant κ-caseins and recombinant β-caseins with different PTMs, -a mixture of casein and γ-casein, a mixture of two or more recombinant β-caseins and recombinant κ-caseins with different PTMs, a mixture of two or more recombinant β-caseins and γ-caseins with different PTMs, a mixture of two or more recombinant γ-caseins and recombinant κ-caseins with different PTMs, a mixture of two or more recombinant γ-caseins and β-caseins with different PTMs, a mixture of two or more recombinant γ-caseins and β-caseins with different PTMs, a mixture of two or more recombinant κ-caseins with different PTMs and / or two or more recombinant β-caseins with different PTMs and / or two or more recombinant γ-caseins with different PTMs); or any combination thereof;

[0205] In some embodiments, the dairy protein component included in the food products provided herein comprises one or more (e.g., two, three, four or more) recombinant whey proteins (e.g., recombinant β-lactoglobulin, recombinant α-lactalbumin, a mixture of recombinant β-lactoglobulin and recombinant α-lactalbumin, a mixture of two or more recombinant β-lactoglobulins with different PTMs, a mixture of two or more recombinant α-lactalbumins with different PTMs, a mixture of two or more recombinant β-lactoglobulins and recombinant α-lactalbumins with different PTMs, a mixture of two or more recombinant α-lactoglobulins and β-lactoglobulins with different PTMs, a mixture of two or more recombinant β-lactoglobulins with different PTMs, and a mixture of two or more recombinant α-lactalbumins with different PTMs);One or more (e.g., two, three, four, or more) recombinant caseins (e.g., recombinant κ-casein, recombinant β-casein, recombinant γ-casein, a mixture of recombinant κ-casein and recombinant β-casein, a mixture of recombinant κ-casein and recombinant γ-casein, a mixture of recombinant β-casein and recombinant γ-casein, a mixture of two or more recombinant κ-caseins with different PTMs, a mixture of two or more recombinant β-caseins with different PTMs, a mixture of two or more recombinant γ-caseins with different PTMs, a mixture of two or more recombinant κ-caseins and recombinant β-caseins with different PTMs, a mixture of two or more recombinant κ-caseins and recombinant β-caseins with different PTMs, - a mixture of casein and γ-casein, a mixture of two or more recombinant β-caseins and recombinant κ-caseins with different PTMs, a mixture of two or more recombinant β-caseins and γ-caseins with different PTMs, a mixture of two or more recombinant γ-caseins and recombinant κ-caseins with different PTMs, a mixture of two or more recombinant γ-caseins and β-caseins with different PTMs, a mixture of two or more recombinant γ-caseins and β-caseins with different PTMs, a mixture of two or more recombinant κ-caseins with different PTMs and / or two or more recombinant β-caseins with different PTMs and / or two or more recombinant γ-caseins with different PTMs); or any combination thereof;

[0206] The milk protein component may further comprise one or more naturally occurring milk proteins. In some embodiments, the milk protein component included in the food products provided herein comprises one or more (e.g., two, three, four or more) naturally occurring whey proteins (e.g., naturally occurring β-lactoglobulin, naturally occurring α-lactalbumin, a mixture of naturally occurring β-lactoglobulin and naturally occurring α-lactalbumin, a mixture of two or more naturally occurring β-lactoglobulins with different PTMs, a mixture of two or more naturally occurring α-lactalbumins with different PTMs, a mixture of two or more naturally occurring β-lactoglobulins and naturally occurring α-lactalbumins with different PTMs, a mixture of two or more naturally occurring α-lactoglobulins and naturally occurring β-lactoglobulins with different PTMs, a mixture of two or more naturally occurring α-lactoglobulins and naturally occurring β-lactoglobulins with different PTMs, a mixture of two or more naturally occurring β-lactoglobulins with different PTMs, and a mixture of two or more naturally occurring α-lactalbumins with different PTMs);One or more (e.g., two, three, four, or more) naturally occurring caseins (e.g., naturally occurring κ-casein, naturally occurring β-casein, naturally occurring γ-casein, a mixture of naturally occurring κ-casein and naturally occurring β-casein, a mixture of naturally occurring κ-casein and naturally occurring γ-casein, a mixture of naturally occurring β-casein and naturally occurring γ-casein, a mixture of two or more naturally occurring κ-caseins with different PTMs, a mixture of two or more naturally occurring β-caseins with different PTMs, a mixture of two or more naturally occurring γ-caseins with different PTMs, a mixture of two or more naturally occurring κ-caseins and naturally occurring β-caseins with different PTMs, a mixture of two or more naturally occurring κ-caseins and naturally occurring β-caseins with different PTMs, a mixture of two or more naturally occurring κ-caseins and naturally occurring β-caseins with different PTMs, a mixture of two or more naturally occurring κ-caseins and and native γ-casein, a mixture of two or more native β-caseins and native κ-caseins with different PTMs, a mixture of two or more native β-caseins and native γ-caseins with different PTMs, a mixture of two or more native γ-caseins and native κ-caseins with different PTMs, a mixture of two or more native γ-caseins and native κ-caseins with different PTMs, a mixture of two or more native γ-caseins and native β-caseins with different PTMs, a mixture of two or more native κ-caseins and native β-caseins with different PTMs, a mixture of two or more native β-caseins with different PTMs and / or two or more native γ-caseins with different PTMs; or any combination thereof;

[0207] In some such embodiments, the milk protein component included in the food products provided herein consists of whey protein and casein in a weight ratio of between about 10 to about 1 to about 1 to about 1 to about 1 to about 10 (e.g., about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 to 1, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, or about 1 to 10).

[0208] In some such embodiments, the milk protein component included in the food products provided herein is comprised of recombinant whey protein and native whey protein in a weight ratio of between about 10 to about 1 to about 1 to about 1 to about 1 to about 10 (e.g., about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 to 1, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, or about 1 to 10).

[0209] In some such embodiments, the milk protein component included in the food products provided herein consists of recombinant casein and natural casein in a weight ratio of between about 10 to about 1 to about 1 to about 1 to about 1 to about 10 (e.g., about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 to 1, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, or about 1 to 10).

[0210] In some such embodiments, the milk protein component included in the food products provided herein consists of recombinant whey protein and recombinant casein in a weight ratio of between about 10 to about 1 to about 1 to about 1 to about 1 to about 10 (e.g., about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 to 1, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, or about 1 to 10).

[0211] In some such embodiments, the milk protein component included in the food products provided herein is comprised of native whey protein and native casein in a weight ratio of between about 10 to about 1 to about 1 to about 1 to about 1 to about 10 (e.g., about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 to 1, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, or about 1 to 10).

[0212] In some such embodiments, the dairy protein component included in the food products provided herein is comprised of recombinant dairy protein and native dairy protein in a weight ratio of between about 10 to about 1 to about 1 to about 1 to about 1 to about 10 (e.g., about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 to 1, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, or about 1 to 10).

[0213] In all embodiments, the milk protein component consists of only a subset of whey proteins, or a subset of caseins, or a mixture of a subset of whey proteins and a subset of caseins. In some embodiments, the subset of whey proteins consists of β-lactoglobulin and / or α-lactalbumin. In some embodiments, the subset of caseins consists of κ-casein and / or β-casein and / or γ-casein. In various embodiments, the mixture of the subset of whey proteins and the subset of caseins consists of a combination of β-lactoglobulin and / or α-lactalbumin with κ-casein and / or β-casein and / or γ-casein. In some such embodiments, the mixture consists of β-lactoglobulin and κ-casein. In other such embodiments, the mixture consists of α-lactalbumin and κ-casein. In still other such embodiments, the mixture consists of β-lactoglobulin and α-lactalbumin and κ-casein.

[0214] Non-dairy Protein Ingredients In some embodiments, a food product (e.g., dairy product, egg product) comprises a non-dairy protein component, wherein the non-dairy protein component comprises a recombinant non-dairy protein and an esterase activity that is substantially removed or modulated compared to the esterase activity of a non-dairy protein component not produced according to the methods provided herein (i.e., not produced by a method that includes at least one step in which the activity of the esterase is substantially removed or modulated).

[0215] The non-dairy protein ingredients included in the food products provided herein may include non-dairy proteins from any source, as well as mixtures of non-dairy proteins from a variety of sources, non-limiting examples of which include animals, plants, algae, fungi, and microorganisms.

[0216] Non-limiting examples of animals include insects (eg, flies), mammals (eg, cows, sheep, goats, rabbits, pigs, humans), and birds (eg, chickens).

[0217] Non-limiting examples of plants include cycads, ginkgo, conifers, cypress, juniper, arborvitae, cedarwood, pine, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, cornflower, marigold, mugwort, safflower, chamomile, lettuce, wormwood, calendula, citronella, sage, thyme, chaseed, mustard, olive, coffee, chili pepper, eggplant, paprika, cranberry, kiwi, vegetable plants (e.g., carrot, celery), Tagetes, tansy, tarragon, sunflower, burdock, basil, and others. le, hyssop, lavender, lemon verbena, marjoram, mentha, patchouli, mentha piperita, peppermint, rosemary, sesame, spearmint, primrose, samara, chili pepper, pimento, potato, sweet potato, tomato, blueberry, night cherries, petunia, morning glory, lilac, jasmine, honeysuckle, snapdragon, psyllium, sage, buckwheat, amaranth, chard, quinoa, spinach, rhubarb, jojoba, cypserea, chlorella, marula, hazelnut, canola, kale, bok choy Rhino, rutabaga, frankincense, myrrh, elemi, thyme, pumpkin, squash, urticae, manioc, Dalbergia, legumes (e.g. alfalfa, lentil, bean, clover, pea, mucuna pruriens, frijoles borroya, black beans, bush clover, licorice, lupin, mesquite, carob, soybean, peanut, tamarind, wisteria, cassia, chickpea / garbanzo, fenugreek, green pea, yellow pea, snow pea, lima bean, broad bean), geranium, flax, pomegranate tree, cotton, okra, neem, i Fig, Mulberry, Clove, Eucalyptus, Tea Tree, Niaouli, Fruit plants (e.g. apple, apricot, peach, plum, pear, berry), Strawberry, Blackberry, Raspberry, Cherry, Prune, Rose, Tangerine, Citrus fruits (e.g. grapefruit, lemon, lime, orange, bitter orange, mandarin), Mango, Citrus bergamot, Bouquet, Grapes, Broccoli, Brussels sprouts, Camelina, Cauliflower, Rape, Rapeseed (Canola), Turnip, Cabbage, Cucumber, Watermelon, Honeydew melon, Zucchini,Birch, walnut, cassava, baobab, allspice, almond, breadfruit, sandalwood, macadamia, taro, moonflower, aloe vera, garlic, onion, shallot, vanilla, yucca, vetiver, alpinia speciosa, barley, cereals, yellow turmeric, ginger, lemongrass, oats, palm, pineapple, rice, rye, sorghum, triticale, turmeric, yam, bamboo, barley, cajeput, canna, cardamom, corn, oats, wheat, cinnamon, sassafras sedge, lindera benzoin, bay laurel, avocado, ylang ylang, mace, nutmeg, moringa, jute, oregano, coriander leaf, chervil, chives, aggregate fruit, cereal plants, herbal plants, leafy vegetables, non-cereal legume plants, nut plants, succulents, terrestrial plants, aquatic plants, dalbergia, millet, drupes, schisandra, flowering plants, non-flowering plants, cultured plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, brushes, lianas, cacti, tropical plants, subtropical plants, temperate plants, and their derivatives and hybrids.

[0218] Non-limiting examples of algae include green algae (e.g., Chlorella vulgaris, Chlorella pyrenoidosa), brown algae (e.g., Alaria marginata, Analipus japonicus, Ascophyllum nodosum, Ecklonia sp., Eisenia bicyclis, Hizikia fusiforme, Kjellmaniella gyrata, Laminaria angustata, Laminaria longirruris, Laminaria Longissima, Laminaria ochotensis, Laminaria niger ... ochotensis, Laminaria claustonia, Laminaria saccharina, Laminaria digitata, Laminaria japonica, Macrocystis pyrifera, Petalonia fascia, Scytosiphon lome), red algae (e.g. Chondrus crispus, Chondrus ocellatus, Eucheuma cottonii, Eucheuma spinosum, Furcellaria fastigiata, Gracilaria bursa-pastoris, bursa-pastoris, Gracilaria lichenoides, Gloiopeltis furcata, Gigartina accicularisacicularis, Gigartina bursa-pastoris, Gigartina pistillata, Gigartina radula, Gigartina skottsbergii, Gigartina stellata, Palmaria palmata, Porphyra columbina, Porphyra crispata, Porphyra deutata, Porphyra perforata, Porphyra suborbiculata, Porphyra tenella tenera, Porphyridium cruentum, Porphyridium purpureum, Porphyridium aerugineum, Rhodella maculate, Rhodella reticulata, Rhodella violacea, Rhodymenia palmata), and derivatives and hybrids thereof.

[0219] Non-limiting examples of fungi include Aspergillus nidulans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida albicans, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Candida versatilis, Candida versatilis, Chrysosporium lucknowense, Debaryomyces hansenii, Endothia parasitica, Eremothecium ashbyii, Fusarium gramineum, Fusarium moniliforme, Fusarium venenatum, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces marxianus var. lactis var. lactis, Kluyveromyces thermotolerans, Morteirella vinaceae var. raffinoseutilizer, Mucor mieheimiehei, Mucor miehei var. Cooney et Emerson, Mucor pusillus Lindt, Myceliophthora thermophile, Neurospora crassa, Penicillium roquefortii, Physcomitrella patens, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens membranaefaciens, Pichia minuta, Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Rhizopus niveus, Saccharomyces bayanus, Saccharomyces beticus beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguoususexiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Sporidiobolus johnsonii, Sporidiobolus salmonicolor, Sporobolomyces roseus, Trichoderma reesei, Xanthophyllomyces dendrorhous, Yarrowia lipolytica lipolytica, Zygosaccharomyces rouxii, and their derivatives and hybrids.

[0220] Non-limiting examples of microorganisms include those from the phylum Firmicutes, Cyanobacteria (blue-green algae), Oscillatoriophcideae, Bacillales, Lactobacillales, Oscillatoriales, Bacillaceae, Lactobacillaceae, Acetobacter suboxydans, Acetobacter xylinum, Actinoplane missouriensis, Arthrospira platensis, Arthrospira maxima, Bacillus cereus, and the like. cereus, Bacillus coagulans, Bacillus subtilus, Bacillus cerus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactococcus lactis, Lactococcus lactis Lancefield Group N, Lactobacillus reuteri, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroidesmesenteroides, Micrococcus lysodeikticus, Spirulina, Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus, Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes olivochromogenes, Streptomyces rubiginosus, Tetrahymena thermophile, Tetrahymena hegewischi, Tetrahymena hyperangularis, Tetrahymena malaccensis, Tetrahymena pigmentosa, Tetrahymena pyriformis, Tetrahymena vorax, Xanthomonas campestris, and derivatives and hybrids thereof.

[0221] In some embodiments, the non-dairy protein is an animal protein. Non-limiting examples of such proteins include collagen.

[0222] In some embodiments, the non-dairy protein is a plant protein. Non-limiting examples of such non-dairy plant proteins include pea protein and potato protein.

[0223] In some embodiments, the non-dairy protein is a fungal protein. Non-limiting examples of such proteins include glucoamylase, xylase, amylase, glucanase, members of the SUN family (Sim1p, Uth1p, Nca3p, Sun4p), elongation factor 1-alpha, mitochondrial leucyl-tRNA synthetase, alpha-amylase, alpha-galactosidase, cellulase, endo-1,4-beta-xylase, endoglucanase, exo-1,4-beta-xylosidase, glucoamylase, peptidase, aspergillopepsin-1, 1,4-beta-D-glucan cellobiohydrolase A, alpha-galactosidase A, alpha-galactosidase B, alpha-galactosidase D, alpha-glucuronidase A, beta-galactosidase C, glucan 1,3-beta-glucosidase A, hydrophobin, and glucan endo-1,3-beta-glucosidase eglC.

[0224] In some embodiments, the non-dairy proteins are derived from non-dairy proteins secreted by a host cell (e.g., any of the natural or recombinant host cells of an animal, plant, algae, fungus, or microorganism disclosed herein). Suitable secreted non-dairy proteins may be identified, for example, by obtaining a secretome (i.e., secreted proteins obtained, for example, by culturing animal, plant, algae, fungus, or microbial cells in liquid culture, removing the cells from the cell culture (e.g., by centrifugation), and optionally concentrating the remaining culture medium; or by genomic sequencing and in silico identifying secreted proteins (see, for example, Mattanovich et al. 2009. Microbial Cell Factories 8:29), whole cell extracts, or fractionated whole cell extracts; optionally partially digesting, glycosylating, phosphorylating, or otherwise enzymatically treating the proteins; and then screening them in an assay (e.g., a high throughput assay) for proteins with similar, identical, or different properties, for example compared to the dairy proteins). Suitable secreted non-dairy proteins may also be identified by screening calcium-rich fractions for proteins with similar, identical, or different properties compared to soy proteins, e.g., dairy proteins. Non-limiting examples of suitable secreted non-dairy proteins are provided in PCT application PCT / US2017 / 48730, filed August 25, 2017. In some embodiments, the non-dairy protein is a secreted fungal protein (i.e., a protein secreted by a fungus, e.g., any of the fungi disclosed herein).

[0225] In some embodiments, the non-dairy protein component comprises a recombinant non-dairy protein. In some such embodiments, the recombinant non-dairy protein has a non-native PTM and / or does not have an epitope capable of eliciting an immune response in a human or animal.

[0226] Other Ingredients The food product provided herein may further comprise one or more other ingredients.For example, in some embodiments, the food product provided herein further comprises lipid.Non-limiting examples of lipid include lipid selected from the group consisting of fat, oil, monoglyceride, diglyceride, triglyceride, phospholipid, and free fatty acid.

[0227] Non-limiting examples of oils include vegetable oils (e.g., sunflower oil, coconut oil, mustard oil, peanut oil, canola oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, rapeseed oil, safflower oil, sesame oil, soybean oil, almond oil, beech nut oil, Brazil nut oil, cashew oil, hazelnut oil, macadamia nut oil, mongo nut oil, pecan oil, pine nut oil, pistachio nut oil, walnut oil, avocado oil, grape oil), microbial derived oils, algae derived oils, fungal derived oils, marine animal oils (e.g., Atlantic fish oil, Pacific fish oil, Mediterranean fish oil, light pressed fish oil, alkali treated fish oil, heat treated fish oil, light and heavy brown fish oil, and the like). oil), bonito oil, sardine oil, tuna oil, sea bass oil, halibut oil, marlin oil, barracuda oil, cod oil, menhaden oil, sardine oil, anchovy oil, capelin oil, Atlantic cod oil, Atlantic herring oil, Atlantic mackerel oil, Atlantic menhaden oil, salmon oil, and shark oil, squid oil, cuttlefish oil, octopus oil, krill oil, seal oil, and whale oil), non-essential oils, essential oils, natural oils, non-hydrogenated oils, partially hydrogenated oils, hydrogenated oils (e.g., hydrogenated coconut oil), crude oils, semi-refined (also called alkali refined) oils, interesterified oils, and refined oils. In some embodiments, long-chain oils (e.g., sunflower oil, corn oil, olive oil, soybean oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, canola oil, safflower oil, linseed oil, palm oil, palm kernel oil, palm fruit oil, coconut oil, babassu oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, genetically engineered sunflower oil that overexpresses 400% oleic acid) are combined with short-chain triglycerides to produce transesterified fatty acid esters. Various combinations of triglycerides and long-chain oils can be incorporated to produce a number of different flavor profiles.

[0228] Non-limiting examples of mono- and diglycerides include plant-derived mono- and diglycerides (e.g., mono- and diglycerides from sunflower, coconut, peanut, cottonseed, olive, palm, rapeseed, safflower, sesame seed, soybean, almond, beech nut, Brazil nut, cashew, hazelnut, macadamia nut, mongongo nut, pecan, pine nut, pistachio, walnut, and avocado). Mono- and diglycerides may contain acyl chains of any of the free fatty acids listed herein. Additional examples of mono- and diglycerides are known in the art.

[0229] Non-limiting examples of free fatty acids include butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, omega-fatty acids (e.g., arachidonic acid, omega-3-fatty acids, omega-6-fatty acids, omega-7-fatty acids, omega-9-fatty acids), fatty acids having an even number of carbons between 4 and 16 carbons in length, monosaturated acids (especially those having 18 carbons), fatty acids having low interfacial tension (e.g., less than 20 carbons), and fatty acids having an even number of carbons between 4 and 16 carbons in length (e.g., less than 20 carbons). Full, Less than 15, Less than 11, Less than 9, Less than 7, Less than 5, Less than 3, Less than 2, Less than 1, or Less than 0.5 dynes / cm, 0.1 to 20, 1 to 15, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 2 to 7, 0.1 to 5, 0.3 to 2, or 0.5 to 1 dynes / cm, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4 .0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0) fatty acids esterified at sn-3, butyric acid (4:0) or caproic acid (6:0), medium chain fatty acids (8:0-14:0) esterified at the sn-1 and sn-2 positions, as well as 16:0, fatty acids with stearic acid (18:0) at the sn-1 position, fatty acids with oleic acid (18:1) at the sn-1 and sn-3 positions, fatty acids with a variety of carbon atoms (e.g., from 8 to 40, 10 to 38, 12 to 36, 14 to 34, 16 to 32, 18 to 30, or 20 to 28 carbon atoms), at least one unsaturated bond (i.e., a carbon-carbon double or triple bond; e.g., at least 2 , at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 carbon-carbon double and / or triple bonds), fatty acids containing conjugated unsaturated bonds (i.e., at least one pair of carbon-carbon double and / or triple bonds are attached to each other without a methylene (CH2) group between them (e.g., 4CH:CHiCH:CHi)), and derivatives of the above fatty acids (e.g., esters (e.g., methyl and ethyl esters), salts (e.g., sodium and potassium salts), triglyceride derivatives, diglyceride derivatives, monoglyceride derivatives). Free fatty acids may be saturated or unsaturated. In some embodiments, the free fatty acids are not derived from or produced by a mammal. Additional examples of free fatty acids are known in the art.

[0230] Non-limiting examples of phospholipids include lecithin phospholipids (e.g., soybean lecithin phospholipid, sunflower lecithin phospholipid, cotton lecithin phospholipid, rapeseed lecithin phospholipid, rice bran lecithin phospholipid, corn lecithin phospholipid, wheat flour lecithin phospholipid), cardiolipin, ceramide phosphocholine, ceramide phosphoethanolamine, glycerophospholipid, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphosphingolipid, and phosphatidylserine. In some embodiments, the phospholipid is not derived from or produced by a mammal.

[0231] Non-limiting examples of triglycerides include tributyrin, a short chain triglyceride, a short chain triglyceride containing three oleic acids; a short chain triglyceride containing hexanoic acid; a short chain triglyceride containing hexanoic acid and butyric acid; a short chain triglyceride containing hexanoic acid and decanoic acid; and a short chain triglyceride containing one butyric acid, one hexanoic acid, and one octanoic acid.

[0232] In some embodiments, the food products provided herein have an ester content of less than 0.3%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, or less than 0.05% by weight; between 0.05% and 3%, 2.6%, 2.2%, 1.8%, 1.4%, 1.0%, 0.6%, 0.2%, or 0.1% by weight; between 0.1% and 3%, 2.6%, 2.2%, 1.8%, 1.4%, 1.0%, 0.6%, or 0.2% by weight; between 0.2% and 3%, 2.6%, 2.2%, between 1.8%, 1.4%, 1.0%, or 0.6% by weight; between 0.6% and 3%, 2.6%, 2.2%, 1.8%, 1.4%, or 1.0% by weight; between 1.0% and 3%, 2.6%, 2.2%, 1.8%, or 1.4% by weight; between 1.4% and 3%, 2.6%, 2.2%, or 1.8% by weight; between 1.8% and 3%, 2.6%, or 2.2% by weight; between 2.2% and 3%, or 2.6% by weight; or between 2.6% and 3% by weight.

[0233] In some embodiments, the food products provided herein have a free lipid content within a desired range as determined by an expert human sensory panel (e.g., having an aroma and / or taste that is determined to be not objectionable by an expert human sensory panel; having a flavor profile that is determined to be desirable by an expert human sensory panel).

[0234] In some embodiments, the food product provided herein further comprises carbohydrate.Non-limiting examples of carbohydrate include starch, flour, and edible fiber.Non-limiting examples of starch include maltodextrin, inulin, fructooligosaccharide, pectin, carboxymethylcellulose, guar gum, corn starch, oat starch, potato starch, rice starch, pea starch, and wheat starch.Non-limiting examples of flour include amaranth flour, oat flour, quinoa flour, rice flour, rye flour, sorghum flour, soy flour, wheat flour, and corn flour. Non-limiting examples of edible fibers include bamboo fiber, barley bran, carrot fiber, citrus fiber, corn bran, soluble dietary fiber, insoluble dietary fiber, oat bran, pea fiber, rice bran, head husks, soy fiber, soy polysaccharides, wheat bran, and wood pulp cellulose. In some embodiments, at least a portion of the carbohydrate is derived from a plant. In some such embodiments, at least a portion of the carbohydrate is derived from a pea.

[0235] In some embodiments, the food products provided herein further comprise natural or artificial sweeteners.Non-limiting examples of sweeteners include stevia, aspartame, cyclamate, saccharin, sucralose, mogroside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, rasititol, mabinlin, malititol, mannitol, miraculin, monatin, monellin, osladin, pentazin, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucine, neohesperidin dihydrochalcone, neotame, P-4000, and carbohydrate-free sweeteners.

[0236] In some embodiments, the food products provided herein further comprise minerals, non-limiting examples of which include fat-soluble minerals, water-soluble minerals, calcium, phosphorus, potassium, sodium, citrate, chloride, phosphate, magnesium, potassium, zinc, iron, molybdenum, manganese, and copper.

[0237] In some embodiments, the food products provided herein further comprise an emulsifier. Non-limiting examples of emulsifiers include anionic emulsifiers, nonionic emulsifiers, cationic emulsifiers, amphoteric emulsifiers, bioemulsifiers, steric emulsifiers, Pickering emulsifiers, glycolipids (e.g., trehalose lipids, sophorolipids, rhamnolipids, mannosylerythriol lipids), oligopeptides (e.g., gramicidin S, polymyxins), lipopeptides (e.g., surfactin), phospholipids, fatty acids, neutral lipids, polymeric biosurfactants, amphiphilic polysaccharides, lipopolysaccharides, proteins (e.g., pea protein, soy protein, chickpea protein, algae protein, yeast protein, potato protein, etc.), and the like. Protein, lentil protein), mannoprotein, sodium phosphate, calcium stearoyl lactylate, mono- and diacetyl tartaric acid esters of monoglycerides, phospholipids, sorbitan monostearate, magnesium stearate, sodium / potassium / calcium salts of fatty acids, calcium stearoyl lactylate, poly-glycerol esters, sorbitan fatty acid esters, acetate esters of monoglycerides, lactate esters of monoglycerides, citrate esters of monoglycerides, polyglycerol esters of fatty acids, polyglycerol polyricinoleate, propane-1,2-Diol esters, sugar esters, sucrose esters of fatty acids, monoglycerides, acetylated monoglycerides, lactate monoglycerides, diglycerides, phosphate monoglycerides, diacetyl tartaric acid esters, sodium / calcium stearoyl-2-lactylate, ammonium phosphatides, polysorbates, polysorbate-80, carboxymethylcellulose (CMC), modulated cellulose, citric acid esters, locust bean gum, guar gum, liposan, emulsan, lecithin, surfactants (e.g., sorbitan trioleate (Span 85), sorbitan tristearate (Span 65), sorbitan sesquioleate (Arlacel 83), glyceryl monostearate, sorbitan monooleate (Span 80), sorbitan monostearate Examples of suitable polyoxyethylene sorbitan monostearate include sorbitan monolaurate (Span 20), sorbitan tristearate (Tween 65), sorbitan trioleate (Tween 85), polyethylene glycol 400 monostearate, polysorbate 60 (Tween 60), polyoxyethylene monostearate, polysorbate 80 (Tween 80), polysorbate 40 (Tween 40), polysorbate 20 (Tween 20), PEG 20 tristearate, PEG 20 trioleate, PEG 20 monostearate, PEG 20 monooleate, PEG 20 monopalmitate, and PEG 20 monolaurate sorbitan monolaurate, and derivatives and mixtures thereof.

[0238] In some embodiments, the food products provided herein further comprise an antioxidant. Non-limiting examples of antioxidants include natural antioxidants (e.g., alpha-tocopherol (e.g., tocopherol found in cow's milk); low molecular weight thiols (e.g., low molecular weight thiols found in cow's milk); retinol (e.g., retinol found in cow's milk); TBHQ; carotenoids (e.g., carotenoids found in cow's milk); vitamin E; bitter bark (Azadirachta indica) extract; riboflavin; rosemary extract; phenolic diterpenes found in rosemary extract (e.g., carnosol, carnosic acid); sage extract; ascorbic acid and its salts; lactic acid and its salts; grape residue silage; phenolic compounds found in grape residue silage (e.g., ferulic acid); soybean (Glycine max) extract; soybean extract found in isoflavone or polyphenolic compounds; garlic (Allium sativum) extract; sativum) extract; sulfur, phenol, flavonoid, or terpenoid compounds found in garlic extract; fennel (Foeniculum vulgare Mill.) extract; chamomile (Matricaria recutita L.) extract; brown algae (e.g., Ascophyllum nodosum, Fucus vesiculosus); immature pink pepper essential oil (GEO); mature pink pepper essential oil (MEO); green tea extract; butylated hydroxyanisole (E320); butylated hydroxytoluene (E321); and catechins (e.g., epigallocatechin gallate, epicatechin gallate, epigallocatechin, C catechin, epicatechin, catechin found in green tea extract). In some embodiments, the food products provided herein contain less than 2,000 ppm by weight of antioxidants.

[0239] Food Product Characteristics In some embodiments, the pH of the food products provided herein is between 2 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, or 2.5; between 2.5 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3; between 3 and 10, 9.5, 9, 8. Between .5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, or 3.5;Between 3.5 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, or 4;Between 4 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, or 4.5;Between 4.5 and 10, 9.5, 9, 8.5, 8, 7.5, 7, between 6.5, 6, 5.5, or 5;between 5 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, or 5.5;between 5.5 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, or 6;between 6 and 10, 9.5, 9, 8.5, 8, 7.5, 7, or 6.5;between 6.5 and 10, 9.5, 9, 8.5, 8, 7.5, or 7;7 between 7.5 and 10, 9.5, 9, 8.5, or 8; between 8 and 10, 9.5, 9, or 8.5; between 8.5 and 10, 9.5, or 9; between 9 and 10, or 9.5; or between 9.5 and 10, wherein the pH is measured in a solution of deionized water containing 4% by weight protein.

[0240] In some embodiments, the food products provided herein have an α-tocopherol content of between 0.04 and 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1; between 0.1 and 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2; between 0.2 and 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3; between 0.3 and 0.999, 0.9, between 0.8, 0.7, 0.6, 0.5, or 0.4; between 0.4 and 0.999, 0.9, 0.8, 0.7, 0.6, or 0.5; between 0.5 and 0.999, 0.9, 0.8, 0.7, or 0.6; between 0.6 and 0.999, 0.9, 0.8, or 0.7; between 0.7 and 0.999, 0.9, or 0.8; between 0.8 and 0.999, or 0.9; or between 0.9 and 0.999.

[0241] In some embodiments, the food products provided herein are homogenized food products (i.e., food products that have been homogenized) and have a molecular weight of between 0.11 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, 4 μm, 2 μm, 1 μm, or 0.5 μm; between 0.5 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, 4 μm, 2 μm, or 1 μm; between 1 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, 4 μm, 2 μm, or 1 μm. between 2 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, or 4 μm; between 4 μm and 15 μm, 12 μm, 10 μm, 8 μm, or 6 μm; between 6 μm and 15 μm, 12 μm, 10 μm, or 8 μm; between 8 μm and 15 μm, 12 μm, or 10 μm; between 10 μm and 15 μm, or 12 μm; or between 12 μm and 15 μm.

[0242] In some embodiments, the food products provided herein are coarse suspension food products (i.e., food products that have been emulsified without the use of high pressure homogenization (e.g., in a colloid mill, ultrasonicator, rotor-stator unit); e.g., salad dressings, mayonnaise) and have a particle size of between 1 μm and 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, or 2 μm; between 3 μm and 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, or 4 μm; between 4 μm and 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, or 5 μm; between 5 μm and 10 μm, 9 μm, 8 μm, 7 μm, or 6 μm; between 6 μm and 10 μm, 9 μm, 8 μm, or 7 μm; between 7 μm and 10 μm, 9 μm, or 8 μm; between 8 μm and 10 μm, or 9 μm; or between 9 μm and 10 μm.

[0243] Food Product Preparation A variety of recipes exist for preparing food products, and any such recipes may be used in the methods provided herein for producing the food products provided herein. Fermentation broths or preparations containing recombinant ingredients may be used in such recipes in place of, or in addition to, conventionally used food ingredients.

[0244] While the invention has been described in conjunction with certain specific embodiments thereof, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. EXAMPLES

[0245] The following examples are included to illustrate specific embodiments of the present invention. The techniques disclosed in the examples represent techniques discovered by the inventors to function well in the practice of the present invention; however, those skilled in the art should understand, in light of this disclosure, that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention. As a result, all matters described or shown in the examples should be considered as illustrative and not in a limiting sense. EXAMPLES

[0246] Removal of esterase activity via purification Recombinant Trichoderma reesei host cells capable of producing recombinant β-lactoglobulin were generated by transforming protoplasts of a Trichoderma reesei strain with a polynucleotide encoding β-lactoglobulin under the control of an inducible promoter targeted for genomic integration. Transformants were selected on medium containing a marker that selected for integration of the recombinant polynucleotide.

[0247] The recombinant host cells were fermented in a culture medium suitable for the growth of the recombinant host cells and the production and secretion of recombinant β-lactoglobulin.

[0248] The fermentation broth was harvested and the biomass and cell debris were removed by centrifugation to obtain a clarified fermentation broth. Recombinant β-lactoglobulin was isolated from the clarified fermentation broth based on charge (i.e., ion complexation), thermolability / thermostability, and / or hydrophobicity.

[0249] For example, recombinant β-lactoglobulin has been isolated by separation based on charge (i.e., ionic complexation with particles of opposite charge to recombinant β-lactoglobulin at a particular pH and / or ionic strength (e.g., acid sodium salt), followed by isolation of the complex and elution of recombinant β-lactoglobulin from the isolated complex via adjustment of pH and / or ionic strength), followed by separation based on hydrophilicity (i.e., loading the recombinant β-lactoglobulin preparation onto a phenyl sepharose column at a suitable pH and / or ionic strength (e.g., 0.8M Na2SO4) to bind esterase to the phenyl sepharose and allow collection of soluble β-lactoglobulin in the flow-through). The β-lactoglobulin preparation was optionally spray-dried to obtain a β-lactoglobulin powder preparation.

[0250] In another example, recombinant β-lactoglobulin was isolated by separation based on charge (i.e., ionic complexation with particles having an opposite charge to recombinant β-lactoglobulin at a particular pH and / or ionic strength (e.g., acid sodium salt), followed by isolation of the complex and elution of recombinant β-lactoglobulin from the isolated complex via pH and / or ionic strength adjustment), followed by separation based on heat lability / thermostability (i.e., heating (e.g., at 80-85° C. for 20-60 min) at a suitable pH and / or ionic strength (e.g., pH 3 and conductivity less than 2 mS / cm, e.g., obtained by diafiltration) to precipitate esterase activity, followed by removal of the precipitate by centrifugation to obtain soluble β-lactoglobulin). The β-lactoglobulin preparation was optionally spray-dried to obtain a β-lactoglobulin powder preparation.

[0251] The esterase activity contained in the β-lactoglobulin preparations was determined by adding the β-lactoglobulin powder preparation (3% w / w) to coconut and / or sunflower oil and homogenizing at 200 bar to form an emulsion. The mixtures were assayed for the presence of free fatty acids using an FFA kit (product no. KA1667; Abnova, Taipei City, Taiwan).

[0252] As shown in Figures 1-4, the β-lactoglobulin preparation obtained by purification had esterase activity substantially removed. EXAMPLES

[0253] Removal of esterase activity via incubation Recombinant Trichoderma reesei host cells capable of producing recombinant β-lactoglobulin were generated by transforming protoplasts of a Trichoderma reesei strain with a polynucleotide encoding β-lactoglobulin under the control of an inducible promoter targeted for genomic integration. Transformants were selected on medium containing a marker that selected for integration of the recombinant polynucleotide.

[0254] The recombinant host cells were fermented in three cultures containing culture media suitable for the growth of the recombinant host cells and the production and secretion of recombinant β-lactoglobulin. The three cultures differed from each other only in the type of antifoam agent contained in the culture medium, which were selected from the list consisting of Industrol DF204, Polyglycol P-2000, and Erol DF6000K.

[0255] Fermentation broth was harvested from each culture, and biomass and cell debris were removed by centrifugation to obtain clarified fermentation broth. Esterase activity contained in the clarified fermentation broth was determined by using pNPL (product no. 61716; Sigma-Aldrich, St. Louis, MO) and 4MUo assay (product no. 75164; Sigma-Aldrich, St. Louis, MO) essentially per manufacturer's protocol. For the 4Muo assay, 20 μL of clarified fermentation broth was combined with 20 μL of 0.1 M sodium acetate buffer pH 4.3, and 40 μL of 0.1 mM 4MU-o in a white 96-well plate, and the mixture was incubated at 43° C. for 60 minutes before it was quenched with 80 μL of 0.1 M Borax solution pH 9.22, and fluorescence was read by excitation at 365 nm and emission reading at 450 nm. For the pNPL assay, 40 μL of clarified fermentation broth at pH 4.3 was combined with 40 μL of 1 mM pNPL solution in a 96-well PCR plate, and the mixture was incubated at 43°C for 60 min before it was quenched with an equal volume of 0.1 M Borax solution pH 9.22 and fluorescence was read by emission reading at 400 nm.

[0256] As shown in FIG. 5, Tukey-Kramer analysis demonstrated that clarified fermentation broths obtained from cultures utilizing Industrol DF204 or Polyglycol P-2000 as the antifoam produced significantly less esterase activity than clarified fermentation broths obtained from cultures utilizing Erol DF6000K as the antifoam. EXAMPLES

[0257] Elimination of esterase activity via genetic modification A recombinant Trichoderma reesei host cell capable of producing recombinant β-lactoglobulin and containing substantially ablated cutinase (e.g., cut1 (UniProt No. G0RH85)) activity (a "cutinase knockout recombinant host cell") was generated by transforming a protoplast of a Trichoderma reesei strain capable of producing recombinant β-lactoglobulin (a "corresponding recombinant host cell") with a polynucleotide (a targeting vector) genetically engineered to integrate by homologous recombination at the cutinase locus and to replace the cutinase open reading frame with a selectable marker.

[0258] The general structure of the targeting vector is shown in Figure 6. The targeting vector contained a selectable marker (the pyr4 gene, allowing growth without uracil addition) flanked by polynucleotide sequences homologous to upstream and downstream polynucleotide sequences flanking the cutinase open reading frame in the Trichoderma reesei genome. The upstream and downstream sequences are provided herein as SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0259] Transformants were selected on minimal medium and then screened by PCR to identify the cutinase knockout recombinant host cells. The cutinase knockout recombinant host cells and the corresponding recombinant host cells were fermented in a culture medium suitable for the growth of the recombinant host cells and the production and secretion of recombinant β-lactoglobulin. The fermentation broth was harvested and the biomass and cell debris were removed by centrifugation to obtain a clarified fermentation broth. The recombinant β-lactoglobulin was isolated from the clarified fermentation broth based on charge (i.e., electrostatic interaction).

[0260] Cutinase activity in β-lactoglobulin preparations obtained from the cutinase knockout strain and the parental strain was assayed using the FFA assay.

[0261] As shown in Figure 7, in the FFA assay, β-lactoglobulin preparations obtained from the corresponding recombinant host cells produced free fatty acids over time, which is indicative of the presence of lipase activity in the β-lactoglobulin preparation. Such production of free fatty acids (i.e. esterase activity) was substantially eliminated in β-lactoglobulin preparations obtained from cutinase knockout recombinant host cells. EXAMPLES

[0262] An ice cream composition comprising recombinant β-lactoglobulin produced in a filamentous fungal cell and further comprising reduced esterase activity. The following samples were prepared and incubated at 4° C. for 16 hours:

[0263] [Table 3]

[0264] As shown in Figure 9A, the use of recombinant β-lactoglobulin produced by filamentous fungal cells resulted in the formation of an undesirable gel composition. Furthermore, the preparation produced an unpleasant odor and taste.

[0265] As shown in FIG. 9B, heat treatment of recombinant β-lactoglobulin prevented gelation (and reduced the production of unpleasant odors and tastes).

[0266] As shown in FIG. 9C, the addition of triacylglycerol lipase negated the effect of heat treatment of recombinant β-lactoglobulin in that the preparation reformed a gel composition (with an unpleasant odor and taste).

[0267] As shown in Figure 9D, the use of bovine β-lactoglobulin purified from whey did not provide an undesirable gel composition, nor did it produce unpleasant odors and tastes.

[0268] As shown in FIG. 9E, addition of triacylglycerol lipase to bovine β-lactoglobulin resulted in gel formation (and produced an unpleasant odor and taste).

[0269] As shown in FIG. 9F, use of a recombinant β-lactoglobulin preparation with esterase activity substantially removed provided a desirable ice cream texture along with desirable aroma and taste.

[0270] Taken together, the data suggest that preparations of recombinant β-lactoglobulin produced by filamentous fungal cells may contain esterase activity that has adverse effects on the texture, aroma and taste of food compositions. EXAMPLES

[0271] Identification of esterase activities that provide desirable sensory profiles Additional recombinant host cells capable of producing a recombinant component (e.g., a recombinant milk protein) (e.g., additional recombinant Trichoderma reesei host cells, additional recombinant Aspergillus niger host cells, additional recombinant Trichoderma citrinoviride host cells, additional recombinant Myceliophthora thermophila host cells, additional recombinant Myceliophthora The recombinant host cells (E.g., A. thermophila host cells) can be transformed with other esterases (e.g., carboxylester hydrolases (e.g., UniProt Nos. G0RBM4, G0RIU1), phospholipase C (e.g., UniProt Nos. G0REM9), acetyl esterases (e.g., A. thermophila host cells) using targeting vectors similar to those disclosed in Example 3 but including upstream and downstream targeting sequences specific to the targeted esterase locus, or using other mutagenesis schemes to introduce mutations (e.g., one or more nucleotide changes, deletions, or insertions) into nucleotide sequences that regulate expression of the esterase, or into an open reading frame encoding the esterase to produce recombinant host cells containing modulated (i.e., higher or lower) or substantially eliminated esterase activity, such as ... The recombinant host cells are produced as described in Example 3 by targeting the transcription factors that regulate the expression of esterases (e.g., UniProt No. G0RHJ4), and transcription factors that regulate the expression of esterases (e.g., UniProt No. G0RX49), and any other esterases disclosed herein, combinations of two or more such other esterases, and combinations of one or more such other esterases with cutinase (e.g., UniProt No. G0RH85). Recombinant ingredient preparations are obtained from such recombinant host cells according to the methods described herein and used to prepare food products. The food products are tested for desired sensory properties (e.g., desired odor, desired taste, desired texture, desired emulsification) to identify suitable esterase activities or combinations of esterase activities that provide the food product with a desired sensory profile.The suitable esterase activity or combination of esterase activities that provides the desired sensory profile for a particular food product may depend on the particular composition of the food product (eg, the particular lipid profile of the food product).

[0272] All publications, patents, patent applications, sequences, database entries, and other references mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, patent application, sequence, database entry, or other reference was specifically and individually indicated. In case of conflict, the present specification, including definitions, shall control. The terminology and descriptions used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the invention.

Claims

1. A recombinant microbial host cell capable of producing a recombinant component and comprising a genetic modification that reduces or essentially eliminates the activity or expression of an esterase relative to the activity or expression of an esterase contained in a corresponding recombinant microbial host cell.

2. The recombinant microbial host cell of claim 1 , wherein the esterase comprises a cutinase.

3. 3. The recombinant microbial host cell of claim 2, wherein the cutinase comprises an amino acid sequence that is at least 80% identical to a sequence selected from UniProt sequences G0RH85, A2QBP1, A0A2T4BJB5, A5ABE6, A2R2W3, G0R6X2, and G2QH51.

4. 4. The recombinant microbial host cell of claim 3, wherein the cutinase comprises an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequence G0RH85.

5. 2. The recombinant microbial host cell of claim 1, wherein the esterase further comprises a carboxylester hydrolase.

6. 2. The recombinant microbial host cell of claim 1, wherein the esterase further comprises a triacylglycerol lipase.

7. 2. The recombinant microbial host cell of claim 1, wherein the esterase further comprises a phospholipase A2.

8. The recombinant microbial host cell of claim 1 , wherein the esterase further comprises a lysophospholipase.

9. 2. The recombinant microbial host cell of claim 1, wherein the esterase further comprises an acetyl esterase.

10. 2. The recombinant microbial host cell of claim 1 , wherein the esterase further comprises a phospholipase C.

11. 2. The recombinant microbial host cell of claim 1, wherein the esterase further comprises a GDSL lipase.

12. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RIU1, A2R1N7, G0RBM4, A0A2T4BBP9, G2Q379, A0A2T4AZ21, A2R8Z3, A2QPC2, G0RCG3, A0A2T4BCL7, A0A2T4B416, and A0A2T4BNI9.

13. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0R9X3 and G0RHJ4.

14. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RX90, G2Q0K1, A0A2T4B235, A2QC75, G0RLB0, G0RLB7, G0RWS1 and A2QF42.

15. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RJ76, G0RKL4 and G0RRK3.

16. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0REM9, A0A2T4BFY3, A2QAD7, G0RGQ7, G0RJC6, G0RQN5, G0RTR6, G0RBZ6 and G0RDK5.

17. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0R707, A2QM14 and A2R709.

18. 2. The recombinant microbial host cell of claim 1, further comprising an esterase comprising an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RBJ0, G0RJY0 and G0RMI3.

19. The recombinant microbial host cell of claim 1, wherein the genetic modification is a genetic modification in a control sequence or a functional portion thereof that drives expression of a transcription factor that regulates expression of the esterase, or in a coding sequence that encodes such a transcription factor or a functional portion thereof.

20. 20. The recombinant microbial host cell of claim 19, wherein the transcription factor comprises an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RX49, G0RHG1, A0A2T4B416, A0A2T4BJU6, A2R2J1, A2R903, G2Q2Z5, and G2Q8I6.

21. 2. The recombinant microbial host cell of claim 1, wherein the recombinant microbial host cell is selected from the group consisting of a recombinant bacterial host cell, a recombinant yeast host cell, and a recombinant filamentous fungal host cell.

22. 22. The recombinant microbial host cell of claim 21 , wherein the recombinant filamentous fungal host cell is derived from a filamentous fungal host cell selected from the group consisting of an Aspergillus niger host cell, a Myceliophthora thermophila host cell, a Trichoderma reesei host cell, and a Trichoderma citrinoviride host cell.

23. The recombinant microbial host cell of claim 1 , wherein the recombinant component is a recombinant protein.

24. 24. The recombinant microbial host cell of claim 23, wherein the recombinant protein is selected from the group consisting of a recombinant plant protein, a recombinant algal protein, a recombinant fungal protein, a recombinant microbial protein, and a recombinant animal protein.

25. 25. The recombinant microbial host cell of claim 24, wherein the recombinant animal protein is a recombinant milk protein.

26. 26. The recombinant microbial host cell of claim 25, wherein the recombinant milk protein is a recombinant whey protein.

27. 27. The recombinant microbial host cell of claim 26, wherein the recombinant whey protein is recombinant beta-lactoglobulin.

28. 27. The recombinant microbial host cell of claim 26, wherein the recombinant whey protein is recombinant alpha-lactalbumin.

29. 26. The recombinant microbial host cell of claim 25, wherein the recombinant milk protein is recombinant casein.

30. 30. The recombinant microbial host cell of claim 29, wherein the recombinant casein is recombinant κ-casein.

31. 30. The recombinant microbial host cell of claim 29, wherein the recombinant casein is β-casein.

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