Production of phospholipids in microorganisms and uses thereof

Abstract

The present invention relates to extracted microbial lipids, lipid-containing microbial cells, and extracts thereof. The present invention also relates to the use of these lipids, cells, and extracts in foods, feeds, and beverages.

Description

[Technical field]

[0001] The present invention relates to extracted microbial lipids, lipid-containing microbial cells, and extracts thereof. The present invention also relates to the use of these lipids, cells, and extracts in foods, feeds, and beverages. [Background technology]

[0002] With the world population projected to reach 9 billion by 2050, the demand for meat and dairy products for human nutrition is expected to continue to grow. However, global meat and dairy production accounts for 70% of freshwater consumption, 38% of total arable land use, and 19% of global greenhouse gas emissions. There is growing interest in finding alternative sources of protein and fat with a lower environmental footprint. There is also a growing market worldwide for non-animal sources of high-quality protein and fat, e.g., plant sources, which are considered more sustainable and environmentally friendly. Cultural and religious reasons also contribute to the growing market for non-animal proteins. However, many current plant-based alternatives to meat and dairy use fats made from blends of vegetable oils such as coconut, soy, and palm oil, which may impart insufficient flavor and functionality. Food and beverage products incorporating lipids from animal sources are still often preferred by consumers, as fats and oils add flavor, lubricity, and texture to foods and contribute to a feeling of fullness upon ingestion.

[0003] The aroma and flavor characteristics of cooked meat are important factors for the eating quality of meat and are highly correlated with consumer acceptance and preference. The aroma and flavor characteristics are derived from a large number of volatile and non-volatile compounds that are generated during the heating of meat, e.g., by cooking or roasting (see, e.g., reviews by Dashdorj et al. (2015) and Mottram (1998)). These compounds result from several types of chemical reactions, namely, Maillard reactions of amino acids or peptides with reducing sugars, lipid oxidation, interactions between Maillard reaction products and lipid oxidation products, and the decomposition of other compounds, such as some sulfur-containing compounds, during cooking or roasting. The reaction products, especially the volatile products, are organic and of low molecular weight, including aldehydes, ketones, alcohols, esters, aliphatic hydrocarbons, thiazoles, oxazoles, and pyrazines, as well as oxygen-containing heterocyclic compounds, such as lactones and alkylfurans. Many of these compounds are not evolved during cooking of meat substitutes made with plant proteins and fats such as coconut, soy, and palm oil, leading to reduced consumer acceptance of these non-animal products.

[0004] There remains a need for alternative non-animal sources of lipids capable of providing meat-like flavor and aroma for human food and nutrition. Summary of the Invention

[0005] This application is based, at least in part, on the surprising determination that certain microbial polar lipids (e.g., phospholipids) can impart meat-related flavors and / or aromas to foods. The inventors have produced and / or extracted lipids from microorganisms that contain ω6 fatty acids in the polar lipids. These resemble certain animal fat compositions (e.g., beef tallow and lard), but differ from animal fats in the types and ratios of ω6 and other fatty acids, and in the types and ratios of phospholipid classes. Despite these differences, the inventors have discovered that when heated in the presence of sugars, amino acids, or other compounds, the extracted lipids mimic the functionality of meat lipids and produce meat-like aromas and / or flavors.

[0006] As determined herein, extracted microbial lipids comprising primarily polar lipids comprising a total fatty acid (TFA) content including the ω6 fatty acid arachidonic acid (ARA), optionally also including γ-linolenic acid (GLA) and dihomo-γ-linolenic acid (DGLA), and optionally also including eicosadienoic acid (EDA), docosatetraenoic acid (DTA) and / or docosapentaenoic acid-ω6 (DPA-ω6), produce meat-like aromas and / or flavors when heated in the presence of sugars and amino acids, albeit in amounts and ratios different from the polar lipids present in meat. Advantageously, in some embodiments, the extracted microbial lipids also contain relatively low levels of saturated fatty acids such as palmitic acid, thereby providing a healthier alternative to meat lipids or lipids that more closely mimic meat lipids.

[0007] Thus, provided herein are, for example, extracted microbial lipids; compositions comprising extracted microbial lipids, amino acids and sugars (e.g., flavor compositions that can be added to foods or food consumable components to form foods); foods and feeds comprising extracted microbial lipids, amino acids and sugars (e.g., foods intended as meat substitutes, such as plant-based burgers, sausages, etc.), and processes and methods for producing compositions, foods and feeds using extracted microbial lipids. As a result of the presence of the extracted lipids, sugars and amino acids in the compositions and foods and feeds, the compositions and foods and feeds of the present disclosure have a meat-like flavor and / or aroma when heated (e.g., produce two or more meat-related volatile compounds).

[0008] In one aspect, there is provided a composition comprising an amino acid or derivative, a sugar, and extracted microbial lipids comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, wherein the polar lipids are present in the extracted microbial lipid in greater amounts than the non-polar lipids, (a) The polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of the ω6 fatty acids are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids include arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA), ARA is present in an amount of about 10% to about 60% (or at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or at least about 55%) of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 0.1% to about 5% of the total fatty acid content of the polar lipids, and GLA is present in an amount of about 1% to about 10% of the total fatty acid content of the polar lipids; (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); When the composition is heated, one or more compounds having meat-associated flavors and / or aromas are produced.

[0009] In some embodiments, ARA is present in an amount of about 20% to about 50% (e.g., about 25% to about 50%, or about 30% to about 50%) of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 1% to about 5% of the total fatty acid content of the polar lipids, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipids.

[0010] In other embodiments, ARA is present in an amount of about 10% to about 20% of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 0.5% to about 5% of the total fatty acid content of the polar lipids, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipids.

[0011] Also provided is a composition comprising an amino acid or derivative, a sugar, and an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, wherein the polar lipids are present in the extracted microbial lipid in greater amounts than the non-polar lipids, (a) the polar lipids comprise a total fatty acid (TFA) content that includes ω6 fatty acids, the ω6 fatty acids being present in an amount of about 30% to about 70% (or at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65%) of the total fatty acid content of the polar lipids, at least a portion of the ω6 fatty acids being esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA); (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); When the composition is heated, one or more compounds having meat-associated flavors and / or aromas are produced.

[0012] In some examples, ω6 fatty acids are present in an amount of about 40% to about 70%, about 40% to about 60%, or about 50% to about 60% of the total fatty acid content of the polar lipids. In certain embodiments, ARA is present in an amount of about 20% to about 50% (e.g., about 25% to about 50%, or about 30% to about 50%) of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 1% to about 5% of the total fatty acid content of the polar lipids, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipids.

[0013] In some embodiments, omega-3 fatty acids are absent from the polar lipid or are present in a total amount of less than about 3% by weight of the TFA content of the polar lipid, and / or the polar lipid lacks C16:2, C16:3 omega-3, EPA, and DHA.

[0014] In one embodiment, the polar lipid comprises myristic acid (C14:0) in an amount less than about 2% by weight of the total fatty acid content of the polar lipid.

[0015] In one embodiment, the phospholipids comprising ω6 fatty acids comprise two, three or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS), optionally one or more of phosphatidic acid (PA), phosphatidylglycerol (PG) and cardiolipin (Car), preferably at least PC and PE, or at least PC, PE, PS and PI, each comprising one or at least two or more of ARA, DGLA and GLA.

[0016] In one embodiment, the phospholipids comprising omega-6 fatty acids include phosphatidylcholine (PC) and phosphatidylethanolamine (PE), each of which comprises one or at least two or more of ARA, DGLA, and GLA.

[0017] In one embodiment, the phospholipids comprising ω6 fatty acids comprise phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA), each comprising one or at least two of ARA, DGLA, and GLA, wherein ARA is present in PC in an amount of about 14% to about 20% of the total fatty acid content in PC, ARA is present in PE in an amount of about 15% to about 20% of the total fatty acid content in PE, and ARA is present in PA in an amount of about 15% to about 20% of the total fatty acid content in PA.

[0018] In one embodiment, stearic acid is present at a level of less than about 7%, or less than about 6%, or less than about 5%, preferably less than 4%, or less than 3% of the total fatty acid content of the polar lipids.

[0019] In one embodiment, the extracted microbial lipid is extracted fungal lipid or extracted eukaryotic microbial lipid.

[0020] In one embodiment, the extracted microbial lipid is extracted yeast lipid, preferably Saccharomyces cerevisiae, Yarrowia lipolytica, or Pichia pastoris lipid.

[0021] In another embodiment, the extracted microbial lipid is extracted Mortierella spp (e.g., M. alpina) lipid.

[0022] In one embodiment, the following: (a) at least one of EDA, DTA, and DPA-ω3 is also present in the polar lipid; (b) At least one of the following applies: the ratio of PC to PE, or the ratio of PC to phospholipids other than PC, is less than 3:1, less than 2:1, less than 1.5:1, less than 1.25:1, less than 1:1, between 3:1 and 1:1, between 2:1 and 1:1, or between 3:1 and 0.5:1.

[0023] In one embodiment, the saturated fatty acid content of the polar lipids comprises one or more or all of lauric acid (C12:0), myristic acid (C14:0), C15:0 fatty acid, C20:0, C22:0, and C24:0, preferably C14:0 and C24:0 or C14:0, C15:0 and C24:0, more preferably C14:0, C15:0 and C24:0, but excluding C20:0 and C22:0.

[0024] In one embodiment, lauric acid and myristic acid are absent from the polar lipid, or lauric acid and / or myristic acid are present in the polar lipid, and the sum of the amount of lauric acid and myristic acid in the polar lipid is less than about 2%, or less than about 1%, preferably less than about 0.5%, and more preferably less than about 0.2% of the total fatty acid content of the polar lipid.

[0025] In one embodiment, C15:0 is not present in polar lipids, or C15:0 is present in polar lipids in an amount less than about 3%, preferably less than about 2%, or less than about 1% of the total fatty acid content of the polar lipid.

[0026] In one embodiment, palmitic acid is present in the polar lipid in an amount of about 10% to about 20% of the fatty acid content of the polar lipid.

[0027] In one embodiment, palmitoleic acid is present in the polar lipid in an amount of from about 3% to about 45%, or from about 3% to about 25%, or from about 3% to about 20%, or from about 3% to about 15% of the total fatty acid content of the polar lipid.

[0028] In another embodiment, oleic acid is present in the polar lipid in an amount of from about 3% to about 60%, or from about 3% to about 40%, or from about 3% to about 25%, or from about 20% to about 60% of the total fatty acid content of the polar lipid.

[0029] In another embodiment, vaccenic acid is not present in the polar lipid, or vaccenic acid is present in the polar lipid in an amount less than about 2%, preferably less than about 1%, or less than about 0.5% of the total fatty acid content of the polar lipid.

[0030] In one embodiment, linoleic acid is present in the polar lipid in an amount of about 3% to about 20% of the total fatty acid content of the polar lipid.

[0031] In another embodiment, eicosadienoic acid is not present in the polar lipid, or eicosadienoic acid is present in the polar lipid in an amount between about 3% and about 12%, or between about 3% and about 8%, or between about 3% and about 6%, or less than about 3% of the total fatty acid content of the polar lipid.

[0032] In further embodiments, C20:0 and C22:0 are not present in the polar lipids, or C20:0 and / or C22:0 are present in the polar lipids, and the sum of the amounts of C20:0 and C22:0 in the polar lipids is less than about 1.0%, less than about 0.5%, preferably less than 0.2% of the total fatty acid content of the polar lipids.

[0033] In another embodiment, C24:0 is not present in the polar lipids, or C24:0 is present in the polar lipids in an amount less than about 1.0%, less than 0.5%, preferably less than 0.3%, or less than 0.2% of the total fatty acid content of the polar lipids.

[0034] In another embodiment, C17:1 is not present in the polar lipids, or C17:1 is present in the polar lipids in an amount less than about 5%, preferably less than about 4% or less than about 3%, more preferably less than about 2% of the total fatty acid content of the polar lipids.

[0035] In another embodiment, monounsaturated fatty acids that are C20 or C22 fatty acids are not present in the polar lipid, or C20:1 and / or C22:1 are present in the polar lipid, and the sum of the amounts of C20:1 and C22:1 in the polar lipid is less than about 1.0%, less than about 0.5%, preferably less than 0.2% of the total fatty acid content of the polar lipid.

[0036] In another embodiment, (i) the content of ω6 fatty acids in the polar lipids that are C20 or C22 fatty acids is about 5% to about 60%, preferably about 10% to about 60%, of the total fatty acid content of the polar lipids, and / or (ii) the content of ω6 fatty acids in the polar lipids that are ω6 fatty acids having 3, 4 or 5 carbon-carbon double bonds is about 5% to about 70%, preferably about 10% to about 70%, more preferably about 40% to about 70%, or about 45% to about 70%, or about 50% to about 70% of the total fatty acid content of the polar lipids.

[0037] In another embodiment, C16:3ω3 is absent from polar lipids, or both C16:2 and C16:3ω3 are absent from polar lipids.

[0038] In another embodiment, the extracted microbial lipids contain PC and / or lack cyclopropane fatty acids, preferably lacking C15:0c, C17:0c, and C19:0c.

[0039] In another embodiment, the extracted lipids are obtained from a genetically modified microorganism. For example, the genetically modified microorganism is (i) synthesizing, or increasing the synthesis of, one or more omega-6 fatty acids in a microorganism; (ii) increasing total fatty acid synthesis and / or accumulation in the microorganism; (iii) increasing total polar lipid synthesis and / or accumulation in the microorganism; (iv) a decrease in triacylglycerol (TAG) synthesis and / or accumulation in a microorganism, or an increase in TAG catabolism in a microorganism, preferably an increase in TAG lipase activity; (v) reducing the catabolism of total fatty acids in microorganisms; or any combination thereof.

[0040] In another embodiment, the genetic modification provides at least two of (i) to (v), preferably (iv) and (v), or (i), (iv) and (v).

[0041] In one embodiment, when the composition is heated, the heat is at least about 100°C, preferably at least about 120°C, and more preferably at least about 140°C.

[0042] Also provided is a composition comprising an amino acid or derivative, a sugar, and an extracted Mortierella spp. lipid comprising esterified fatty acids in the form of either (i) polar lipids without any non-polar lipids, or (ii) polar lipids and non-polar lipids, where the polar lipids are present in the extracted microbial lipid in greater amounts than the non-polar lipids. In some embodiments, the extracted Mortierella spp. lipid is extracted Mortierella alpina lipid. The composition may also further comprise another food, feed, or beverage ingredient.

[0043] In some embodiments of the compositions of the present invention, the sugar, sugar alcohol, sugar acid, or sugar derivative is selected from ribose, xylose, glucose, fructose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, altodextrins, glycogen, galactose, lactose, ribitol, gluconic acid and glucuronic acid, amylose, amylopectin, or any combination thereof, preferably the sugar is ribose or xylose.

[0044] In a further embodiment, the amino acid or derivative thereof is selected from cysteine, cystine, cysteine ​​sulfoxide, allysine, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, or any combination thereof, preferably, the amino acid or derivative thereof is a sulfur-containing amino acid or derivative.

[0045] In other embodiments, the composition further comprises one or more esterified or non-esterified fatty acids from a source other than the extracted microbial lipids, cells, or extracts.

[0046] In some embodiments, the composition is in the form of a powder, solution, suspension, or emulsion.

[0047] In one embodiment, the composition comprises less than 5%, less than 10%, less than 15%, or less than 20% (w / w or w / v) protein.

[0048] In one embodiment, the composition comprises at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, or at least about 50 mg of extracted microbial lipids per gram of dry composition or slurry, or per ml of liquid composition.

[0049] In one embodiment, the composition comprises from about 10 mg to about 100 mg of extracted microbial lipids, or from about 15 mg to about 50 mg of extracted microbial lipids per gram of dry composition or slurry, or per ml of liquid composition.

[0050] Also provided is a food, feed, or beverage comprising an ingredient comprising a composition described herein and at least one other food, feed, or beverage ingredient.

[0051] In another aspect, there is provided a food, feed or beverage comprising extracted Mortierella spp. lipids (e.g. extracted M. alpina lipids), wherein the lipids comprise esterified fatty acids in the form of either (i) polar lipids, absent any non-polar lipids, or (ii) polar lipids and non-polar lipids, wherein the polar lipids are present in the extracted microbial lipids in greater amounts than the non-polar lipids, and wherein the food, feed or beverage further comprises an amino acid or derivative, a sugar, and at least one other food, feed or beverage ingredient.

[0052] In a further aspect, there is provided a food, feed or beverage comprising an ingredient which is an extracted microbial lipid as defined above and herein, the food, feed or beverage further comprising an amino acid or derivative, a sugar and at least one other food, feed or beverage ingredient.

[0053] In another aspect, a food, feed, or beverage is provided comprising a lipid and at least one other food, feed, or beverage component, wherein the lipid is the product of a reaction between the extracted microbial lipid of the invention, an amino acid or derivative, and a sugar under conditions sufficient to produce at least two compounds having a meat-related flavor and / or aroma.

[0054] In some embodiments, the sugar, sugar alcohol, sugar acid, or sugar derivative in the food, feed, or beverage is selected from ribose, xylose, glucose, fructose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, altodextrin, glycogen, galactose, lactose, ribitol, gluconic acid and glucuronic acid, amylose, amylopectin, or any combination thereof, preferably the sugar is ribose or xylose.

[0055] In one embodiment, the amino acid or derivative thereof in the food, feed or beverage is selected from cysteine, cystine, cysteine ​​sulfoxide, allicin, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, or any combination thereof, preferably the amino acid or derivative thereof is a sulfur containing amino acid or derivative.

[0056] In one embodiment, at least one other food, feed, or beverage ingredient comprises a protein (eg, a microbial protein or a plant protein), and optionally the composition comprises at least 10% protein by weight.

[0057] In some embodiments, the food, feed, or beverage has no animal-derived components, while in other embodiments, the food, feed, or beverage contains components that are derived from animals, such as components that contain meat.

[0058] Also provided is a food or feed comprising at least two meat-related flavour and / or aroma compounds derived from the extracted microbial lipids or compositions of the invention as defined herein, wherein the food, feed or beverage comprises a greater amount of the at least two compounds having meat-related flavour and / or aroma than the amount in a corresponding food, feed or beverage produced using a corresponding lipid or composition lacking polar lipids comprising omega-6 fatty acids.

[0059] In one embodiment, the corresponding lipids of the corresponding food, feed or beverage may comprise lipids other than polar lipids comprising omega-6 fatty acids (e.g. non-polar lipids).

[0060] In one embodiment, the corresponding lipids of the corresponding food, feed, or beverage are free of esterified fatty acids in the form of either (i) polar lipids without any non-polar lipids, or (ii) polar lipids and non-polar lipids, where the polar lipids are present in the extracted microbial lipids in greater amounts than the non-polar lipids, wherein: The polar lipids of (a)(i) and (ii) comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of the ω6 fatty acids are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids include arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA), wherein ARA is present in an amount of about 10% to about 60% of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 0.1% to about 5% of the total fatty acid content of the polar lipids, and GLA is present in an amount of about 1% to about 10% of the total fatty acid content of the polar lipids; (b) the polar lipids have a total saturated fatty acid content including palmitic acid and stearic acid; (c) the polar lipids have a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis).

[0061] In one embodiment, the corresponding lipids of the corresponding food, feed, or beverage are free of esterified fatty acids in the form of either (i) polar lipids without any non-polar lipids, or (ii) polar lipids and non-polar lipids, where the polar lipids are present in the extracted microbial lipids in greater amounts than the non-polar lipids, wherein: The polar lipids of (a)(i) and (ii) comprise a total fatty acid (TFA) content that includes ω6 fatty acids, the ω6 fatty acids being present in an amount of about 30% to about 70% of the total fatty acid content of the polar lipid, at least a portion of the ω6 fatty acids being esterified in the form of phospholipids in the polar lipid, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA); (b) the polar lipids have a total saturated fatty acid content including palmitic acid and stearic acid; (c) the polar lipids have a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis).

[0062] In one embodiment, the food or feed is a meat substitute.

[0063] In certain embodiments, the application of heat to a food, feed, or beverage produces one or more compounds, preferably volatile compounds, having meat-associated flavors and / or aromas.

[0064] With respect to the compositions, foods, feeds, or beverages described herein, the application of heat to the composition, food, feed, or beverage can result in the production of 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethylbenzaldehyde, 2,3,4,5-tetramethylcyclopent-2-ene-1 In one embodiment, two or more volatile compounds selected from 1,3-bis(1,1-dimethylethyl)-benzene, 2-octen-1-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, trans-2-octen-1-ol, 1-nonanol, 1,3-bis(1,1-dimethylethyl)-benzene, 2-octen-1-ol, adamantanol-like compounds, hexanal, 2-pentylfuran, 1-octen-3-ol, 2-pentylthiophene, and 1,3,5-titriane may be produced.

[0065] In some examples, application of heat to the composition, food, feed, or beverage produces two or more volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, 1-octanol, trans-2-octen-1-ol, and 1-nonanol.

[0066] In a further embodiment, application of heat to the composition, food, feed, or beverage produces two or more volatile compounds selected from 1-pentanal, 3-octanone, 2-octen-1-ol, 1-nonanol, and 1-octanol, and optionally 1,3-bis(1,1-dimethylethyl)-benzene.

[0067] In one embodiment, application of heat to the composition, food, feed, or beverage produces two or more volatile compounds selected from 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethyl-benzaldehyde, and 2,3,4,5-tetramethylcyclopent-2-en-1-ol.

[0068] In one embodiment the food, feed or beverage is, or has been, optionally heated at a temperature of at least about 100°C, preferably at least about 120°C, more preferably at least about 140°C.

[0069] Also provided is a method of producing a food, feed or beverage, the method comprising combining a composition of the invention with at least one other food, feed or beverage ingredient.

[0070] In another aspect, a method of producing a food, feed, or beverage is provided, the method comprising combining extracted microbial lipid as defined herein, optionally heated at a temperature of at least about 100°C, at least about 120°C, or at least about 140°C, with sugars, amino acids, or derivatives, and at least one other food, feed, or beverage ingredient.

[0071] In a further aspect, there is provided a method of preparing a food, feed or beverage for consumption, the method comprising heating the food, feed or beverage of the invention to cause chemical reactions between fatty acids, sugars and amino acids in the food, feed or beverage.

[0072] Also provided is a method of increasing meat-related flavor and / or aroma in a food, feed, or beverage, comprising heating an extracted microbial lipid as defined herein or a food, feed, or beverage comprising a composition of the invention and at least one other food, feed, or beverage ingredient under conditions sufficient to produce meat-related flavor and / or aroma compounds.

[0073] In some embodiments of the above method, the food, feed, or beverage is heated to a temperature of at least about 100°C, preferably at least about 120°C, and more preferably at least about 140°C.

[0074] There is also provided the use of an extracted microbial lipid as defined herein, or a composition of the invention, to produce a food, feed or beverage ingredient, or a food, feed or beverage.

[0075] Also provided were isolates of Mortierella sp. i) yNI0125, deposited with the National Measurement Institute Australia under V21 / 019953 on 12 October 2021; ii) yNI0126, deposited with the National Measurement Institute Australia under V21 / 019951 on 12 October 2021; iii) yNI0127, deposited with the National Measurement Institute Australia under V21 / 019952 on 12 October 2021; and iv) Selected from yNI0132, deposited with the National Measurement Institute Australia under V21 / 019954 on October 12, 2021.

[0076] In another aspect, the invention provides a lipid-containing microbial cell extract of the invention or a microbial cell extract produced from a microbial cell of the invention, comprising a polar lipid comprising omega-6 fatty acids esterified in the form of a phospholipid. The extract may be produced by any means known in the art, including, for example, culturing the microbial cells, disrupting the cell wall (e.g., heating the cells or lysing the cell wall), and optionally centrifuging and / or concentrating the resulting lysate (e.g., evaporating).

[0077] In another aspect, the present invention provides a method for extracting lipids from a microbial cell of the present invention, e.g. (a) obtaining a microbial cell of the invention, and (b) extracting lipids from microbial cells; thereby producing extracted lipids.

[0078] Suitable methods for extracting lipids from microbial cells are described herein.For example, lipids can be extracted by any means known in the art, such as, but not limited to, exposing cells to organic solvents, pressurizing cells, or treating cells with microwave irradiation, sonication, high speed homogenization, high pressure homogenization, bead beating, autoclaving, pyrolysis, or any combination thereof.

[0079] In one embodiment, the method further comprises culturing the cells.

[0080] In one embodiment, the cells are cultured in a medium comprising one or more of omega-6 fatty acids, preferably LA, GLA, DGLA, EDA, ARA, DTA, or DPA omega-6.

[0081] In one embodiment, the omega-6 fatty acid is a free fatty acid or a fatty acid salt.

[0082] In one embodiment the cells are cultured in a medium lacking ω6 fatty acids, preferably lacking ω6 other than LA, or in a medium comprising oleic acid and / or glycerol, preferably oleic acid and glycerol.

[0083] In one embodiment, the method further comprises modifying or purifying the lipids, preferably by one or more of reducing the amount of one or more non-polar lipids and / or free fatty acids, increasing the amount of one or more ω6 fatty acids in the total fatty acid content of the lipids, increasing the amount of total ω6 fatty acids in the total fatty acid content of the lipids, and reducing the amount of total saturated fatty acids in the total fatty acid content of the lipids, or modifying the ratio of one or more of PC:PE, PC:PI, or PC:PS. The ratio of one or more of PC:PE, PC:PI, or PC:PS can also be altered by adjusting the culture conditions prior to lipid extraction.

[0084] In one embodiment, the method further comprises purifying polar lipids from the extracted microbial lipids, preferably reducing the amount of one or more of TAGs, DAGs, free fatty acids, proteins, carbohydrates, waxes, pigments, or volatile compounds. For example, purification of polar lipids can be carried out using known solvent extraction and fractionation methods.

[0085] In another aspect, the present invention provides a process for culturing microbial cells, the process comprising: (a) obtaining a microbial cell of the invention; (b) expanding the number of cells by culturing the cells in a suitable medium.

[0086] In another aspect, the present invention provides a process for producing a microbial cell that produces a lipid of the present invention, preferably producing an increased amount of said lipid compared to a precursor microbial cell, the process comprising the step of introducing one or more genetic modifications and / or exogenous polynucleotides as defined above into a precursor microbial cell.

[0087] In one embodiment, the process comprises: (i) generating progeny cells from the cells containing the introduced genetic modification and / or exogenous polynucleotide; (ii) mutagenizing the population of progenitor cells; (iii) introducing one or more exogenous polynucleotides, whereby the exogenous polynucleotides are integrated into the genome of the microbial cell, preferably at one or more predefined locations; (iv) determining the fatty acid composition of the cell or its progeny; and (v) selecting progeny cells that contain the lipid of the present invention.

[0088] In another aspect, the invention provides a composition comprising one or more or all of the lipids of the invention, the microbial cells of the invention, or the microbial cell extracts of the invention, and one, two, or all three of: (i) a sugar, sugar alcohol, sugar acid, or sugar derivative; (ii) an amino acid or derivative thereof containing a free amino group; and (iii) a sulfur-containing compound other than a sulfur-containing amino acid.

[0089] In one embodiment, the present invention provides a composition for producing a food-like aroma and / or flavor when heated, the composition comprising: a) a microbial biomass containing phospholipids and / or extracted lipids, preferably comprising phospholipids extracted from a microorganism; b) one or more sugars, sugar alcohols, sugar acids, or sugar derivatives; c) one or more amino acids or derivatives or salts thereof.

[0090] In some embodiments, the composition includes both a phospholipid-containing microbial biomass and phospholipids extracted from the microorganism. In one embodiment, the dry weight ratio of microbial biomass to extracted lipid / phospholipid is 10:1 to 2:1, 2:1 to 1:1, 1:1 to 1:2, or 1:2 to 1:10. In one embodiment, the extracted lipid / phospholipid is from a different microorganism than the microbial biomass.

[0091] Such compositions may, in some embodiments, be used to increase the meat-related flavor and / or aroma of a food, feed, or beverage. The composition may be a powder, solution, suspension, emulsion, or other suitable form. Additionally, the composition may be packaged in a packet, shaker, or other container that allows a user to easily add the composition to a food, feed, or beverage, or an ingredient thereof.

[0092] In one embodiment, the composition further comprises another food, feed or beverage ingredient.

[0093] In one embodiment, the sugar, sugar alcohol, sugar acid, or sugar derivative is selected from ribose, xylose, glucose, fructose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, altodextrins, glycogen, galactose, lactose, ribitol, gluconic acid and glucuronic acid, amylose, amylopectin, or any combination thereof, preferably the sugar is ribose or xylose.

[0094] In one embodiment, the amino acid or derivative thereof is selected from cysteine, cystine, cysteine ​​sulfoxide, allysine, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, or any combination thereof, preferably, the amino acid or derivative thereof is a sulfur-containing amino acid or derivative.

[0095] In one embodiment, the composition further comprises one or more esterified or non-esterified fatty acids from a source other than the extracted microbial lipids, cells, or extracts.

[0096] In one embodiment, the composition is a dry composition. In another embodiment, the composition is a liquid composition. In one embodiment, the composition is in the form of a powder, solution, suspension, or emulsion.

[0097] In another aspect, the invention provides a food, feed or beverage comprising one or more or all of the components of the lipid of the invention, the microbial cell of the invention, the microbial cell extract of the invention, or the composition of the invention, and at least one other food, feed or beverage ingredient.

[0098] In another aspect, the invention provides a food, feed, or beverage comprising an ingredient that is a Mortierella sp. or a homogenate thereof and at least one other food, feed, or beverage ingredient. In one embodiment, the Mortierella sp. is viable. In one embodiment, the Mortierella sp. is dead, e.g., the cells may be heat treated to render them incapable of replicating. In one embodiment, the food, feed, or beverage comprises at least 1%, at least 5%, at least 10%, 1%, and 20%, or between 1% and 50% of the Mortierella sp. or homogenate thereof. In one embodiment, the Mortierella sp. is genetically modified as defined herein. In one embodiment, the Mortierella sp. is not genetically modified.

[0099] In another aspect, the invention provides a food, feed, or beverage comprising an ingredient that is a Yarrowia sp. or a homogenate thereof (e.g., a Yarrowia cell as described herein, such as a Yarrowia cell comprising polar lipids as defined above or herein) and at least one other food, feed, or beverage ingredient. In one embodiment, the Yarrowia sp. is live. In one embodiment, the Yarrowia sp. is dead, e.g., the cells may have been heat treated to render them incapable of replicating. In one embodiment, the food, feed, or beverage comprises at least 1%, at least 5%, at least 10%, 1%-20%, or 1%-50% of the Yarrowia sp. or homogenate thereof. In one embodiment, the Yarrowia sp. is genetically modified as defined herein. In one embodiment, the Yarrowia sp. is not genetically modified.

[0100] In one embodiment any of the foods, feeds or beverages of the present invention are packaged for ready sale.

[0101] In another aspect, the invention provides a method of producing a food, feed, or beverage, the method comprising combining one or more or all of the lipids of the invention, the microbial cells of the invention, the microbial cell extracts of the invention, or the compositions of the invention with at least one other food, feed, or beverage ingredient, or heating the lipids, cells, extracts, or compositions. For example, the lipids, microbial cells, microbial cell extracts, or compositions can be combined with other food or feed or beverage ingredients by mixing, applying it to the surface of the other ingredients, or immersing / soaking the other ingredients. In one embodiment, the food, feed, or beverage is prepared by (a) heating a composition comprising the lipids of the invention and / or the microbial cells of the invention, and (b) mixing the product from (a) with other food, feed, or beverage ingredients.

[0102] In another aspect, the invention provides a method of preparing a food, feed or beverage for consumption, the method comprising heating a food, feed or beverage of the invention to effect a chemical reaction between fatty acids, sugars and amino acids in the food or feed, in one embodiment the chemical reaction comprises the Maillard reaction.

[0103] In another aspect, the present invention provides a method for increasing meat-related flavor and / or aroma of a food, feed, or beverage when the food, feed, or beverage is heated, the method comprising: (a) contacting or combining a lipid of the present invention, a microbial cell of the present invention, a microbial cell extract of the present invention, or a composition of the present invention with the food, feed, or beverage, and, optionally, (b) heating the food, feed, or beverage. Alternatively, the food, feed, or beverage is prepared by (a) heating a composition comprising the lipid of the present invention and / or the microbial cell of the present invention, and (b) contacting or mixing the product from (a) with other food, feed, or beverage ingredients. In some embodiments, the step of contacting or combining the food product, beverage product, or feed with the composition comprises one or more or all of mixing the food, beverage product, or feed with the composition, coating the food, beverage product, or feed with the composition, basting the food, beverage product, or feed with the composition, dipping the food, beverage product, or feed with the composition. In some embodiments, the method further comprises the step of grinding, mincing, rolling, chopping, extruding, or drying the food product, beverage product, or feed after or simultaneously with the step of contacting the food product, beverage product, or feed with the composition, or any combination of these further steps.

[0104] In another aspect, the invention provides a method for increasing meat-related flavor and / or aroma in a food, feed or beverage, comprising heating a food, feed or beverage comprising one or more or all of the lipid of the invention, the microbial cell of the invention, the microbial cell extract of the invention, or the composition of the invention, and at least one other food, feed or beverage ingredient under conditions sufficient to produce meat-related flavor and / or aroma compounds.

[0105] In one embodiment the food, feed or beverage ingredient is heated to a temperature of at least about 100° C., preferably at least about 120° C., more preferably at least about 140° C. In one embodiment the heating step is for at least 5 minutes. In one embodiment the heating step is for a period of between 5 minutes and 75 minutes, preferably between 5 minutes and 45 minutes.

[0106] In one embodiment, the meat-related flavor and / or aroma is beef-like, chicken-like, pork-like, or fish-like. In a preferred embodiment, the composition provides or increases an umami flavor or aroma in a food or beverage product. In a preferred embodiment, the composition does not provide a bitter or sour taste to a food product, beverage product, or feed.

[0107] In another aspect, the invention provides the use of one or more or all of the lipid of the invention, the microbial cell of the invention, the microbial cell extract of the invention, or the composition of the invention to produce a food, feed or beverage ingredient, or a food, feed or beverage, or to increase the meat-related flavor and / or aroma of a food, feed or beverage.

[0108] Any embodiment herein is intended to apply mutatis mutandis to any other embodiment unless stated otherwise.

[0109] The present invention is not limited in scope by the specific embodiments described herein, which are for purposes of illustration only, and functionally equivalent products, compositions, and methods are expressly intended to be within the scope of the invention as described herein.

[0110] Throughout this specification, unless otherwise indicated or the context dictates otherwise, references to a single step, composition of matter, group of steps, or group of composition of matter are deemed to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of composition of matter.

[0111] The invention will now be described by way of the following non-limiting examples and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0112] [Figure 1] Biosynthetic pathway of polyunsaturated fatty acids. [Diagram 2] Growth curves of S. cerevisiae cultured in YPD medium for up to 7 days [Diagram 3] Graphical representation of volatile compounds identified by GC-MS in reaction mixtures containing YL ARA and YL polar lipid preparations as shown in Table 32. The graph shows the area percentage (%) of the total compounds identified for each reaction mixture. Bars not shown for some compound IDs mean that the compound was not detected in that mixture under the specified analytical conditions. [Figure 4-1] Graphical representation of volatile compounds identified by HS-SPME-GCMS in reaction mixtures containing ARA-PC or 18:0 / 18:1-PC (Con) polar lipids applied at 2.5 or 5.0 mg. The graph shows the percentage (%) of each compound relative to the total area of ​​compounds identified for each reaction mixture. Bars not shown for a compound mean that the compound was not detected in that mixture under the specified analytical conditions. [Figure 4-2] Graphical representation of volatile compounds identified by HS-SPME-GCMS in reaction mixtures containing ARA-PC or 18:0 / 18:1-PC (Con) polar lipids applied at 2.5 or 5.0 mg. The graph shows the percentage (%) of each compound relative to the total area of ​​compounds identified for each reaction mixture. Bars not shown for a compound mean that the compound was not detected in that mixture under the specified analytical conditions. [Figure 4-3]Graphical representation of volatile compounds identified by HS-SPME-GCMS in reaction mixtures containing ARA-PC or 18:0 / 18:1-PC (Con) polar lipids applied at 2.5 or 5.0 mg. The graph shows the percentage (%) of each compound relative to the total area of ​​compounds identified for each reaction mixture. Bars not shown for a compound mean that the compound was not detected in that mixture under the specified analytical conditions. [Diagram 5] Schematic diagram for making gene constructs to introduce inactivating deletions in genes of interest such as microbial FAD2 and URA3. Panel A. DNA synthesis of a 2 kb fragment with 1,000 bp 5' upstream and 1,000 bp 3' downstream regions of the gene of interest, bounded with a SacII site between the two regions. The positions of the restriction enzyme and lox sites are indicated by vertical lines. CDS: protein coding region of the gene of interest. B. Amplification of hygromycin (Hph) or nourseothricin (Nat1) antibiotic resistance genes using primers adapted to the SacII sites. C. Assembly of the gene construct by insertion of a SacII-terminated antibiotic resistance gene cassette into the DNA fragment of A. Not drawn to scale. [Figure 6] Schematic diagram of the construction of genetic constructs for introducing gene deletions into microorganisms. Panel A. PCR amplification of the 5' upstream and 3' downstream regions of the gene of interest and ligation to generate a 2 kb fragment. Oligonucleotide primers are indicated with small horizontal arrows, restriction enzyme sites and lox sites are indicated with vertical lines. CDS: protein coding region of the gene of interest. B. Amplification of hygromycin (Hph) or nourseothricin (Nat1) resistance genes using primers adapted to the flanking AsiSI sites. Assembly of genetic constructs for introduction into microorganisms such as CYlipolytica. [Figure 7] Schematic structure of a phospholipid. One of the hydroxyls can be replaced with a different head group, such as choline, serine, or inositol. [Figure 8] FIG. 1 is a schematic diagram of the phospholipid synthesis pathway. [Figure 9]1 shows meaty taste results of a sensory evaluation of samples containing various concentrations of Maillard reaction matrix and Mortierella alpina biomass. [Figure 10] 1 shows the hedonic results of a sensory evaluation of samples containing different concentrations of Maillard reaction matrix and Mortierella alpina biomass. [Figure 11] Figure 1 shows the combined meaty taste and pleasantness results of the sensory evaluation of samples containing different concentrations of Maillard reaction matrix and Mortierella alpina biomass. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0113] Sequence Listing Commentary [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0114] General Technology and Standards Definitions Unless expressly defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, fermentation, molecular genetics, protein chemistry, non-meat foods, and biochemistry).

[0115] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described, for example, in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TABrown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors),DNA Cloning:A Practical Approach,Volumes 1-4,IRL Press (1995 and 1996),and FMAusubel et al.(editors),Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience (1988,including all updates until present),Ed Harlow and David Lane(editors) Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory,(1988),and JEColigan et al.(editors) It is described and explained throughout the literature, such as Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0116] The term "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y" and shall be interpreted as providing explicit support for both meanings or either meaning.

[0117] As used herein, unless otherwise specified, the term about refers to + / - 20%, more preferably + / - 10%, more preferably + / - 5%, more preferably + / - 1% of the specified value.

[0118] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0119] Selected Definitions As used herein, "lipid" is any of a class of organic compounds that are or contain fatty acids, which may be esterified or non-esterified, or derivatives thereof, and are insoluble in water, but soluble in organic solvents, such as chloroform. As used herein, the term "extracted lipid" refers to a lipid composition extracted from microbial cells. The extracted lipid may be a relatively crude composition, obtained, for example, by lysing the cells, or a more purified composition in which most, if not all, of one or more of the water, nucleic acids, proteins, and carbohydrates derived from the cells have been removed. Examples of purification methods are described below. In one embodiment, the extracted lipid comprises at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w / w) lipid by weight of the composition. In an embodiment, the extracted lipid comprises about 10% to 95% lipid by weight, for example, about 10% to about 50% or about 50% to 95% lipid by weight. The lipid may be solid or liquid at room temperature (25° C.), or a mixture of the two, and if liquid, is considered an oil, and if solid, is considered a fat. In one embodiment, the extracted lipid of the present invention is not blended with another lipid produced from another source, for example, an animal lipid. Alternatively, the extracted lipid may be blended with a different lipid.

[0120] As used herein, the term "polar lipid" refers to amphipathic lipid molecules with a hydrophilic head and a hydrophobic tail, including phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol), cephalin, sphingolipids (sphingomyelin and glycosphingolipids), phosphatidic acid, cardiolipin, and glyceroglycolipids. Phospholipids are composed of the main structural units of fatty acids, glycerol, phosphoric acid, and amino alcohols. They are generally considered to be structural lipids that play an important role in the structure of plant, microbial, and animal membranes. Due to their chemical structure, polar lipids exhibit bipolarity and exhibit solubility or partial solubility in both polar and nonpolar solvents.

[0121] As used herein, the term "phospholipid" refers to an amphipathic molecule with a hydrophilic head and a hydrophobic tail, with a glycerol backbone esterified to the phosphate "head" group and two fatty acids providing the hydrophobic tail. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine. Due to their charged head group at neutral pH, phospholipids are polar lipids and have some solubility in solvents such as chloroform as well as ethanol. Phospholipids are the major components of all cell membranes. They can form lipid bilayers due to their amphipathic properties. Well-known phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin.

[0122] As used herein, the term "non-polar lipid" refers to fatty acids and their derivatives that are soluble in organic solvents but insoluble in water. The fatty acids may be free fatty acids and / or esterified forms. Examples of esterified forms include, but are not limited to, triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG). Non-polar lipids also include sterols, sterol esters, and wax esters. Non-polar lipids are also known as "neutral lipids" and, in some contexts, are also referred to as "oils". Non-polar lipids may be liquid or solid at room temperature, depending on the degree of unsaturation of the fatty acids in the non-polar lipid. Typically, the more saturated the fatty acid content, the higher the melting temperature of the lipid.

[0123] As used herein, the term fatty acid refers to a carboxylic acid consisting of an aliphatic hydrocarbon chain and a terminal carboxyl group. The hydrocarbon chain can be either saturated or unsaturated. Unsaturated fatty acids include monounsaturated fatty acids, which have only one carbon-carbon double bond, and polyunsaturated fatty acids (PUFAs), which have at least two carbon-carbon double bonds, typically 2-6 carbon-carbon double bonds. The fatty acids may be free fatty acids (FFAs) or esterified to a glycerol or glycerol phosphate molecule, a CoA molecule, or other head group known in the art, and are preferably esterified as part of a polar lipid, such as a phospholipid.

[0124] As used herein, the term "total fatty acid (TFA) content" or variations thereof refers to the total amount of fatty acids, for example, in extracted lipids or cells, on a weight basis. TFAs can be expressed as a percentage of the weight of cells or other fractions, for example, as a percentage of polar lipids. Unless otherwise specified, weights related to cell weight are dry cell weights (DCW). In one embodiment, TFA content is measured by converting fatty acids to fatty acid methyl esters (FAMEs) or fatty acid butyl esters (FABEs) and measuring the amount of FAMEs or FABEs by GC using the addition of a known amount of a unique fatty acid standard as a quantitative standard in the GC. Typically, the amount and fatty acid composition of lipids containing only fatty acids in the C10-C24 range are determined by conversion to FAMEs, while lipids containing fatty acids in the C4-C10 range are determined by conversion to FABEs. Thus, TFAs represent the weight of only fatty acids in the lipid, not the weight of fatty acids and their linked moieties.

[0125] "Saturated fatty acids" do not contain double bonds or other functional groups along the acyl chain. The term "saturated" refers to hydrogen in that all carbons (except the carboxylic acid [-COOH] group) contain as many hydrogens as possible.

[0126] "Unsaturated fatty acids" are of similar configuration to saturated fatty acids, except that there are one or more alkene functional groups along the chain, each replacing a singly bonded "-CH2-CH2-" portion of the chain with a doubly bonded "-CH=CH-" portion (i.e., a carbon that is doubly bonded to another carbon). The two next carbon atoms in the chain attached to either side of the double bond can occur in a cis or trans configuration, preferably in a cis configuration.

[0127] As used herein, the term monounsaturated fatty acid refers to a fatty acid that contains at least 12 carbon atoms in its carbon chain and only one alkene group (carbon-carbon double bond) in the chain. Monounsaturated fatty acids include C12:1Δ9, C14:1Δ9, C16:1Δ9 (palmitoleic acid), C18:1Δ9 (oleic acid) and C18:1Δ11 (vaccenic acid).

[0128] As used herein, the term "polyunsaturated fatty acid" or "PUFA" refers to a fatty acid that contains at least 12 carbon atoms in its carbon chain and at least two alkene groups (carbon-carbon double bonds). Usually, the number of carbon atoms in the carbon chain of a fatty acid refers to an unbranched carbon chain. Unless otherwise stated, if the carbon chain is branched, the number of carbon atoms excludes those in the side chain. The polar lipids of the present invention, such as those in the extracts or cells of the present invention, contain at least one omega-6 fatty acid with unsaturation (carbon-carbon double bond) at the sixth carbon-carbon bond from the methyl end of the fatty acid. Examples of ω6 fatty acids include, but are not limited to, arachidonic acid (ARA, C20:4Δ5,8,11,14; ω6), dihomo-γ-linolenic acid (DGLA, C20:3Δ8,11,14; ω6), eicosadienoic acid (EDA, C20:2Δ11,14; ω6), docosatetraenoic acid (DTA, C22:4Δ7,10,13,16; ω6), docosapentaenoic acid-ω6 (DPA-ω6, C22:5Δ4,7,10,13,16; ω6), γ-linolenic acid (GLA, C18:3Δ6,9,12; ω6), and linoleic acid (LA, C18:2Δ9,12; ω6). In some embodiments, the polar lipids of the invention, such as the extracts or cells of the invention, comprise at least one ω3 fatty acid having an unsaturation (carbon-carbon double bond) at the third carbon-carbon bond from the methyl end of the fatty acid. In some embodiments, the polar lipids of the invention, such as the extracts or cells of the invention, do not comprise any ω3 fatty acids, such as one or more of C16:3ω3, ALA, EPA and DHA. Examples of omega-3 fatty acids include, but are not limited to, alpha-linolenic acid (ALA, C18:3Δ9,12,15; ω3), hexadecatrienoic acid (C16:3ω3), eicosapentaenoic acid (EPA, C20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA, C22:5Δ7,10,13,16,19, ω3), docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19, ω3), eicosatetraenoic acid (ETA, C20:4Δ8,11,14,17; ω3), and eicosatrienoic acid (ETrA, C20:3Δ11,14,17; ω3).In some embodiments, the polar lipids of the invention, such as extracts or cells of the invention, do not comprise one or more or all of the following omega-3 fatty acids: C16:3 omega-3, EPA and DHA.

[0129] As used herein, "C12:0" refers to lauric acid.

[0130] As used herein, "C14:0" refers to myristic acid.

[0131] As used herein, "C15:0" refers to n-pentadecanoic acid.

[0132] As used herein, "C16:0" refers to palmitic acid.

[0133] As used herein, "C17:1" refers to heptadecenoic acid.

[0134] As used herein, "C16:1Δ9" refers to palmitoleic acid, or -hexadec-9-enoic acid.

[0135] As used herein, "C18:0" refers to stearic acid.

[0136] As used herein, "C18:1Δ9," sometimes abbreviated as "C18:1," refers to oleic acid.

[0137] As used herein, "C18:1Δ11" refers to vaccenic acid.

[0138] As used herein, "C20:0" refers to eicosanoic acid.

[0139] As used herein, "C20:1" refers to eicosenoic acid.

[0140] As used herein, "C22:0" refers to docosanoic acid.

[0141] As used herein, "C22:1" refers to erucic acid.

[0142] As used herein, "C24:0" refers to tetracosanoic acid.

[0143] A "triacylglyceride", "triacylglycerol", or "TAG" is a glyceride in which glycerol is esterified with three fatty acids, which may be the same (e.g., triolein) or, more commonly, different. All three of the fatty acids may be different, or two of the fatty acids may be the same and the third different. In the Kennedy pathway of TAG synthesis, DAG is formed as described below, and then the third acyl group is esterified to the glycerol backbone by the activity of diglyceride acyltransferase (DGAT). TAG is a form of non-polar lipid. The three acyl groups esterified in the TAG molecule are considered to be esterified at the sn-1, sn-2, and sn-3 positions, relative to their positions in the glycerol backbone of the TAG molecule. Although the sn-1 and sn-3 positions are chemically identical, biochemically the acyl groups esterified at the sn-1 and sn-3 positions differ in that separate and distinct acyltransferase enzymes catalyze the esterification.

[0144] "Diacylglyceride", "diacylglycerol", or "DAG" is a glyceride in which glycerol is esterified with two fatty acids, which may be the same or preferably different. As used herein, DAG contains hydroxyl groups at the sn-1, 3, or sn-2 positions, and therefore DAG does not include phosphorylated glycerolipid molecules such as PA or PC. In the Kennedy pathway of DAG synthesis, the precursor sn-glycerol-3-phosphate (G3P) is esterified to two acyl groups, each derived from a fatty acid coenzyme A ester, at the sn-1 position in a first reaction catalyzed by glycerol-3-phosphate acyltransferase (GPAT), to form LysoPA, followed by a second acylation at the sn-2 position catalyzed by lysophosphatidic acid acyltransferase (LPAAT) to form phosphatidic acid (PA). This intermediate is then dephosphorylated by PAP to form DAG.

[0145] As used herein, an "oil" is a composition that contains primarily lipids and is liquid at room temperature.

[0146] As used herein, an "oleaginous" cell or microorganism is a cell or microorganism that can store at least 20% of its cell mass, e.g., 20%-70%, lipid on a dry weight basis. The lipid content may depend on the culture conditions, as known in the art. It is understood that as long as a microorganism can synthesize and accumulate at least 20% lipid on a dry cell weight basis under at least one set of culture conditions, it is considered an oleaginous cell, even if it accumulates less than 20% lipid under different conditions. As used herein, a "microorganism derived from an oleaginous microorganism" is a microorganism derived from a precursor oleaginous microorganism by one or more genetic modifications. A microorganism derived from an oleaginous microorganism may itself be an oleaginous microorganism, or may produce less than 20% lipid and not be an oleaginous microorganism. The genetic modifications may be introduced by human intervention or may occur naturally, as long as at least one of the genetic modifications is introduced by human intervention. In one embodiment, the genetic modifications to generate the derived microorganism include one or more genetic modifications that result in reduced synthesis and / or accumulation of TAG.

[0147] As used herein, a "heterotrophic" cell is a cell that can utilize organic material as a carbon source for metabolism and growth. Heterotrophic organisms may also be capable of growing autotrophically under suitable conditions.

[0148] As used herein, "fermentation" refers to a metabolic process that brings about chemical changes in organic substrates through the action of enzymes within cells under conditions that either lack oxygen or where the oxygen level is reduced relative to air.

[0149] As used herein, "meat-like flavor and / or aroma" or "meat-related flavor and / or aroma" refers to flavors and / or aromas that are identical to or similar to one or more meats, such as beef, steak, chicken, e.g., roast chicken or chicken skin, pork, lamb, duck, venison, chicken, or other meat soups, meat broths, or liver. Such aromas are typically detected by human volunteers, e.g., by a qualified sensory panel. Meat-like or meat-related flavors and / or aromas can also be detected by evaluating volatile compounds that arise after cooking of a composition or food product. Volatile compounds exhibiting meat-like or meat-related aromas and flavors are known in the art and include, but are not limited to, those exemplified herein, such as 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethylbenzaldehyde, 2,3,4,5-tetramethylphenyl ether, 2,4-dimethylbenzaldehyde, 2,5,6-trimethylphenyl ether ... These include tetramethylcyclopent-2-en-1-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, trans-2-octen-1-ol, 1-nonanol, 1,3-bis(1,1-dimethylethyl)-benzene, 2-octen-1-ol, adamantanol-like compounds, hexanal, 2-pentylfuran, 1-octen-3-ol, 2-pentylthiophene, and 1,3,5-titriane.

[0150] microbial lipids Microbial lipids, particularly extracted microbial lipids, are provided which are suitable for use in compositions, foods, feeds and beverages to impart a meat-like aroma and / or flavor to the compositions, foods, feeds and beverages when the compositions, foods, feeds and beverages are heated.

[0151] In one aspect, there is provided an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, with the polar lipids preferably being present in the extracted microbial lipid in greater amounts than the non-polar lipids; (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content comprising ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, and the ω6 fatty acids comprise two, three, four or more fatty acids selected from the group consisting of arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6) and γ-linolenic acid (GLA); (b) the phospholipids in the polar lipids comprise at least two, preferably three or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), each of which comprises one or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA, and optionally one or more of phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin (Car), each of which comprises one or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA; (c) the polar lipids have a total saturated fatty acid content including palmitic acid and stearic acid; (d) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); (e) omega-3 fatty acids are absent from the polar lipid or are present in a total amount of less than about 3% by weight of the TFA content of the polar lipid, and / or the polar lipid lacks C16:2, C16:3 omega-3, EPA, and DHA.

[0152] In another aspect, there is provided an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, with the polar lipids preferably being present in the extracted microbial lipid in greater amounts than the non-polar lipids; (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content comprising ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6), or γ-linolenic acid (GLA), or any combination thereof; (b) the phospholipids in the polar lipids comprise phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), each of which comprises one or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA, and optionally one or more of phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin (Car), each of which comprises one or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA; (c) the polar lipids have a total saturated fatty acid content including palmitic acid and stearic acid; (d) Polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9cis).

[0153] In another aspect, the present invention provides an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, with the polar lipids preferably being present in the extracted microbial lipid in greater amounts than the non-polar lipids; (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content comprising ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6), or γ-linolenic acid (GLA), or any combination thereof; (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); (d) omega-3 fatty acids are absent from the polar lipid or are present in a total amount of less than about 3% by weight of the TFA content of the polar lipid, and / or the polar lipid lacks C16:2, C16:3 omega-3, EPA, and DHA.

[0154] In another aspect, the present invention provides an extracted microbial lipid comprising esterified omega-6 fatty acids in the form of polar lipids, (a) the polar lipids comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6), or γ-linolenic acid (GLA), or any combination thereof; (b) the phospholipids in the polar lipids comprise phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), each of which comprises one or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA, and optionally one or more of phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin (Car), each of which comprises one or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA; (c) the polar lipids have a total saturated fatty acid content including palmitic acid and stearic acid; (d) Polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9cis).

[0155] In another aspect, the present invention provides an extracted microbial lipid comprising esterified omega-6 fatty acids in the form of polar lipids, (a) the polar lipids comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising one or two or all three of eicosadienoic acid (EDA), docosatetraenoic acid (DTA), and docosapentaenoic acid-ω6 (DPA-ω6); (b) gamma-linolenic acid (GLA) is either absent from or present in polar lipids; (c) the polar lipids have a total saturated fatty acid content including palmitic acid and stearic acid; (d) Polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9cis).

[0156] In another aspect, an extracted microbial lipid is provided that comprises esterified omega-6 fatty acids in the form of polar lipids, (a) the polar lipids comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising two, three, four or more fatty acids selected from the group consisting of arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6) and γ-linolenic acid (GLA); (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); (d) The polar lipids lack C16:2, C16:3ω3, EPA, and DHA.

[0157] In another aspect, the present invention provides an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, with the polar lipids preferably being present in the extracted microbial lipid in greater amounts than the non-polar lipids; (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content that comprises ω6 fatty acids, at least a portion of the ω6 fatty acids are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids of the polar lipids comprise an amount of arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6) or γ-linolenic acid (GLA), or any combination thereof, each amount expressed as a weight percent of the total fatty acid content of the polar lipid, and the sum of the amounts of ARA, DGLA, EDA, DTA, DPA-ω6 and GLA is at least about 10%; (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis);

[0158] In another aspect, there is provided an extracted yeast lipid comprising esterified fatty acids in the form of either (i) polar lipids without any non-polar lipids, or (ii) polar lipids and non-polar lipids, the polar lipids of (a)(i) and (ii) comprise a total fatty acid (TFA) content comprising ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids of the polar lipids comprising an amount of arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6) or γ-linolenic acid (GLA), or any combination thereof, wherein the sum of the amounts of ARA, DGLA, EDA, DTA, DPA-ω6 and GLA is preferably at least about 5% by weight, more preferably at least about 10% by weight of the TFA content of the polar lipid; (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis);

[0159] In another aspect, there is provided an extracted Saccharomyces cerevisiae lipid comprising esterified omega-6 fatty acids in the form of polar lipids, (a) the polar lipids comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, and the ω6 fatty acids are one, two, three, four or more fatty acids selected from the group consisting of arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-ω6 (DPA-ω6) and γ-linolenic acid (GLA); (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis);

[0160] Also provided is an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, where the polar lipids are present in the extracted microbial lipid in greater amounts than the non-polar lipids; (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of the ω6 fatty acids are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids include arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA), ARA being present in an amount of about 10% to about 60% of the total fatty acid content of the polar lipids, DGLA being present in an amount of about 0.1% to about 5% of the total fatty acid content of the polar lipids, and GLA being present in an amount of about 1% to about 10% of the total fatty acid content of the polar lipids; (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); When the composition is heated, one or more compounds having meat-associated flavors and / or aromas are produced.

[0161] In the above embodiments, ARA may be present in an amount of about 20% to about 50% of the total fatty acid content of the polar lipids, DGLA may be present in an amount of about 1% to about 5% of the total fatty acid content of the polar lipids, and GLA may be present in an amount of about 3% to about 10%. In certain embodiments, ARA is present in an amount of about 25% to about 50%, or about 30% to about 50%. In other embodiments, ARA is present in an amount of about 10% to about 25% (or 10% to 20%) of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 0.5% to about 5%, and GLA is present in an amount of about 3% to about 10%.

[0162] Also provided is an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, where the polar lipids are present in the extracted microbial lipid in greater amounts than the non-polar lipids; (a) the polar lipids comprise a total fatty acid (TFA) content that includes ω6 fatty acids, the ω6 fatty acids being present in an amount of about 30% to about 70% of the total fatty acid content of the polar lipids, at least a portion of the ω6 fatty acids being esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA); (b) the polar lipids have a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content including oleic acid and palmitoleic acid (C16:1Δ9 cis); When the composition is heated, one or more compounds having meat-associated flavors and / or aromas are produced.

[0163] In some embodiments, the omega-6 fatty acids are present in an amount of about 40% to about 70%, about 40% to about 60%, or about 50% to about 60% of the total fatty acid content of the polar lipids. In one embodiment, ARA is present in an amount of about 20% to about 50% (e.g., 25% to about 50%, or about 30% to about 50%) of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 1% to about 5%, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipids.

[0164] The ratio of polar lipids to non-polar lipids in the extracted microbial lipids of the present invention can be at least 1.1:1, at least 1.5:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, between 1.1:1 and 10:1, between 1.1:1 and 5:1, or between 1.1:1 and 25.1:1.

[0165] In one embodiment, when the polar lipid comprises DPA-ω6, one or more or all of GLA, DGLA, EDA, ARA, and DTA are also present.In one embodiment, when the polar lipid comprises DPA-ω6, one or more or all of ARA, EPA, and DHA are also present.

[0166] In one embodiment, the polar lipid comprises EDA and one, two, or all three of arachidonic acid (ARA), dihomo-gamma-linolenic acid (DGLA), and gamma-linolenic acid (GLA) esterified in the polar lipid, and the level of EDA in the polar lipid is at least about 1% of the total fatty acid content of the polar lipid.

[0167] In one embodiment, the polar lipid lacks one, two, three, or all four of C16:2, C16:3ω3, EPA, and DHA. In a preferred embodiment, the polar lipid lacks C16:3ω3, EPA, and DHA. In a further embodiment, the polar lipid also lacks alpha-linolenic acid (ALA) or has less than 2% or less than 1% ALA. In a further embodiment, the polar lipid also lacks EPA or has less than 2% or less than 1% EPA. In a further embodiment, the polar lipid also lacks DHA or has less than 2% or less than 1% DHA.

[0168] In one embodiment, omega-3 fatty acids are present in a total amount of less than about 2% by weight, less than about 1% by weight, or between 3% and 0.1% by weight of the TFA content of the polar lipids.

[0169] In one embodiment, the extracted lipid comprises three, four or more fatty acids selected from the group consisting of ARA, DGLA, EDA, DTA, DPA-ω6 and GLA, such as a combination of ARA, DGLA and GLA, or a combination of fatty acids other than ARA, DGLA and GLA, preferably a combination of ARA, DGLA, GLA and at least one of EDA, DTA and DPA-ω6. In one embodiment, the total amount of ARA, DGLA, EDA, DTA, DPA-ω6 and GLA is about 10% to about 70%, or about 10% to about 75%, or about 10% to about 80%, each amount expressed as a percentage of the total fatty acid content of the polar lipid. In one embodiment, the ω6 fatty acid present in the greatest amount of the total fatty acid content of the polar lipid is neither LA nor ARA. In one embodiment, when the ω6 fatty acid present in the greatest amount is GLA or DGLA, the polar lipid comprises one or more of EDA, DTA or DPA-ω6.

[0170] In one embodiment, the phospholipids in the polar lipids comprise at least two, at least three, or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS), each of which comprises one, two, three, or four or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA, and optionally one or more or all of phosphatidic acid (PA), phosphatidylglycerol (PG) and cardiolipin (Car), each of which comprises one, two, three, or four or more of ARA, DGLA, EDA, DTA, DPA-ω6, and GLA.

[0171] In one embodiment, the polar lipid comprises myristic acid (C14:0) in an amount less than about 2% by weight of the total fatty acid content of the polar lipid. In a preferred embodiment, the polar lipid comprises myristic acid (C14:0) in an amount less than about 1% by weight of the total fatty acid content of the polar lipid.

[0172] In some embodiments, stearic acid is present at a level of less than about 14%, or less than about 12%, or less than about 10% of the total fatty acid content of the polar lipids. In preferred embodiments, stearic acid is present at a level of less than about 7%, or less than about 6%, or less than about 5%, preferably less than 4%, or less than 3% of the total fatty acid content of the polar lipids.

[0173] In some embodiments, ARA is present in an amount of about 10% to about 60%, about 10% to about 30%, about 10% to about 25%, about 15% to about 60%, about 20% to about 60%, or about 30% to about 60% by weight of the TFA content of the polar lipid. In preferred embodiments, ARA is present in an amount of about 20% to about 60%, or about 30% to about 60%, or about 40% to about 60%, or about 50% to about 60% by weight of the TFA content of the polar lipid.

[0174] In one embodiment, the extracted microbial lipid is extracted eukaryotic microbial lipid. In one embodiment, the extracted microbial lipid is extracted fungal microbial lipid.

[0175] In one embodiment, the extracted microbial lipid is an extracted fungal lipid, for example from a filamentous fungus or mold, or a eukaryotic microbial lipid. In one embodiment, the extracted fungal lipid is a lipid of a Mortierella sp., such as Mortierella alpina or Mortierella elongata. In one embodiment, the extracted fungal lipid is from the Genus Mucor, for example from the species Mucor hiemalis.

[0176] In one embodiment, the extracted microbial lipid is extracted yeast lipid, preferably Saccharomyces cerevisiae, Yarrowia lipolytica, or Pichia pastoris lipid.

[0177] In one embodiment, the polar lipids include one or more or all of EDA, DTA, and DPA-ω6.

[0178] In one embodiment, when the polar lipid comprises DGLA and ARA, or GLA, DGLA and ARA, the following: (a) at least one of EDA, DTA, and DPA-ω3 is also present in the polar lipid; (b) At least one of the following applies: the ratio of PC to PE, or the ratio of PC to phospholipids other than PC, is less than 3:1, less than 2:1, less than 1.5:1, less than 1.25:1, less than 1:1, between 3:1 and 1:1, between 2:1 and 1:1, or between 3:1 and 0.5:1.

[0179] In one embodiment, GLA is present in the polar lipid in an amount that is (i) less than the sum of the amounts of ARA, DGLA, EDA, DTA, and DPA-ω6 in the polar lipid, or (ii) less than the amount of ARA, less than the amount of DGLA, less than the amount of EDA, less than the amount of DTA, and less than the amount of DPA-ω6 in the polar lipid, or any combination thereof.

[0180] In embodiments, the saturated fatty acid content of the polar lipids comprises one or more or all of lauric acid (C12:0), myristic acid (C14:0), C15:0 fatty acid, C20:0, C22:0, and C24:0, preferably C14:0 and C24:0 or C14:0, C15:0 and C24:0, more preferably C14:0, C15:0 and C24:0, but not including C20:0 and C22:0.

[0181] In embodiments, lauric acid and myristic acid are absent from the polar lipid, or lauric acid and / or myristic acid are present in the polar lipid, and the sum of the amount of lauric acid and myristic acid in the polar lipid is less than about 2%, or less than about 1%, preferably less than about 0.5%, and more preferably less than about 0.2% of the total fatty acid content of the polar lipid.

[0182] In embodiments, C15:0 is not present in the polar lipids, or C15:0 is present in the polar lipids in an amount less than about 3%, preferably less than about 2%, or less than about 1% of the total fatty acid content of the polar lipids.

[0183] In embodiments, palmitic acid is present in the polar lipid in an amount from about 3% to about 45%, or from about 10% to about 40%, or from about 20% to about 45% of the total fatty acid content of the polar lipid.

[0184] In embodiments, palmitoleic acid is present in the polar lipid in an amount of from about 3% to about 45%, or from about 3% to about 25%, or from about 3% to about 20%, or from about 3% to about 15% of the total fatty acid content of the polar lipid.

[0185] In some embodiments, oleic acid is present in the polar lipid in an amount of from about 3% to about 60%, or from about 3% to about 40%, or from about 3% to about 25%, or from about 20% to about 60% of the total fatty acid content of the polar lipid.

[0186] In several embodiments, vaccenic acid is not present in the polar lipid, or vaccenic acid is present in the polar lipid in an amount less than about 2%, preferably less than about 1%, or less than about 0.5% of the total fatty acid content of the polar lipid.

[0187] In embodiments, linoleic acid is present in the polar lipid in an amount from about 3% to about 45%, or from about 3% to about 30%, or from about 3% to about 20% of the total fatty acid content of the polar lipid.

[0188] In some embodiments, gamma-linoleic acid is absent from the polar lipid, or gamma-linoleic acid is present in the polar lipid in an amount between about 3% and about 12%, or between about 3% and about 8%, or between about 3% and about 6%, or less than about 3% of the total fatty acid content of the polar lipid.

[0189] In some embodiments, eicosadienoic acid is not present in the polar lipid, or eicosadienoic acid is present in the polar lipid in an amount between about 3% and about 12%, or between about 3% and about 8%, or between about 3% and about 6%, or less than about 3% of the total fatty acid content of the polar lipid.

[0190] In some embodiments, dihomo-gamma-linolenic acid is not present in the polar lipid, or dihomo-gamma-linolenic acid is present in the polar lipid, preferably in an amount less than about 2%, between 0.1% and about 2%, between about 10% and about 60%, between about 12% and about 60%, or between about 15% and about 60% of the total fatty acid content of the polar lipid.

[0191] In several embodiments, C20:0 and C22:0 are not present in the polar lipids, or C20:0 and / or C22:0 are present in the polar lipids and the combined amount of C20:0 and C22:0 in the polar lipids is less than about 1.0%, or less than about 0.5%, preferably less than 0.2% of the total fatty acid content of the polar lipids.

[0192] In several embodiments, C24:0 is not present in the polar lipids, or C24:0 is present in the polar lipids in an amount less than about 1.0%, less than about 0.5%, preferably less than 0.3%, or less than 0.2% of the total fatty acid content of the polar lipids.

[0193] In several embodiments, C17:1 is not present in the polar lipids, or C17:1 is present in the polar lipids in an amount less than about 5%, preferably less than about 4% or less than about 3%, more preferably less than about 2% of the total fatty acid content of the polar lipids.

[0194] In embodiments, monounsaturated fatty acids that are C20 or C22 fatty acids are not present in the polar lipids, or C20:1 and / or C22:1 are present in the polar lipids, and the sum of the amounts of C20:1 and C22:1 in the polar lipids is less than about 1.0%, less than about 0.5%, preferably less than 0.2% of the total fatty acid content of the polar lipids.

[0195] In some embodiments, (i) the content of ω6 fatty acids in the polar lipids that are C20 or C22 fatty acids is about 5% to about 60%, preferably about 10% to about 60%, of the total fatty acid content of the polar lipids, and / or (ii) the content of ω6 fatty acids in the polar lipids that are ω6 fatty acids having 3, 4 or 5 carbon-carbon double bonds is about 5% to about 70%, preferably about 10% to about 70%, more preferably about 40% to about 70%, or about 45% to about 70%, or about 50% to about 70% of the total fatty acid content of the polar lipids.

[0196] In some embodiments, C16:3ω3 is absent from polar lipids, or both C16:2 and C16:3ω3 are absent from polar lipids.

[0197] In embodiments, the extracted lipids contain PC and / or are devoid of cyclopropane fatty acids, preferably devoid of C15:0c, C17:0c, and C19:0c.

[0198] In one embodiment, the weight of the extracted microbial lipid is at least 100 mg, preferably at least 1 g. In one embodiment, the extracted microbial lipid is in a liquid form having a volume of at least 1 ml, preferably at least 10 ml.

[0199] Fatty acid biosynthesis Biosynthesis of ω6 fatty acids in organisms such as microalgae, mosses, and fungi usually occurs as a series of oxygen-dependent desaturation and elongation reactions (FIG. 1). Polynucleotides encoding these enzymes can be used to genetically engineer microorganisms to produce the extracted lipids of the present invention. The desaturase and elongase proteins, and the genes encoding them, that may be used in the present invention are either those known in the art or homologs or derivatives thereof. All of the desaturase enzymes shown to be involved in ω6 fatty acid biosynthesis belong to the group of so-called "front-end" desaturases. Preferred proteins, or combinations of proteins, are those encoded by the genetic constructs provided herein, such as the amino acid sequences provided as SEQ ID NOs: 1-19.

[0200] The activity of either an elongase or desaturase for use in the present invention may be tested by expressing the gene encoding the enzyme in a microbial cell, such as a yeast cell, and determining whether the cell has an increased ability to produce ω6 fatty acids compared to a comparable cell in which the enzyme is not expressed.

[0201] Although certain enzymes are specifically described herein as "bifunctional," the absence of such a term does not necessarily mean that the particular enzyme has no activities other than those specifically defined.

[0202] Desaturase As used herein, the term "desaturase" refers to an enzyme capable of introducing a carbon-carbon double bond into the acyl group of a fatty acid substrate, typically in an esterified form, for example, as an acyl-CoA ester. The acyl group may be esterified to a phospholipid, such as phosphatidylcholine (PC), or to an acyl carrier protein (ACP), or preferably to CoA. Desaturases may generally be classified accordingly into three groups. In one embodiment, the desaturase is a front-end desaturase.

[0203] As used herein, the term "front-end desaturase" refers to members of a class of enzymes that introduce a double bond between a carboxyl group and an existing unsaturated moiety of the acyl chain of a lipid, which are structurally characterized by the presence of an N-terminal cytochrome b5 domain along with a typical fatty acid desaturase domain containing three highly conserved histidine boxes (Napier et al., 1997).

[0204] As used herein, "Δ5-desaturase" refers to a protein capable of carrying out a desaturase reaction to introduce a carbon-carbon double bond into the fifth carbon-carbon bond from the carboxyl terminus of a fatty acid substrate. In one embodiment, the fatty acid substrate is DGLA and the enzyme produces ARA. In one embodiment, the Δ5-desaturase has greater activity on ω6 fatty acids as compared to the corresponding ω3 fatty acids. In one embodiment, the "Δ5-desaturase" is capable of converting DGLA-CoA to ARA-CoA, i.e., an acyl-CoA desaturase. In one embodiment, the "Δ5-desaturase" is capable of converting DGLA esterified at the sn-2 position of PC. Examples of Δ5-desaturases are listed in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a). In one embodiment, the Δ5-desaturase comprises amino acids having a sequence provided in SEQ ID NO: 15, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO: 15. In another embodiment, the Δ5-desaturase comprises amino acids having a sequence provided in SEQ ID NO: 16, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO: 16. In another embodiment, the Δ5-desaturase is from Pavlova salina or Mortierella alpina.

[0205] As used herein, "Δ6-desaturase" refers to a protein capable of carrying out a desaturase reaction that introduces a carbon-carbon double bond at the sixth carbon-carbon bond from the carboxyl terminus of a fatty acid substrate. Preferably, the Δ6-desaturase has greater activity on ω6 fatty acids when compared to the corresponding ω3 fatty acids. In one embodiment, the fatty acid substrate is LA and the enzyme produces GLA. In one embodiment, the "Δ6-desaturase" is capable of converting LA-CoA to GLA-CoA, i.e., is an acyl-CoA desaturase. In one embodiment, the "Δ6-desaturase" is capable of converting LA esterified at the sn-2 position of PC. In a further embodiment, the Δ6-desaturase has activity on both the fatty acid substrate LA-CoA and LA bound to the sn-2 position of PC. Preferably, the Δ6-desaturase has greater activity on LA-CoA than on LA-PC. A Δ6-desaturase can also have activity as a Δ5-desaturase, in which case it is called a Δ5 / Δ6 bifunctional desaturase, so long as it has greater Δ6-desaturase activity towards LA than Δ5-desaturase activity towards DGLA. Examples of Δ6-desaturases are listed in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a). Preferred Δ6-desaturases are from Mortierella alpina or Ostreococcus tauri.

[0206] In one embodiment, the Δ6-desaturase is further characterized by having greater Δ6-desaturase activity towards linoleic acid (LA, C18:2Δ9,12,ω6) than α-linolenic acid (ALA, C18:3Δ9,12,15,ω3) as fatty acid substrate.

[0207] In one embodiment, the Δ6-desaturase has no detectable Δ5-desaturase activity towards ETA. In another embodiment, the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:5 or SEQ ID NO:6, or a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO:5 or SEQ ID NO:6. The Δ6-desaturase may or may not also have Δ8-desaturase activity.

[0208] As used herein, "Δ8-desaturase" refers to a protein capable of carrying out a desaturase reaction to introduce a carbon-carbon double bond into the eighth carbon-carbon bond from the carboxyl terminus of a fatty acid substrate. The Δ8-desaturase can convert at least EDA to DGLA. In one embodiment, the Δ8-desaturase can convert EDA-CoA to DGLA-CoA, i.e., is an acyl-CoA desaturase. In one embodiment, the Δ8-desaturase can convert EDA esterified at the sn-2 position of PC. Preferably, the Δ8-desaturase has greater activity on EDA-CoA than on EDA-PC. The Δ8-desaturase can also have activity as a Δ6-desaturase, and is referred to as a Δ6 / Δ8 bifunctional desaturase, insofar as it has greater Δ8-desaturase activity on EDA than on Δ6-desaturase activity on LA. In one embodiment, the Δ8-desaturase comprises amino acids having a sequence as provided in SEQ ID NO: 14, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO: 14. In one embodiment, the Δ8-desaturase is a Pavlova salina Δ8-desaturase.

[0209] As used herein, "Δ12-desaturase" refers to a protein capable of carrying out a desaturase reaction to introduce a carbon-carbon double bond at the 12th carbon-carbon bond from the carboxyl terminus of a fatty acid substrate. Δ12-desaturases typically convert either oleoyl-phosphatidylcholine or oleoyl-CoA to linoleoyl-phosphatidylcholine (C18:1-PC) or linoleoyl-CoA (C18:1-CoA), respectively. The subclass that uses PC-linked substrates is referred to as phospholipid-dependent Δ12-desaturases, and the latter subclass is referred to as acyl-CoA-dependent Δ12-desaturases. Plant and fungal Δ12-desaturases are generally of the former subclass, whereas animal Δ12-desaturases are generally of the latter subclass, with the exception of some lower animal Δ12-desaturases, such as the C. elegans Δ12-desaturase, e.g., the Δ12-desaturases encoded by genes cloned from insects by Zhou et al. (2008). Many other Δ12-desaturase sequences can be readily identified by searching sequence databases. In one embodiment, the Δ12-desaturase comprises amino acids having a sequence as provided in any one of SEQ ID NOs:1-4, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NOs:1-4. In one embodiment, the Δ12-desaturase is a Lachancea kluyveri, Y. lipolytica, Acheta domesticus, or Fusarium moniliforme Δ12-desaturase. In a preferred embodiment, the Δ12-desaturase is a fungal Δ12-desaturase or a fungal. As used herein, "fungal Δ12-desaturase" refers to a Δ12-desaturase from a fungal source, including an oomycete source, or a variant whose amino acid sequence is at least 95% identical thereto. Genes encoding a number of desaturases have been isolated from fungal sources. US 7,211,656 describes a Δ12 desaturase from Saprolegnia diclina.WO2009016202 describes fungal desaturases from Helobdella robusta, Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosiga brevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola, Naegleria gruben, Nectria haematococca, Nematostella vectensis, Phycomyces blakesleeanus, Trichoderma resii, Physcomitrella patens, Postia placenta, Selaginella moellendorffii and Microdochium nivale. WO2005 / 012316 describes Δ12-desaturases from Thalassiosira pseudonana and other fungi. WO2003 / 099216 describes genes encoding fungal Δ12-desaturases isolated from Neurospora crassa, Aspergillus nidulans, Botrytis cinerea, and Mortierella alpina.

[0210] As used herein, "Δ4-desaturase" refers to a protein capable of performing a desaturase reaction to introduce a carbon-carbon double bond at the fourth carbon-carbon bond from the carboxyl terminus of a fatty acid substrate. The Δ4-desaturase is capable of converting at least DTA to DPA-ω6 (C22:5Δ4,7,10,13,16). Preferably, the Δ4-desaturase has greater activity on ω6 fatty acids when compared to the corresponding ω3 fatty acids. In one embodiment, the Δ4-desaturase is capable of converting DTA-CoA to DPAω6-CoA, i.e., is an acyl-CoA desaturase. In one embodiment, the Δ4-desaturase is capable of converting DTA esterified at the sn-2 position of PC to DPAω6-PC. The desaturation step to produce DPAω6 from DTA is catalyzed in non-mammalian organisms by a Δ4-desaturase, and genes encoding this enzyme have been isolated from the freshwater protist Euglena gracilis and the marine species Thraustochytrium sp. (Qiu et al., 2001; Meyer et al., 2003). In one embodiment, the Δ4-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:18, or a Pavlova spp. Δ4-desaturase, such as a Pavlova salina Δ4-desaturase, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO:18. In one embodiment, the Δ4-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:19, or a Thraustochytrium sp. Δ4-desaturase, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO:19.

[0211] In one embodiment, a desaturase for use in the invention has greater activity on an acyl-CoA substrate than on the corresponding acyl-PC substrate. In another embodiment, a desaturase for use in the invention has greater activity on an acyl-PC substrate than on the corresponding acyl-CoA substrate, but has some activity on both substrates. As mentioned above, "corresponding acyl-PC substrate" refers to a fatty acid esterified at the sn-2 position of phosphatidylcholine (PC), which fatty acid is the same fatty acid as the acyl-CoA substrate. In one embodiment, the greater activity is at least two-fold greater. To test which substrate a desaturase acts on, i.e., on an acyl-CoA or acyl-PC substrate, an assay can be performed in yeast cells as described in Domergue et al. (2003 and 2005). The availability of an acyl-CoA substrate for a desaturase can also be inferred when an elongase is expressed together with the desaturase, e.g., where the elongase catalyzes the elongation of the product of the desaturase, there is a high enzymatic conversion efficiency, such as at least about 90%.

[0212] Elongated gauze Biochemical evidence suggests that fatty acid elongation consists of four steps: condensation, reduction, dehydration, and a second reduction. In the context of the present invention, "elongase" refers to a polypeptide that catalyzes the condensation step in the presence of other members of the elongation complex under suitable physiological conditions. It has been shown that heterologous or homologous expression in cells of only the condensation component ("elongase") of the elongation protein complex is required for the elongation of each acyl chain. Thus, the introduced elongase can successfully recruit the reduction and dehydration activities from the transgenic host to perform a successful acyl elongation. The specificity of the elongation reaction with respect to chain length and degree of unsaturation of the fatty acid substrate is believed to reside within the condensation component. This component is also believed to be rate-limiting in the elongation reaction.

[0213] As used herein, a "Δ6-elongase" is capable of converting at least GLA to DGLA. In one embodiment, the elongase comprises amino acids having a sequence as provided in SEQ ID NO: 13, a biologically active fragment thereof, or an amino acid sequence that is at least 60% identical to SEQ ID NO: 13. In one embodiment, the Δ6-elongase is from Physcomitrella patens (Zank et al., 2002; Accession No. AF428243) or Thalassiosira pseudonana (Ruiz-Lopez et al., 2012). In a preferred embodiment, the Δ6-elongase is from Pyramimonas cordata. In a further embodiment, the Δ6-elongase has greater activity on ω6 substrates than the corresponding ω3 substrates.

[0214] As used herein, a "Δ9-elongase" is capable of converting at least LA to EDA. In one embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in any one of SEQ ID NOs: 9-12, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to any one of SEQ ID NOs: 9-12. In a further embodiment, the Δ9-elongase has greater activity towards an ω6 substrate than the corresponding ω3 substrate.

[0215] As used herein, the term "having greater activity on an ω6 substrate than on a corresponding ω3 substrate" refers to the relative activity of an enzyme on substrates that differ due to the action of an ω3 desaturase.

[0216] Elongases for use in the present invention have activity only on acyl-CoA substrates and not on the corresponding acyl-PC substrates.

[0217] Other genes In addition to expression of the desaturases and elongases described above, production of ω6 fatty acids in polar lipids of microbial cells can be enhanced by genetic modifications to regulate expression of one or more endogenous genes involved in microbial fatty acid biosynthesis, catabolism, and regulation. Exemplary such microbial genes are provided in Table 1.

[0218] In some embodiments, the genetic modification to increase production of ω6 fatty acids in polar lipids provides for increased expression and / or activity of one or more genes in Table 1. In some embodiments, the genetic modification provides for increased expression and / or activity of fatty acid synthesis genes (see Table 1 for examples). In some embodiments, the genetic modification provides for increased expression and / or activity of phospholipid synthesis genes (see Table 1 for examples). In some embodiments, the genetic modification provides for increased expression and / or activity of lipid synthesis regulatory genes (see Table 1 for examples).

[0219] In some embodiments, the genetic modification to increase production of omega-6 fatty acids in polar lipids reduces or prevents expression and / or activity of one or more genes in Table 1. In some embodiments, the genetic modification reduces or prevents expression and / or activity of lipid catabolic genes (see Table 1 for examples). [ka] [ka] [ka] [ka] [ka]

[0220] Phospholipid synthesis in microorganisms As the main structural components of biological membranes, phospholipids play important roles in cell morphology and organelle function, with some also acting as secondary messengers. Phospholipids are amphipathic molecules with a glycerol backbone and two fatty acids esterified to the phosphate head groups (Figure 7). Due to their charged head groups at neutral pH, they are polar lipids and exhibit some solubility in solvents such as ethanol in addition to solvents such as chloroform. The most common fatty acids esterified to the glycerophosphate backbone of phospholipids in eukaryotic microorganisms such as S. cerevisiae include palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), and oleic acid (C18:1) (Carman and Gil-Soo, 2011). The major phospholipids found in whole-cell extracts from S. cerevisiae are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). Phosphatidylglycerol (PG) and cardiolipin (CL) are minor phospholipids in whole S. cerevisiae cell extracts, but are the major phospholipids in mitochondrial lipids (Zhang et al., 2014). Other yeasts, such as Y. lipolytica and Schizosaccharomyces pombe, have similar phospholipid compositions (Fernandez et al., 1986; Fakas 2017). In contrast, the phospholipid composition of prokaryotes, such as Escherichia coli, is composed mainly of PE, PG, and CL, and these phospholipids contain primarily fatty acids 16:0, 16:1, and 18:1Δ11 (De Siervo 1969). E. coli and many other bacteria lack PC.

[0221] The enzymes and corresponding genes involved in the synthesis of phospholipids in microorganisms are listed in Table 1, and the pathway of phospholipid synthesis is outlined in Figure 8. The enzymes and genes involved in phospholipid synthesis in yeast have been extensively characterized in S. cerevisiae (Carman and Zeimetz, 1996). The specific synthesis of phospholipids begins with the synthesis of phospholipid phosphatidic acid (PA), which is generated from glycerol-3-phosphate or dihydroxyacetone phosphate after a fatty acyl-coenzyme A (CoA)-dependent reaction catalyzed by glycerol-3-phosphate acyltransferase and lysophospholipid acyltransferase (Athenstaedt and Daum, 1997; Athenstaedt et al., 1999; Zheng and Zou, 2001). All major phospholipid classes in S. cerevisiae are synthesized from a common precursor: cytidine diphosphate diacylglycerol (CDP-DAG). CDP-DAG is synthesized in a reaction catalyzed by CDP-DAG synthase, which converts PA to CDP-DAG using cytidine triphosphate (CTP) as the CDP donor (Carter and Kennedy 1966; Shen et al., 1996). CDP-DAG is a key intermediate for the synthesis of all major and minor phospholipids in S. cerevisiae, as well as in all other yeasts. In one reaction, CDP-DAG donates its phosphatidyl moiety to inositol to form PI in a reaction catalyzed by PI synthase (Nikawa and Yamashita, 1984). The inositol used in this reaction is derived from glucose-6-phosphate via reactions catalyzed by inositol-3-phosphate synthase (Klig and Henry, 1984; Dean-Johnson and Henry, 1989) and inositol-3-phosphate phosphatase (Murray and Greenberg, 2000). The inositol used in the synthesis of PI can also be utilized from exogenously supplied inositol in the medium by inositol permease.CDP-DAG may also donate its phosphatidyl moiety to glycerol-3-phosphate to form phosphatidylglycerophosphate (PGP) in a reaction catalyzed by PGP synthase (Chang et al., 1998). PGP is then dephosphorylated to PG by PGP phosphatase (Osman et al., 2010). Cardiolipin (CL) synthase catalyzes the reaction between PG and another molecule of CDP-DAG to produce CL (Chang et al., 1998). The final enzyme that utilizes CDP-DAG is PS synthase (Letts et al., 1983), which catalyzes the formation of PS by replacing CMP with serine from CDP-DAG (Kanfer and Kennedy, 1964). PS is then decarboxylated to PE by the PS decarboxylase enzyme (Trotter et al., 1993). PE is then converted to PC by a three-step S-adenosylmethionine (AdoMet)-dependent methylation reaction, the first of which is catalyzed by PE methyltransferase and the last two by phospholipid methyltransferase (Kodaki and Yamashita, 1987).

[0222] PE and PC can also be synthesized from exogenously supplied ethanolamine and choline through the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway (Nikawa et al., 1987). Exogenously supplied ethanolamine and choline are phosphorylated by ethanolamine kinase and choline kinase with ATP to form phosphoethanolamine and phosphocholine, respectively (Kim et al., 1999; Hosaka et al., 1989). These intermediates are then activated with CTP to form CDP-ethanolamine and CDP-choline, respectively, through phosphoethanolamine cytidylyltransferase and phosphocholine cytidylyltransferase (Min-Seok et al., 1996; Tsukagoshi et al., 1987). Ethanolamine phosphotransferase and choline phosphotransferase then convert CDP-ethanolamine and CDP-choline in a reaction with DAG to form PE and PC (Hjelmstad and Bell 1988; Hjelmstad and Bell, 1991). CTP, required for the synthesis of CDP-DAG, CDP-ethanolamine, and CDP-choline, is derived from UTP by the action of the CTP synthase enzyme. DAG, used for the synthesis of PE and PC via the Kennedy pathway, is derived from PA by the PA phosphatase encoded by PAH1 (Han et al., 2006). DAG generated in the PA phosphatase reaction can be converted back to PA by DAG kinase (Han et al., 2008a; Han et al., 2008b) or used for the synthesis of the neutral lipid TAG by the acyltransferase enzymes encoded by DGA1 and LRO1. Furthermore, additional acyltransferase enzymes involved in the synthesis of ergosterol esters can also acylate DAG to form TAG.

[0223] The Kennedy pathway plays a key role in the synthesis of PE and PC when enzymes in the CDP-DAG pathway are non-functional or defective (Carman and Henry, 1999; Greenberg and Lopes, 1996). For example, mutants defective in the three-step methylation of PE require choline supplementation for growth and synthesize PC via the CDP-choline branch of the Kennedy pathway. Mutants defective in the synthesis of PS or PE can synthesize PC when supplemented with ethanolamine or choline, respectively. Ethanolamine is incorporated into PE via the CDP-ethanolamine branch of the Kennedy pathway, and PE is then methylated to form PC. Mutants defective in the CDP-DAG pathway can also synthesize PE or PC when supplemented with lysoPE, lysoPC, or PC with short acyl chains. LysoPE and lysoPC transported into cells are acylated to PE and PC, respectively, by lysophospholipid acyltransferase, which also utilizes lysoPA as a substrate. Furthermore, Kennedy pathway mutants defective in both the CDP-choline and CDP-ethanolamine branches can synthesize PC only through the CDP-DAG pathway, however, unlike the CDP-DAG pathway mutants, they do not exhibit auxotrophy and have an essentially normal phospholipid complement.

[0224] When cells are grown in the absence of ethanolamine and choline, evidence supports that the CDP-DAG pathway is primarily responsible for the synthesis of PE and PC (Carman and Henry 1989). However, when these precursors are not supplemented in the culture medium, the Kennedy pathway may contribute to the synthesis of PE and PC. For example, PC synthesized by the CDP-DAG pathway is always hydrolyzed to choline and PA by phospholipase D. Choline is then incorporated back into PC via the CDP-choline branch of the Kennedy pathway, and PA is converted to other phospholipids via the intermediates CDP-DAG and DAG.

[0225] The details provided above about S. cerevisiae phospholipid synthesis, as well as the genes and enzymes involved, were found to also apply to the oleaginous yeast Yarrowia lipolytica. Another common yeast, S. pombe, uses a pathway for PL biosynthesis that is very similar to that of S. cerevisiae. However, there is one major difference between S. pombe and S. cerevisiae. S. pombe is a natural inositol auxotroph and cannot form L-myo-inositol 3-phosphate from its precursor glucose 6-phosphate, and therefore cannot grow in the absence of inositol. As a result, the PI content of S. pombe cells is strongly dependent on the concentration of inositol in the growth medium. The inositol auxotrophy of S. pombe is due to the lack of inositol-3-phosphate synthase, encoded by the INO1 gene of S. cerevisiae, as evidenced by the observation that expression of Pichia pastoris inositol-3-phosphate synthase in S. pombe can convert this natural inositol auxotroph into an inositol prototroph.

[0226] Phospholipids in E. coli and other Gram-negative bacteria are used to build the inner and outer membranes. E. coli has only three major phospholipid species in its membranes, with PE constituting the majority of phospholipids (75%), and PG and CL forming the remaining portion at 15-20% and 5-10%, respectively. Bacterial phospholipid synthesis begins with the acylation of glycerol 3-phosphate (G3P) to form lysophosphatidic acid (lysoPA). This detergent-like intermediate undergoes a second acylation to form phosphatidic acid (PA), the main precursor of bacterial phospholipids. The major PL in E. coli is synthesized from PA by enzymes of the CDP-DAG pathway, as described for S. cerevisiae. In summary, an acyl transfer module deposits PA into the membrane, where it is activated to CDP-DAG by CDP-DAG synthase. This intermediate is used for both PE synthesis via PS synthase and PS decarboxylase (Psd). PG is formed from the same intermediate by PGP synthase, the phosphorylated intermediate is dephosphorylated by PGP phosphatase, and finally, CL is produced by the condensation of two PG molecules by CL synthase.

[0227] microbial cells A wide variety of different microbial cells can be used in the present invention. In one embodiment, microbial cells exist as single-celled organisms, although such cells may aggregate with one another. Examples of microbial cells of the present invention include bacterial cells and eukaryotic cells, such as fungal and algal cells. Eukaryotic organisms are preferred over bacterial (prokaryotic) microorganisms. As used herein, the terms "microbial cell", "microbe" and "microorganism" mean the same thing.

[0228] In one embodiment, the microbial cells are suitable for fermentation, but can also be cultured under ambient oxygen concentrations. In another embodiment, the microbial cells are oleaginous cells, preferably oleaginous eukaryotes, or are preferably derived from prooleaginous microorganisms, such as proeukaryotic oleaginous microorganisms. In another embodiment, the microbial cells are heterotrophic cells, preferably heterotrophic eukaryotes. Preferably, the microbial cells have at least two of these, and more preferably are characterized by all of these characteristics.

[0229] In one embodiment, the cell of the present invention is a yeast cell. Examples of yeast cells useful in the present invention include, but are not limited to, Saccharomyces sp., such as Saccharomyces cerevisiae, Yarrowia sp., such as Yarrowia lipolytica, Pichia sp., such as Pichia pastoris, Candida sp., such as Candida rugosa, Aspergillus sp., such as Aspergillus niger, Cryptococcus sp., such as Cryptococcus curvatus, Lipomyces sp., such as Lipomyces starkeyi, Rhodosporidium sp., such as Rhodosporidium toruloides, Rhodotorula sp., such as Rhodotorula glutinis, and Trichosporon sp., such as Trichosporon fermentans.

[0230] In one embodiment, the fungal cell is a mold cell. Examples of mold cells useful in the present invention include, but are not limited to, Cunninghamella sp., such as Cunninghamella echinulate, Mortierella sp., such as Mortierella alpina, Mortierella elongata, and Mortierella exigua, Mucorales sp., such as Mucorales fungi, and Trichoderma sp., such as Trichoderma harzianum.

[0231] In one embodiment, the cell is a bacterial cell. Examples of bacterial cells useful in the present invention include, but are not limited to, Acinetobacter, such as Acinetobacter baylyi, Alcanivorax sp., such as Alcanivorax borkumensis, Gordonia sp., such as DG, Mycobacterium sp., such as Mycobacterium tuberculosis, Nocardia sp., such as Nocardia globerula, Rhodococcus sp., such as Rhodococcus opacus, and Streptomyces sp., such as Streptomyces coelicolor.

[0232] In one embodiment, the cell is an algae cell, such as a microalgae cell or a Bacillariophyceae cell. Examples of algal cells useful in the present invention include, but are not limited to, Prototheca sp., such as Prototheca moriformis, Thraustochytrium spp., Chlorella sp., such as Chlorella protothecoides, Chlorella vulgaris, or Chlorella ellipsoidea, Schizochytrium sp., such as Schizochytrium strain FCC-1324, Dunaliella sp., Haematococcus sp., such as Haematococcus pluvialis, Neochloris sp., such as Neochloris oleabundans, e.g., strain UTEX #1185, Pseudochlorococcum sp., Scenedesmus sp., such as Scenedesmus obliquus, Tetraselmis sp., such as Tetraselmis chui or Tetraselmis tetrathele, Chaetoceros sp., such as Chaetoceros calcitrans, Chaetoceros gracilis, or Chaetoceros muelleri, sp., Nitzschia sp. such as Nitzschia cf. pusilla, Phaeodactylum sp. such as Phaeodactylum tricornutum, Skeletonema sp. such as CS 252 strain, Thalassiosira sp. such as Thalassiosira pseudonana, Crypthecodinium sp. such as Crypthecodinium cohnii, Isochrysis sp. such as Isochrysis zhangjiangensis, Nannochloropsis sp. such as Nannochloropsis oculata (e.g. NCTU-3 strain), Pavlova sp. such as Pavlova salina, Rhodomonas sp. and Thalassiosira sp. such as Thalassiosira weissflogii.

[0233] In one embodiment, the cell is a genetically modified microorganism.

[0234] In some embodiments, the genetically modified microorganism comprises: (i) synthesizing, or increasing the synthesis of, one or more omega-6 fatty acids in a microorganism; (ii) increasing total fatty acid synthesis and / or accumulation in the microorganism; (iii) increasing total polar lipid synthesis and / or accumulation in the microorganism; (iv) a decrease in triacylglycerol (TAG) synthesis and / or accumulation in a microorganism, or an increase in TAG catabolism in a microorganism, preferably an increase in TAG lipase activity, or (v) reducing the catabolism of total fatty acids in microorganisms; or any combination thereof.

[0235] The genetic modification may include the introduction of an exogenous polynucleotide, the mutation or deletion of a gene or regulatory sequence, or any other known genetic modification. Suitable techniques for genetically modifying microorganisms are described herein.

[0236] In one embodiment, the genetic modification provides at least two of (i) to (v) above, preferably (iv) and (v), or (i), (iv) and (v).

[0237] In one embodiment, the genetic modification is selected from the group consisting of: (i) one or more exogenous polynucleotides encoding a Δ12 desaturase, a Δ6 desaturase, a Δ6 elongase, a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, a Δ5 elongase, a Δ4 desaturase, or any combination thereof; (ii) one or more genetic modifications that result in increased expression and / or activity of acetyl-CoA synthetase, ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthase subunit beta, or fatty acid synthase subunit alpha, or any combination thereof; (iii) one or more genetic modifications that result in increased expression and / or activity of CDP-DAG synthase, phosphatidylinositol synthase, phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, phospholipid methyltransferase, phosphatidylinositol transfer protein, phosphatidylinositol / phosphatidylcholine transfer protein, phosphatidylinositol phosphatase, phosphatidic acid cytidylyltransferase, or diacylglycerol kinase (DGK); (iv) one or more genetic modifications that result in a decrease in expression and / or activity of DGAT1, DGAT2, LRO1, ARE1, or ARE2; (v) one or more genetic modifications that result in decreased expression and / or activity of cholesterol esterase / TAG lipase, TAG lipase, phospholipase B, phospholipase D, acyl-CoA oxidase, acyl-CoA oxidase 2, acyl-CoA oxidase 3, acyl-CoA oxidase 5, multifunctional oxidative protein, or peroxisomal oxoacylthiolase.

[0238] Preferred combinations of enzymes encoded by polynucleotides in (i) according to the Δ6 desaturase pathway are (a) a Δ12 desaturase and a Δ6 desaturase to produce GLA, (b) a Δ12 desaturase, a Δ6 desaturase and a Δ6 elongase to produce GLA and DGLA, (c) a Δ12 desaturase, a Δ6 desaturase, a Δ6 elongase and a Δ5 desaturase to produce GLA, DGLA and ARA, (d) a Δ12 desaturase, a Δ6 desaturase, a Δ6 elongase, a Δ5 desaturase and a Δ5 elongase to produce GLA, DGLA, ARA and DTA, and (e) a Δ12 desaturase, a Δ6 desaturase, a Δ6 elongase, a Δ5 desaturase, a Δ5 elongase and a Δ4 desaturase to produce GLA, DGLA, ARA, DTA and DPAω6. Preferred combinations of enzymes encoded by polynucleotides of (i) according to the Δ9 elongase pathway are: (f) a Δ12 desaturase and a Δ9 elongase to produce EDA; (g) a Δ12 desaturase, a Δ9 elongase and a Δ8 desaturase to produce EDA and DGLA; (h) a Δ12 desaturase, a Δ9 elongase, a Δ8 desaturase and a Δ5 desaturase to produce EDA, DGLA and ARA; (i) a Δ12 desaturase, a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase and a Δ5 elongase to produce EDA, DGLA, ARA and DTA; and (j) a Δ12 desaturase, a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, a Δ5 elongase and a Δ4 desaturase to produce EDA, DGLA, ARA, DTA and DPAω6. In each of combinations (a)-(j), the Δ12 desaturase can be omitted if the microbial cell has an endogenous Δ12 desaturase that converts oleic acid to LA with sufficient activity to allow for the production of sufficient ω6 fatty acids. One of skill in the art can readily determine whether to use an exogenous Δ12 desaturase.

[0239] Preferred combinations of enzymes encoded by polynucleotides in (iii) are (a) one or more or all three of diacylglycerol kinase, phosphatidic acid cytidylyltransferase, and phosphatidylserine synthase, (b) diacylglycerol kinase, phosphatidic acid cytidylyltransferase, phosphatidylserine synthase, and phosphatidylserine decarboxylase, (c) phosphatidic acid cytidylyltransferase, phosphatidylserine synthase, and phosphatidylserine decarboxylase, and (d) phosphatidylserine synthase and phosphatidylserine decarboxylase. To generate more PC, polynucleotides encoding phosphatidylethanolamine methyltransferase, or phosphatidylethanolamine methyltransferase and phospholipid methyltransferase can be added to any of the combinations (a) to (d) or used by themselves.

[0240] A preferred combination of genetic modifications (iv) is one that reduces the activity of DGAT1 and LRO1, or the activity of all three of DGAT1, DGAT2 and LRO1.

[0241] A more preferred embodiment of the above preferred embodiment comprises a genetic modification that reduces the activity of a regulator of lipid synthesis, for example the addition of a null mutation in any one of the genes SAP190, TOR2 or most preferably OPI1.

[0242] In one embodiment, the genetically modified microorganism comprises one or more genetic modifications that increase the amount of at least two phospholipids selected from the group consisting of PC, PE, PS, and PI, compared to the corresponding wild-type microorganism, each amount being expressed as a percentage of the total polar lipid content.The genetic modifications to achieve this include the genetic modifications in the previous paragraph.

[0243] In some embodiments, the at least two phospholipids are PC and PE, PC and PS, or PC and PI, or PC and PE, PC and PS, or PC and PI are present in an altered ratio relative to the polar lipids from the corresponding wild-type microorganism.

[0244] In another aspect, the present invention provides a microbial cell comprising the lipid of the present invention. The microbial cell may be in suspension, for example in aqueous suspension, frozen, dried, or any other suitable form. The microbial cell may be live or dead, or a mixture of live and dead cells, for example at least 99% of the cells may be dead. The cells may be heat treated to render them incapable of replicating.

[0245] In embodiments, the microbial cells comprise or consist of eukaryotic cells, fungal cells, bacterial cells or algal cells, live microbial cells, dead microbial cells, or any mixture thereof.

[0246] In embodiments, the microbial cells are one or more or all of: (i) those suitable for fermentation; (ii) oleaginous cells; (iii) non-oleaginous cells, preferably non-oleaginous cells derived from oleaginous cells by genetic modification; and (iv) heterotrophic cells.

[0247] In some embodiments, the microbial cells are yeast cells, including but not limited to Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Trichoderma spp., Candida rugose, Aspergillus niger, Crypthecodinium cohnii, and any mixtures thereof.

[0248] In one embodiment, the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and any mixture thereof.

[0249] In one embodiment, the microbial cells comprise algal cells selected from the group consisting of Prototheca moriformis, Thraustochytrium spp., Chlorella protothecoides, Schizochytrium sp., such as strain FCC-1324, and any mixtures thereof.

[0250] In one embodiment, the fungal cell is of a filamentous fungus or mold species, for example, Mortierella sp., such as Mortierella alpina or Mortierella elongata. In one embodiment, the fungal cell is from the Genus Mucor, for example, from the species Mucor hiemalis. Examples of Mortierella sp. include those of the present invention.

[0251] In one embodiment, the microbial cell is a microbial cell other than Mortierella alpina.

[0252] In one embodiment, the microbial cell comprises a genetic modification that results in increased production of ω6 fatty acids in polar lipids. In one embodiment, the microbial cell comprises one or more of the genetic modifications listed above in relation to the lipids of the present invention.

[0253] In one embodiment, the microbial cell comprises a genetic modification that results in a reduction of endogenous Δ12 desaturase expression and / or activity. In one embodiment, the genetic modification is a mutation in a gene encoding an endogenous Δ12 desaturase, preferably a null mutation in the FAD2 gene. In one embodiment, the null mutation is a gene deletion. Surprisingly, the inventors observed that when ω6 fatty acids were supplied to the culture medium, the amount of ω6 fatty acids, such as ARA, incorporated into the polar lipid fraction of yeast was increased in a fad2 null mutant compared to the corresponding wild-type strain. In one embodiment, the amount of ω6 fatty acids endogenously produced in a fad2 mutant microbial cell is increased compared to the corresponding FAD2 wild-type cell.

[0254] In one embodiment, the microbial cell comprises one or more genetic modifications that result in reduced triacylglycerol (TAG) synthesis. In one embodiment, the one or more genetic modifications comprise one or more mutations that reduce the expression and / or activity of the DGA1, DGA2, LRO1 or ARE1 gene, respectively, and preferably comprise null mutations in any one or more or all of the DGA1, DGA2, LRO1 and ARE1 genes. In one embodiment, the null mutation is a deletion of at least a portion of the gene.

[0255] In embodiments, the microbial cell comprises a mutation, preferably a null mutation, that reduces the expression and / or activity of: a) at least DGA1 and DGA2, b) at least DGA1 and LRO1; c) at least DGA1, DGA2, and LRO1, or d) at least DGA1, DGA2, LRO1, and ARE1.

[0256] In one embodiment, the microbial cell comprises one or more exogenous polynucleotides encoding one or more desaturases and / or one or more elongases.

[0257] In embodiments, the microbial cells comprise at least: a) Δ12 desaturase, b) Δ5 elongase, c) a Δ5 elongase and a Δ4 desaturase; d) a Δ6 desaturase, and optionally a Δ12 desaturase; e) a Δ9 elongase, and optionally, a Δ12 desaturase; f) a Δ6 desaturase, a Δ6 elongase, and optionally, a Δ12 desaturase; g) a Δ9 elongase, a Δ8 desaturase, and optionally a Δ12 desaturase; h) a Δ6 desaturase, a Δ6 elongase, a Δ5 desaturase, and optionally, a Δ12 desaturase; i) a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, and optionally, a Δ12 desaturase; j) Δ6 desaturase, Δ6 elongase, Δ5 desaturase, Δ5 elongase, and optionally, a Δ12 desaturase; k) a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, a Δ5 elongase, and, optionally, a Δ12 desaturase; l) a Δ6 desaturase, a Δ6 elongase, a Δ5 desaturase, a Δ5 elongase, a Δ4 desaturase, and, optionally, a Δ12 desaturase, or m) one or more exogenous polynucleotides encoding a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, a Δ5 elongase, a Δ4 desaturase, and, optionally, a Δ12 desaturase; Each polynucleotide is operably linked to one or more promoters capable of directing the expression of that polynucleotide in a microbial cell.

[0258] In embodiments, the microbial cell comprises one or more exogenous polynucleotides encoding a Δ6 desaturase, a Δ6 elongase, a Δ5 desaturase, and optionally a Δ12 desaturase, each polynucleotide operably linked to one or more promoters capable of directing expression of that polynucleotide in the microbial cell. The microbial cell may comprise two or more Δ6 desaturase genes, two or more Δ6 elongase genes, two or more Δ5 desaturase genes, and / or two or more Δ12 desaturase genes, in each case encoding either the same or different enzymes.

[0259] In embodiments, the microbial cell comprises one or more exogenous polynucleotides encoding a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, and optionally a Δ12 desaturase, each polynucleotide operably linked to one or more promoters capable of directing expression of that polynucleotide in the microbial cell. The microbial cell may further comprise one or more exogenous polynucleotides encoding a Δ6 desaturase and a Δ6 elongase. The microbial cell may comprise two or more Δ8 desaturase genes, two or more Δ9 elongase genes, two or more Δ5 desaturase genes, and / or two or more Δ12 desaturase genes, in each case encoding either the same or different enzymes.

[0260] In one embodiment, one or more exogenous polynucleotides are integrated into the genome of the cell. In one embodiment, the exogenous polynucleotides are integrated into a single site in the microbial cell genome. In an alternative embodiment, the exogenous polynucleotides are not integrated into a single site in the microbial cell genome, but instead, one or more polynucleotides are integrated into one site and one or more other polynucleotides are integrated into another site in the genome. The polynucleotides may be integrated into three or more sites in the genome.

[0261] In one embodiment, one or more or all of the desaturases and / or elongases have greater activity on ω6 fatty acids as compared to the corresponding ω3 fatty acids.

[0262] The above desaturases may act on CoA- or PC-linked substrates, or both. In one embodiment, one or more or all of the desaturases, preferably the Δ6-desaturase and / or the Δ5-desaturase and / or the Δ12-desaturase, have greater activity on acyl-CoA substrates than the corresponding acyl-PC substrates.

[0263] In embodiments, the Δ12 desaturase comprises an amino acid sequence set forth as any one of SEQ ID NOs:1-4, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to any one or more of SEQ ID NOs:1-4.

[0264] In embodiments, the Δ12 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:1, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:1.

[0265] In embodiments, the Δ12 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:2, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:2.

[0266] In embodiments, the Δ12 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:3, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:3.

[0267] In embodiments, the Δ12 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:4, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:4.

[0268] In embodiments, the Δ6 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:5 or SEQ ID NO:6, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:5 or SEQ ID NO:6.

[0269] In embodiments, the Δ6 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:5, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:5.

[0270] In embodiments, the Δ6 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:6, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:6.

[0271] In embodiments, the Δ9 elongase comprises an amino acid sequence set forth as any one of SEQ ID NOs:7-12, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to any one or more of SEQ ID NOs:7-12.

[0272] In embodiments, the Δ9 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:7, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:7.

[0273] In embodiments, the Δ9 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:8, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:8.

[0274] In embodiments, the Δ9 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:9, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:9.

[0275] In embodiments, the Δ9 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:10, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:10.

[0276] In embodiments, the Δ9 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:11, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:11.

[0277] In embodiments, the Δ9 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:12, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:12.

[0278] In embodiments, the Δ6 elongase comprises an amino acid having the sequence set forth as SEQ ID NO:13, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:13.

[0279] In embodiments, the Δ8 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:14, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:14.

[0280] In embodiments, the Δ5 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:15 or SEQ ID NO:16, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:15 or SEQ ID NO:16.

[0281] In embodiments, the Δ5 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:15, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:15.

[0282] In embodiments, the Δ5 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:16, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:16.

[0283] In embodiments, the Δ5 elongase comprises an amino acid having a sequence set forth as SEQ ID NO:17, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:17.

[0284] In embodiments, the Δ4 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:18 or SEQ ID NO:19, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:18 or SEQ ID NO:19.

[0285] In embodiments, the Δ4 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:18, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:18.

[0286] In embodiments, the Δ4 desaturase comprises an amino acid having a sequence set forth as SEQ ID NO:19, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:19.

[0287] In another aspect, the present invention provides a DNA construct, or a combination of DNA constructs, encoding one or more of the above-mentioned desaturase and elongase enzymes, preferably integrated into the genome of a microbial cell. In some embodiments, the DNA construct is a vector.

[0288] In another aspect, the present invention provides an isolated strain of Mortierella sp. comprising an internal transcribed spacer (ITS) region having a nucleotide sequence set forth in any one of SEQ ID NOs: 105-110, 112, 121, 126-146, or a nucleotide sequence at least 90%, at least 95%, or at least 99% identical to one or more of SEQ ID NOs: 105-110, 112, 121, 126-146. In one embodiment, the Mortierella sp. is Mortierella alpina comprising an internal transcribed spacer (ITS) region having a nucleotide sequence set forth in any one of SEQ ID NOs: 105-110 or 112, or a nucleotide sequence at least 90%, at least 95%, or at least 99% identical to one or more of SEQ ID NOs: 105-110 or 112. In one embodiment, the Mortierella sp. is Mortierella elongata, comprising an internal transcribed spacer (ITS) region having a nucleotide sequence set forth in SEQ ID NO:121 or SEQ ID NO:134, or a nucleotide sequence that is at least 90%, at least 95%, or at least 99% identical to one or both of SEQ ID NO:121 or SEQ ID NO:134.

[0289] In one embodiment, the isolated strain is i) yNI0125, deposited with the National Measurement Institute Australia under V21 / 019953 on 12 October 2021; ii) yNI0126, deposited with the National Measurement Institute Australia under V21 / 019951 on 12 October 2021; iii) yNI0127, deposited with the National Measurement Institute Australia under V21 / 019952 on 12 October 2021; and iv) Selected from yNI0132, deposited with the National Measurement Institute Australia under V21 / 019954 on October 12, 2021.

[0290] In another aspect, the present invention provides an isolated strain of Mucor hiemalis comprising an internal transcribed spacer (ITS) region having a nucleotide sequence set forth in any one of SEQ ID NOs: 104, 113-120, or 122-125, or a nucleotide sequence that is at least 90%, at least 95%, or at least 99% identical to one or more of SEQ ID NOs: 104, 113-120, or 122-125.

[0291] In another aspect, the present invention provides an isolated fungal strain comprising an internal transcribed spacer (ITS) region having a nucleotide sequence as set forth in SEQ ID NO:111, or a nucleotide sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:111.

[0292] Polypeptides The terms "polypeptide" and "protein" are commonly used interchangeably.

[0293] A polypeptide or a class of polypeptides may be defined by the degree of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by the identity to one reference amino acid sequence being greater than the identity to another reference amino acid sequence. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970, GCG program) with parameters of gap creation penalty=5 and gap extension penalty=0.3. The query sequence is at least 100 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids long, and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over the entire length of the reference amino acid sequence. A polypeptide or a class of polypeptides may have the same enzymatic activity as the reference polypeptide, or may have an activity different from the activity of the reference polypeptide, or may lack the activity of the reference polypeptide. Preferably, the polypeptide has an enzymatic activity that is at least 10%, at least 50%, at least 75%, or at least 90% of the activity of the reference polypeptide.

[0294] The polynucleotides defined herein may encode biologically active fragments of enzymes such as desaturases or elongases. As used herein, a biologically active fragment is a portion of a polypeptide defined herein that maintains a defined activity of the full-length reference polypeptide, e.g., has desaturase and / or elongase activity or other enzymatic activity. As used herein, a biologically active fragment does not include the full-length polypeptide. A biologically active fragment can be any size portion, so long as it maintains the defined activity. Preferably, a biologically active fragment maintains at least 10%, at least 50%, at least 75%, or at least 90% of the activity of the full-length protein.

[0295] With respect to defined polypeptides or enzymes, it will of course be understood that % identity figures higher than those provided herein encompass preferred embodiments. Thus, in light of the minimum % identity figures, where applicable, it is preferred that the polypeptide / enzyme comprises an amino acid sequence which is at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant specified SEQ ID NO. In one embodiment, for each of the ranges recited above, the % identity does not include 100%, ie, the amino acid sequence differs from that of the designated SEQ ID NO.

[0296] Amino acid sequence variants / mutants of the polypeptides defined herein can be prepared by introducing appropriate nucleotide changes into the nucleic acids defined herein or by in vitro synthesis of the desired polypeptide. Such variants / mutants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. Deletions, insertions, and substitutions can be combined to arrive at the final construct, provided that the final peptide product possesses the desired enzymatic activity.

[0297] Mutant (altered) peptides can be prepared using any technique known in the art. For example, the polynucleotides defined herein can be subjected to in vitro mutagenesis or DNA shuffling techniques, as broadly described by Harayama (1998). Products derived from the mutated / altered DNA can be readily screened using the techniques described herein to determine whether they have, for example, desaturase or elongase activity.

[0298] In designing amino acid sequence variants, the location of the mutation sites and the nature of the mutation will depend on the property to be altered. The mutation sites can be modified individually or sequentially, for example, by (1) substituting conservative amino acid selections first, followed by more drastic selections depending on the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the positioned site.

[0299] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues, and typically about 1 to 5 contiguous residues.

[0300] Substitutional variants have at least one amino acid residue in a polypeptide molecule removed and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include those that are not conserved among naturally occurring desaturases or elongases. These sites are preferably substituted in a relatively conservative manner to maintain enzymatic activity. Such conservative substitutions are shown in Table 2 under the heading of "exemplary substitutions."

[0301] In preferred embodiments, mutant / variant polypeptides have only one or two or three or four, or no more than one or two or three or four, conservative amino acids compared to the naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 2. As one of skill in the art would recognize, such minor changes could reasonably be expected not to alter the activity of the polypeptide when expressed in a recombinant cell. [ka]

[0302] Polynucleotides The present invention also provides for the use of polynucleotides, which may be, for example, genes, isolated polynucleotides, or chimeric gene constructs, such as chimeric DNA. It may be double-stranded or single-stranded DNA or RNA of genomic or synthetic origin, and may be combined with carbohydrates, lipids, proteins, or other materials to perform a specific activity as defined herein. The term "polynucleotide" is used interchangeably herein with the term "nucleic acid molecule."

[0303] In one embodiment, the polynucleotide does not occur in nature. Examples of non-naturally occurring polynucleotides include, but are not limited to, polynucleotides that have been codon-optimized for expression in a microbial cell, e.g., mutated by using the methods described herein, and polynucleotides in which an open reading frame encoding a protein is operably linked to a promoter with which it is not naturally associated, i.e., a promoter that is heterologous with respect to the open reading frame, such as, for example, in the constructs described herein.

[0304] As used herein, "chimeric DNA" or "chimeric gene construct" or the like refers to any DNA molecule that is not a natural DNA molecule in its natural location, also referred to herein as a "DNA construct". Typically, chimeric DNA or chimeric genes contain regulatory and transcriptional sequences or protein coding sequences that are not found operably linked together in nature, i.e., heterologous to each other. Thus, chimeric DNA or chimeric genes may contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but arranged in a manner different from that found in nature.

[0305] "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, "recombinant nucleic acid molecule", "recombinant polynucleotide", or variations thereof, refers to a nucleic acid molecule constructed or modified by recombinant DNA technology. Terms such as "foreign polynucleotide" or "exogenous polynucleotide" or "heterologous polynucleotide" refer to any nucleic acid that is introduced into the genome of a cell by experimental manipulation. A foreign or exogenous gene may be a gene inserted into a non-native organism, a native gene introduced into a new location in a native host, or a chimeric gene. A "transgene" is a gene that has been introduced into a genome by a transformation procedure. The terms "genetic modification", "genetic mutation", "transgenic", and variations thereof include introducing a gene into a cell by transformation or transduction, mutating a gene in a cell, deleting a gene, and altering or modulating the regulation of a gene by genetic changes in the genome in a cell or organism in which these actions have been performed, or in their progeny. As used herein, "genomic region" refers to a location in the genome where a transgene, or a group of transgenes (also referred to herein as a cluster), has been inserted into a cell or its ancestor. Such regions contain only nucleotides incorporated by human intervention, such as by the methods described herein.

[0306] The term "exogenous" in reference to a polynucleotide refers to a polynucleotide when present in a cell in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally contain the polynucleotide. However, the cell may be a cell that contains a non-endogenous polynucleotide that results in an altered amount of production of the encoded polypeptide. Exogenous polynucleotides include polynucleotides that have not been separated from other components of the transgenic (recombinant) cell or cell-free expression system in which it is present, as well as polynucleotides that are produced in such cells or cell-free systems and then purified from at least some other components. An exogenous polynucleotide (nucleic acid) may be a contiguous stretch of nucleotides present in nature, or may contain two or more contiguous stretches of nucleotides from different sources (natural and / or synthetic) that have been combined to form a single polynucleotide. Typically, such chimeric polynucleotides contain at least an open reading frame encoding a polypeptide operably linked to a promoter suitable for driving transcription of the open reading frame in the cell of interest.

[0307] It will be understood that, with respect to defined polynucleotides, % identity figures higher than those provided above encompass preferred embodiments. Thus, where applicable, in light of minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence that is at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant specified SEQ ID NO. In one embodiment, for each of the ranges recited above, the % identity does not include 100%, ie, the nucleotide sequence differs from that of the designated SEQ ID NO.

[0308] The polynucleotide may have one or more mutations, which are deletions, insertions, or substitutions of nucleotide residues, compared to a natural molecule. The polynucleotides having mutations relative to the reference sequence may be either naturally occurring (i.e., isolated from a natural source) or synthetic (e.g., by performing site-directed mutagenesis or DNA shuffling on the above-mentioned nucleic acid). It is therefore clear that the polynucleotide may be either derived from a natural source or from a recombinant source. Preferred polynucleotides are those that have a coding region that is codon-optimized for translation in a microbial cell, as is known in the art.

[0309] Recombinant Vectors Recombinant expression can be used to generate the recombinant microorganism of the invention. Recombinant vectors contain heterologous polynucleotide sequences, i.e., polynucleotide sequences not naturally found adjacent to the polynucleotide molecules defined herein, preferably derived from a species other than the species from which the polynucleotide molecule is derived. Vectors can be either RNA or DNA, and are typically plasmids. Plasmid vectors typically contain additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic cells, such as pYES-, pUC-, pSK-, pGEM-, pSP-, or pBS-derived vectors. Suitable yeast expression vectors include the pPIC series of vectors, Yeast Integrating Plasmids (YIp), Yeast Replicative Plasmids (YRp), Yeast Centromeric Plasmids (YCp), and Yeast Episomal Plasmids (YEp). The additional nucleic acid sequences include an origin of replication to provide autonomous replication of the vector, a selectable marker gene, preferably encoding antibiotic or herbicide resistance, a unique multiple cloning site that provides multiple sites for inserting nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of microbial cells. A recombinant vector may comprise a combination of two or more polynucleotides defined herein, for example, three, four, five, or six polynucleotides defined herein, preferably a chimeric gene construct as described herein, each polynucleotide operably linked to an expression control sequence operable in the cell.

[0310] As used herein, "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or regulates the transcription of the coding sequence in an appropriate cell. Generally, a promoter transcriptional regulatory element operably linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory elements, such as enhancers, do not need to be physically adjacent or located in close proximity to the coding sequence that they enhance transcription. For example, an intron within a 5'UTR sequence or an intron near the 5' end of a protein coding region may contain a transcriptional enhancer that results in increased expression levels, such as the FBAIN promoter region.

[0311] To facilitate identification of transformants, the nucleic acid construct desirably includes a selectable or screenable marker gene as, or in addition to, the foreign or exogenous polynucleotide. By "marker gene" is meant a gene that confers a distinct phenotype to cells expressing the marker gene, thus allowing such transformed cells to be distinguished from cells that do not possess the marker. A selectable marker gene confers a trait that can be "selected" based on resistance to a selection agent (e.g., herbicide, antibiotic, radiation, heat, or other treatment that damages non-transformed cells). A screenable marker gene (or reporter gene) confers a trait that can be identified through observation or testing, i.e., by "screening" (e.g., β-glucuronidase, luciferase, GFP, or other enzymatic activity not present in non-transformed cells). The marker gene and the nucleotide sequence of interest do not need to be linked. The choice of the actual marker is not critical, so long as the marker is functional (i.e., selective) in combination with the selected cells.

[0312] Examples of selectable markers are markers that confer antibiotic resistance, such as hygromycin, nourseothricin, ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably hygromycin or kanamycin resistance.

[0313] The recombinant yeast of the invention can contain a reporter gene encoding either a galactosidase or a selectable growth marker.

[0314] A "galactosidase" can be any enzyme capable of cleaving terminal galactose residues from a variety of substrates and cleaving the substrate to produce a detectable signal. In one embodiment, the galactosidase is a β-galactosidase, such as bacterial (e.g., from E. coli) LacZ. In an alternative embodiment, the galactosidase is an α-galactosidase, such as yeast (e.g., S. cerevisiae) Mel-1. β-galactosidase activity can be detected using substrates for the enzyme, such as X-gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside), which forms an intense blue product upon cleavage; ONPG (o-nitrophenyl galactoside), which forms a water-soluble yellow dye with an absorbance maximum of about 420 nm upon cleavage; and CPRG (chlorophenol red-β-D-galactopyranoside), which gives a water-soluble red product upon cleavage that can be measured spectrophotometrically. Alpha-galactosidase activity can be detected using substrates for the enzyme such as o-nitrophenyl alpha-D-galactopyranoside, which upon cleavage forms an indigo dye, and chlorophenol red-alpha-D-galactopyranoside, which upon cleavage produces a water-soluble red product that can be measured spectrophotometrically. Kits for detecting galactosidase expression in yeast are commercially available, for example, the β-galactosidase (LacZ) expression kit from Thermo Scientific.

[0315] Preferably, the selectable growth marker is a nutritional marker or an antibiotic resistance marker.

[0316] Exemplary yeast selectable nutritional markers include, but are not limited to, LEU2, TRP1, HIS3, HIS4, URA3, URA5, SFA1, ADE2, MET15, LYS5, LYS2, ILV2, FBA1, PSE1, PDI1 and PGK1. Those skilled in the art will appreciate that any gene whose chromosomal deletion or inactivation results in an inviable host, a so-called essential gene, can be used as a selection marker if the functional gene is provided, for example, on a plasmid, as demonstrated for PGK1 in pgk1 yeast strains. Suitable essential genes can be found in the Stanford Genome Database (SGD) (http:: / / db.yeastgenome.org). Any essential gene product that, when deleted or inactivated, does not result in an auxotrophic (biosynthetic) requirement (e.g., PDI1, PSE1, PGK1 or FBA1) can be used as a selectable marker on a plasmid, for example, in a yeast host cell that is unable to produce that gene product in the absence of the plasmid, to achieve improved plasmid stability without the drawback of having to culture the cells under specific selective conditions. "Auxotrophic (biosynthetic) requirement" includes deficiencies that can be complemented by additions or modifications to the growth medium.

[0317] Expression An expression vector can direct gene expression in a microbial cell. As used herein, an expression vector is a vector that can transform a host cell and result in the expression of one or more specific polynucleotide molecules. An expression vector useful in the present invention contains regulatory sequences, such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with recombinant cells and control the expression of the polynucleotide molecules of the present invention. In particular, a polynucleotide or vector useful in the present invention includes a transcription control sequence. A transcription control sequence is a sequence that controls the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are sequences that control transcription initiation, such as promoter sequences and enhancer sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. The choice of regulatory sequence used depends on the target microbial cell. A variety of such transcription control sequences are known to those skilled in the art.

[0318] Yeast cells are typically transformed by chemical methods (e.g., as described by Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Kawai et al., 2010). Cells are usually treated with lithium acetate to produce approximately 10 4 Achieve a transformation efficiency of colony forming units (transformed cells). Other standard procedures for transforming yeast include i) the spheroplast method, which, as the name suggests, relies on the generation of yeast spheroplasts, ii) the biolistic method, which bombards DNA-coated metal microprojectiles into the cells, and iii) the glass bead method, which relies on agitation of yeast cells with glass beads and DNA delivered to the cells. Of course, any suitable means of introducing nucleic acids into yeast cells can be used.

[0319] It is well known that transformation of organisms such as yeast with exogenous plasmids can result in clonal differences in the penetrance of the transformed genes due to differences in copy number or other factors. Therefore, to maximize the chances of identifying suitable receptor=ligand pairs during screening, it is desirable to screen two or more independent clonal isolates for each transformed receptor. Different clonal isolates may be screened individually or combined into a single well for screening. The latter option may be particularly advantageous when nutritional reporters rather than colorimetric reporters are used.

[0320] "Constitutive promoter" refers to a promoter that directs the expression of an operably linked transcription sequence in a cell without the need to be induced by a specific growth condition. Examples of constitutive promoters useful in the yeast cells of the present invention include, but are not limited to, yeast PGK (phosphoglycerate kinase) promoter, yeast ADH-1 (alcohol dehydrogenase) promoter, yeast ENO (enolase) promoter, yeast glyceraldehyde 3-phosphate dehydrogenase promoter (GPD) promoter, yeast PYK-1 (pyruvate kinase) promoter, yeast translation elongation factor-1-alpha promoter (TEF) promoter, and yeast CYC-1 (cytochrome c-oxidase promoter) promoter. In a preferred embodiment, the yeast promoter is a S. cerevisiae promoter. In another embodiment, the constitutive promoter may not be derived from yeast. Examples of such promoters useful in the present invention include, but are not limited to, the cauliflower mosaic virus 35S promoter, the glucocorticoid response element, and the androgen response element. A constitutive promoter may be a naturally occurring molecule or a variant thereof that contains, for example, one, two, or three nucleotide substitutions that do not abolish (and preferably enhance) promoter function.

[0321] Recombinant DNA techniques can be used to improve expression of transformed polynucleotide molecules, for example, by manipulating the copy number of polynucleotide molecules in a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resulting transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing expression of a polynucleotide molecule as defined herein include, but are not limited to, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of stability sequences to the mRNA, replacement or modification of transcriptional control signals (e.g., promoters, operators, enhancers), replacement or modification of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of the polynucleotide molecule to accommodate the codon usage of the host cell, and deletion of sequences that destabilize the transcript.

[0322] Other gene modification techniques Any method can be used to introduce nucleic acid molecules into microbial cells, and many such methods are well known. For example, transformation and electroporation are common methods for introducing nucleic acids into yeast cells (see, e.g., Gietz et al., 1992; Ito et al., 1983; and Becker et al., 1991).

[0323] In one embodiment, the integration of the gene of interest into a specific chromosomal site in the microbial cell occurs via homologous recombination. According to this embodiment, an integration cassette containing a module including at least one marker gene and / or gene to be integrated (internal module) is flanked on both sides by DNA fragments that are homologous to DNA fragments (recombinogenic sequences) at the ends of the targeted integration site. After transformation of the microbial cell with the cassette by a suitable method, the chromosomal region between the two sites of the genome corresponding to the recombinogenic sequences of the integration cassette can be replaced by the internal module by homologous recombination between the recombinogenic sequences (Orr-Weaver et al., 1981).

[0324] In one embodiment, an integration cassette for integrating a gene of interest into a microbial cell comprises a heterologous gene under the control of a suitable promoter, together with a selection marker flanked by recombination sequences for integration of the heterologous gene into the microbial cell chromosome. In one embodiment, the heterologous gene comprises any of the fatty acid biosynthetic genes described herein.

[0325] If deletion of an endogenous gene is desired, the recombination cassette can contain a selectable marker (without any other heterologous gene sequences) flanked by DNA fragments that are homologous to DNA fragments at the ends (and / or adjacent sequences) of the endogenous gene targeted for deletion. Other suitable methods for deleting or mutating endogenous genes (e.g., using site-specific or RNA-guided nucleases) are described below.

[0326] The selectable marker gene can be any marker gene used in microbial cells, including, but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinant sequence can be optionally selected depending on the desired integration site suitable for the desired application.

[0327] In another embodiment, integration of the gene into the chromosome of the microbial cell can occur via random integration (Kooistra et al., 2004).

[0328] Furthermore, in some embodiments, the particular introduced marker gene is removed from the genome using techniques well known to those skilled in the art, for example, loss of the URA3 marker can be obtained by plating URA3-containing cells in FOA (5-fluoroorotic acid)-containing medium and selecting FOA-resistant colonies (Boeke et al., 1984).

[0329] The exogenous nucleic acid molecule contained in the microbial cell of the present disclosure can be maintained in the cell in any form. For example, the exogenous nucleic acid molecule can be integrated into the genome of the cell or maintained in an episomal state that can be stably inherited ("inherited") by daughter cells. Such extrachromosomal genetic elements (plasmids, mitochondrial genomes, etc.) can additionally contain a selection marker that ensures the presence of such genetic elements in daughter cells. Furthermore, the microbial cell can be stably or transiently transformed. Furthermore, the microbial cell described herein can contain a single copy or multiple copies of the specific exogenous nucleic acid molecule described above.

[0330] Genome editing using site-specific nucleases Genome editing uses engineered nucleases that are composed of sequence-specific DNA binding domains fused to non-specific DNA cleavage modules. These chimeric nucleases allow efficient and precise genetic modifications (including deletions, mutations, and insertions) by stimulating the cell's endogenous cellular DNA repair mechanisms to induce targeted DNA double-strand breaks that repair the induced breaks. Such mechanisms include, for example, error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR).

[0331] In the presence of a donor plasmid with extended homology arms, HDR can result in the introduction of single or multiple transgenes to correct or replace existing genes. In the absence of a donor plasmid, NHEJ-mediated repair results in targeted small insertion or deletion mutations that cause gene disruption.

[0332] Engineered nucleases useful in the methods of the invention include zinc finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs).

[0333] Typically, nuclease-encoding genes are delivered into cells by plasmid DNA, viral vectors, or in vitro transcribed mRNA. The use of fluorescent surrogate reporter vectors also allows enrichment of ZFN-modified and TALEN-modified cells. As an alternative to the ZFN gene delivery system, cells can be contacted with purified ZFN proteins that can cross cell membranes and induce endogenous gene disruption.

[0334] Zinc finger nucleases (ZFNs) comprise a DNA-binding domain and a DNA-cleavage domain, where the DNA-binding domain is comprised of at least one zinc finger and is operably linked to the DNA-cleavage domain, where the zinc finger DNA-binding domain is at the N-terminus of the protein and the DNA-cleavage domain is located at the C-terminus of the protein.

[0335] ZFN must have at least one zinc finger.In a preferred embodiment, ZFN will have at least three zinc fingers to have sufficient specificity to be useful for target gene recombination in host cell.Typically, ZFN with more than three zinc fingers will have gradually greater specificity with each additional zinc finger.

[0336] The zinc finger domain can be derived from any class or type of zinc finger. In certain embodiments, the zinc finger domain is derived from a Cis zinc finger domain, most commonly represented by, for example, the zinc finger transcription factors TFIIIA or Sp1. 2 His 2 In a preferred embodiment, the zinc finger domain comprises three Cis 2 His 2ZFNs include zinc finger-type nucleotides. The DNA recognition and / or binding specificity of ZFNs can be altered to achieve targeted recombination at any selected site in cellular DNA. Such alterations can be achieved using known molecular biology and / or chemical synthesis techniques (see, e.g., Bibikova et al., 2002).

[0337] ZFN DNA cleavage domains are derived from a class of non-specific DNA cleavage domains, such as the DNA cleavage domains of type II restriction enzymes, such as FokI (Kim et al., 1996). Other useful endonucleases can include, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI.

[0338] To target genetic recombination or mutation according to a preferred embodiment of the present invention, two 9 bp zinc finger DNA recognition sequences must be identified in the host microbial cell DNA. These recognition sites are in opposite orientation to each other and are separated by about 6 bp of DNA. ZFNs are then generated by designing and generating combinations of zinc fingers that specifically bind to DNA at the target locus, and then linking the zinc fingers to a DNA cleavage domain.

[0339] ZFN activity can be improved by the use of transient low-temperature culture conditions to increase nuclease expression levels (Doyon et al., 2010) and by co-delivery of site-specific nucleases and DNA end-processing enzymes (Certo et al., 2012). The specificity of ZFN-mediated genome editing can be improved by using zinc finger nickases (ZFNickases), which stimulate HDR without activating the error-prone NHE-J repair pathway (Kim et al., 2012; Wang et al., 2012; Ramirez et al., 2012; McConnell Smith et al., 2009).

[0340] Transcription activator-like (TAL) effector nucleases (TALENs) contain a TAL effector DNA-binding domain and an endonuclease domain.

[0341] TAL effectors are proteins of plant pathogenic bacteria that are injected into plant cells by pathogens, where they translocate to the nucleus and function as transcription factors to activate specific plant genes. The primary amino acid sequence of a TAL effector determines the nucleotide sequence to which it binds. Thus, target sites can be predicted for TAL effectors, and TAL effectors can be engineered and generated for the purpose of binding to specific nucleotide sequences.

[0342] Fused to the nucleic acid sequence encoding the TAL effector is a nuclease or a portion of a nuclease, typically a sequence encoding a non-specific cleavage domain from a type II restriction endonuclease such as FokI (Kim et al., 1996). Other useful endonucleases can include, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. The fact that some endonucleases (e.g., FokI) only function as dimers can be exploited to enhance the target specificity of the TAL effector. For example, in some cases, each FokI monomer may be fused to a TAL effector sequence that recognizes a different DNA target sequence, and the inactive monomers will join together to generate a functional enzyme only if the two recognition sites are in close proximity. By requiring DNA binding to activate the nuclease, highly site-specific restriction enzymes can be created.

[0343] Sequence-specific TALEN can recognize a specific sequence in the preselected target nucleotide sequence present in cells.Thus, in some embodiments, the target nucleotide sequence can be scanned for nuclease recognition sites, and a specific nuclease can be selected based on the target sequence.In other cases, TALEN can be engineered to target a specific cellular sequence.

[0344] Genome editing using programmable RNA-guided DNA endonucleases Unlike the site-specific nucleases mentioned above, the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system offers an alternative to ZFNs and TALENs for inducing targeted gene modifications. In bacteria, the CRISPR system provides adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage.

[0345] CRISPR systems rely on CRISPR RNA (crRNA) and transactivating chimeric RNA (tracrRNA) for sequence-specific silencing of invading foreign DNA. There are three types of CRISPR / Cas systems. In type II systems, Cas9 functions as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition. CRISPR RNA base-pairs with tracrRNA to form a 2-RNA structure that guides Cas9 endonuclease to the complementary DNA site for cleavage.

[0346] CRISPR loci are a distinct class of interspersed short sequence repeats (SSRs) that were first recognized in E. coli (Ishino et al., 1987; Nakata et al., 1989). Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (Groenen et al., 1993; Hoe et al., 1999; Masepohl et al., 1996; Mojica et al., 1995).

[0347] CRISPR loci differ from other SSRs by the structure of their repeats, called short regularly interspaced repeats (SRSRs) (Janssen et al., 2002; Mojica et al., 2000). The repeats are short elements that occur in clusters and are regularly spaced by unique intervening sequences that always have a constant length (Mojica et al., 2000). The repeat sequences are highly conserved among strains, but the number of interspersed repeats and the sequence of the spacer regions vary from strain to strain (van Embden et al., 2000).

[0348] Common structural features of CRISPR loci have been described by Jansen et al. (2002) as: (i) the presence of multiple short direct repeats that show little or no sequence variation within a given locus, (ii) the presence of unique spacer sequences between repeats of similar size, (iii) the presence of a consensus leader sequence of several hundred base pairs in most species harboring multiple CRISPR loci, (iv) the absence of long open reading frames within the locus, and (v) the presence of one or more cas genes.

[0349] CRISPRs are typically short partial palindromic sequences of 24-40 bp containing internal and terminal inverted repeats of up to 11 bp. Although isolated elements have been detected, they are generally arranged in clusters of repeating units (up to about 20 or more per genome) spaced by unique intervening 20-58 bp sequences. CRISPRs are usually homogeneous within a given genome and are largely identical. However, there are examples of heterogeneity, for example in Archaea (Mojica et al., 2000).

[0350] As used herein, the term "cas gene" generally refers to one or more cas genes associated with or adjacent to adjacent CRISPR loci or associated in the vicinity thereof. A comprehensive review of the Cas protein family is provided in Haft et al. (2005). The number of cas genes in a given CRISPR locus may vary between species.

[0351] cell culture Effective culture conditions are known to those skilled in the art and include, but are not limited to, suitable media, bioreactors, temperature, pH and oxygen conditions that allow lipid production. Suitable media refers to any medium in which cells are cultured to produce lipids as defined herein. Such media typically include aqueous media with assimilable carbon, nitrogen and phosphate sources, as well as other nutrients such as appropriate salts, minerals, metals and vitamins. The cells defined herein can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri dishes. Cultivation can be carried out at temperatures, pH and oxygen content suitable for the recombinant cells. Such culture conditions are within the expertise of those skilled in the art.

[0352] lipid extraction The extraction of lipids from microbial cells of the present invention uses methods similar to those known in the art for lipid extraction from oleaginous microorganisms, for example as described in Patel et al. (2018). In one embodiment, the extraction is performed by solvent extraction, in which an organic solvent (e.g., hexane or a mixture of hexane and ethanol) is mixed with at least the biomass, preferably after the biomass has been dried and ground, but can also be performed under wet conditions. The solvent dissolves the lipids within the cells, and the solution is then separated from the biomass by physical action (e.g., sonication). Sonication is one of the most widely used pretreatment methods to disrupt the cellular integrity of microbial cells. Other pretreatment methods can include microwave irradiation, high-speed homogenization, high-pressure homogenization, bead beating, autoclaving, and pyrolysis. The organic solvent can then be separated (e.g., by distillation) from the non-polar lipids. This second separation step yields the non-polar lipids from the cells, and, if conventional steam recovery is used, a reusable solvent.

[0353] In solvent extraction, an organic solvent (e.g., hexane or a mixture of hexane and ethanol) is mixed with at least the biomass of microbial cells, preferably after the biomass has been dried and ground. The solvent dissolves the lipids in the biomass and the like, and then this solution is separated from the biomass by mechanical action (e.g., using the process described above). This separation step can also be performed by filtration (e.g., using a filter press or similar device) or centrifugation, etc. The organic solvent can then be separated from the non-polar lipids (e.g., by distillation). This second separation step yields the non-polar lipids from the microbial cells and, if conventional steam recovery is used, a reusable solvent.

[0354] The lipids extracted from the microbial cells of the present invention may be subjected to conventional oil processing procedures. As used herein, the term "purified", when used in connection with the lipids of the present invention, typically means that the extracted lipids have been subjected to one or more processing steps that increase the purity of the lipid components. For example, the purification steps may include one or more or all of the group consisting of degumming, deodorizing, bleaching, drying, and / or fractionation of the extracted oil. However, as used herein, the term "purified" does not include transesterification processes or other processes that change the fatty acid composition of the lipids or oils of the present invention to change the fatty acid composition of the total fatty acid content. In other words, in a preferred embodiment, the fatty acid composition of the purified lipid is essentially the same as the fatty acid composition of the unpurified lipid.

[0355] Degumming Degumming is an initial step in the purification of lipids in liquid form (oil), whose main objective is to separate from the oil the majority of phospholipids, which may be present as approximately 1-2% of the total extracted lipid. Addition of about 2% water, typically containing phosphoric acid, to the crude oil at 70-80°C separates the majority of the phospholipids along with trace metals and pigments. The insoluble material removed is primarily a mixture of phospholipids, also known as lecithin. Degumming can be performed by adding concentrated phosphoric acid to the crude extracted lipid to convert non-hydratable phosphatides to a hydratable form and chelate any trace metals present. The gums are separated from the oil by centrifugation. Recovered gums containing omega-6 fatty acids other than LA alone are encompassed by the present invention.

[0356] Alkaline Refining Alkaline refining is one of the refining processes for treating lipids in the form of oils, and is sometimes called neutralization. It is usually performed after degumming and prior to bleaching. After degumming, the oil can be treated by adding an amount of alkaline solution sufficient to titrate out all of the fatty acids and phosphoric acids and remove the soaps thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate, and ammonium hydroxide. This process is typically carried out at room temperature and removes the free fatty acid fraction. The soaps are removed by centrifugation or by extraction into a solvent for the soaps, and the neutralized oil is washed with water. If necessary, any excess alkali in the oil can be neutralized with a suitable acid, such as hydrochloric acid or sulfuric acid.

[0357] bleaching Bleaching is a refining process in which the oil is heated to 90-120°C for 10-30 minutes in the presence of bleaching earth (0.2-2.0%) and in the absence of oxygen, with nitrogen or steam, or by operating in vacuum. This step in oil processing is designed to remove undesirable colorants, and the process also removes oxidation products, trace metals, sulfur compounds, and traces of soaps.

[0358] Deodorization Deodorization is the treatment of fats and oils at high temperatures (200-260°C) and low pressures (0.1-1 mm Hg). It is typically accomplished by introducing steam into the oil at a rate of about 0.1 ml / min per 100 ml of oil. After sparging for about 30 minutes, the oil is cooled under vacuum. Typically, the oil is transferred to a glass container, flushed with argon, and then stored refrigerated. This treatment improves the color of the oil and removes most of the volatile or odorous compounds, including any remaining free fatty acids, monoacylglycerols, and oxidation products.

[0359] Transesterification As used herein, "transesterification" refers to the process of exchanging fatty acids within and between TAGs (esterification), or transferring a fatty acid to another alcohol to form an ester. This may involve first releasing the fatty acid from the TAG as a free fatty acid, or it may directly produce a fatty acid ester, preferably a fatty acid methyl or ethyl ester. In the transesterification reaction of TAG with an alcohol, such as methanol or ethanol, the alkyl group of the alcohol forms an ester bond with the acyl group (including SCFA) of the TAG.

[0360] Foods, Feeds, Beverages, and Compositions The present invention includes compositions that can be used as food or beverage ingredients, foods or beverages for human consumption, or feeds for animal consumption, preferably at least foods for human consumption. The compositions can also be added to foods, beverages, or feeds to increase the "meatiness" of the aroma and / or flavor of the food, beverage, or feed (e.g., to increase the amount of volatile compounds known to have meat-related aromas). For the purposes of the present invention, a food, beverage, or feed is a preparation for human or animal consumption that, when taken into the body, (a) functions to nourish or grow tissues or provide energy, and / or (b) maintains, restores, or supports proper nutritional status or metabolic function. A food or beverage ingredient is a composition that can be used as a component of a food or beverage with at least one other ingredient other than water, such as, for example, macronutrients, proteins, carbohydrates, vitamins, and / or minerals.

[0361] Suitable food / feed products include meat substitutes, soup bases, stew bases, snack foods, bouillon powders, bouillon cubes, flavor packets, or frozen foods. Meat substitutes can be assembled, for example, as hot dogs, hamburgers, minced meat, sausages, steaks, fillets, roasts, breasts, thighs, wings, meatballs, meatloaf, bacon, strips, fingers, nuggets, cutlets, or cubes. Ingredients and methods for producing foods, feeds, and beverages containing meat substitutes are well known in the art (see, for example, WO2008 / 124370, WO2013 / 010042, WO2015 / 153666, and WO2017 / 070303), and the extracted microbial lipids, microbial cells, and / or compositions of the present invention can be used to produce foods, feeds, and beverages containing the extracted microbial lipids, microbial cells, and / or compositions of the present invention.

[0362] The food, beverage, or feed of the invention includes, for example, the extracted lipid of the invention, the microbial cells of the invention, or both the extracted lipid and the microbial cells of the invention, the microbial cell extract, or the composition of the invention. In some embodiments, the extracted lipid and / or the microbial cells are heated, prior to incorporation into the food product, under conditions suitable to produce one or more (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, or 31) volatile compounds exhibiting meat-like or meat-related aromas and flavors, e.g., 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2,5-diethyl-1-heptanol, 2,6-diethyl-1-heptanol, 2,7-diethyl-1-heptanol, 2,8-diethyl-1-heptanol, 2,9-diethyl-1-heptanol, 2,10-diethyl-1-heptanol, 2,11-diethyl-1-heptanol, 2,12-diethyl-1-heptanol, 2,13-diethyl-1-heptanol, 2,14-diethyl-1-heptanol, 2,15-diethyl-1-heptanol, 2,16-diethyl-1-heptanol, 2,17-diethyl-1-heptanol, 2,18- -nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethylbenzaldehyde, 2,3,4,5-tetramethylcyclopent-2-en-1-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, trans-2-octen-1-ol, 1-nonanol, 1,3-bis(1,1-dimethylethyl)-benzene, 2-octen-1-ol, adamantanol-like compounds, hexanal, 2-pentylfuran, 1-octen-3-ol, 2-pentylthiophene, and 1,3,5-titriane. In some examples, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, 1-octanol, trans-2-octen-1-ol, and 1-nonanol are produced. In other embodiments, one or more (e.g., 2, 3, 4, or 5) volatile compounds selected from 1-pentanal, 3-octanone, 2-octen-1-ol, 1-nonanol, and 1-octanol, and optionally 1,3-bis(1,1-dimethylethyl)-benzene are produced.As will be appreciated, the amounts and ratios of various fatty acids (and in particular ω6 fatty acids (e.g., ARA, GLA, DGLA, EDA, DTA and / or DPA-ω6) in the extracted microbial lipid will change when one or more of these volatile compounds are produced from the reaction between fatty acids, sugars and amino acids on the polar lipid. As a result, the lipid remaining after the reaction may have a different fatty acid profile compared to the "initial" extracted microbial lipid. Thus, in some embodiments, the food, beverage, or feed of the invention comprises lipid, which is the product of a reaction between the extracted microbial lipid of the invention, amino acids or derivatives, and sugars under conditions suitable to produce at least two compounds having meat-related flavors and / or aromas. In particular embodiments, the conditions include heating, for example, at a temperature of at least about 100°C, 110°C, 120°C, 130°C, or 140°C, for a period of time (e.g., as further described below) and with sugars and amino acids or derivatives in an amount or concentration sufficient to produce the volatile compounds.

[0363] The food product may be in either solid or liquid form, for example in the form of a powder, solution, suspension, slurry, or emulsion. Additionally, the composition may contain edible macronutrients, proteins, carbohydrates, vitamins, and / or minerals in amounts desired for a particular application. The amounts of these components will vary depending on whether the composition is intended for use in normal individuals or individuals with special needs, such as those suffering from metabolic disorders, etc.

[0364] Examples of suitable ingredients with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates, and proteins. Examples of such edible fats other than the lipids of the present invention include, but are not limited to, palm oil, canola oil, soybean oil, corn oil, sunflower seed oil, safflower seed oil, cottonseed oil, coconut oil, borage oil, fungal oil, black current oil, and mono- and diglycerides. Examples of such carbohydrates include, but are not limited to, glucose, a mixture of glucose and fructose, edible lactose, and hydrolyzed starch. In addition, examples of proteins that can be utilized in the nutritional composition of the present invention include, but are not limited to, soy protein, mycoprotein (e.g., Rhiza mycoprotein), seitan, pea protein, potato protein, electrodialysis whey, electrodialysis skim milk, milk whey, or hydrolyzates of these proteins. In some embodiments, the protein is a textured or structured protein product comprising a protein fiber network and / or aligned protein fibers that produce a meat-like texture, which can be obtained from a dough after application of mechanical energy (e.g., extrusion, rotating, stirring, shaking, shear, pressure, turbulence, collision, congesting, beating, friction, wave), radiant energy (e.g., microwave, electromagnetic), thermal energy (e.g., heating, steam texturing), enzymatic activity (e.g., transglutaminase activity), chemical reagents (e.g., pH adjusters, kosmotropic salts, chaotropic salts, gypsum, surfactants, emulsifiers, fatty acids, amino acids), other methods that result in protein denaturation and alignment of protein fibers, or a combination of these methods, followed by fixation of the fibrous and / or aligned structure (e.g., by rapid temperature and / or pressure changes, rapid dehydration, chemical fixation, oxidation-reduction), and optional post-treatment (e.g., hydration, soaking, drying, coloring) after the fibrous and / or aligned structure is generated and fixed.

[0365] With regard to vitamins and minerals, the following may be added to the food, beverage, or feed of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complex. Iron may be provided in the form of iron bound to heme or in a form other than iron bound to heme, preferably in the form of a ferrous salt. Other such vitamins and minerals may also be added.

[0366] Additional ingredients include food grade oils such as canola oil, corn oil, sunflower oil, soybean oil, olive oil, or coconut oil, seasonings such as edible salt (e.g., sodium chloride or potassium chloride) or herbs (e.g., rosemary, thyme, basil, sage, or mint), flavorings, proteins (e.g., soy protein isolate, wheat gluten, pea vicilin, and / or pea legumin), protein concentrates (e.g., soy protein concentrate), emulsifiers (e.g., lecithin), gelling agents (e.g., k-carrageenan or gelatin), fiber (e.g., bamboo filler or inulin), or minerals (e.g., iodine, zinc, and / or calcium).

[0367] The foods and feeds described herein may also contain natural colouring agents, such as turmeric or beet juice, or artificial colouring agents, such as azo dyes, triphenylmethane, xanthene, quinine, indigoid, titanium dioxide, Red No. 3, Red No. 40, Blue No. 1, or Yellow No. 5.

[0368] The foods and feeds described herein may also include meat shelf life extenders such as carbon monoxide, nitrites, sodium metabisulfite, bombal, vitamin E, rosemary extract, green tea extract, catechins, and other antioxidants.

[0369] The components utilized in the food, beverage or feed of the present invention may be of semi-purified or refined origin, by which is meant materials prepared by purification of natural materials or by de novo synthesis.

[0370] In one embodiment, the food, beverage, or feed has no animal-derived components. Thus, in a preferred embodiment, at least some of the ingredients are plant materials or plant-derived materials. In some embodiments, the food, beverage, or feed can be soy-free, wheat-free, yeast-free, MSG-free, and / or protein hydrolysate-free, and can taste meaty, with a very tasty flavor and no or reduced levels of rancidity or off-flavors.

[0371] In addition, the microbial lipids, microbial cells, and / or compositions of the present invention can be used to modify the taste and / or aroma profile of other foods (e.g., textured plant proteins such as meat replicas, meat substitutes, tofu, mock duck, or gluten-based plant products, textured soy proteins, pork, fish, lamb, or poultry products such as chicken or turkey products) and can be applied to other foods before or during cooking. In some embodiments, the microbial lipids, microbial cells, and / or compositions described herein can be used to provide a non-meat or poultry product with a particular meat-like taste and odor, such as the taste and odor of beef.

[0372] In some embodiments, the compositions, foods, feeds, and beverages described herein comprise the components necessary to induce the Maillard reaction upon heating of the composition. For example, the compositions may comprise one or both of (i) a sugar, sugar alcohol, sugar acid, or sugar derivative, and (ii) and an amino acid or derivative thereof.

[0373] Suitable sugars, sugar alcohols, sugar acids, and sugar derivatives include glucose, fructose, ribose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, maltodextrin, glycogen, galactose, lactose, ribitol, gluconic acid, and glucuronic acid, amylose, amylopectin, and xylose, and combinations thereof.

[0374] Suitable amino acids and derivatives thereof include cysteine, cystine, cysteine ​​sulfoxide, allysine, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, and tyrosine.

[0375] The compositions, foods, feeds and beverages may also include one or more other flavor precursors including oils (e.g., vegetable oils), free fatty acids, alpha-hydroxy acids, dicarboxylic acids, nucleosides, nucleotides, vitamins, peptides, protein hydrolysates, extracts, phospholipids, lecithin, and organic molecules.

[0376] The foods, feeds, beverages, and compositions described herein can be packaged in a variety of ways, including being sealed in individual packets or shakers so that the compositions can be sprinkled or spread onto the food before or during cooking.

[0377] The foods, beverages, and feeds described herein can be evaluated for flavor and aroma using trained human panelists. Evaluation can include looking, feeling, chewing, smelling, and tasting the product, preferably at least smelling the food, beverage, or feed, to determine the product's appearance, color, integrity, texture, flavor, mouthfeel, etc. Panelists can be presented with samples under red or white light. Scales can be used to evaluate the overall acceptability or quality of the food, or specific quality attributes such as meatiness, texture, and flavor. Foods, feeds, and beverages can also be presented to animals, such as pet animals, to evaluate their appeal to the animals.

[0378] In some embodiments, the food, beverage, or feed described herein can be compared to another product (e.g., meat or meat substitute) based on olfactometer readings. In various embodiments, the olfactometer can be used to evaluate odor concentration and odor threshold, odor threshold exceedance compared to a reference gas, hedonic scale score to determine the degree of evaluation, or relative odor intensity.

[0379] In some embodiments, the identified volatile chemicals can be evaluated using GCMS. For example, a person can evaluate the smelling experience of the chemicals responsible for a particular peak. This information can be used to further refine the profile of flavor and aroma compounds produced by the composition of the present invention.

[0380] Characteristic flavor and aroma components are mainly generated during the cooking process by chemically reactive molecules including amino acids, fats and sugars found in plants and meat. Thus, in some embodiments, food, beverages or feeds are tested for similarity to meat during or after cooking. In some embodiments, human ratings, human evaluations, olfactometer readings, or GC-MS measurements, or a combination thereof, are used to create an olfactory map of the food or feed. Similarly, an olfactory map of a food, beverage or feed, for example a meat replica, can be created. These maps can be compared to assess how similar the cooked food or feed is to meat.

[0381] The exact amount of microorganisms and / or extracted lipids, preferably phospholipids, in the composition or food, beverage or feed of the present invention may vary depending, for example, on the identity of the microorganism, the form and moisture content of the microbial biomass, the total lipid or phospholipid content and the composition of the total fatty acid content or the composition of the polar lipids contained in the microbial biomass or its extract, the intensity of the desired flavor and / or aroma, and the intended use of the composition. In some embodiments, the composition of the present invention comprises at least about 25 mg of microbial biomass, in particular at least about 50 mg, preferably at least about 60 mg, more preferably at least about 70 mg of microbial biomass, e.g., dry biomass, per gram of dry composition or slurry, or per ml of liquid composition. In certain embodiments, the composition of the present invention comprises from about 25 mg to about 250 mg of microbial biomass, e.g., from about 25 mg to about 200 mg of microbial biomass, e.g., dry biomass. In certain embodiments, the composition of the present invention comprises from about 25 mg to about 150 mg, e.g., from about 50 mg to about 150 mg of dry biomass. In particularly preferred embodiments, the present invention provides about 50 mg to about 100 mg of dry biomass, such as about 75 mg of dry biomass. According to some embodiments, the compositions of the present invention comprise about 50 mg to about 200 mg of wet biomass, preferably about 50 mg to about 150 mg of wet biomass. According to some specific embodiments, the compositions of the present invention comprise about 75 mg to about 125 mg of wet biomass.

[0382] According to some embodiments, the composition may contain, for example, at least about 5 mg of lipids, preferably phospholipids, extracted from microorganisms, for example, at least about 10 mg or at least about 15 mg of lipids, preferably phospholipids, extracted from microorganisms, per gram of dry composition or slurry, or per mL of liquid composition. According to some embodiments, the composition contains about 10 mg to about 100 mg, about 10 mg to about 80 mg, about 10 to about 70 mg, about 10 to about 60 mg, particularly preferably about 10 to about 50 mg of lipids, preferably phospholipids, extracted from microorganisms. According to some embodiments, the composition of the present invention provides at least about 15 mg, for example at least about 20 mg of lipids, preferably phospholipids, extracted from microorganisms. According to some embodiments, the food, feed, or beverage may contain, for example, at least about 0.1 mg of lipid, preferably phospholipid, extracted from a microorganism, for example, at least about 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or at least about 10 mg of lipid, preferably phospholipid, extracted from a microorganism, per gram of dry composition or slurry, or per mL of liquid composition. According to some embodiments, the composition contains about 0.1 mg to about 100 mg, 0.5 mg to about 80 mg, about 1 mg to about 50 mg, about 1 mg to about 30 mg, about 5 mg to 60 mg, or about 5 mg to about 30 mg of lipid, preferably phospholipid, extracted from a microorganism.

[0383] In some embodiments, the composition comprises at least about 25 mg of microbial biomass, such as dry biomass, and at least about 5 mg of lipids, preferably phospholipids, extracted from the microorganisms, per gram of dry composition or slurry, or per ml of liquid composition. In some embodiments, the composition comprises at least about 70 mg of microbial biomass, such as dry biomass, and at least about 10 mg of lipids, preferably phospholipids, extracted from the microorganisms. In some embodiments, the composition comprises about 25 mg to about 150 mg of dry microbial biomass and about 10 mg to about 100 mg of lipids, preferably phospholipids, extracted from the microorganisms, for example, about 50 mg to about 100 mg of microbial dry biomass and about 15 mg to about 50 mg of lipids, preferably phospholipids, extracted from the microorganisms. In some embodiments, the composition comprises about 50 mg to about 150 mg of microbial wet biomass and about 10 mg to about 100 mg of lipids, preferably phospholipids, extracted from the microorganisms, for example, about 75 mg to about 125 mg of microbial wet biomass and about 15 mg to about 50 mg of lipids, preferably phospholipids, extracted from the microorganisms.

[0384] The compositions, foods, feeds, and beverages of the present invention comprise one or more sugars, sugar alcohols, sugar acids, or sugar derivatives, for example, in an amount sufficient to promote the production of meat-like or meat-related aroma compounds. Suitable sugars, sugar alcohols, sugar acids, or sugar derivatives will be known to those skilled in the art. In this context, the sugars, sugar alcohols, sugar acids, or sugar derivatives are suitable for use in the Maillard reaction in food, beverage, or feed applications. In this context, the sugars, sugar alcohols, sugar acids, or sugar derivatives are components of the compositions of the present invention that are separate from the microbial biomass or components thereof, and amino acids or derivatives or salts thereof, even if the microbial biomass or components thereof themselves contain sugars, sugar alcohols, sugar acids, or sugar derivatives. Suitable sugars, sugar alcohols, sugar acids, and sugar derivatives include glucose, fructose, ribose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, maltodextrin, glycogen, galactose, lactose, ribitol, gluconic acid, and glucuronic acid, amylose, amylopectin, or xylose. In particularly preferred embodiments, the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives include one or more of ribose, glucose (dextrose), a combination of glucose and fructose, and xylose. In particularly preferred embodiments, the compositions of the present invention include ribose, which has been found in the examples of the present application to provide compositions that, in some cases, produce a more meaty flavor and / or aroma than compositions containing glucose alone as the sugar. In certain embodiments, the compositions of the present invention include both glucose and ribose, and in the examples of this application, it has been found that a combination of ribose and glucose, in some cases, provides a composition that produces a more meaty flavor and / or aroma than a composition containing ribose alone.

[0385] According to some embodiments, the one or more sugars, sugar alcohols, sugar acids or sugar derivatives are present in the composition in a total amount of about 5 mmol to about 200 mmol, such as about 5 mmol to about 100 mmol, for example, about 5 mmol to about 80 mmol, for example, about 5 mmol to about 70 mmol, for example, about 10 mmol to about 70 mmol, for example, about 15 mmol to about 70 mmol, for example, about 30 mmol to about 60 mmol, per kg of dry composition or slurry, or per L of liquid composition, measured based on the weight or volume of the composition excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms. In some embodiments, the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives are present in the composition in an amount of at least about 5 mmol, such as at least about 10 mmol, such as at least about 15 mmol, such as at least about 20 mmol, per kg of dry composition or slurry, or per L of liquid composition, measured based on the weight or volume of the composition excluding / before the addition of biomass and / or extracted lipids. In a preferred embodiment, the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives comprise ribose and / or glucose.

[0386] In some embodiments, the one or more sugars, sugar alcohols, sugar acids or sugar derivatives are present in the food, feed or beverage in a total amount of about 0.1 mmol to about 100 mmol, about 0.5 mmol to about 30 mmol, about 1 mmol to about 20 mmol, about 1 mmol to about 10 mmol, about 7 mmol to about 20 mmol, about 7 mmol to about 15 mmol per kg of dry composition or slurry, or per L of liquid food (e.g., beverage), measured based on the weight or volume of the food, feed or beverage excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms. In a preferred embodiment, the one or more sugars, sugar alcohols, sugar acids or sugar derivatives comprise ribose and / or glucose.

[0387] As used herein, sugar "derivative" refers to a sugar that is modified from a naturally occurring sugar, for example, by modification of a substituent such as a hydroxyl group. For example, a sugar derivative may be modified to include an alternative substituent such as an amino group, an acid group, a phosphate group, an acetate group, etc. Sugar derivatives include, but are not limited to, amino sugars, deoxy sugars, glycosylamines, and sugar phosphates.

[0388] In some embodiments, the compositions, foods, feeds, and beverages of the present invention comprise one or more amino acids or their derivatives or salts in an amount sufficient to promote the production of, for example, meat-like or meat-related aroma compounds. In this regard, the amino acids or their derivatives or salts are suitable for use in the Maillard reaction in food, beverage, or feed applications. In this regard, the amino acids or their derivatives or salts are separate components from the microbial biomass or its components, and the sugars, sugar alcohols, sugar acids, or sugar derivatives, even if the microbial biomass or its components themselves contain amino acids or their derivatives or salts. In certain embodiments, the one or more amino acids or their derivatives or salts contain free amino groups. Thus, in some embodiments, reference to amino acids or derivatives refers to free amino acids that are not present in the context of peptides or proteins. Suitable amino acids and their derivatives include cysteine, cystine, cysteine ​​sulfoxide, allicin, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamate or glutamic acid, glutamine, glycine, proline, serine, and tyrosine. In a particularly preferred embodiment, the amino acid is cysteine ​​and / or cystine. In some preferred embodiments, the composition, food, feed, or beverage comprises glutamic acid or its salt, and in the examples of the present application, it has been found that the presence of glutamic acid in some cases provides a more meaty / fishy flavor and / or aroma. In some particularly preferred embodiments, the composition, food, feed, or beverage comprises glutamic acid or a salt thereof in addition to one or more other amino acids or derivatives or salts thereof, for example, the composition, food, feed, or beverage may comprise glutamic acid or a salt thereof and cysteine ​​or a salt thereof. In preferred embodiments, the one or more amino acids or derivatives or salts thereof comprise sulfur-containing amino acids or salts. Amino acid salts suitable for human or animal consumption and therefore suitable for incorporation into the compositions of the present invention are well known to those skilled in the art and may be readily selected by those skilled in the art.

[0389] As used herein, an amino acid "derivative" refers to an amino acid that includes chemical modifications, for example, by introducing a group into the side chain of the amino acid, such as a nitro group in tyrosine or an iodine in tyrosine, by converting a free carboxylic acid group into an ester or amide group, by converting an amino group into an amide by acylation, by acylation of a hydroxy group to obtain an ester, by alkylation of a primary amine to obtain a secondary amine, or by attachment of a hydrophilic moiety to the amino acid side chain. Other derivatives may be obtained by oxidation or reduction of the side chain of the amino acid. Modification of an amino acid may also include derivatization of an amino acid by addition and / or removal of chemical groups to / from the amino acid, and may include the use of amino acid analogs, such as phosphorylated amino acids, or unnatural amino acids, such as N-alkylated amino acids (e.g., N-methyl amino acids), D-amino acids, β-amino acids, or γ-amino acids. Exemplary derivatives may include derivatives obtained by attachment of a derivative moiety, i.e., a substituent, to the amino acid. The term "derivative" in relation to an amino acid will be readily understood by those skilled in the art.

[0390] According to some embodiments, the one or more amino acids or derivatives or salts thereof are each present in the composition in a total amount of about 5 mmol to about 200 mmol, such as about 5 mmol to about 100 mmol, for example, about 5 mmol to about 80 mmol, for example, about 5 mmol to about 70 mmol, for example, about 10 mmol to about 70 mmol, for example, about 15 mmol to about 70 mmol, for example, about 30 mmol to about 60 mmol, per kg of dry composition or slurry, or per L of liquid composition, wherein the amount is calculated based on the weight or volume of the composition excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms. In some embodiments, the one or more amino acids or derivatives or salts thereof are present in the composition in an amount of at least about 5 mmol, such as at least about 10 mmol, such as at least about 15 mmol, such as at least about 20 mmol, per kg of dry composition or slurry, or per L of liquid composition, calculated based on the weight or volume of the composition excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms. In preferred such embodiments, the one or more amino acids comprise cysteine ​​and / or cystine.

[0391] According to some embodiments, the one or more amino acids or derivatives or salts thereof are each present in the food, feed or beverage in a total amount of about 0.5 mmol to about 40 mmol, about 0.5 mmol to about 30 mmol, about 1 mmol to about 10 mmol, about 1.5 mmol to about 10 mmol, about 0.5 to about 5 mmol, about 1 mmol to about 5 mmol, or about 5 to about 10 mmol per kg of dry composition or slurry, or per L of liquid food (e.g., beverage), calculated based on the weight or volume of the food, feed or beverage excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms. In a preferred embodiment, the one or more amino acids comprise cysteine ​​and / or cystine.

[0392] The one or more sugars, sugar alcohols, sugar acids, or sugar derivatives, and one or more amino acids, or derivatives or salts thereof, are present in a composition of the present disclosure, or a food, beverage product, or feed of the present disclosure, in an amount sufficient to produce a food-like aroma, such as a meaty aroma, when heat is applied to the composition, food, beverage product, or feed. In certain embodiments, the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives, and one or more amino acids or derivatives or salts thereof in a composition of the disclosure or a food, beverage product, or feed of the disclosure are capable of reducing the amount of any of the following compounds when heat is applied to the composition, food, beverage product, or feed: 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethylbenzaldehyde, 2,3,4,5-tetramethylcyclohexane, 2,4,5-tetramethylcyclohexane, 2,5,6 ... One or more volatile compounds selected from lopent-2-en-1-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, trans-2-octen-1-ol, 1-nonanol, 1,3-bis(1,1-dimethylethyl)-benzene, 2-octen-1-ol, adamantanol-like compounds, hexanal, 2-pentylfuran, 1-octen-3-ol, 2-pentylthiophene, and 1,3,5-titriane are present in an amount sufficient to produce, for example, two or more, three or more, four or more, or five or more of the foregoing compounds. In some particular embodiments, the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives, and one or more amino acids or derivatives or salts thereof are present in a composition of the disclosure or a food, beverage product, or feed of the disclosure in an amount sufficient to produce one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, 1-octanol, trans-2-octen-1-ol, and 1-nonanol, when heat is applied to the composition, food, beverage product, or feed.In other embodiments, one or more (e.g., 2, 3, 4, or 5) volatile compounds selected from 1-pentanal, 3-octanone, 2-octen-1-ol, 1-nonanol, and 1-octanol, and optionally 1,3-bis(1,1-dimethylethyl)-benzene are produced.

[0393] In some embodiments, the compositions of the invention comprise glutamic acid or a salt or derivative thereof in addition to one or more other amino acids or derivatives or salts thereof, and the glutamic acid is present in an amount of about 2 mmol to about 100 mmol, such as about 2 mmol to about 50 mmol, for example, about 2 mmol to about 40 mmol, for example, about 2 mmol to about 40 mmol, for example, about 5 mmol to about 40 mmol, for example, about 5 mmol to about 30 mmol, per kg of dry composition or slurry, or per L of liquid composition, calculated based on the volume of the composition excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms. In some embodiments, glutamic acid or a salt thereof is present in an amount of at least about 1 mmol, such as at least about 2 mmol, such as at least about 3 mmol, such as at least about 4 mmol, such as at least about 5 mmol, such as at least about 7 mmol, such as at least about 10 mmol, per kg of dry composition or slurry, or per L of liquid composition, measured based on the weight or volume of the composition excluding / before the addition of microbial biomass and / or extracted lipids. In some embodiments, the glutamate salt is monosodium glutamate.

[0394] In some embodiments, a food, feed, or beverage of the invention comprises glutamic acid or a salt or derivative thereof in addition to one or more other amino acids or derivatives or salts thereof, and the glutamic acid is present in an amount of about 0.1 mmol to about 20 mmol, about 0.3 mmol to about 15 mmol, about 0.5 mmol to about 10 mmol, about 0.5 mmol to about 5 mmol, or about 1 mmol to about 5 mmol per kg of dry composition or slurry, or per L of liquid composition (e.g., beverage), calculated based on the volume of the food, feed, or beverage excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms.

[0395] In some embodiments, the composition comprises glutamic acid or a salt thereof and a further amino acid selected from cysteine ​​and cystine or a salt or derivative thereof, and the glutamic acid or salt thereof is present in an amount of about 2 mmol to about 100 mmol per kg of dry composition or slurry, or per L in the case of a liquid composition, such as about 2 mmol to about 50 mmol, for example, about 2 mmol to about 40 mmol, for example, about 2 mmol to about 40 mmol, for example, about 5 mmol to about 40 mmol, for example, about and cysteine ​​or cystine is present in an amount of about 5 mmol to about 200 mmol, 5 mmol to about 100 mmol, for example, about 5 mmol to about 80 mmol, for example, about 5 mmol to about 70 mmol, for example, about 10 mmol to about 70 mmol, for example, about 15 mmol to about 70 mmol, for example, about 30 mmol to about 60 mmol, said amounts being calculated based on the weight or volume of the composition excluding / before the addition of biomass and / or extracted lipid. In some embodiments, the composition comprises glutamic acid or a salt thereof and a further amino acid selected from cysteine ​​and cystine or a salt or derivative thereof, wherein glutamic acid or a salt thereof is present in an amount of at least about 1 mmol, such as at least about 2 mmol, for example at least about 3 mmol, such as at least about 4 mmol, for example at least about 5 mmol, such as at least about 7 mmol, for example at least about 10 mmol per kg of dry composition or slurry or per L of liquid composition, and cysteine ​​or cystine is present in an amount of at least about 5 mmol, such as at least about 10 mmol, for example at least about 15 mmol, for example at least about 20 mmol, wherein the amounts are calculated based on the weight or volume of the composition excluding / before the addition of biomass and / or extracted lipids, including phospholipids.

[0396] A preferred composition, food, feed, or beverage of the present invention comprises iron as an additional separate component. Iron can enhance the meat-like flavor and / or aroma produced by the composition, food, feed, or beverage of the present invention. In some embodiments, the iron is in the form of an iron salt, preferably a ferrous salt. Any iron salt suitable for ingestion may be used, and such salts, such as chelated forms of iron, are well known to those skilled in the art. In some embodiments, the iron source is iron(II) fumarate. Iron(II) fumarate is available, for example, as iron tablets from APOHEALTH Pty Ltd (NSW, Australia). The iron source is a component other than the microbial biomass or its components, even if the microbial biomass or its components contain iron.

[0397] In certain embodiments, the compositions of the invention comprise an amount of iron equivalent to up to about 100 mg, up to about 50 mg, about 20 to about 50 mg, or about 30 to about 40 mg of elemental iron per kg of dry composition or slurry, or per L of liquid composition, calculated based on the volume of the composition excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms.

[0398] In a particularly preferred embodiment, the compositions, foods, and feeds of the present invention comprise an aqueous component. The presence of some moisture in the composition promotes the production of a food-like flavor and / or aroma upon heating. In some embodiments, the aqueous component comprises an aqueous buffer, such as, for example, a phosphate buffer. In certain embodiments, the compositions of the present invention comprise an aqueous component apart from any water incidentally contained in other components, such as any moisture present in the microbial biomass. The compositions of the present invention are preferably not dry or substantially dry, having less than 10% moisture by weight. In one embodiment, the composition is a dry composition. In another embodiment, the composition is a liquid composition. In one embodiment, the composition is in the form of a powder, solution, suspension, slurry, or emulsion. In some embodiments, the composition is provided as a composition excluding the aqueous component (i.e., a dry composition), and the aqueous component is added to the composition before heating.

[0399] In some embodiments, the compositions of the present invention may further comprise an aqueous buffer. The buffer maintains the pH of the composition and provides moisture to the composition that promotes the production of food-like flavors and / or aromas upon heating, as described above. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a buffer with a pH of about 5.0 to about 7, such as about 5 to about 6, such as about 5.3 or about 6.0. In a particular embodiment, the buffer is a phosphate buffer with a pH of about 6.0.

[0400] The composition, food, feed or beverage of the present invention may further comprise one or more additional components. Such components may be, for example, flavor precursors intended to participate in the Maillard reaction that occurs when the composition is heated. For example, such additional components may include oils, such as vegetable oils, free fatty acids, alpha-hydroxy acids, dicarboxylic acids, nucleosides, nucleotides, vitamins, peptides, protein hydrolysates, extracts, phospholipids, lecithins, and organic molecules.

[0401] In some embodiments, the composition, food, feed, or beverage further comprises thiamine. Thiamine can enhance the meat-like aroma and / or flavor produced by the composition of the present invention. In some embodiments, thiamine can be present in the composition in an amount of about 0.5 to about 5 mmol, about 1 to about 4 mmol, or about 1 to about 3 mmol, or at least about 0.2 mmol, such as at least about 0.3 mmol, such as at least about 0.4 mmol, such as at least about 0.5 mmol, such as at least about 0.7 mmol, per kg of dry composition or slurry, or per L of liquid composition. In certain embodiments, thiamine is present in an amount of about 1.5 mmol to about 2.5 mmol, such as about 2 mmol, or in an amount calculated based on the weight or volume of the composition excluding / before the addition of lipids, preferably phospholipids, extracted from the microbial biomass and / or microorganisms. In some embodiments, thiamine may be present in a feed or beverage in an amount of about 0.1 to about 5 mmol, about 0.1 to about 1 mmol, about 0.5 to about 5 mmol, or about 1 to about 3 mmol per kg of dry composition or slurry, or per L of liquid composition (e.g., beverage), calculated based on the weight or volume of the composition excluding / before the addition of microbial biomass and / or lipids, preferably phospholipids, extracted from the microorganisms.

[0402] In some embodiments, the composition, food, feed, or beverage further comprises a yeast extract. In the food science arts, "yeast extract" is generally understood to refer to the water-soluble portion of autolyzed yeast and is commercially available from various suppliers. See, for example, Sigma Aldrich, Cat. No. Y1625 Yeast Extract. Yeast extract does not contain whole yeast cell biomass. The presence of yeast extract can enhance the meat-like aroma and / or flavor produced by the composition when heated. The yeast extract can be a typical flavorless yeast extract or, for example, a beef flavored or roasted chicken skin flavored yeast extract. In some embodiments, the composition is suitable for producing a food-like aroma and / or flavor that is a meat-like aroma and / or flavor, and the composition comprises a yeast extract. The presence of yeast extract can enhance the meat-like aroma and / or flavor produced by the composition of the present invention, as observed in the following examples.

[0403] In some embodiments, the yeast extract is present in an amount of about 100 mg to about 200 gm, or about 200 mg to about 100 gm, or about 10 g to about 200 g, for example, about 15 g to about 200 g, for example, about 20 g to about 200 g, for example, about 30 g to about 200 g, for example, about 40 g to about 200 g, for example, about 50 g to about 200 g, for example, about 50 g to about 180 g, for example, about 60 g to about 180 g, per kg of dry composition or slurry, or per L of liquid composition, calculated based on the volume of the composition excluding / before the addition of microbial biomass and / or phospholipids extracted from a microorganism. In some embodiments, the yeast extract is present in the composition in an amount of at least about 5 g, such as at least about 7 g, such as at least about 10 g, such as at least about 15 g, such as at least about 20 g, such as at least about 25 g, such as at least about 30 g, such as at least about 40 g, such as at least about 50 g, such as at least about 60 g per kg of dry composition or slurry, or per L of liquid composition. In a particular embodiment, the yeast extract is present in the composition in an amount of at least about 30 g per kg of dry composition or slurry, or per L of liquid composition.

[0404] In some embodiments, the composition, food, feed, or beverage does not contain yeast extract. The presence of yeast extract may enhance the meat-like aroma and / or flavor produced by the composition, food, feed, or beverage, so yeast extract may be omitted if an alternative food-like flavor and / or aroma is desired, such as, for example, vegetable or herbal aroma and / or flavor. The absence of yeast extract may reduce the potential masking of a desired aroma and / or flavor, such as vegetable aroma and / or flavor, by the meat-like aroma and / or flavor enhanced by the presence of yeast extract. Thus, in some embodiments, the food-like aroma and / or flavor is a fishy aroma and / or flavor, vegetable, and / or herbal aroma and / or flavor, and the composition, food, feed, or beverage does not contain yeast extract.

[0405] In some embodiments, the composition, food, feed, or beverage further comprises one or more herbs and / or spices. As shown in the examples below, compositions comprising herbs, such as fenugreek (Trigonella foenum-graecum), have been found to enhance the vegetable, soupy, and / or herbal flavors and / or aromas produced by the compositions of the present invention in some cases. These herbal, vegetable, and / or soupy flavors and / or aromas can, in some embodiments, partially or completely mask the meaty / fishy aromas and / or flavors, allowing for adjustment of the overall aroma and / or flavor produced by the compositions of the present invention. As used herein, herbs and / or spices refer to plant parts or extracts having aromatic properties suitable for use in foods or beverages. Typically, herbs are understood to refer to the leafy, green, or flowering parts of the plant, while spices are understood to refer to other parts of the plant, usually dried, including seeds, bark, roots, and fruits. The herbs or spices may be in the form of whole plant parts, or chopped, crushed, or rolled plant parts, or in a dried form, for example as a powder. In certain embodiments, the one or more herbs and / or spices include fenugreek. Fenugreek is also said to contain several bioactive components, which may provide health benefits to the consumer. In some embodiments, the one or more herbs and / or spices include fenugreek leaves.

[0406] In one embodiment, the composition, food, feed or beverage of the present invention comprises (a) phospholipids and / or microbial biomass comprising phospholipids extracted from a microorganism, (b) glucose and / or ribose, (c) cysteine ​​and / or cystine, (d) an iron source, e.g., an iron salt, (e) glutamic acid or a salt thereof, (f) thiamine, (g) an aqueous component, e.g., an aqueous buffer, e.g., a phosphate buffer, having a pH of about 5 to about 6, e.g., about 5.3 or about 6.0, and (h) optionally, a yeast extract. In one embodiment, the composition comprises (b) ribose and (c) cysteine.

[0407] The composition, food, feed, or beverage of the present invention produces a food-like flavor and / or aroma, preferably a meat-like flavor and / or aroma, when heated. Heating refers to raising the temperature of the composition, for example above room temperature, and to any temperature and for any time sufficient to produce a food-like flavor and / or aroma. In this context, the temperature is raised high enough and long enough for the Maillard reaction to occur between amino groups and sugars in the composition, and additional reactions with lipids, preferably phospholipids, or their degradation products in the composition, food, feed, or beverage to produce a food-like flavor and / or aroma. Selection of a suitable temperature and duration for the heating step is easily performed by one skilled in the art. As used herein, "heated" or "heating" or the like should be understood to mean heating under conditions sufficient to produce a food-like aroma, unless otherwise specified. Heat can be applied to the composition of the present invention before contacting the food, or after application to the food, or both. Such heating of the compositions, foods comprising the compositions, or foods, feeds, or beverages of the present invention can be done, for example, in an oven, a frying pan, a wok, or the like, or on a barbecue. The exact temperature to which the composition, food, feed, or beverage should be heated to produce a food-like flavor and / or aroma, preferably a meat-like flavor and / or aroma, may vary, for example, depending on the precise composition, food, feed, or beverage, and the time the composition, food, feed, or beverage is heated, and the amount of composition, food, feed, or beverage heated, including in some embodiments compositions that produce a food-like flavor and / or aroma when the composition or food is heated, for example, to a temperature of at least about 100° C., at least about 110° C., at least about 120° C., at least about 130° C., or at least about 140° C. In this regard, the temperature should not be so high that the food burns or has a burnt flavor and / or aroma. In certain embodiments, the composition, food, feed, or beverage produces a food-like flavor and / or aroma when heated to about 140°C.

[0408] Similarly, the compositions and foods of the present invention produce food-like flavors and / or aromas, preferably meat-like flavors and / or aromas, when heated for various periods of time, depending, for example, on the temperature to which the composition is heated, the exact nature of the composition, and the amount of the composition heated. Nevertheless, in some embodiments, the composition, food, feed, or beverage may produce food-like flavors and / or aromas when heated for at least 5 minutes or at least 10 minutes, for example, at least 15 minutes, at least about 30 minutes, or at least about 45 minutes. In some embodiments, the composition, food, feed, or beverage may produce food-like flavors and / or aromas when heated for at least about 1 hour, for example, about 1 hour. Heat is preferably applied for a length of time that does not produce a burnt flavor and / or aroma, as will be appreciated by those skilled in the art.

[0409] In one embodiment, the composition, food, feed or beverage of the present invention produces a food-like flavor and / or aroma, preferably a meat-like flavor and / or aroma, when heated at a temperature of at least about 100° C. for at least 5 minutes or at least 10 minutes, at a temperature of at least about 100° C. for at least 30 minutes, at a temperature of at least about 120° C. for at least 30 minutes, at a temperature of at least about 130° C. for at least 1 hour, or at least about 140° C. In a preferred embodiment, the composition, food, feed or beverage produces a food-like flavor and / or aroma when heated at about 140° C. for about 1 hour.

[0410] According to some embodiments, the compositions, foods, feeds or beverages of the present invention may produce food-like flavors and / or aromas when heated at temperatures and for times different from those outlined above, however, it will be appreciated that in some embodiments, the compositions may produce stronger and / or more desirable food-like flavors and / or aromas when heated to the temperatures and / or for the times discussed above.

[0411] The food-like flavor and / or aroma produced by the composition, food, feed, or beverage of the present invention may, according to some preferred embodiments, comprise a meat-like flavor and / or aroma. In certain embodiments, the food-like flavor and / or aroma may be the aroma of cooked meat or meat-based food. For example, the food-like flavor and / or aroma may be beef, steak, chicken, e.g., roast chicken or chicken skin, pork, lamb, duck, venison, chicken, or other meat soup, meat broth, liver, or generally "meat-like". Such aromas are typically detected by human volunteers, e.g., by a qualified sensory panel. In this context, a composition, food, feed, or beverage is said to produce a food-like or meat-like flavor and / or aroma if, in a double-blind test of the composition, food, or beverage, at least one-third, e.g. at least one-half, of the number of volunteers in the taste / sniff panel detect the food-like or meat-like flavor and / or aroma. Similarly, a food or beverage comprising the composition of the present invention has an increased food-like or meat-like flavor and / or aroma when at least one-third, e.g., at least one-half, of the number of volunteers in a tasting / smelling panel detect an increased food-like or meat-like flavor and / or aroma in a double-blind study compared to a corresponding food or beverage lacking the composition of the present invention. It will be appreciated that in some cases, there is some variability in how various flavors and / or aromas are perceived by different subjects who experience those aromas, and subjects may describe the exact flavor and / or aroma slightly differently. In one embodiment, the number of volunteers is at least 6, e.g., at least 10, at least 25, at least 50, or between 6 and 50.

[0412] In some embodiments, heating the composition, food, feed, or beverage may be accomplished by the addition of one or more of the following: 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethylbenzaldehyde, 2,3,4,5-tetramethylcyclopent-2-en-1-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, trans-2-octene-1 In some particular embodiments, the heating produces volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, 1-octanol, trans-2-octen-1-ol, and 1-nonanol.

[0413] The food-like flavors and / or aromas produced by the composition, food, feed or beverage of the present invention according to some embodiments include fish-like flavors and / or aromas, such as cooked fish flavors and / or aromas, such as fried fish flavors and / or aromas, vegetable and / or herb flavors and / or aromas, such as cooked vegetable and / or herb flavors and / or aromas, such as soup, mushroom, onion, vegetable, herb or roasted vegetable flavors and / or aromas. In some embodiments, the composition, food, feed or beverage comprises ribose and the food-like flavors and / or aromas include meat-like flavors and / or aromas, such as cooked meat-like flavors and / or aromas, and / or fish-like flavors and / or aromas, such as cooked fish or fried fish flavors and / or aromas.

[0414] In some embodiments the composition, food, feed or beverage comprises glutamic acid, e.g. glutamic acid in addition to a further amino acid such as cysteine ​​or a salt or derivative thereof, and the food-like flavor and / or aroma comprises, e.g. a meaty flavor and / or aroma, e.g. a cooked meat flavor and / or aroma, and / or a fishy flavor and / or aroma, e.g. a cooked fish or fried fish flavor and / or aroma.

[0415] In some embodiments, the composition, food, feed, or beverage comprises a yeast extract and the food-like flavor and / or aroma comprises a meaty flavor and / or aroma, e.g., a cooked meat-like flavor and / or aroma. In some embodiments, the composition does not comprise a yeast extract and the food-like flavor and / or aroma comprises a fishy flavor and / or aroma, e.g., a cooked fish or fried fish-like, vegetable and / or herbal aroma and / or flavor.

[0416] In some preferred embodiments, the microorganism is a Saccharomyces spp., Yarrowia spp., Mortierella spp., or Mucor spp., such as Saccharomyces cerevisiae, Yarrowia lipolytica, Mortierella alpina, or Mucor hiemalis, such as Saccharomyces cerevisiae strain D5A Yarrowia lipolytica strain W29, Mortierella alpina, or Mucor hiemalis, and the food-like flavor and / or aroma comprises a meat-like flavor and / or aroma, such as a chicken-like flavor and / or aroma, such as a cooked chicken flavor and / or aroma, such as a roast chicken, chicken skin, or chicken soup flavor and / or aroma. In a preferred embodiment, the microbial biomass is a Mortierella spp., such as Mortierella alpina, and the food-like flavor and / or aroma comprises a beef-like flavor and / or aroma.

[0417] In some embodiments the composition, food, feed, or beverage comprises one or more herbs and / or spices, such as fenugreek, e.g. fenugreek leaves, and the food-like flavor and / or aroma comprises vegetable, soup-like and / or herbal flavor and / or aroma.

[0418] It will be appreciated that in some cases there is some variability in how various flavors and aromas are perceived by different subjects experiencing those aromas, and subjects may describe the exact flavors and aromas slightly differently.

[0419] In certain embodiments, the compositions, foods, feeds, or beverages of the present invention may produce a food-like flavor as well as a food-like aroma. Such food-like flavors may correspond to the food-like aromas disclosed herein. Thus, references to aromas herein may be understood to refer, according to certain aspects, instead to aromas and / or flavors, where appropriate, and vice versa.

[0420] The compositions, foods, feeds or beverages of the present invention are suitable for human or animal consumption, typically at least for human consumption.

[0421] In some embodiments, the compositions of the invention are incorporated into a food or beverage product or feed before or during heating, such that when the food or beverage product is heated, e.g., during cooking, the compositions produce associated food-like aromas via Maillard and related reactions. In some embodiments, the compositions of the invention are heated before being incorporated into or added to a food or beverage product or feed.

[0422] The present invention further relates to a method of producing a food, beverage product, or feed comprising combining the composition of the present invention with one or more additional ingestible ingredients. The present invention further relates to a method of producing a food, beverage product, or feed comprising combining the microbial lipids of the present invention with amino acids and sugars and one or more additional ingestible ingredients. Each of the embodiments described above in relation to the composition of the present invention also applies to the food, beverage, and feed of the present invention, the methods of making them, and the use of the food, beverage, and feed. Suitable additional ingredients that may be included in such food, beverage products, or feed are discussed below. For example, the composition can be combined with other ingestible ingredients by mixing, applying it to the surface of the other ingredients, or immersing / soaking the other ingredients. In one embodiment, the food, feed, or beverage product is prepared by (a) heating the composition of the present invention and (b) mixing the product from (a) with other food, feed, or beverage ingestible ingredients, or by (a) mixing the composition of the present invention with other food, feed, or beverage ingestible ingredients, and (b) heating the mixture resulting from (a).

[0423] The food, drink product, or feed may be in either solid or liquid form and may be intended to be frozen, refrigerated, or kept at room temperature before cooking. In some embodiments, the food, drink product, or feed is provided as a dry product, excluding the aqueous component, which is added to the composition before, during, or after heating, especially before heating. The food or drink product or feed may contain edible macronutrients, proteins, carbohydrates, vitamins, and / or minerals in amounts desired for a particular application. The amounts of these ingredients will vary depending on whether the composition is intended for use in normal individuals, or for individuals with special needs, such as individuals suffering from metabolic disorders, or for vegetarians or vegans.

[0424] According to a preferred embodiment, the food or beverage product of the present invention does not contain any animal-derived components. In a preferred embodiment, at least a portion of the ingredients are plant materials or plant-derived materials. Such an embodiment is advantageously suitable for a vegan or vegetarian diet. In some embodiments, the food or beverage product or feed may be soy-free, wheat-free, yeast-free, MSG-free, and / or protein hydrolyzed product-free. The food or beverage product or feed preferably has a food-like taste or aroma, such as a meaty or fishy aroma, imparted by the composition of the present invention. EXAMPLES

[0425] Example 1. Materials and Methods Media and Chemicals YPD medium is a rich medium containing 10 g / L yeast extract (Sigma Aldrich, Catalog No. Y1625), 20 g / L peptone (Sigma Aldrich, Catalog No. P0556), and 20 g / L glucose (Sigma Aldrich, Catalog No. G7021). YPD plates also contain 20 g / L agar. SD-Ura medium contained yeast synthetic dropout medium (Sigma Catalog No. Y1501).

[0426] Unless otherwise stated, chemicals were sourced as follows: L-cysteine ​​(Sigma, Cat. No. 168149), D-(-) ribose (Sigma, Cat. No. R7500), thiamine hydrochloride (Sigma, Cat. No. 47858), ferrous fumarate (Fe 2+ , Apohealth, NSW, Australia, code number MH / Drugs / 25-KD / 617), L-glutamic acid monosodium salt hydrate (Sigma, catalog number G5889), potassium phosphate dihydrogen (Sigma, catalog number 1048731000).

[0427] Large-scale culture media Unless otherwise stated, the medium used to prepare seed cultures for large-scale cultures (2 L or more) contained 10.64 g / L potassium dihydrogen orthophosphate (KH 2 PO 4 ), 4.0 g / L diammonium hydrogen orthophosphate ((NH 4 ) 2 HPO 4 The medium was a defined medium (DM) with basal medium (BM) containing 1.7 g / L citric acid (monohydrate), and 1.7 g / L citric acid (monohydrate). These components were dissolved in approximately 70% of the required volume of water purified by reverse osmosis, adjusted to pH 6.0 with 2M NaOH, and made up to the required volume using purified water. The BM was sterilized at 121°C for 20 minutes and cooled to room temperature. The following components were then added separately: 30 ml / L 660 g / L glucose (autoclaved), 10 ml / L 1M magnesium sulfate heptahydrate (autoclaved) to a final concentration of 20 g / L, 10 ml / L trace metals solution (see below, filter sterilized), 10 ml / L 15 g / L thiamine hydrochloride (filter sterilized), 3 ml / L 10% (v / v) Sigma Antifoam 204 (autoclaved).

[0428] Fermentation medium (FM) for 2L and 10L cultures also used BM as the base medium. The required volume was added to the bioreactor and sterilized at 121°C for 60 minutes in a fluid cycle for autoclavable bioreactors or 30 minutes for stationary steam bioreactors, and cooled to 31°C. The following components were added per liter of base medium: 121 ml / L of 660 g / L glucose (autoclaved), 5 ml / L of 1M magnesium sulfate heptahydrate (autoclaved) for a final concentration of 80 g / L, 5 ml / L of trace metals solution (see below, filter sterilized), 5 ml / L of 15 g / L thiamine hydrochloride (filter sterilized), and 50 ml / L of 200 g / L ammonium chloride (filter sterilized). Glucose, magnesium, trace metals solution, and thiamine solution were mixed and added together to the bioreactor. Once the medium was formulated, the pH was checked and was usually just below 6.0. Using a pH controller, ammonia solution was added to the medium to bring the pH to 6.0.

[0429] The trace metal solution (TM) contains 2.0 g of CuSO per liter. 4 .5H 2 0, 0.08 g NaI, 3.0 g MnSO 4 .H 2 0.2 g NaMoO 4 .2H 2 0, 0.02 g H 3 BO 3 , 0.5 g CoCl 2 .6H 2 0, 7.0 g ZnCl 2 , 22.0 g FeSO 4 .7H 2 0, 0.50 g CaSO 4 .2H 2 The solution contained 10 mL of 0.5% CO, 1 ml of HO, and 1 ml of sulfuric acid. The reagents were added in the order listed. The addition of sulfuric acid resulted in dissolution of the calcium sulfate. The trace metals solution was sterilized by filtration through a 0.2 μm filter and stored in aluminum foil wrapped bottles at 2-8°C.

[0430] One pH adjustment reagent is phosphoric acid solution (10% w / v), 118 ml of 85% H3 PO 4 to 882 ml of purified water. The solution was sterilized by autoclaving.

[0431] The other was an ammonia solution (10% v / v), which was prepared by adding 330 ml of 30% ammonia solution to 670 ml of purified water. The solution was considered to be self-sterilizing. An antifoam solution was prepared by mixing 100 ml of Sigma antifoam 204 with 900 ml of purified water to obtain a concentration of 10%. The mixture was sterilized by autoclaving.

[0432] A feed solution was prepared by adding 134 ml of filter-sterilized 200 g / L ammonium chloride to 1 L of 660 g / L glucose and sterilized by autoclaving.

[0433] Microbial strains and cloning vectors For experiments on the production of lipids, including phospholipids, S. cerevisiae strains INVSc1 (ThermoFisher, Cat. No. C81000) and D5A (ATCC 200062) were used as host strains. When various lipid-modifying genes were tested in yeast by adding transgenes, the pYES2 plasmid was used as a base vector for gene introduction. INVSc1 and pYES2 were obtained from Invitrogen (Cat. No. V825-20). The genotype of INVSc1 was MATa his3Δ1 leu2 trp1-289 ura3-52 / MATα his3Δ1 leu2 trp1-289 ura3-52, and its phenotype was His-, Leu-, Trp-, and Ura-. The pYES2 vector had unique HindIII and XhoI restriction enzyme sites that were used for the insertion of DNA fragments encoding various proteins described herein. The pYES2 expression vector contained the URA3 gene as a selectable marker gene for introduction into yeast strains that are Ura-, a 2μ origin of replication for high copy maintenance, and an inducible Gal1 promoter for expression of protein coding regions in yeast. The plasmid also contained an ampicillin resistance gene for selection in E. coli during cloning experiments.

[0434] Several strains of Yarrowia lipolytica were obtained from the American Type Culture Collection (Manassas VA, USA): strain JM23 (ATCC 90812) with the genotype leu235 lys512 ura318 xpr2::LYS5B, strain IFP29 (ATCC 20460) with the genotype leu235 lys512 ura318 xpr2::LYS5B, and the wild-type strain W29 (Casaregola et al., 2000). Strains Y2047 (ATCC PTA-7186, US7588931) and Y2096 (ATCC PTA-7186) were obtained from ATCC.

[0435] Escherichia coli strains DH5α and BL21 were obtained from ThermoFisher Scientific (catalog number 18265017, EC0114).

[0436] The fungal strain described herein as yNI0121 (Mucor hiemalis) has been deposited under the Budapest Treaty at the National Measurement Institute, Port Melbourne, VIC 3207, Australia on February 4, 2021 and is designated under the following deposit number: yNI0121 deposit accession number V22 / 001757. The fungal strains described herein as yNI0125 (Mortierella elongata), yNI0126 (Mortierella sp.), yNI0127 (Mortierella sp.) and yNI0132 (Mortierella alpina) have been deposited under the Budapest Treaty at the National Measurement Institute, Port Melbourne, VIC 3207, Australia on October 12, 2021 and are designated under the following deposit numbers: yNI0125 deposit accession number V21 / 019953, yNI0126 deposit accession number V21 / 019951, yNI0127 deposit accession number V21 / 019952, and yNI0132 deposit accession number V21 / 019954.

[0437] Growth of S. cerevisiae and Y. lipolytica cultures for lipid analysis. To provide inoculum for cultures for fatty acid production, extraction, and analysis, small-scale cultures of Y. lipolytica or S. cerevisiae were grown in 5 ml of YPD medium for 24 hours at 29°C. For experiments, the inoculum culture was diluted to an optical density at 600 nm (OD600) of 0.1 in growth medium with a volume of, for example, 50 to 2000 ml. Cultures were grown in polypropylene tubes for 10 ml cultures or in glass flasks for larger volumes, with the vessel having a volume at least 5 times larger than the culture volume. The vessel was sealed with 3M Micropore Surgical Tape (Cat. No. 1530-1) tape and incubated in a shaker at 200 rpm for aeration at a specified temperature of 29°C unless otherwise specified.

[0438] When SD-Ura medium was used, carbon sources such as 2% glycerol or raffinose (w / v) (MP Chemicals, USA, Cat. No. 4010022) were used. Cultures were incubated overnight at 28°C with shaking for aeration. Inoculum cultures were diluted to provide an initial OD600 of 0.1 into 10 ml of SD-Ura medium containing 2% (w / v) glycerol or raffinose and 1% tergitol (v / v) (Sigma Aldrich Cat. No. NP40S) medium, or other volumes as specified. Cultures in 50 ml tubes or 250 ml flasks were incubated in a shaker at 200 rpm for aeration at 28°C. OD600 was checked at 15 or 30 min time intervals. When OD600 reached 0.3, exogenous compounds as potential substrates (if present) were added along with 2% galactose to induce transgenes from the GAL1 promoter as required.

[0439] For transformants such as pYES2 derivatives, large-scale cultures of S. cerevisiae cells were grown in a volume of 3 L. These were inoculated from glycerol stocks. Starter cultures were grown in 10 ml of SD-Ura medium containing 2% (w / v) raffinose for two nights. Cells were transferred to 3 L of SD-Ura medium containing 2% (w / v) raffinose and 1% tergitol (NP-40) until the OD600 was 0.1 and grown at 28°C with shaking at 200 rpm. OD600 was checked at time intervals of 15 and 30 min. When the OD600 reached 0.3, galactose was added to a final concentration of 2% (w / v) to induce the transgene. If necessary, sodium butyrate was added to the culture to a final concentration of 2 mg / ml. The flasks were then loosely closed with sterile aluminum foil. Cultures were grown in an incubator for 48 h, after which the cells were harvested by centrifugation.

[0440] To provide the inoculum, cultures of E. coli were grown from glycerol stocks in 5 ml LB medium for 24 h. Cultures were diluted in LB medium in polypropylene tubes or glass flasks to an OD of 0.1 and incubated at 37°C in a shaker at 200 rpm for aeration unless otherwise specified.

[0441] Providing lipid substrates to cells In substrate feeding experiments, both yeast and bacterial inoculum cultures were diluted with their respective growth medium containing 1% tergitol at OD600 of 0.1 and incubated with shaking for a period of time, typically 2 hours. Lipid substrates such as fatty acids, oils, or oil hydrolysates were then added to the medium and the cultures were further incubated for different periods of time. Fatty acid substrates were obtained from NuChek Prep: e.g., gamma-linolenic acid (GLA, Catalog No. U-63-A), dihomo-gamma-linolenic acid (DGLA, Catalog No. U-69-A), arachidonic acid (ARA, Catalog No. U-71-A), docosatetraenoic acid-N6 (DTA, Catalog No. U-83-A), and docosapentaenoic acid-ω6 (DPAω6, Catalog No. U-102-AX). Fatty acids were dissolved in ethanol and fed to the cultures to a final concentration of 0.5 mg / ml. When used, ARA-containing oil was obtained from Jinan Boss Chemical Industry Co., Ltd. (China) and has 50% ARA in its total fatty acid content. The oil was dissolved in ethanol and applied to the cultures to a final concentration of up to 5.0 mg / ml.

[0442] When compounds were added as potential carbon sources (feeding assays), the following compounds were obtained from Sigma Aldrich: ethanolamine (catalog number 110167), choline chloride (C7017), myo-inositol (13011), butyric acid (B103500), sodium butyrate (B5887), tributyrin (W222305), or palmitic acid (76119). Butyric acid dissolved in water was provided to S. cerevisiae to a final concentration of up to 2 mg / ml. When provided to Y. lipolytica cultures, butyric acid (B103500) was prepared in 50% glycerol and added to the culture to a final concentration of 2 mg / ml.

[0443] Oil preparations were also provided to some Y. lipolytica cultures: castor oil (Aussie Soap Supplies, AU, Cat. No. SKU: CB100), tributyrin (Sigma Aldrich, Cat. No. W222305), and long chain polyunsaturated fatty acids (GreenOMEGA 3 capsules, Green nutritionals, AU). These oils were emulsified in 70% NP40 and added to the medium at a final concentration of 2 mg / ml. In this case, the final NP40 concentration was 7% (v / v).

[0444] 2L Fermentation Parameters The following parameters were used for a 3 L (total volume) Sartorius Biostat B autoclavable bioreactor with a maximum working volume of 2 L culture. The volume of the starting medium was 1 L. The initial temperature set point was 31° C. and was not changed during the process. The temperature controller configuration was min: -100%, max: 100%, XP: 4%, TI: 300 sec, TD: 75 sec, Death: 0.0%, cascade control using a dissolved oxygen controller, min agitator speed: 500 rpm, max agitator speed: 1200 rpm, pH adjustment set point: 6.0, pH controller configuration: min: -100%, max: 100%, XP: 30%, TI 30 sec, TD: 0 sec, Death: 0.2% (corresponding to 0.02 pH units). The acid and base used for the automatic pH control were 10% H 3 PO 4 and 10% ammonia solution.

[0445] The initial dissolved oxygen set point was 30%. The dissolved oxygen (DO) electrode was calibrated after sterilization and the medium temperature stabilized at 31°C. 0% saturation was calibrated using pure nitrogen, a stirrer speed of 100 rpm, and a nitrogen flow rate of 0.1 L / min, and saturation was established at a stirrer speed of 500 rpm and an air flow rate of 0.5 L / min. Cascade control used a stirrer in a two-step cascade followed by gas mixing to provide oxygen enrichment of the air stream. Oxygen enrichment was used to reduce the air flow rate, thereby reducing foaming which can be detrimental to the process as yeast cells tend to float on the foam. Air flow was constant at 0.5 L / min with minimum oxygen enrichment at 0% and maximum oxygen enrichment at 50%. The dissolved oxygen controller configuration was set as follows: Death: 0%, Min: 0% (510 rpm), Max: 100% (1425 rpm), XP: 90%, TI: 50 sec, TD: 0 sec.

[0446] For foam control, automated chemical foam control was achieved using 10% Sigma Antifoam 204, adding 10 ml of 10% (v / v) Sigma Antifoam 204 before inoculation, 20 ml at 7 hours after inoculation, and 30 ml at 31 hours after inoculation. The foam controller configuration was cycle: 10 seconds, pulse: 5 seconds, sensitivity: 04.

[0447] Using the secondary seed culture, the target inoculum OD600 calculated based on the starting volume of the basal medium was 0.20. For fed-batch mode, feeding of the feeding solution was started 14 hours after inoculation with a feed flow rate of 20 ml / h. At the completion of each process, the vessel was drained and the cells were harvested by centrifugation.

[0448] 10L Fermentation Parameters The same parameters were used for a 15 L Sartorius Biostat C10 stationary steam bioreactor with a maximum working volume of 10 L culture, with the following differences: To calibrate the dissolved oxygen (DO) electrode, 0% saturation was calibrated using pure nitrogen with a stirrer speed of 100 rpm and a nitrogen flow rate of 1 L / min, and saturation was established with a stirrer speed set at 500 rpm and an air flow rate of 3 L / min. For cascade control, the air flow was constant at 3.0 L / min. The dissolved oxygen controller configuration was set as follows: HTime Stirrer: 0 min, Death: 0.5%, Min: 34% at 510 rpm, Max: 95% at 1425 rpm, XP: 150%, TI: 100 sec, TD: 0 sec, HTime GasMix: 0 min, Death: 0.5%, Min: 0% (no oxygen supplement), Max: 50%, XP: 5%, TI: 200 sec, TD: 0 sec.

[0449] For 2L fermentations, the target inoculation OD600 was 0.20 when a secondary seed culture was used. For fed-batch mode, feeding of the feed solution was started 14 hours after inoculation with a feed flow rate of 100 ml / h. At the completion of each process, unless otherwise stated, 24 hours after inoculation, the cultures were heat inactivated at 105°C for 5 minutes and then cooled to 31°C before harvesting the cells by centrifugation.

[0450] Seed culture for large-scale cultivation For the primary seed culture, a frozen glycerol stock of the yeast strain was used to inoculate 100 mL of DM in a 1 L plastic baffled Erlenmeyer flask with a vented cap. This was incubated for 24 ± 2 hours at 28 °C with shaking at 200 rpm for aeration. At the end of incubation, the optical density at 600 nm (OD600) was measured. The secondary seed culture was prepared by using the primary seed culture to inoculate 500 mL of DM in a 2 L plastic baffled Erlenmeyer flask with a vented cap to a starting OD600 of 0.04. The second seed culture was incubated for 16 ± 2 hours at 28 °C with shaking at 200 rpm. At the end of incubation, the OD600 was measured. This culture was used to inoculate the large-scale fermentation.

[0451] Cell harvesting, washing, and lyophilization Cells from small scale cultures were harvested, for example, by centrifugation in 50 ml tubes at 4600 g for 15 min, washed twice with 10 ml and finally with 1 ml MilliQ water. For the final wash, the cell suspension was transferred to a pre-weighed 2 ml Eppendorf tube, centrifuged and the cell pellet was freeze-dried (VirTis Bench Top freeze dryer, SP Scientific) for measuring dry cell weight, before being weighed and lipid extraction. When lipid substrates such as ARA, DGLA, γ-linolenic acid (GLA), butyric acid or palmitic acid were added to the growth medium, the cell pellet was washed successively with 1 ml of 1% tergitol (v / v), 1 ml of 0.5% tergitol and a final wash with 1 ml of water to remove remaining substrates from outside the cells and freeze-dried as described above. When oil was added to the growth medium, cells were harvested by centrifugation as above, but the cell pellet was washed successively with 5 ml of 10% tergitol (v / v), 5 ml of 5% tergitol, 5 ml of 1% tergitol, 5 ml of 0.5% tergitol, and a final wash of 5 ml of water to remove residual oil from the exterior of the cells. In some cases, microscopic observation after staining with Bodipy confirmed the absence of stained oil in the cell wall. The final wash allowed the pellet to be transferred to a pre-weighed 2 ml Eppendorf tube and lyophilized before being weighed and subjected to lipid extraction.

[0452] Lipid extraction from yeast cells Total cellular lipids were extracted from yeast cells such as S. cerevisiae or Y. lipolytica using a method modified from Bligh and Dyer (1959). Approximately 50 mg of lyophilized cells were homogenized using a Bullet Blender Blue (Next Advance, Inc. USA) with 0.6 ml of a mixture of chloroform / methanol (2 / 1, v / v) and 0.5 g of zirconium oxide beads (cat. no. ZROB05, Next Advance, Inc., USA) in a 2 ml Eppendorf tube at speed 6 for 5 min. The mixture was then sonicated in a sonication water bath for 5 min and 0.3 ml of 0.1 M KCl was added. The mixture was shaken for 10 min and centrifuged at 10,000 g for 5 min. The lower organic phase containing lipids was transferred to a glass vial and the remaining lipids were extracted from the upper phase containing cell debris by mixing with 0.4 ml of chloroform for 20 min and centrifuging. The lower phase was collected and combined with the first extract in a glass vial. The solvent was evaporated from the lipid sample under a stream of nitrogen gas, and the extracted lipid was resuspended in a measured amount of chloroform. If necessary, the lipid sample was stored at -20°C until further analysis.

[0453] Lipid extraction from larger biomass To extract total lipids from larger biomasses, a different method of cell homogenization was used using a larger volume of solvent, unless otherwise stated. Approximately 1.5 g of freeze-dried cells distributed in six 50 ml Cellstar polypropylene tubes (6 × tube A) (cat. no. 227261, Greiner bio-one) were homogenized for 3 min in 9 ml chloroform / methanol (2 / 1, v / v) per tube using an Ultra-Turrax T25 homogenizer (IKA Labortechnik Staufen, Germany). After adding 3 ml of 1 M KCl to each tube, homogenization was carried out for another 2 min. Each tube was centrifuged at 6,000 g for 3 min. The lower phase was transferred to a new tube (tube B) and the solvent was evaporated under a stream of nitrogen at room temperature. The upper phase was mixed with 1 g of glass beads in a Vibramax mixer for 10 min and mixed with vigorous vortexing for 1 min. 6 ml of chloroform was added to each tube and mixed again for 3 min. After centrifugation, the lower phase was transferred to tube B and the solvent was evaporated under a stream of nitrogen gas at room temperature. To extract the remaining lipids, the upper phase of tube A was mixed with an additional 6 ml of chloroform and mixed for 3 min. After centrifugation, the lower phase was transferred again to tube B. 3 ml of methanol and 3 ml of 0.1 M KCl were added to tube B and mixed for 3 min. The lower phase was transferred to a Falcon tube and the solvent was evaporated under a stream of nitrogen gas at room temperature. The extracted lipids were dissolved in chloroform / methanol (2 / 1, v / v) and stored at -20°C.

[0454] Lipid fractionation by thin-layer chromatography To separate different lipid types such as TAG, DAG, free fatty acids, and polar lipids such as phospholipids (PL), total lipids were fractionated on thin-layer chromatography (TLC) plates (Silica gel 60, Cat. No. 1.05626.0001, MERCK, Darmstadt, Germany) using hexane:diethyl ether:acetic acid (70 / 30 / 1 v / v / v) as the solvent system. Samples of lipid standards such as 18-6A (Nu-Chek Prep Inc, USA), which contains TAG, DAG, FFA, and MAG, were run in adjacent lanes to identify different lipid spots. When differentiating between various TAGs containing short chain fatty acids (SCFAs), a standard containing triheptadecanoin (Nu-chek, USA, Cat. No. T-155), a triglyceride mixture C2-C10 containing equal amounts of triacetin (TAG 6:0), tributyrin (TAG 12:0), tricaprylin (TAG 18:0), and tridecanoin (TAG 30:0) (Sigma Aldrich, Cat. No. 17810-1AMP-S) were run in adjacent lanes to identify the TAG lipid spots. After chromatography, the plate was sprayed with a primulin (Cat. No. 206865, Sigma, Taufkirchen, Germany) solution prepared at a concentration of 5 mg / 100 ml in acetone:water (80 / 20 v / v), and the lipid bands were visualized under UV light. The silica with lipids from each spot was scraped off and transferred to a tube. The lipid fraction was extracted from the silica for derivatization using either methylation, propylation, or butylation.

[0455] Large-scale fractionation of PL and TAG from total lipids PL and TAG were fractionated from approximately 100 mg of total lipids extracted from approximately 10 g of cell dry weight by loading the lipids on an 18 cm line on each of eight TLC plates (Silica gel 60, Cat. No. 1.05626.0001, MERCK, Darmstadt, Germany) and chromatographed using a solvent mixture consisting of hexane / diethyl ether / acetic acid (70:30:1, v:v:v). To aid in the identification of lipid bands, aliquots of lipid standards (18-6A; NuCheck Inc, USA) containing TAG, DAG, FFA, and MAG were run in parallel. After staining the plates with primulin and visualization under UV light, the PL band located at the origin and the TAG band with the same mobility as the TAG standard were collected and transferred to Falcon tubes. The lipid / silica samples were extracted with a mixture of 6 ml chloroform and 3 ml methanol, mixed vigorously for 5 min, then 3 ml water was added and mixed for another 5 min. After centrifugation at 3,000g for 5 min, the lower organic phase was transferred to a new tube. After centrifugation at 3000rcf for 5 min, the lower phase was transferred to a Falcon tube. The upper phase was mixed with 5ml of chloroform for 5 min to extract the remaining lipids. After centrifugation, the lower phase was combined with the first extract. The solvent was evaporated under a stream of nitrogen gas. The extracted lipids, TAG or PL were dissolved in a small amount of chloroform and filtered through a 0.2 μm microspin filter (Chromservis, EU, Cat. No. CINY-02) to remove any particulates. The fatty acid composition and amount of each PL and TAG fraction was determined by preparation and GC analysis of FAMEs. Such preparations were used, for example, to separate different polar lipid classes such as PC, PE, PI, and PS, or in the Maillard reaction for aroma testing or detection of volatile compounds as reaction products.

[0456] Lipid derivatization to fatty acid methyl esters (FAMEs) For analysis by GC, fatty acid methyl esters (FAMEs) were prepared from total extracted lipids or purified TAG or PL fractions by treatment with 0.7 ml of 1N methanol-HCl (Sigma Aldrich, Cat. No. 90964) in 2 ml glass vials with PTTE-lined screw caps for 2 h at 80°C. A known amount of heptadecanoin (Nu-Chek Prep, Inc., Cat. No. N-7-A, Waterville, MN, USA) dissolved in toluene was added to each sample before processing as an internal standard for quantification. After cooling the vials, 0.3 ml of 0.9% NaCl (w / v) and 0.1 ml hexane were added and the mixture was vortexed for 5 min. The mixture was centrifuged at 1700 g for 5 min and the upper hexane phase containing FAMEs was analyzed by GC.

[0457] Analysis and quantification of FAMEs by GC Individual FAMEs were identified and quantified by GC using an Agilent 7890A GC (Palo Alto, California, USA) equipped with a 30 m SGE-BPX70 column (70% cyanopropyl polysilphenylene-siloxane, 0.25 mm internal diameter, 0.25 μm film thickness), split / splitless injector and an Agilent Technologies 7693 series autosampler and injector, and a flame ionization detector (FID). Samples were injected in split mode (50:1 ratio) with an oven temperature of 150°C. The column temperature was programmed at 150°C for 1 min, increased to 210°C at 3°C / min, held for 2 min, reached 240°C at 50°C / min, and then held at 240°C for 0.4 min. The injector temperature was set at 240°C and the detector was set at 280°C. Helium was used as the carrier gas at a constant flow rate of 1.0 ml / min. FAME peaks were identified based on the retention times of FAME standards (GLC-411, GLC-674, NuChek Inc., USA). Peaks were integrated with Agilent Technologies ChemStation software (Rev B.04.03(16), Palo Alto, California, USA) based on the known amounts of external standard GLC-411 (Nucheck) and C17:0-ME internal standard response. The resulting data provide fatty acid composition on a weight basis, in terms of the percentage (wt%) of each fatty acid in a total fatty acid content of 100%. These percentages on a weight basis can be easily converted to percentages on a molar basis (mol%) based on the known molecular weight of each fatty acid.

[0458] Saponification of triacylglycerol Free fatty acids were released from TAG by incubating 1 mg of TAG in 0.2 ml of 3 M KOH at 80 °C for 3 min. After the sample was cooled to room temperature, 100 μl of hexane was added to the mixture. The mixture was vortexed for 5 min, centrifuged at 1700 g for 5 min, and the upper organic phase was collected for GC analysis.

[0459] Lipid derivatization to ethyl or propyl esters To convert the fatty acids in TAG to fatty acid ethyl esters (FAEEs), 2 mg of TAG was incubated in 1N HCl / ethanol solution at 80°C for 2 h. After the sample was cooled to room temperature, 100 μl of hexane was added to the mixture. The mixture was vortexed for 5 min, centrifuged at 1700g for 5 min, and the upper organic phase was collected for GC analysis. To convert the fatty acids in TAG to fatty acid propyl esters, 2 mg of TAG was incubated in 1N HCl / propanol instead of 1N HCl / ethanol and otherwise treated similarly.

[0460] Derivatization of fatty acids in TAG to butyl esters The TAG fraction was extracted from the silica of the TAG spot on the TLC plate as follows: 0.6 ml of chloroform:methanol (2:1, v / v) was added to the silica scraped off the TLC plate. The mixture was shaken and centrifuged at 10,000 g for 5 min. 0.3 ml of 0.1 M KCl was then added and the mixture was shaken for 5 min. The mixture was centrifuged at 10,000 g for 5 min and the lower organic phase was collected in a 2 ml GC vial. The silica / aqueous phase was extracted a second time, this time with 0.3 ml of chloroform mixed for 10 min followed by centrifugation at 10,000 g for 5 min. The lower organic phase was again collected and pooled in the same GC vial as the first extract. The pooled extract containing the TAG was filtered through a 0.2 μm microspin filter (Chromservis, EU, Cat. No. CINY-02) to remove traces of silica particles. The filtered TAG extract was then transferred to a GC vial with a flat insert and thoroughly dried under a stream of nitrogen.

[0461] The purified TAG was then derivatized to butyl esters using 60 μl of butanoic acid:1N HCl (Sigma Aldrich, Cat. No. 87472) as described in Mannion et al. (2018) with some modifications. Valeric acid (C5:0) (Sigma Aldrich, Cat. No. 75054) was added as an internal standard in an amount of 23.25 μg for SCFA and MCFA quantification, and 5 μg of heptanoic acid (Nu-Chek PREP, Inc., Cat. No. N-7-A Waterville, MN, USA) was added as an internal standard for LCFA quantification. The mixture was vortexed and heated at 80° C. for 2 h. The reaction was then stopped by adding 0.03 ml of water and 0.03 ml of hexane and mixed thoroughly for 10 min. After centrifugation at 1700g for 5 min, the upper organic phase was transferred to a new tube with a flat insert containing 0.1 ml of saturated NaCl for a second wash to remove traces of butanol. The mixture was mixed for 5 min, centrifuged at 1700g for 5 min, and the organic phase was transferred to a new GC vial with a conical insert and quickly capped for GC-FID analysis as described below.

[0462] Analysis and Quantitation of Butyl Esters This method was suitable for the quantification of short chain fatty acids (SCFA, C2-C8) as well as medium chain fatty acids (MCFA, C10-C14) and long chain fatty acids (LCFA, C16-C18) in lipid samples, including purified TAG preparations. It was the preferred method for the quantification of SCFA. FABEs prepared as described above were analyzed on an Agilent 7890A GC using a 30 m BPX70 column (0.25 mm ID, 0.25 μm film thickness, SGE, Australia). The column temperature was set at 40°C for 1 min, followed by increasing the temperature to 210°C at a rate of 3°C / min and holding for 2 min. The column temperature was further increased to 240°C at a rate of 100°C / min and held at this temperature for 0.5 min. Helium was used as the carrier gas with a flow rate of 1.031 ml / min. The injector temperature was programmed at 240°C with an inlet pressure of 11.8 psi. Samples were injected in split mode at a ratio of 50:1. The FID detector temperature was 280 °C with flow rates of 40 ml / min hydrogen gas, 400 ml / min air, and 25 ml / min make-up gas (He). FABE peaks were identified based on the retention times of a FABE standard mixture prepared with equal amounts of analytical grade C4-C18:1 fatty acids. The peak areas of the FABE mixture were used to determine response factors for individual FABE peaks in the GC and applied to correct for the area percentages of the FABE peaks.

[0463] In a variation of the GC method for quantification of FABE, referred to herein as the "C8C24 method," several column parameters were adjusted. The column temperature was set at 40° C. for 13 min, then the temperature was increased to 210° C. at a rate of 320° C. / min and held for 2 min. The column temperature was further increased to 240° C. at a rate of 10° C. / min and held at this temperature for 0.53 min. The injector temperature, FID detector temperature, and helium flow rate were the same as before.

[0464] Peak identity by GC-MS The identity of unknown or unclear peaks in the GC-FID chromatograms was confirmed by gas chromatography-mass spectrometry (GC-MS) analysis. Samples were run on the GC-MS operating in electron ionization mode at 70 eV to confirm peak identities and to identify extra peaks that may correspond to possible contamination, decomposition products, or reagent signals. A Shimadzu GC-MS QP2010 Plus (Shimadzu, Japan) system connected to an HTX-Pal liquid autosampler was used with the following parameters: injection volume of 1 μl or 2 μl using a 15:1 split / splitless inlet at a temperature of 250 °C. The oven temperature program used was the same as for the GC-FID. MS ion source and interface temperatures were 200 °C and 250 °C, respectively. Data were collected at a scan rate of 1000 and a scan range of 40-500 m / z. Peak separation was provided by a Stabilwax or Stabilwax-DA (Restek / Shimadzu) capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) using He as the carrier gas at 30 cm / sec. Mass spectral correlation was performed using the NIST library, retention index, and matching retention time of available standards. Identified SCFAs were set to be present when the signal-to-noise ratio was greater than 10:1. Instrumental and procedural blanks were run for quality control purposes.

[0465] Analysis of volatile compounds by solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GCMS). Reagents and Chemical Standards Analytical standards were obtained from Sigma Aldrich (USA) and represented various chemical classes of compounds in the Maillard reaction: 1-octen-3-ol, methional, 2(5H)-furanone, 2-methyl-3-heptanone, 1-pentanol, pentanal, hexanal, nonanal, 1-heptanal, octanal, trans-2-nonenal, isovaleric acid, 2-pentyl-furan, and 2,4,6-trimethyl-pyridine. Standard stock and working solutions were prepared in methanol (LCMS grade). A series of n-alkane (C8–C20) mixtures were purchased from Supelco (USA) and diluted with hexane for injection into the GC-MS.

[0466] Headspace HS-SPME-GCMS method HS-SPME was performed using a 50 / 30 μm divinylbenzene-carboxene-polydimethylsiloxane (DVB / CAR / PDMS) Stableflex fiber (Supelco, USA), 10 mm long, for an automated autosampler. Various amounts of sample (0.5, 1, 2, and 7.4 ml) were used to test the best volume required for analysis. Samples were conditioned at 50°C for 10 min in 10 ml headspace magnetic cap vials before extraction. Volatiles were extracted at 50°C for 20 min under stirring using a Combi-Pal autosampler HTX PAL (CTC Analytics). Samples were desorbed for 1 min at an injector temperature of 240°C in splitless mode. Each fiber was conditioned in a needle heater with helium flow for 8 min at 240°C before and after sample desorption to reduce carryover from the previous sample. Volatile compounds desorbed from the fibers were analyzed by a Shimadzu QP2010 Plus GC-MS equipped with a Restek Stabilwax column (30 m × 0.25 mm × 0.25 μm). The carrier gas was helium at a constant flow rate of 1 ml / min. The oven ramp program was started at 40 °C, held for 3 min, heated to 240 °C at a rate of 4 °C / min and held for 2 min. Ion fragmentation was acquired in EI mode at 70 eV and scanned in full scan mode from 40 to 400 m / z. Volatiles were identified by comparing the linear retention index calculated using the NIST mass spectral library and a series of n-alkanes (C8-C20) as external references. Authentic standards purchased from different compound classes and blanks (empty HS vials) were also analyzed for analytical quality control. Mass spectral matches were considered only with a minimum similarity index of 80%. Peaks with a signal-to-noise ratio of 2 were selected to identify volatile compounds such as Maillard reaction compounds.

[0467] Recombinant DNA methods Derivatives of pYES2 with single genes inserted for testing in yeast were generated by inserting the protein coding region between the unique HindIII and XhoI sites or other restriction enzyme sites in the plasmid as needed by standard cloning methods. E. coli strain DH5α was used for cloning and plasmid propagation and DNA preparation according to standard methods.

[0468] The GoldenGate (GG) method (Larroude et al., 2018) allows for rapid and efficient combinatorial assembly of multiple expression cassettes in a single vector and was therefore used to generate multigene constructs for testing in S. cerevisiae or Y. lipolytica. According to Celinska et al. (2017) and Larroude et al. (2018), the GG DNA parts and donor vectors, also called L0 vectors, were obtained from Addgene, USA. The DNA parts included promoters (GGE146, GGE151, and GGE294), terminators (GGE014, GGE015, GGE080, GGE020, and GGE021), and the backbone assembly vector (destination vector) was GGE114.

[0469] Protein coding regions for insertion into vectors by GG assembly were codon-optimized for S. cerevisiae or Y. lipolytica using Twist Bioscience and GeneArt online software (Twist Bioscience: www.twistbioscience.com / products / genes, ThermoFisher / GeneArt: www.thermofisher.com / au / en / home / life-science / cloning / gene-synthesis / geneart-gene-synthesis.html) and synthesized either by Twist Bioscience or GeneArt (ThermoFisher, USA) or in the laboratory. Internal BsaI restriction enzyme sites were avoided in the codon-optimized nucleotide sequences of protein coding regions, since BsaI sites were used in the GG assembly method. NotI restriction enzyme sites were also avoided within the nucleotide sequences, since NotI was used to linearize the gene constructs for transformation of Y. lipolytica. When one, two, or three genes were inserted into a single vector, individual components were designed with 4-nucleotide overhangs immediately 5' of each translation initiation codon (ATG) and 3' of the translation termination codon, with the sequence of each 4-nucleotide overhang designed according to the component's position in the backbone vector GGE114 according to Table 3. An external BsaI site with the appropriate 4-nt overhang was added to the 5' end of each DNA strand.

[0470] To avoid false positives when performing GG reactions with the GG backbone vector GGE114 carrying the ampicillin selection marker gene, the protein coding region was synthesized in a cloning vector carrying the kanamycin selection marker gene. E. coli strain DH5α was used for cloning and plasmid propagation according to standard methods. Antibiotics were used as appropriate to select transformed cells, e.g., ampicillin was added at 100 μg / mL to select for constructs carrying the ampicillin selection marker gene.

[0471] The destination vector GGE114 contains a red fluorescent protein (RFP) chromophore, which serves as a color-based visual marker for negative cloning in E. coli as described by Larroude et al. (2018). Vector GGE114 was a preassembled destination vector that contained InsUp and InsDown fragments and a common brick ZETA sequence instead of the URA3 marker, with the aim of reducing the number of fragments assembled into the backbone vector pYES2, which contains a 2μ origin for high copy maintenance in addition to the bacterial replicon, when using this combination. In this case, the RFP was between the URA3 marker and ZETA down. In the presence of BsaI enzyme, the RFP was released and one, two, or three transcription units (TUs; promoter-protein coding region terminator) were inserted.

[0472] The GG assembly reaction mix contained equimolar amounts (50 ng) of GG backbone vectors such as GGE114 and other DNA components (donor vectors) in a final volume of 7.5 μl by adding 0.75 μl 10xT4 ligase buffer, 0.75 μl 10xBSA (bovine serum albumin), 0.75 μl BsaI HF-V2 (NEB), 0.5 μl T4 ligase (NEB). The reaction mixture was incubated at 37 °C for 3 min, followed by 25 cycles of 16 °C for 4 min, then 1 cycle of 50 °C for 5 min and 80 °C for 5 min. 2-3 μl of the sample was introduced into competent cells of E. coli strain DH5α by standard methods. Colonies lacking RFP were confirmed to contain the desired gene insert by colony PCR with appropriate primers and verified by restriction enzyme digestion. Glycerol stocks were made and stored at -80 °C. [ka]

[0473] Transformation of S. cerevisiae A rapid method was used for the introduction of pYES2-based genetic constructs into S. cerevisiae without the use of competent cells. A loop filled with S. cerevisiae cells was scraped off a fresh plate and the cells were resuspended in 100 μl of transformation buffer (Sigma Aldrich, Cat. No. T0809). Approximately 1 μg of plasmid DNA containing 10 μl of 10 mg / ml salmon testis DNA boiled for 5 min before use was added to the cell suspension along with 600 μl of plate buffer (Sigma Aldrich, Cat. No. P8966) and mixed well. The mixture was incubated for 16 h at room temperature at minimum speed in a rotor wheel. The mixture was then heat shocked at 42°C for 15 min, spun at 3500 rpm for 3 min, and the cell pellet was resuspended in 200 μl of sterile water. To select for transformants, aliquots up to 100 μl were plated on synthetic dropout selection medium lacking uracil (SD-URA, Sigma Aldrich, Cat. No. Y1501). Plates were incubated at 28° C. for 3 days or until colonies appeared. Two or more colonies were picked from each plate and tested for the presence of the gene construct by colony PCR to identify transformants.

[0474] Transformation of Y. lipolytica for integration of expression cassettes The DNA of the gene construct containing the expression cassette (transcription unit) for insertion into Y. lipolytica by homologous recombination was digested with NotI or other appropriate restriction enzymes to release the expression cassette. The linearized DNA was introduced into competent cells of the selected Ura-Y. lipolytica strain, prepared using the Frozen-EZ Yeast Transformation II kit (Zymo Research, California, USA). Briefly, 5 μl (2 ug) of the NotI-digested linearized expression vector was mixed with 50 μl of competent cells and 500 μl of EZ3 solution from the kit and mixed thoroughly. The negative control transformation included competent cells that did not contain any DNA of the gene construct. The mixture was incubated at 28° C. for 2 hours, and then 100 μl was spread on SD-Ura plates. The plates were incubated at 28° C. for 2 days. When the recipient strain was auxotrophic, lacking a functional URA3 gene, only transformants that received the vector carrying the URA gene grew on these plates. In Y. lipolytica transformation, many colonies were observed. + Colonies were selected and confirmed to be transformed by colony PCR for a phenotype corresponding to the introduced gene construct and intended genetic modification.

[0475] Gene expression analysis Expression of the transgene was analyzed using a DNase RQ1 kit (Promega catalog no. M6101) and Qiagen columns (Qiagen RNAse-free DNAse), and RNA was purified from cells and used oligo-dT primer (200–500 ng), dNTPs (10 mM), Superscript III reverse transcriptase, and 0.1 M DTT for reverse transcription using standard methods.

[0476] Example 2. Polar lipid content and composition of animal fats The flavor and aroma of cooked animal products such as meat are derived from a wide variety of compounds including peptides, amino acids, sugars, vitamins such as thiamine, and lipids, including phospholipids (Dashdorj et al., 2015; Resconi et al., 2013). The main source of volatiles in cooked meat is from the Maillard reaction between amino acids and sugars and from the thermal decomposition of lipids. There are published reports on the content and composition of phospholipids in livestock products, e.g., Ashes et al. (1992), Margetak et al. (2012) and Resconi et al. (2013), but most of these reports relate more generally to polar lipids or phospholipids, or are only a part of the fatty acid composition, and do not report on the presence of trace fatty acids such as fatty acids with odd carbon numbers, conjugated fatty acids such as CLA, or trans fatty acids, which are reported to be prevalent in foods of animal origin.

[0477] The present inventors have more thoroughly investigated the fatty acid composition of phospholipid fractions from several animal sources, including beef and pork.

[0478] Extraction of lipids from animal sources Pork and Wagyu beef samples were purchased from a local market in Canberra, Australia. To extract lipids from the meat, a 2 g sample of minced meat was homogenized for 3 min in 6 ml of chloroform / methanol (2 / 1, v / v) solvent using an Ultra-Turrax homogenizer (IKA Labortechnik Staufen, Germany) in a 50 ml conical-bottom polypropylene tube (Falcon tube, no. 227280, Greiner bio-one GmbH, Germany) (Tube A). 2 ml of 1 M KCl was added to the homogenate and the mixture was homogenized for another 3 min and then mixed in a vibramax for 10 min. After centrifugation at 3,000 g for 5 min, the lower organic phase was transferred to a new Falcon tube (Tube B). The extraction was repeated with 4 ml of chloroform, homogenization, and centrifugation as before. The lower organic phase was collected and added to Tube B. The solvent was evaporated from tube B under a stream of nitrogen at room temperature. The extraction was repeated again with 4 ml of chloroform and the lower phase was transferred to tube B. Two ml each of methanol and 0.1 M KCl were added to tube B, mixed for 3 minutes and centrifuged. The lower phase was transferred to a new tube, tube C. The upper phase was mixed with 2 ml of fresh chloroform, centrifuged and the lower phase was again transferred to tube C. The solvent was evaporated from tube C with nitrogen. The extracted lipids were dissolved in chloroform, transferred to a glass vial and stored at -20°C. A sample of the extracted lipids was set aside as "total lipids" for analysis.

[0479] A lipid product commercially available as lard (York Foods) was purchased and analyzed.

[0480] Determination of lipid fractions and fatty acid composition To separate different lipid types, such as polar lipids including TAG, DAG, free fatty acids, and phospholipids (PL), total lipids extracted from pork, Wagyu beef, and lard were fractionated on TLC plates and collected as described in Example 1. The lipid fractions were extracted from silica spots, converted to FAMEs, and then analyzed and quantified by GC as described in Example 1.

[0481] Fractionation of polar lipids to separate PL classes To investigate the composition of PL classes in the extracted polar lipids, e.g. to determine the ratio of different PL amounts or their fatty acid composition, polar lipid extracts from beef, pork, and lard were applied to the origin of TLC plates (silica gel 60, catalogue no. 1.05626.0001, MERCK, Darmstadt, Germany) and chromatographed using a solvent mixture of chloroform / methanol / acetic acid / water (90:15:10:3, v:v:v:v). Lipid bands were visualized by spraying the plates with a 0.002% (w / v) solution of primulin in 80% acetone / water (v / v) and viewing under UV light. Phospholipid classes were identified with reference to known phospholipid standards applied to adjacent lanes on the TLC plate. Phospholipid standards, namely PC (Cat. No. 850375), PE (Cat. No. 850725), PS (Cat. No. 840035), PI (Cat. No. 850149), PG (Cat. No. 840475), PA (Cat. No. 840875) and LPC (Cat. No. 845875), were purchased from Avanti Polar lipids (USA) and cardiolipin was purchased from Sigma (Cat. No. 1649). Separation was achieved for all of these phospholipid classes on the TLC plate. This procedure also separated any galactolipids from PL. Silica containing the individual bands were collected in glass vials for further characterization of the lipid classes, including their relative amounts, fatty acid composition, and fatty acid distribution at the sn-1 and sn-2 positions of the PL molecule.

[0482] Fatty acid methyl esters (FAMEs) were prepared from total extracted lipids or purified TAG or polar lipid fractions as described in Example 1 and analyzed by GC. Peak areas were integrated with Agilent Technologies ChemStation software (Rev B.04.03(16)) and the lipid content and fatty acid composition in each sample were calculated based on the area of ​​the internal standard (heptadecanoin).

[0483] Confirmation of peak identity by GC-MS The identity of some of the FAME peaks was confirmed by GC-MS, especially when the peak identity was not clear from the retention time in the GC chromatogram, such as for minor or rare fatty acids, to identify extra peaks that may correspond to solvent, degradation products, or reagent signals. Analysis was performed as described in Example 1.

[0484] Fatty acid composition of TAG and polar lipid fractions from pork, beef, and lard The fatty acid composition of the extracted lipids, as well as the total fatty acid (TFA) content of the TAG and polar lipid fractions, was determined by GC quantification of FAMEs for pork, Wagyu beef and lard samples as described in Example 1. The fatty acid composition data is presented in Table 4 and is expressed as a percentage of the total fatty acid content by weight for each fatty acid. The standard deviation was generally less than 0.1.

[0485] As expected for animal lipids, all fractions contained a high percentage of saturated fatty acids (SFA), with palmitic acid at about 17% to 28% and stearic acid at about 12% to 18%. Monounsaturated fatty acids (MUFA) were also present in significant amounts, with about 40% in TAG and about 18% oleic acid in polar lipids. Palmitoleic acid (C16:1Δ9) was also present in lower amounts in all fractions, at about 1-3%. Other monounsaturated fatty acids present were C16:1Δ7, C17:1Δ9, C18:1Δ11, and the unusual fatty acid C20:1Δ8. Polyunsaturated fatty acids (PUFA) of both the ω3 and ω6 classes were also present in all fractions, with ALA, EPA, and DPA for the ω3 fatty acids, and DGLA, ARA, and DTA for the ω6 fatty acids. Several minor fatty acids, including odd-chain fatty acids C15:0 and C17:0, as well as minor peaks with retention times close to those of conjugated linoleic acid (CLA), were also observed but were not confirmed by GC-MS. Meat is known to contain both branched and straight-chain fatty acids with odd numbers of carbons (Taormina et al., 2020). A minor peak with a retention time close to that of C15:1 was shown by GC-MS to be decanal-dimethyl acetal, which is not a fatty acid. Similarly, a minor peak with a retention time close to that of C17:1t was proven by GC-MS to be related to decanal-dimethyl acetal and 1,1-demethoxy-dodecane. These peaks were therefore excluded from quantification. The presence of both trans fatty acids and...

Claims

1. 1. A composition comprising an amino acid or derivative, a sugar, and an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, the polar lipids being present in the extracted microbial lipid in an amount greater than the non-polar lipids, (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content that includes ω6 fatty acids, at least a portion of which are esterified in the form of phospholipids in the polar lipids, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA), wherein ARA is present in an amount of about 10% to about 60% of the total fatty acid content of the polar lipid, DGLA is present in an amount of about 0.1% to about 5% of the total fatty acid content of the polar lipid, and GLA is present in an amount of about 1% to about 10% of the total fatty acid content of the polar lipid; (b) the polar lipids comprise a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content comprising oleic acid and palmitoleic acid (C16:1Δ9 cis); A composition, wherein when the composition is heated, one or more compounds having a meat-associated flavor and / or aroma are produced.

2. ARA is present in an amount of about 20% to about 50%, e.g., about 25% to about 50%, or about 30% to about 50% of the total fatty acid content of the polar lipid, DGLA is present in an amount of about 1% to about 5% of the total fatty acid content of the polar lipid, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipid; or 2. The composition of claim 1, wherein ARA is present in an amount of about 10% to about 20% of the total fatty acid content of the polar lipid, DGLA is present in an amount of about 0.5% to about 5% of the total fatty acid content of the polar lipid, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipid.

3. 1. A composition comprising an amino acid or derivative, a sugar, and an extracted microbial lipid comprising esterified fatty acids in the form of either (i) polar lipids, free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, the polar lipids being present in the extracted microbial lipid in an amount greater than the non-polar lipids, (a) the polar lipids of (i) and (ii) comprise a total fatty acid (TFA) content that comprises ω6 fatty acids, the ω6 fatty acids being present in an amount of about 30% to about 70% of the total fatty acid content of the polar lipid, at least a portion of the ω6 fatty acids being esterified in the form of phospholipids in the polar lipid, the ω6 fatty acids comprising arachidonic acid (ARA), dihomo-γ-linolenic acid (DGLA), and γ-linolenic acid (GLA); (b) the polar lipids comprise a total saturated fatty acid content that includes palmitic acid and stearic acid; (c) the polar lipids comprise a total monounsaturated fatty acid content comprising oleic acid and palmitoleic acid (C16:1Δ9 cis); A composition, wherein when the composition is heated, one or more compounds having a meat-associated flavor and / or aroma are produced.

4. the ω6 fatty acids are present in an amount of about 40% to about 70%, about 40% to about 60%, or about 50% to about 60% of the total fatty acid content of the polar lipids; and / or 4. The composition of claim 3, wherein ARA is present in an amount of about 20% to about 50%, e.g., about 25% to about 50%, or about 30% to about 50% of the total fatty acid content of the polar lipids, DGLA is present in an amount of about 1% to about 5% of the total fatty acid content of the polar lipids, and GLA is present in an amount of about 3% to about 10% of the total fatty acid content of the polar lipids.

5. 5. The composition of any one of claims 1 to 4, wherein omega-3 fatty acids are absent from the polar lipid or are present in a total amount of less than about 3% by weight of the TFA content of the polar lipid, and / or the polar lipid is devoid of C16:2, C16:3 omega-3, EPA and DHA.

6. the phospholipids comprising the ω6 fatty acid comprise two, three or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS), optionally one or more of phosphatidic acid (PA), phosphatidylglycerol (PG) and cardiolipin (Car), preferably at least PC and PE, or at least PC, PE, PS and PI, each comprising one or at least two or more of ARA, DGLA and GLA; The phospholipids comprising the ω6 fatty acids comprise phosphatidylcholine (PC) and phosphatidylethanolamine (PE), each of which comprises one or at least two or more of ARA, DGLA and GLA; and / or 6. The composition of claim 1, wherein the phospholipids comprising the ω6 fatty acids comprise phosphatidylcholine (PC) and phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA), each comprising one or at least two or more of ARA, DGLA, and GLA, wherein ARA is present in PC in an amount of about 14% to about 20% of the total fatty acid content of the PC, ARA is present in PE in an amount of about 15% to about 20% of the total fatty acid content of the PE, and ARA is present in PA in an amount of about 15% to about 20% of the total fatty acid content of the PA.

7. The extracted microbial lipid is an extracted fungal lipid or a eukaryotic microbial lipid; Optionally, 7. A composition according to any one of claims 1 to 6, selected from extracted yeast lipids, preferably Saccharomyces cerevisiae, Yarrowia lipolytica or Pichia pastoris lipids, and extracted Mortierella spp lipids.

8. below: (d) at least one of EDA, DTA, and DPA-ω3 is also present in the polar lipid; (e) at least one of the following applies: the ratio of PC to PE, or the ratio of PC to phospholipids other than PC, is less than 3:1, less than 2:1, less than 1.5:1, less than 1.25:1, less than 1:1, between 3:1 and 1:1, between 2:1 and 1:1, or between 3:1 and 0.5:1; and / or the saturated fatty acid content of the polar lipids comprises one or more or all of lauric acid (C12:0), myristic acid (C14:0), C15:0 fatty acid, C20:0, C22:0 and C24:0, preferably C14:0 and C24:0 or C14:0, C15:0 and C24:0, more preferably C14:0, C15:0 and C24:0 but not including C20:0 and C22:0; stearic acid is present at a level of less than about 7%, or less than about 6%, or less than about 5%, preferably less than 4%, or less than 3% of the total fatty acid content of the polar lipid; the polar lipid comprises myristic acid (C14:0) in an amount less than about 2% by weight of the total fatty acid content of the polar lipid; lauric acid and myristic acid are absent from the polar lipid, or lauric acid and / or myristic acid are present in the polar lipid, and the sum of the amount of lauric acid and myristic acid in the polar lipid is less than about 2%, or less than about 1%, preferably less than about 0.5%, more preferably less than about 0.2% of the total fatty acid content of the polar lipid; C15:0 is absent from the polar lipid or C15:0 is present in the polar lipid in an amount of less than about 3%, preferably less than about 2%, or less than about 1% of the total fatty acid content of the polar lipid; palmitic acid is present in said polar lipid in an amount from about 10% to about 20% of the total fatty acid content of said polar lipid; palmitoleic acid is present in the polar lipid in an amount from about 3% to about 45%, or from about 3% to about 25%, or from about 3% to about 20%, or from about 3% to about 15% of the total fatty acid content of the polar lipid; oleic acid is present in said polar lipid in an amount from about 3% to about 60%, or from about 3% to about 40%, or from about 3% to about 25%, or from about 20% to about 60% of the total fatty acid content of said polar lipid; Vaccenic acid is not present in the polar lipids or vaccenic acid is present in the polar lipids in an amount less than about 2%, preferably less than about 1%, or less than about 0.5% of the total fatty acid content of the polar lipids; linoleic acid is present in said polar lipid in an amount from about 3% to about 20% of the total fatty acid content of said polar lipid; eicosadienoic acid is absent from said polar lipid, or eicosadienoic acid is present in said polar lipid in an amount from about 3% to about 12%, or from about 3% to about 8%, or from about 3% to about 6%, or less than about 3% of the total fatty acid content of said polar lipid; C20:0 and C22:0 are absent from the polar lipid, or C20:0 and / or C22:0 are present in the polar lipid, and the sum of the amount of C20:0 and C22:0 in the polar lipid is less than about 1.0%, less than about 0.5%, preferably less than 0.2% of the total fatty acid content of the polar lipid; C24:0 is absent from the polar lipid or C24:0 is present in the polar lipid in an amount less than about 1.0%, less than 0.5%, preferably less than 0.3%, or less than 0.2% of the total fatty acid content of the polar lipid; C17:1 is absent from the polar lipid or C17:1 is present in the polar lipid in an amount of less than about 5%, preferably less than about 4% or less than about 3%, more preferably less than about 2% of the total fatty acid content of the polar lipid; no monounsaturated fatty acids are present in the polar lipid which are C20 or C22 fatty acids, or C20:1 and / or C22:1 are present in the polar lipid, and the sum of the amount of C20:1 and C22:1 in the polar lipid is less than about 1.0%, less than about 0.5%, preferably less than 0.2% of the total fatty acid content of the polar lipid; (i) the content of ω6 fatty acids in the polar lipids that are C20 or C22 fatty acids is from about 5% to about 60%, preferably from about 10% to about 60%, of the total fatty acid content of the polar lipid; and / or (ii) the content of ω6 fatty acids in the polar lipids that are ω6 fatty acids having 3, 4 or 5 carbon-carbon double bonds is from about 5% to about 70%, preferably from about 10% to about 70%, more preferably from about 40% to about 70%, or from about 45% to about 70%, or from about 50% to about 70% of the total fatty acid content of the polar lipid; C16:3ω3 is absent from said polar lipids, or both C16:2 and C16:3ω3 are absent from said polar lipids; and / or 8. The composition of any one of claims 1 to 7, wherein the extracted microbial lipids comprise PC and / or are devoid of cyclopropane fatty acids, preferably devoid of C15:0c, C17:0c, and C19:0c.

9. The extracted lipids are obtained from a genetically modified microorganism; Optionally The genetically modified microorganism comprises: (i) synthesizing, or increasing the synthesis of, one or more ω6 fatty acids in the microorganism; (ii) increasing total fatty acid synthesis and / or accumulation in the microorganism; (iii) increasing total polar lipid synthesis and / or accumulation in the microorganism; (iv) a decrease in triacylglycerol (TAG) synthesis and / or accumulation in said microorganism, or an increase in TAG catabolism in said microorganism, preferably an increase in TAG lipase activity; (v) reducing the catabolism of total fatty acids in the microorganism; or any combination thereof, Optionally, The composition according to any one of claims 1 to 8, wherein the genetic modifications provide at least two of (i) to (v), preferably (iv) and (v), or (i), (iv) and (v).

10. A composition comprising an amino acid or derivative, a sugar, and an extracted Mortierella spp. lipid, preferably an extracted Mortierella alpina lipid, comprising esterified fatty acids in the form of either (i) polar lipids free of any non-polar lipids, or (ii) polar lipids and non-polar lipids, wherein the polar lipids are present in the extracted microbial lipid in an amount greater than the non-polar lipids.

11. the sugar, sugar alcohol, sugar acid, or sugar derivative is selected from ribose, xylose, glucose, fructose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, altodextrins, glycogen, galactose, lactose, ribitol, gluconic and glucuronic acids, amylose, amylopectin, or any combination thereof, preferably the sugar is ribose or xylose; and / or The composition according to any one of claims 1 to 10, wherein the amino acid or derivative thereof is selected from cysteine, cystine, cysteine ​​sulfoxide, allysine, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, or any combination thereof, preferably, the amino acid or derivative thereof is a sulfur-containing amino acid or derivative.

12. further comprising one or more esterified or non-esterified fatty acids from a source other than the extracted microbial lipids, cells, or extracts; Contains less than 5%, less than 10%, less than 15%, or less than 20% (w / w or w / v) protein; at least about 5 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, or at least about 50 mg of extracted microbial lipids per gram of dry composition or slurry or per ml of liquid composition; and / or 12. The composition of any one of claims 1 to 11, comprising from about 10 mg to about 100 mg of extracted microbial lipids, or from about 15 mg to about 50 mg of extracted microbial lipids per gram of dry composition or slurry, or per ml of liquid composition.

13. 13. A food, feed or beverage comprising an ingredient comprising the composition of any one of claims 1 to 12 and at least one other food, feed or beverage ingredient.

14. A food, feed or beverage comprising extracted Mortierella spp. lipids, such as extracted M. alpina lipids, wherein the lipids comprise esterified fatty acids in the form of either (i) polar lipids without any non-polar lipids, or (ii) polar lipids and non-polar lipids, wherein the polar lipids are present in the extracted microbial lipids in an amount greater than the non-polar lipids, and wherein the food, feed or beverage further comprises an amino acid or derivative, a sugar, and at least one other food, feed or beverage ingredient.

15. 13. A food, feed or beverage comprising an ingredient which is the extracted microbial lipid as defined in any one of claims 1 to 12, said food, feed or beverage further comprising an amino acid or derivative, a sugar and at least one other food, feed or beverage ingredient.

16. 13. A food, feed or beverage comprising a phospholipid and at least one other food, feed or beverage ingredient, wherein the phospholipid is the product of a reaction between the extracted microbial lipid as defined in any one of claims 1 to 12, an amino acid or derivative, and a sugar under conditions sufficient to produce at least two compounds having a meat-related flavor and / or aroma.

17. the sugar, sugar alcohol, sugar acid, or sugar derivative is selected from ribose, xylose, glucose, fructose, sucrose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-bisphosphate, inositol, maltose, molasses, altodextrin, glycogen, galactose, lactose, ribitol, gluconic and glucuronic acid, amylose, amylopectin, or any combination thereof, preferably the sugar is ribose or xylose; the amino acid or derivative thereof is selected from cysteine, cystine, cysteine ​​sulfoxide, allysine, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, or any combination thereof, preferably, the amino acid or derivative thereof is a sulfur-containing amino acid or derivative; the at least one other food, feed, or beverage ingredient comprises a protein, e.g., a microbial protein or a plant protein, optionally comprising at least 10% by weight of the composition; the food, feed or drink does not have any animal-derived components; and / or 17. A food, feed or drink according to any one of claims 14 to 16, wherein the food, feed or drink comprises a component obtained from an animal, optionally wherein the component comprises meat.

18. 13. A food or feed comprising at least two meat-related flavour and / or aroma compounds derived from the extracted microbial lipids as defined in any one of claims 1 to 10 or from a composition according to any one of claims 1 to 12, wherein the food, feed or beverage comprises a greater amount of the at least two compounds having meat-related flavour and / or aroma than the amount in a corresponding food, feed or beverage produced using a corresponding lipid or composition lacking polar lipids comprising omega6 fatty acids.

19. the food, feed or beverage is a meat substitute; and / or 19. A food, feed or beverage according to any one of claims 13 to 18, wherein the application of heat to the food, feed or beverage produces one or more compounds having meat-related flavours and / or aromas, preferably volatile compounds.

20. By applying heat to the composition, food, feed or beverage, 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethylbenzaldehyde, 2,3,4,5-tetramethylcyclopent-2-en-1-ol, 1-octanol, and producing two or more volatile compounds selected from benzene, 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, trans-2-octen-1-ol, 1-nonanol, 1,3-bis(1,1-dimethylethyl)-benzene, 2-octen-1-ol, adamantanol-like compounds, hexanal, 2-pentylfuran, 1-octen-3-ol, 2-pentylthiophene, and 1,3,5-titriane; applying heat to the composition, food, feed, or beverage produces two or more volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, 1-octanol, trans-2-octen-1-ol, and 1-nonanol; applying heat to the composition, food, feed, or beverage produces two or more volatile compounds selected from 1-pentanal, 3-octanone, 2-octen-1-ol, 1-nonanol, and 1-octanol, and optionally 1,3-bis(1,1-dimethylethyl)-benzene; or 20. The composition of any one of claims 1 to 12, or the food, feed, or beverage of any one of claims 13 to 19, wherein application of heat to the composition, food, feed, or beverage produces two or more volatile compounds selected from 1,3-dimethylbenzene, p-xylene, ethylbenzene, 2-heptanone, 2-pentylfuran, octanal, 1,2-octadecanediol, 2,4-diethyl-1-heptanol, 2-nonanone, nonanal, 1-octen-3-ol, 2-decanone, 2-octen-1-ol, (E)-; 2,4-dimethyl-benzaldehyde, and 2,3,4,5-tetramethylcyclopent-2-en-1-ol.

21. 1. A method for producing a food, feed or beverage comprising the steps of: A composition according to any one of claims 1 to 12; or The extracted microbial lipids as defined in any one of claims 1 to 12, optionally heated at a temperature of at least about 100°C, at least about 120°C, or at least about 140°C, and sugars, amino acids or derivatives, combining with at least one other food, feed, or beverage ingredient.

22. An isolated strain of Mortierella sp. i) yNI0125, deposited at the National Measurement Institute Australia under V21 / 019953 on October 12, 2021; ii) yNI0126, deposited at the National Measurement Institute Australia under V21 / 019951 on October 12, 2021; iii) yNI0127, deposited at the National Measurement Institute Australia under V21 / 019952 on October 12, 2021; and iv) An isolate selected from yNI0132 deposited at the National Measurement Institute Australia under V21 / 019954 on October 12, 2021.

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