Microbial production of saturated fats

Abstract

The present invention relates to extracted microbial lipids, microbial cells containing said lipids, 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] FIELD OF THE INVENTION The present invention relates to extracted microbial lipids, microbial cells containing said lipids, and extracts thereof. The invention also relates to the use of these lipids, cells, and extracts in foods, feeds, and beverages. [Background technology]

[0002] Saturated fatty acids are straight chains of carbon atoms consisting of a methylene (CH2) group between the terminal methyl group and the carboxylic acid group. The most common saturated fatty acids in food are lauric acid (C12), palmitic acid (C16), and stearic acid (C18). Despite health concerns about consuming high levels of certain saturated fatty acids, they form an important part of some foods and their manufacturing processes. Coconut oil is the most saturated natural fat (typically about 94% saturated). Other "saturated" fats include palm kernel oil (typically 82% saturated), cocoa butter (typically 60-64% saturated), and palm oil (typically 51% saturated). Lard and beef tallow are also often considered to be in this saturated fat category, even though they typically contain only 40% and 37% saturated fat, respectively (Talbot, 2011). There is a growing need for alternative sources of saturated fatty acids, particularly from non-animal sources, that can be used in foods and food manufacturing processes. Summary of the Invention

[0003] The inventors have generated new extracted microbial lipids that contain high levels of saturated fatty acids and low levels of polyunsaturated fatty acids.

[0004] Thus, in a first aspect, the present invention provides extracted microbial lipids comprising a total fatty acid content comprising a total saturated fatty acid content of saturated fatty acids (SFAs) and a total monounsaturated fatty acid content of monounsaturated fatty acids (MUFAs), wherein at least a portion of the total fatty acid content comprising at least a portion of the SFAs and at least a portion of the MUFAs is esterified in the form of triacylglycerols (TAGs), such that the extracted microbial lipids have a total TAG content, and wherein: (i) the total SFA content of the extracted microbial lipids comprises stearic acid (C18:0), palmitic acid (C16:0), myristic acid (C14:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0), such that at least 50% by weight of the fatty acids of the total fatty acid content of the extracted microbial lipids are SFAs; (ii) 20% by weight to 85% by weight of the total fatty acid content is stearic acid; (iii) the total MUFA content of the extracted microbial lipids comprises oleic acid (C18:1Δ9) and palmitoleic acid (C16:1Δ9), and optionally C16:1Δ7 and / or C17:1; (iv) the total fatty acid (TFA) content of the extracted microbial lipids is devoid of polyunsaturated fatty acids (PUFAs) or comprises a PUFA content comprising linoleic acid (C18:2Δ9,12), wherein the PUFA content is less than 5 wt.% of the total fatty acid content; (v) the extracted microbial lipids either contain polar lipids, including phospholipids, or are devoid of polar lipids; and (vi) the extracted microbial lipids are solid at 25°C.

[0005] In one embodiment, the lipid is solid at one or more or all of 30°C, 35°C, 40°C, 45°C, or 50°C.

[0006] In one embodiment, 20% to 80% by weight, 20% to 75% by weight, 20% to 70% by weight, 20% to 65% by weight, 20% to 60% by weight, 20% to 55% by weight, 20% to 50% by weight, 20% to 45% by weight, 25% to 80% by weight, 25% to 75% by weight, 25% to 70% by weight, 25% to 65% by weight, 25% to 60% by weight, 25% to 55% by weight, 25% to 50% by weight, or 25% to 45% by weight of the TFA content of the lipids, or the TFA content of the TAG content, or both, is stearic acid. In this embodiment, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia Lipids, such as Y. liplytica lipids.

[0007] In one embodiment, the TFA content of the lipids, or the TFA content of the TAG content, or both, contains less than 4 wt%, less than 3 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or less than 0.2 wt%, or 0.2 wt% to 5 wt%, 0.2 wt% to 4 wt%, 0.2 wt% to 3 wt%, 0.2 wt% to 2 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, or 0.5 wt% to 2 wt% linoleic acid (LA), or LA is essentially absent from the TFA content of the lipids and / or the TFA content of the TAG content. In this embodiment, the extracted microbial lipids are preferably extracted yeast lipids, more preferably extracted Yarrowia lipids, e.g., Yarrowia lipolytica lipids.

[0008] In one embodiment, the TFA content of the lipids or the TAG content, or both, contains less than 4 wt%, less than 3 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or less than 0.2 wt%, or 0.2 wt% to 5 wt%, 0.2 wt% to 4 wt%, 0.2 wt% to 3 wt%, 0.2 wt% to 2 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, or 0.5 wt% to 2 wt%, or PUFAs are essentially absent from the TFA content of the lipids and / or the TAG content. In this embodiment, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0009] In one embodiment, the TFA content of the lipids and / or TAGs comprises at least 10% by weight, at least 12% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, 10% to 50% by weight, 10% to 45% by weight, 10% to 40% by weight, 10% to 35% by weight, 10% to 30% by weight, 15% to 50% by weight, 15% to 45% by weight, 15% to 40% by weight, 15% to 35% by weight, 15% to 30% by weight, 20% to 50% by weight, 20% to 45% by weight, 20% to 40% by weight, 20% to 35% by weight, or 20% to 30% by weight of oleic acid. In this embodiment, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0010] In one embodiment, the TFA content of the lipid and / or the TFA content of the TAG comprises at least 5%, at least 8%, at least 10%, or at least 12% palmitic acid. In one embodiment, the palmitic acid content is 5% to 25%, 8% to 25%, 10% to 25%, or 12% to 25%.

[0011] In one embodiment, the lipids are extracted from a microorganism that contains at least one genetic modification and has a higher total SFA content, C18:0 content, C20:0 content, and C22:0 content in the lipid TFA content and / or TAG content compared to a corresponding extracted microbial lipid obtained from a corresponding microorganism lacking at least one genetic modification. Exemplary genetic modifications are discussed herein. In this embodiment, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0012] In one embodiment, the TFA content and / or TAG content of the lipid is 30% to 90% by weight, 30% to 85% by weight, 30% to 80% by weight, 30% to 75% by weight, 30% to 70% by weight, 30% to 65% by weight, 30% to 60% by weight, 30% to 55% by weight, 30% to 50% by weight, 30% to 45% by weight, 30% to 40% by weight, 35% to 90% by weight, 35% to 85% by weight, 35% to 80% by weight, 35% to 75% by weight, 35% to 70% by weight, 35% to 65% by weight, 35% to 60% by weight, 35% to 55% by weight, 35% to 50% by weight, 35% to 45% by weight , 35% to 40% by weight, 40% to 90% by weight, 40% to 85% by weight, 40% to 80% by weight, 40% to 75% by weight, 40% to 70% by weight, 40% to 65% by weight, 40% to 60% by weight , 40% to 55% by weight, 40% to 50% by weight, 40% to 45% by weight, 45% to 90% by weight, 45% to 85% by weight, 45% to 80% by weight, 45% to 75% by weight, 45% to 70% by weight %, 45% to 65% by weight, 45% to 60% by weight, 45% to 55% by weight, 45% to 50% by weight, 50% to 90% by weight, 50% to 85% by weight, 50% to 80% by weight, 50% to 75% by weight %, 50% to 70% by weight, 50% to 65% by weight, 50% to 60% by weight, 55% to 90% by weight, 55% to 85% by weight, 55% to 80% by weight, 55% to 75% by weight, 55% to 70% by weight, 55% to 65% by weight, 55% to 60% by weight, 60% to 90% by weight, 60% to 85% by weight, 60% to 80% by weight, 60% to 75% by weight, or 60% to 70% by weight of SFA. In this embodiment, the extracted microbial lipid is preferably extracted yeast lipid, and more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0013] In one embodiment, when the total SFA content is at least 60% by weight of the TFA content of the lipid and / or the TFA content of the TAG, the stearic acid content is at least 40% by weight.

[0014] In one embodiment, the ratio of total saturated fatty acids containing 18 or more carbons to total saturated fatty acids containing 16 or fewer carbons (L / S-SFA ratio) of the TFA content of the lipid or the TAG content of the lipid, or both, is at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or from about 3 to about 10. In one embodiment, the TFA content of the lipids, the TAG content of the lipids, or the L / S-SFA ratio of both is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, or 2.5 to 4. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, more preferably extracted Yarrowia lipids, e.g., Yarrowia lipolytica lipids.

[0015] In one embodiment, lipids are extracted from a microorganism comprising at least one genetic modification, and the TFA content or TAG content or L / S-SFA ratio of the lipids is at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10 times, or 3 to 10 times greater than the corresponding extracted microbial lipid obtained from a corresponding microorganism lacking at least one genetic modification. In one embodiment, lipids are extracted from a microorganism comprising at least one genetic modification, and the TFA content or TAG content or L / S-SFA ratio of the lipids is increased by 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, or 2.5 to 4 fold compared to the corresponding extracted microbial lipid obtained from a corresponding microorganism lacking at least one genetic modification. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, and more preferably extracted Yarrowia lipids, such as Yarrowia lipolytica lipids. Exemplary genetic modifications are discussed herein.

[0016] In one embodiment, the sum of the content of C20:0, C22:0, and C24:0 fatty acids is between 5% and 25% by weight, 5% and 20% by weight, 5% and 18% by weight, 5% and 16% by weight, 5% and 15% by weight, 5% and 14% by weight, 5% and 13% by weight, 5% and 12% by weight, 5% and 10% by weight, 6% and 25 ... The extracted microbial lipids are preferably extracted yeast lipids, more preferably extracted Yarrowia lipids, such as Yarrowia lipolytica lipids.

[0017] In one embodiment, the C20:0 fatty acid content is from about 1% to about 5% by weight, from about 1% to about 4% by weight, from about 1% to about 3% by weight, from about 2% to about 5% by weight, from about 2% to about 4% by weight, or from about 2% to about 3% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids, or both.

[0018] In one embodiment, the C22:0 fatty acid content is from about 1% to about 5% by weight, from about 1% to about 4% by weight, from about 2% to about 5% by weight, or from about 2% to about 4% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids, or both.

[0019] In one embodiment, the C24:0 fatty acid content is from about 1% to about 6% by weight, from about 1% to about 5% by weight, from about 1% to about 4% by weight, from about 2% to about 6% by weight, from about 2% to about 5% by weight, or from about 2% to about 4% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids, or both.

[0020] In another embodiment, the C20:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, or at least about 3% by weight of the TFA content of the extracted lipid or the TAG content of the extracted lipid, or both; the C22:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, or at least about 3.5% by weight; and / or the C24:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, or at least 4% by weight.

[0021] In one embodiment, (i) 20% to 55% by weight of the TFA content of the lipids or of the TAG content, or both, is stearic acid, and (ii) less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.2%, or 0.2% to 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2% by weight is linoleic acid (LA) or total PUFAs, or both, or LA or PUFAs, or both, are essentially absent from the TFA content of the lipids and / or TAG content. In one embodiment, (i) 20% to 50% by weight of the TFA content of the lipids or of the TAG content, or both, is stearic acid, and (ii) less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.2%, or 0.2% to 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2% by weight is linoleic acid (LA) or total PUFAs, or both, or LA or PUFAs, or both, are essentially absent from the TFA content of the lipids and / or TAG content. In one embodiment, (i) 20% to 45% by weight of the TFA content of the lipids, or the TFA content of the TAG content, or both, is stearic acid, and (ii) less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.2%, or 0.2% to 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, or 0.5% to 2% by weight is linoleic acid (LA) or total PUFAs, or both, or LA or PUFAs, or both, are essentially absent from the TFA content of the lipids and / or TAG content. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, more preferably extracted Yarrowia lipids, e.g., Yarrowia lipolytica lipids.

[0022] In one embodiment, (i) 20% to 55% by weight of the TFA content of the lipid or the TAG content, or both, is stearic acid, and (ii) at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, or 20% to 30% by weight is oleic acid. In one embodiment, (i) 20% to 50% by weight of the TFA content of the lipid or the TAG content, or both, is stearic acid, and (ii) at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, or 20% to 30% by weight is oleic acid. In one embodiment, (i) 20% to 45% by weight of the TFA content of the lipid or the TAG content, or both, is stearic acid, and (ii) at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, or 20% to 30% by weight is oleic acid.In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, and more preferably extracted Yarrowia lipids, such as Yarrowia lipolytica lipids.

[0023] In one embodiment, (i) 20% to 55% by weight of the TFA content of the lipid or of the TAG content, or both, is stearic acid, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, 2.5 to 4. In one embodiment, (i) 20% to 50% by weight of the TFA content of the lipid or the TAG content of the lipid, or both, is stearic acid, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, 2.5 to 4. In one embodiment, (i) 20% to 45% by weight of the lipid or the TAG content of the lipid, or both, is stearic acid, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, 2.5 to 4. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, and more preferably extracted Yarrowia lipids, such as Yarrowia lipolytica lipids.

[0024] In one embodiment, the lipid is extracted from a microorganism comprising at least one genetic modification, and (i) 20% to 55% by weight of the TFA content of the lipid or of the TAG content, or both, is stearic acid, and (ii) the lipid has a higher total SFA content, C18:0 content, C20:0 content, and C22:0 content, or all of a higher total SFA content, C18:0 content, C20:0 content, and C22:0 content, in the TFA content and / or TAG content, compared to a corresponding extracted microbial lipid obtained from a corresponding microorganism lacking said at least one genetic modification. In one embodiment, the lipid is extracted from a microorganism comprising at least one genetic modification, and (i) 20% to 50% by weight of the TFA content of the lipid or of the TAG content, or both, is stearic acid, and (ii) the lipid has a higher total SFA content, C18:0 content, C20:0 content, and C22:0 content, or one or more or all of a higher total SFA content, C18:0 content, C20:0 content, and C22:0 content, in the TFA content and / or TAG content, compared to a corresponding extracted microbial lipid obtained from a corresponding microorganism lacking said at least one genetic modification. In one embodiment, lipids are extracted from a microorganism comprising at least one genetic modification, and (i) 20% to 45% by weight of the TFA content of the lipids or the TAG content, or both, is stearic acid, and (ii) the lipids have a higher total SFA content, C18:0 content, C20:0 content, and / or C22:0 content, in the TFA content and / or TAG content, compared to corresponding extracted microbial lipids obtained from a corresponding microorganism lacking the at least one genetic modification. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, and more preferably extracted Yarrowia lipids, e.g., Yarrowia lipolytica lipids.

[0025] In one embodiment, (i) 20% to 55% by weight of the TFA content of the lipid or the TAG content, or both, is stearic acid, and (ii) the TFA content of the lipid or the TAG content, or both, is C24:0 fatty acid content, is about 1% to about 6% by weight, about 1% to about 5% by weight, about 1% to about 4% by weight, about 2% to about 6% by weight, about 2% to about 5% by weight, or about 2% to about 4% by weight. In one embodiment, (i) 20% to 50% by weight of the TFA content of the lipid or the TAG content, or both, is stearic acid, and (ii) the TFA content of the lipid or the TAG content, or both, is C24:0 fatty acid content, is about 1% to about 6% by weight, about 1% to about 5% by weight, about 1% to about 4% by weight, about 2% to about 6% by weight, about 2% to about 5% by weight, or about 2% to about 4% by weight. In one embodiment, (i) 20% to 45% by weight of the TFA content of the lipids or the TAG content, or both, is stearic acid, and (ii) the C24:0 fatty acid content of the lipids or the TAG content, or both, is about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 2% to about 6%, about 2% to about 5%, or about 2% to about 4% by weight. In these embodiments, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0026] In one embodiment, (i) less than 4 wt.% of the lipid or TAG content or TFA content, or both, is linoleic acid (LA), and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, 10 wt.% to 50 wt.%, 10 wt.% to 45 wt.%, 10 wt.% to 40 wt.%, 10 wt.% to 35 wt.%, 10 wt.% to 30 wt.%, 15 wt.% to 50 wt.%, 15 wt.% to 45 wt.%, 15 wt.% to 40 wt.%, 15 wt.% to 35 wt.%, 15 wt.% to 30 wt.%, 20 wt.% to 50 wt.%, 20 wt.% to 45 wt.%, 20 wt.% to 40 wt.%, 20 wt.% to 35 wt.%, or 20 wt.% to 30 wt.% is oleic acid. In one embodiment, (i) less than 3 wt.% of the TFA content of the lipid or of the TAG content, or both, is linoleic acid (LA), and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, 10 wt.% to 50 wt.%, 10 wt.% to 45 wt.%, 10 wt.% to 40 wt.%, 10 wt.% to 35 wt.%, 10 wt.% to 30 wt.%, 15 wt.% to 50 wt.%, 15 wt.% to 45 wt.%, 15 wt.% to 40 wt.%, 15 wt.% to 35 wt.%, 15 wt.% to 30 wt.%, 20 wt.% to 50 wt.%, 20 wt.% to 45 wt.%, 20 wt.% to 40 wt.%, 20 wt.% to 35 wt.%, or 20 wt.% to 30 wt.% is oleic acid. In one embodiment, (i) less than 2 wt.% of the TFA content of the lipid or of the TAG content, or both, is linoleic acid (LA), and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, 10 wt.% to 50 wt.%, 10 wt.% to 45 wt.%, 10 wt.% to 40 wt.%, 10 wt.% to 35 wt.%, 10 wt.% to 30 wt.%, 15 wt.% to 50 wt.%, 15 wt.% to 45 wt.%, 15 wt.% to 40 wt.%, 15 wt.% to 35 wt.%, 15 wt.% to 30 wt.%, 20 wt.% to 50 wt.%, 20 wt.% to 45 wt.%, 20 wt.% to 40 wt.%, 20 wt.% to 35 wt.%, or 20 wt.% to 30 wt.% is oleic acid.In one embodiment, (i) less than 0.2 wt.% of the TFA content of the lipids or the TAG content, or both, is linoleic acid (LA), or LA is absent from the TFA content of the lipids or the TAG content, or both, and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, between 10 wt.% and 50 wt.%. %, 10% to 45% by weight, 10% to 40% by weight, 10% to 35% by weight, 10% to 30% by weight, 15% to 50% by weight, 15% to 45% by weight, 15% to 40% by weight, 15% to 35% by weight, 15% to 30% by weight, 20% to 50% by weight, 20% to 45% by weight, 20% to 40% by weight, 20% to 35% by weight, or 20% to 30% by weight is oleic acid. In these embodiments, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0027] In one embodiment, (i) Less than 4% by weight of the TFA content of the lipid or of the TAG content, or both, is linoleic acid (LA), and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5-10, 1.5-9, 1.5-8, 1.5-7, 1.5-6, 1.5-5, 1.5-4, 1.75-10, 1.75-9, 1.75-8, 1.75-7, 1.75-6, 1.75-5, 1.75-4, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2.5-10, 2.5-9, 2.5-8, 2.5-7, 2.5-6, 2.5-5, and 2.5-4. In one embodiment, (i) less than 3% by weight of the TFA content of the lipid or of the TAG content, or both, is linoleic acid (LA), and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4. In one embodiment, (i) less than 2% by weight of the TFA content of the lipid or of the TAG content, or both, is linoleic acid (LA), and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4.In one embodiment, (i) less than 0.2 wt.% of the TFA content of the lipid or of the TAG content, or both, is linoleic acid (LA), and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, more preferably extracted Yarrowia lipids, such as Yarrowia lipolytica lipids.

[0028] In one embodiment, (i) less than 4 wt% of the TFA content of the lipid or TAG content or both is PUFA, and (ii) at least 10 wt%, at least 12 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, 10 wt% to 50 wt%, 10 wt% to 45 wt%, 10 wt% to 40 wt%, 10 wt% to 35 wt%, 10 wt% to 30 wt%, 15 wt% to 50 wt%, 15 wt% to 45 wt%, 15 wt% to 40 wt%, 15 wt% to 35 wt%, 15 wt% to 30 wt%, 20 wt% to 50 wt%, 20 wt% to 45 wt%, 20 wt% to 40 wt%, 20 wt% to 35 wt%, or 20 wt% to 30 wt% is oleic acid. In one embodiment, (i) less than 3 wt.% of the TFA content of the lipid or TAG content or both is PUFA, and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, 10 wt.% to 50 wt.%, 10 wt.% to 45 wt.%, 10 wt.% to 40 wt.%, 10 wt.% to 35 wt.%, 10 wt.% to 30 wt.%, 15 wt.% to 50 wt.%, 15 wt.% to 45 wt.%, 15 wt.% to 40 wt.%, 15 wt.% to 35 wt.%, 15 wt.% to 30 wt.%, 20 wt.% to 50 wt.%, 20 wt.% to 45 wt.%, 20 wt.% to 40 wt.%, 20 wt.% to 35 wt.%, or 20 wt.% to 30 wt.% is oleic acid. In one embodiment, (i) less than 2 wt.% of the TFA content of the lipid or TAG content or both is PUFA, and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, 10 wt.% to 50 wt.%, 10 wt.% to 45 wt.%, 10 wt.% to 40 wt.%, 10 wt.% to 35 wt.%, 10 wt.% to 30 wt.%, 15 wt.% to 50 wt.%, 15 wt.% to 45 wt.%, 15 wt.% to 40 wt.%, 15 wt.% to 35 wt.%, 15 wt.% to 30 wt.%, 20 wt.% to 50 wt.%, 20 wt.% to 45 wt.%, 20 wt.% to 40 wt.%, 20 wt.% to 35 wt.%, or 20 wt.% to 30 wt.% is oleic acid.In one embodiment, (i) less than 0.2 wt.% of the TFA content of the lipid or TAG content, or both, is PUFA, or PUFA is absent from the TFA content of the lipid or TAG content, or both, and (ii) at least 10 wt.%, at least 12 wt.%, at least 15 wt.%, at least 20 wt.%, at least 25 wt.%, at least 30 wt.%, 10 wt.% to 50 wt.% of the TFA content of the lipid or TAG content, or both, is PUFA. %, 10% to 45% by weight, 10% to 40% by weight, 10% to 35% by weight, 10% to 30% by weight, 15% to 50% by weight, 15% to 45% by weight, 15% to 40% by weight, 15% to 35% by weight, 15% to 30% by weight, 20% to 50% by weight, 20% to 45% by weight, 20% to 40% by weight, 20% to 35% by weight, or 20% to 30% by weight is oleic acid. In these embodiments, the extracted microbial lipid is preferably extracted yeast lipid, more preferably extracted Yarrowia lipid, e.g., Yarrowia lipolytica lipid.

[0029] In one embodiment, (i) less than 4 wt.% of the TFA content of the lipid or of the TAG content, or both, is PUFA, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4. In one embodiment, (i) less than 3 wt.% of the TFA content of the lipid or of the TAG content, or both, is PUFA, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4. In one embodiment, (i) less than 2 wt.% of the TFA content of the lipid or of the TAG content, or both, is PUFA, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4.In one embodiment, (i) less than 0.2 wt.% of the TFA content of the lipid or the TAG content, or both, is PUFA, or PUFA is absent from the TFA content of the lipid or the TAG content, or both, and (ii) the L / S-SFA ratio of the TFA content of the lipid or the TAG content of the lipid, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, and 2.5 to 4. In these embodiments, the extracted microbial lipids are preferably extracted yeast lipids, more preferably extracted Yarrowia lipids, such as Yarrowia lipolytica lipids.

[0030] In one embodiment, C20:0, C22:0, and C24:0 fatty acids comprise at least 95%, at least 97%, or at least 99% by weight of the TFA content of the extracted lipids or the TFA content of the lipid TAGs, or both, fatty acids having at least 20 carbon atoms or more.

[0031] In a preferred embodiment, the total MUFA content of the extracted microbial lipid or the TAG content in the extracted microbial lipid of the above embodiments comprises (i) oleic acid (C18:1Δ9), palmitoleic acid (C16:1Δ9), and C16:1Δ7, or (ii) oleic acid (C18:1Δ9), palmitoleic acid (C16:1Δ9), C16:1Δ7, and C17:1.

[0032] In a preferred embodiment, the extracted microbial lipids or the TAG content in the extracted microbial lipids are devoid of cyclopropane fatty acids. In one embodiment, the lipid comprises a polar lipid, wherein the TFA content of the polar lipid has one or more characteristics defined herein for the TFA content or TAG content of the extracted lipid.

[0033] In one embodiment, the TAG comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% by weight of the total lipid content of the extracted lipid.

[0034] In one embodiment, the lipids comprise sterols and / or sterol esters, preferably ergosterol and / or ergosterol esters. In one embodiment, the extracted microbial lipids comprise at least 1 wt%, at least 2 wt%, or at least 3 wt% ergosterol and / or ergosterol esters. In one embodiment, the extracted microbial lipids comprise 1 wt% to 10 wt%, 2 wt% to 10 wt%, or 3 wt% to 10 wt% ergosterol and / or ergosterol esters.

[0035] In one embodiment, the lipid has a water content of less than 20% by weight. In one embodiment, the lipid has a volatile solvent content of less than 10%, less than 7.5%, or less than 5% by weight.

[0036] In one embodiment, the lipids comprise TAG molecules comprising a MUFA, preferably oleic acid, esterified at their sn-2 position, wherein the ratio of the number of TAG molecules comprising a MUFA esterified at the sn-2 position to the number of TAG molecules comprising a fatty acid other than a MUFA esterified at their sn-2 position in the extracted lipid (MUFA:other FA ratio at sn-2) is less than about 0.50, less than about 0.30, less than about 0.20, less than about 0.10, less than about 0.05, less than about 0.04, less than about 0.03, or less than about 0.02.

[0037] In one embodiment, lipids are extracted from microorganisms that contain at least one genetic modification and contain polar lipids, including phospholipids, wherein at least two, preferably three, or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) in the extracted lipids have a higher amount of SFA than the corresponding lipids extracted from the corresponding microorganism that lacks at least one genetic modification.Exemplary genetic modifications are discussed herein.

[0038] In one embodiment, the lipid is (a) an exogenous polynucleotide encoding a FATA fatty acylthioesterase; (b) at least one exogenous polynucleotide encoding at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), and (c) a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding a Δ12 desaturase, more preferably a null mutation of an endogenous gene encoding a Δ12 desaturase, most preferably a null mutation of the FAD2 gene; It is extracted from a microorganism containing a genetic modification including wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell.

[0039] In one embodiment, at least one DGAT comprises nucleotides having a sequence set forth in any one of SEQ ID NOs: 144-154, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 144-154.

[0040] In one embodiment, the FATA fatty acylthioesterase comprises a nucleotide having a sequence set forth in any one of SEQ ID NOs: 84 or 86, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 84 or 86.

[0041] In one embodiment, the microorganism has a higher total SFA content, C20:0 content, C22:0 content, and C24:0 content in the lipid TFA content and / or TAG content compared to a corresponding extracted microbial lipid obtained from a corresponding microorganism lacking at least one genetic modification. In one embodiment, the C20:0 content is at least 1%, 1.5%, or 2%, the C22:0 content is at least 1%, 1.5%, or 2%, and the C24:0 content is at least 1.5%, 2%, or 2.5%. In one embodiment, the C20:0 content is about 1.0% to 4%, the C22:0 content is about 1.0% to 4%, and the C24:0 content is about 2% to 5%. In another embodiment, the C20:0 content, C22:0 content, and C24:0 content include the amounts described above.

[0042] In one embodiment, the microorganism further comprises an exogenous polynucleotide encoding an acyl-CoA synthetase (ACS), optionally wherein the ACS comprises a nucleotide having a sequence set forth as any one of SEQ ID NOs: 88-89, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 88-89.

[0043] In another embodiment, the microorganism further comprises an exogenous polynucleotide encoding a lysophosphatidic acid acyltransferase (LPAAT), optionally wherein the LPAAT comprises a nucleotide having a sequence set forth in SEQ ID NO:91, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:91.

[0044] In one embodiment, the extracted microbial lipids are obtained from microbial cells that comprise or consist of eukaryotic cells, fungal cells, bacterial cells, or algal cells, live microbial cells, dead microbial cells, or any mixture thereof.

[0045] In one embodiment, 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.

[0046] In one embodiment, the microbial cell is a yeast cell. Examples of suitable yeast cells include, but are not limited to, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and any mixture thereof. In one embodiment, the yeast cell is Yarrowia lipolytica.

[0047] In one embodiment, the lipid has a weight of at least 5 g, at least 10 g, at least 50 g, or at least 100 g. In one embodiment, the lipid is obtained from microbial cells cultured in the presence of less than 5 g / L, less than 2 g / L, less than 1 g / L stearate, or cultured in the absence of stearate added to the medium. Also provided is a microbial cell comprising a lipid as defined herein.

[0048] In a further aspect, the present invention provides a microbial cell, preferably a yeast cell, having at least one genetic modification, wherein the microbial cell has, in each case compared to a corresponding microbial cell lacking the at least one genetic modification and cultured under the same conditions, (i) an increased saturated fatty acid (SFA) content, an increased SFA content with at least 18 carbons, an increased stearic acid content, an increased C20:0 and C22:0 fatty acid content, an increased C24:0 fatty acid content, an increased L / S-SFA ratio, or any combination thereof, in the total fatty acid (TFA) content of the microbial cell, or the TFA content of TAGs in the microbial cell, or both, and (ii) increased triacylglycerol (TAG) production or accumulation, or both. In a preferred embodiment, the cell exhibits (a) an increased saturated fatty acid (SFA) content, and (b) increased triacylglycerol (TAG) production or accumulation, or both, or (a) an increased SFA content with at least 18 carbons, and (b) increased triacylglycerol (TAG) production or accumulation, or both, or (a) increased stearate content, and (b) increased triacylglycerol (TAG) production or accumulation, or both; or (a) increased content of C20:0 and C22:0 fatty acids, and (b) increased triacylglycerol (TAG) production or accumulation, or both; or (a) increased content of C24:0 fatty acids, and (b) increased triacylglycerol (TAG) production or accumulation, or both; or (a) increased L / S-SFA ratio, and (b) increased triacylglycerol (TAG) production or accumulation, or both. In more preferred embodiments, the cells have (a) an increased saturated fatty acid (SFA) content, (b) an increased stearate content, and (c) increased triacylglycerol (TAG) production or accumulation, or both, or (a) an increased saturated fatty acid (SFA) content, (b) an increased C20:0 and C22:0 content, and (c) increased triacylglycerol (TAG) production or accumulation, or both, or (a) an increased saturated fatty acid (SFA) content, (b) an increased stearate, C20:0, and C22:0 content, and (c) increased triacylglycerol (TAG) production or accumulation, or both. In each more preferred embodiment, the lipid content of the cells may also include an increased L / S-SFA ratio. In one embodiment, the C20:0 content is at least 1%, 1.5%, or 2%, the C22:0 content is at least 1%, 1.5%, or 2%, and the C24:0 content is at least 1.5%, 2%, or 2.5%. In one embodiment, the C20:0 content is 1.0% to 4%, the C22:0 content is 1.0% to 4%, and the C24:0 content is 2% to 5%. In another embodiment, the C20:0 content, C22:0 content, and C24:0 content include the amounts described above.

[0049] In one embodiment, the cellular TFA content or the cellular TAG content, or both, have one or more characteristics defined herein for the lipids of the invention for the TFA content or TAG content of the extracted lipids.

[0050] In one embodiment, the TFA content of the lipids or the TAG content of the cells, or both, 20% to 80% by weight, 20% to 75% by weight, 20% to 70% by weight, 20% to 65% by weight, 20% to 60% by weight, 20% to 55% by weight, 20% to 50% by weight, 20% to 45% by weight, 25% to 80% by weight, 25% to 75% by weight, 25% to 70% by weight, 25% to 65% by weight, 25% to 60% by weight, 25% to 55% by weight, 25% to 50% by weight, or 25% to 45% by weight is stearic acid. In this embodiment, the cells are preferably yeast cells, more preferably Yarrowia cells, such as Yarrowia lipolytica cells.

[0051] In one embodiment, the TFA content of the lipids or TAG content of the cells, or both, comprises less than 4 wt%, less than 3 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or 0.2 wt%, or 0.2 wt% to 5 wt%, 0.2 wt% to 4 wt%, 0.2 wt% to 3 wt%, 0.2 wt% to 2 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, or LA is essentially absent from the TFA of the lipids and / or TAG content of the cells. In one embodiment, the TFA content of the lipids and / or TAG content of the cells is The PUFAs may be present in an amount of less than 4 wt%, less than 3 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or 0.2 wt%, or between 0.2 wt% and 5 wt%, between 0.2 wt% and 4 wt%, between 0.2 wt% and 3 wt%, between 0.2 wt% and 2 wt%, between 0.5 wt% and 5 wt%, between 0.5 wt% and 4 wt%, between 0.5 wt% and 3 wt%, or between 0.5 wt% and 2 wt%, or the PUFAs are essentially devoid of TFAs from the lipid and / or TAG content of the cells. In these embodiments, the cells are preferably yeast cells, more preferably Yarrowia cells, such as Yarrowia lipolytica cells.

[0052] In one embodiment, the TFA content of the lipids of the cells and / or the TFA content of the TAG of the cells comprises at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, or 20% to 30% oleic acid by weight. In this embodiment, the cells are preferably yeast cells, more preferably Yarrowia cells, e.g., Yarrowia lipolytica cells.

[0053] In one embodiment, the cellular TFA content, the cellular TAG content, or the cellular polar lipids, or any combination thereof, lacks at least genetic modification and has an increased L / S-SFA ratio compared to a corresponding microbial cell cultured under the same conditions. In one embodiment, the L / S-SFA ratio of the lipid or TFA content or TAG content or both of the cells is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, or 2.5 to 4. In these embodiments, the cells are preferably yeast cells, more preferably Yarrowia cells, e.g., Yarrowia lipolytica cells.

[0054] In one embodiment, the L / S-SFA ratio is at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or about 3 to about 10 times higher than the TFA content, TAG content, or polar lipid content of a corresponding microbial cell lacking at least one genetic modification and cultured under the same conditions.

[0055] In one embodiment, the microbial cells, preferably yeast cells, (a) FATA fatty acylthioesterase, (b) at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), or (c) at least one genetic modification that is an exogenous polynucleotide encoding FATA and at least one fatty acid acyltransferase, preferably at least DGAT; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell.

[0056] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) FATA fatty acylthioesterase, (b) at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), or (c) FATA and at least one fatty acid acyltransferase, preferably at least DGAT, and (d) optionally providing a microbial cell, preferably a yeast cell, comprising at least one genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding a Δ12 desaturase, more preferably a null mutation of an endogenous gene encoding a Δ12 desaturase; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell.

[0057] In one embodiment, the genetic modification comprises an exogenous polynucleotide encoding one or more diacylglycerol acyltransferase (DGAT), glycerol-3-phosphate acyltransferase (GPAT), or phospholipid:diacylglycerol acyltransferase (PDAT) polypeptides, or any combination thereof. In an embodiment comprising one or more DGATs, at least one or all polynucleotides in the cell encoding a DGAT comprise a nucleotide having a sequence set forth in any one of SEQ ID NOS: 144-154, or a nucleotide sequence at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOS: 144-154. In one embodiment, at least one or all polynucleotides encoding a DGAT in the cell encode a sequence set forth in any one or more of SEQ ID NOS: 115-125, and differ from any one of SEQ ID NOS: 144-154 only by codon degeneracy.

[0058] In one embodiment, a microbial cell, preferably a yeast cell, of the invention comprises one or more exogenous polynucleotides, each encoding a GPAT. In one embodiment, at least one of the GPATs or all of the GPATs comprises amino acids having a sequence set forth in any one of SEQ ID NOs: 126-138, or an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 126-138.

[0059] In one embodiment, at least one or all polynucleotides encoding GPAT comprise nucleotides having a sequence set forth in any one of SEQ ID NOs: 155 to 167, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 155 to 167. In one embodiment, at least one or all polynucleotides encoding GPAT encode a sequence set forth in one or more of SEQ ID NOs: 126 to 138 and differ from one of SEQ ID NOs: 155 to 167 only by codon degeneracy.

[0060] In one embodiment, a microbial cell, preferably a yeast cell, of the invention comprises one or more exogenous polynucleotides, each encoding a phospholipid:diacylglycerol acyltransferase (PDAT). In one embodiment, at least one of the PDATs, or all of the PDATs, comprises amino acids having a sequence set forth in any one of SEQ ID NOs: 139-143, or an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 139-143.

[0061] In one embodiment, at least one or all polynucleotides encoding PDAT comprise nucleotides having a sequence set forth in any one of SEQ ID NOs: 168-172, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 168-172. In one embodiment, at least one or all polynucleotides encoding PDAT encode a sequence set forth in one or more of SEQ ID NOs: 139-143 and differ from one of SEQ ID NOs: 168-172 only by codon degeneracy.

[0062] In one embodiment, the microbial cell, preferably a yeast cell, comprises an exogenous polynucleotide encoding a DGAT and an exogenous polynucleotide encoding a GPAT, two or more DGATs and one GPAT, two or more GPATs and one DGAT, or two or more DGATs and two or more GPATs, wherein at least one of the DGATs and / or at least one of the GPATs is as defined above.

[0063] In one embodiment, the cell comprises an exogenous polynucleotide encoding a DGAT and an exogenous polynucleotide encoding a PDAT, two or more DGATs and one PDAT, two or more PDATs and one DGAT, or two or more DGATs and two or more PDATs, wherein at least one of the DGATs and / or at least one of the PDATs is as defined above.

[0064] In one embodiment, the cell comprises an exogenous polynucleotide encoding a GPAT and an exogenous polynucleotide encoding a PDAT, two or more GPATs and one PDAT, two or more PDATs and one GPAT, or two or more GPATs and two or more PDATs, wherein at least one of the GPATs and / or at least one of the PDATs is as defined above.

[0065] In one embodiment, the cell comprises an exogenous polynucleotide encoding a DGAT, an exogenous polynucleotide encoding a GPAT, and an exogenous polynucleotide encoding a PDAT, or one or more of a plurality of members thereof.

[0066] In one embodiment, the cell comprises an exogenous polynucleotide encoding two or more DGATs, wherein at least one of the DGATs is as defined above. In one embodiment, the cell comprises an exogenous polynucleotide encoding two or more GPATs, wherein at least one of the GPATs is as defined above. In one embodiment, the cell comprises an exogenous polynucleotide encoding two or more PDATs, wherein at least one of the PDATs is as defined above.

[0067] In a further aspect, the present invention provides a microbial cell, preferably a yeast cell, comprising a lipid and at least one genetic modification, which genetic modification is at least one exogenous polynucleotide encoding: (a) FATA fatty acylthioesterase, (b) at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), or (c) FATA and at least one fatty acid acyltransferase, preferably at least DGAT; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell, and wherein: (d) optionally, a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding a Δ12 desaturase, more preferably a null mutation of an endogenous gene encoding a Δ12 desaturase, most preferably a null mutation of the FAD2 gene; and (e) 20% to 80% by weight, 20% to 75% by weight, 20% to 70% by weight, 20% to 65% by weight, 20% to 60% by weight, 20% to 55% by weight, 20% to 50% by weight, 20% to 45% by weight, 25% to 80% by weight, 25% to 75% by weight, 25% to 70% by weight, 25% to 65% by weight, 25% to 60% by weight, 25% to 55% by weight, 25% to 50% by weight, or 25% to 45% by weight of the TFA content of the cells is stearic acid.

[0068] In one embodiment of this aspect, the TFA content of the lipid or TAG content of the cells, or both, comprises less than 4 wt%, less than 3 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or less than 0.2 wt%, or 0.2 wt% to 5 wt%, 0.2 wt% to 4 wt%, 0.2 wt% to 3 wt%, 0.2 wt% to 2 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt% linoleic acid (LA), or LA is essentially absent from the TFA of the lipid and / or TAG content of the cells. In another embodiment of this aspect, the TFA content of the lipid or TAG content of the cells, or both, comprises less than 4 wt%, less than 3 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, or less than 0.2 wt%, or 0.2 wt% to 5 wt%, 0.2 wt% to 4 wt%, 0.2 wt% to 3 wt%, 0.2 wt% to 2 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, or PUFAs are essentially absent from the TFA of the lipid and / or TAG content of the cells. In one embodiment of this aspect, 20 wt% to 55 wt% of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid. In one embodiment of this aspect, 20 wt% to 50 wt% of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid. In one embodiment of this aspect, 20% to 45% by weight of the TFA content of the cellular lipid or of the cellular TAG content, or both, is stearic acid.

[0069] In a further aspect, the present invention provides a microbial cell, preferably a yeast cell, comprising a lipid and at least one genetic modification, which genetic modification is at least one exogenous polynucleotide encoding: (a) FATA fatty acylthioesterase, (b) at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), or (c) FATA and at least one fatty acid acyltransferase, preferably at least DGAT; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell, and wherein: (d) optionally, a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding a Δ12 desaturase, more preferably a null mutation of an endogenous gene encoding a Δ12 desaturase, preferably a null mutation of the FAD2 gene; and (e) The L / S-SFA ratio of the TFA content of the cellular lipid or TAG content, or both, is 1.5 to 10, 1.5 to 9, 1.5 to 8, 1.5 to 7, 1.5 to 6, 1.5 to 5, 1.5 to 4, 1.75 to 10, 1.75 to 9, 1.75 to 8, 1.75 to 7, 1.75 to 6, 1.75 to 5, 1.75 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2.5 to 10, 2.5 to 9, 2.5 to 8, 2.5 to 7, 2.5 to 6, 2.5 to 5, or 2.5 to 4. In one embodiment of this aspect, 20% to 55% by weight of the TFA content of the cellular lipid or TAG content, or both, is stearic acid. In one embodiment of this aspect, 20% to 50% by weight of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid. In one embodiment of this aspect, 20% to 45% by weight of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid.

[0070] In a further aspect, the present invention provides a microbial cell, preferably a yeast cell, comprising a lipid and at least one genetic modification, which genetic modification is at least one exogenous polynucleotide encoding: (a) FATA fatty acylthioesterase, (b) at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), or (c) FATA and at least one fatty acid acyltransferase, preferably at least DGAT; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell; and (d) optionally, a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding a Δ12 desaturase, more preferably a null mutation of an endogenous gene encoding a Δ12 desaturase, most preferably a null mutation of the FAD2 gene; and (e) The total content of C20:0, C22:0, and C24:0 fatty acids in the TFA content or TAG content, or both, of the lipids of the cells is 5% to 25% by weight, 5% to 20% by weight, 5% to 18% by weight, 5% to 16% by weight, 5% to 15% by weight, 5% to 14% by weight, 5% to 13% by weight, 5% to 12% by weight, 5% to 10% by weight, 6% to 25% by weight, 6% to 20% by weight , 6% to 18% by weight, 6% to 16% by weight, 6% to 15% by weight, 6% to 14% by weight, 6% to 13% by weight, 6% to 12% by weight, 6% to 10% by weight, 7% to 25% by weight, 7% to 20% by weight, 7% to 18% by weight, 7% to 16% by weight, 7% to 15% by weight, 7% to 14% by weight, 7% to 13% by weight, 7% to 12% by weight, or 7% to 10% by weight, (f) the C20:0 fatty acid content is between about 1% and about 5% by weight, between about 1% and about 4% by weight, between about 1% and about 3% by weight, between about 2% and about 5% by weight, between about 2% and about 4% by weight, or between about 2% and about 3% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids, or both; (g) the C22:0 fatty acid content is between about 1% and about 5% by weight, between about 1% and about 4% by weight, between about 2% and about 5% by weight, between about 2% and about 4% by weight of the TFA content of the extracted lipid or the TAG content of the extracted lipid, or both; and (h) the C24:0 fatty acid content is between about 1% and about 6%, between about 1% and about 5%, between about 1% and about 4%, between about 2% and about 6%, between about 2% and about 5%, or between about 2% and about 4% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids, or both; or (i) the C20:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, or at least about 3% by weight of the TFA content of the extracted lipid or the TAG content of the extracted lipid, or both; the C22:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, or at least about 3.5% by weight; and the C24:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, or at least 4% by weight.

[0071] In one embodiment of this aspect, 20% to 55% by weight of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid. In one embodiment of this aspect, 20% to 50% by weight of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid. In one embodiment of this aspect, 20% to 45% by weight of the TFA content of the lipid or TAG content of the cells, or both, is stearic acid.

[0072] In an embodiment of the above aspect, the TFA content of the cellular lipids and / or the TFA content of the cellular TAG further comprises at least 10 wt%, at least 12 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, 10 wt% to 50 wt%, 10 wt% to 45 wt%, 10 wt% to 40 wt%, 10 wt% to 35 wt%, 10 wt% to 30 wt%, 15 wt% to 50 wt%, 15 wt% to 45 wt%, 15 wt% to 40 wt%, 15 wt% to 35 wt%, 15 wt% to 30 wt%, 20 wt% to 50 wt%, 20 wt% to 45 wt%, 20 wt% to 40 wt%, 20 wt% to 35 wt%, or 20 wt% to 30 wt% oleic acid.

[0073] In an embodiment of the above aspect, the at least one acyltransferase, preferably at least a DGAT, has at least equal or greater activity with stearyl-CoA molecules as a substrate compared to palmitoyl-CoA. The cell may contain two or more DGATs with this property.

[0074] In an embodiment of the above aspect, one or more or all of the exogenous DGATs comprise amino acids having a sequence set forth in SEQ ID NO: 81 or any one of SEQ ID NOs: 115-125, or an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to SEQ ID NO: 81 or any one or more of SEQ ID NOs: 115-125.

[0075] In an embodiment of the above aspect, at least one of the DGATs is yeast DGA1. In one embodiment, at least one DGA1 comprises amino acids having a sequence set forth in SEQ ID NO:53, 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:53.

[0076] In an embodiment of the above aspect, the FATA comprises amino acids having the sequence set forth in SEQ ID NO: 83 or SEQ ID NO: 85, 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 one or both of SEQ ID NO: 83 and SEQ ID NO: 85.

[0077] In the above embodiments reciting a DGAT, at least one or all polynucleotides in a cell encoding a DGAT can comprise nucleotides having a sequence set forth in any one of SEQ ID NOs: 144-154, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 144-154. In one embodiment, at least one or all polynucleotides encoding a DGAT in a cell encode a sequence set forth in one or more of SEQ ID NOs: 115-125 and differ from one of SEQ ID NOs: 144-154 only by codon degeneracy.

[0078] In one embodiment, a microbial cell, preferably a yeast cell, of the invention comprises one or more exogenous polynucleotides, each encoding a GPAT. In one embodiment, at least one of the GPATs, or all of the GPATs, comprises amino acids having a sequence set forth in any one of SEQ ID NOs: 126-138, or an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 95% identical to any one or more of SEQ ID NOs: 126-138.

[0079] In one embodiment, at least one or all polynucleotides encoding GPAT comprise nucleotides having a sequence set forth in any one of SEQ ID NOs: 155 to 167, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 95% identical to any one or more of SEQ ID NOs: 155 to 167. In one embodiment, at least one or all polynucleotides encoding GPAT encode a sequence set forth in one or more of SEQ ID NOs: 126 to 138 and differ from one of SEQ ID NOs: 155 to 167 only by codon degeneracy.

[0080] In one embodiment, a microbial cell, preferably a yeast cell, of the invention comprises one or more exogenous polynucleotides, each encoding a PDAT. In one embodiment, at least one of the PDATs, or all of the PDATs, comprises amino acids having a sequence set forth in any one of SEQ ID NOs: 139-143, or an amino acid sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 95% identical to any one or more of SEQ ID NOs: 139-143.

[0081] In one embodiment, at least one or all polynucleotides encoding PDAT comprise nucleotides having a sequence set forth in any one of SEQ ID NOs: 168-172, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 95% identical to any one or more of SEQ ID NOs: 168-172. In one embodiment, at least one or all polynucleotides encoding PDAT encode a sequence set forth in one or more of SEQ ID NOs: 139-143 and differ from one of SEQ ID NOs: 168-172 only by codon degeneracy.

[0082] In one embodiment, the microbial cell, preferably a yeast cell, comprises an exogenous polynucleotide encoding a diacylglycerol acyltransferase (DGAT) and an exogenous polynucleotide encoding a glycerol-3-phosphate acyltransferase (GPAT), two or more DGATs and one gPAT, two or more GPATs and one DGAT, or two or more DGATs and two or more GPATs, wherein at least one of the DGATs and / or at least one of the GPATs is as defined above.

[0083] In one embodiment, the cell comprises an exogenous polynucleotide encoding a DGAT and an exogenous polynucleotide encoding a PDAT, two or more DGATs and one PDAT, two or more PDATs and one DGAT, or two or more DGATs and two or more PDATs, wherein at least one of the DGATs and / or at least one of the PDATs is as defined above.

[0084] In one embodiment, the cell comprises an exogenous polynucleotide encoding a GPAT and an exogenous polynucleotide encoding a PDAT, two or more GPATs and one PDAT, two or more PDATs and one GPAT, or two or more GPATs and two or more PDATs, wherein at least one of the GPATs and / or at least one of the PDATs is as defined above.

[0085] In one embodiment, the cell comprises an exogenous polynucleotide encoding a DGAT, an exogenous polynucleotide encoding a GPAT, and an exogenous polynucleotide encoding a PDAT, or one or more of a plurality of members thereof.

[0086] In one embodiment, the cell comprises an exogenous polynucleotide encoding two or more DGATs, wherein at least one of the DGATs is as defined above.

[0087] In one embodiment, the cell comprises an exogenous polynucleotide encoding two or more GPATs, wherein at least one of the GPATs is as defined above.

[0088] In one embodiment, the cell comprises an exogenous polynucleotide encoding two or more PDATs, wherein at least one of the PDATs is as defined above.

[0089] In an embodiment of the above aspect, the microbial cell, preferably a yeast cell, further comprises an exogenous polynucleotide encoding a lysophosphatidic acid acyltransferase (LPAAT), wherein the polynucleotide is operably linked to one or more promoters capable of directing expression of the polynucleotide in the microbial cell. In one embodiment, the LPAAT comprises amino acids having a sequence set forth in SEQ ID NO:90, 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:90.

[0090] In an embodiment of the above aspect, the microbial cell, preferably a yeast cell, further comprises an exogenous polynucleotide encoding lysophosphatidylcholine acyltransferase (LPCAT), monoacylglycerol acyltransferase (MGAT), phospholipase A2 (PLA2), phospholipase C (PLC), phospholipase D (PLD), CDP-choline diacylglycerol choline phosphotransferase (CPT), phosphatidylcholine:diacylglycerol choline phosphotransferase (PDCT), acyl-CoA synthase (ACS), fatty acid desaturase, or any combination of two or more thereof.

[0091] In embodiments of the above aspects, the microbial cell, preferably a yeast cell, more preferably a W. lipolytica cell, comprises a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase or Δ9 desaturase, or reduced expression and / or activity of both the Δ12 desaturase and the Δ9 desaturase, preferably a genetic modification of an endogenous gene encoding the Δ12 desaturase or the Δ9 desaturase, more preferably a null mutation in the endogenous gene encoding the Δ12 desaturase, or a null mutation in the endogenous gene encoding the Δ12 desaturase and a mutation in the endogenous gene encoding the Δ9 desaturase that is not a null mutation. In embodiments of the above aspects, the microbial cell, preferably a yeast cell, further comprises an exogenous polynucleotide encoding a silencing RNA molecule that reduces the expression and / or activity of a fatty acid desaturase gene, and / or a mutation in a fatty acid desaturase gene that reduces the expression and / or activity of a desaturase gene, preferably the Δ9 desaturase gene or the Δ12 desaturase gene. In a preferred embodiment, the mutation in the Δ9 desaturase gene is not a null mutation. In a preferred embodiment, the mutation in the Δ12 desaturase gene is a null mutation. In one embodiment, the cell further comprises an exogenous polynucleotide encoding a silencing RNA molecule that reduces expression and / or activity of an endogenous DGAT, GPAT, or PDAT gene, and / or a mutation in the endogenous DGAT, GPAT, or PDAT gene that reduces expression and / or activity of a desaturase gene.

[0092] In a preferred embodiment of the above aspect, the genetic modification is a mutation in a gene encoding an endogenous Δ12 desaturase, preferably a null mutation in the FAD2 gene.

[0093] In embodiments of the above aspects, the endogenous Δ12 desaturase comprises amino acids having a sequence set forth in SEQ ID NO:1, or an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO:1.

[0094] Thus, in one embodiment, the microbial cells, preferably yeast cells, (a) an exogenous polynucleotide encoding a FATA fatty acylthioesterase; (b) at least one exogenous polynucleotide encoding at least one fatty acid acyltransferase, preferably at least a diacylglycerol acyltransferase (DGAT), and (c) a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding a Δ12 desaturase, more preferably a null mutation of an endogenous gene encoding a Δ12 desaturase, most preferably a null mutation of the FAD2 gene; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in a microbial cell.

[0095] In one embodiment, at least one DGAT comprises nucleotides having a sequence set forth in any one of SEQ ID NOs: 144-154, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 144-154.

[0096] In one embodiment, the FATA fatty acylthioesterase comprises a nucleotide having a sequence set forth in any one of SEQ ID NOs: 84 or 86, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 84 or 86.

[0097] In one embodiment, the microbial cells have a higher amount of one or more or all of the total SFA content, C20:0 content, C22:0 content, and C24:0 content in the TFA content and / or TAG content of the lipid compared to a corresponding extracted microbial lipid obtained from a corresponding microorganism lacking at least one genetic modification.

[0098] In one embodiment, the C20:0 fatty acid content is from about 1% to about 5% by weight, from about 1% to about 4% by weight, from about 1% to about 3% by weight, from about 2% to about 5% by weight, from about 2% to about 4% by weight, or from about 2% to about 3% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids, or both.

[0099] In one embodiment, the C22:0 fatty acid content is from about 1% to about 5%, from about 1% to about 4%, from about 2% to about 5%, or from about 2% to about 4% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids or both.

[0100] In one embodiment, the C24:0 fatty acid content is from about 1% to about 6%, from about 1% to about 5%, from about 1% to about 4%, from about 2% to about 6%, from about 2% to about 5%, or from about 2% to about 4% by weight of the TFA content of the extracted lipids or the TAG content of the extracted lipids or both.

[0101] In another embodiment, the C20:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, or at least about 3% of the TFA content of the extracted lipid or the TAG content of the extracted lipid, or both, the C22:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, or at least about 3.5%, and the C24:0 fatty acid content is at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, or at least 4% by weight.

[0102] In another embodiment, the C20:0 content is from 1.0% to 4%, the C22:0 content is from 1.0% to 4%, and the C24:0 content is from 2% to 5%.

[0103] In one embodiment, the microorganism further comprises an exogenous polynucleotide encoding an acyl-CoA synthetase (ACS), optionally wherein the ACS comprises a nucleotide having a sequence set forth as any one of SEQ ID NOs: 88-89, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 88-89.

[0104] In another embodiment, the microorganism further comprises an exogenous polynucleotide encoding a lysophosphatidic acid acyltransferase (LPAAT), optionally wherein the LPAAT comprises a nucleotide having a sequence set forth in SEQ ID NO:91, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:91.

[0105] In an embodiment of the above aspect, the microbial cell, preferably the yeast cell, comprises a genetic modification that results in reduced gene expression or activity, or both, of an endogenous gene, preferably the DGA2 gene, encoding a DGAT that has a preference for PUFA-CoA as a substrate compared to stearoyl-CoA, or a preference for palmitoyl-CoA compared to stearoyl-CoA, or both.

[0106] In an embodiment of the above aspect, the endogenous DGA2 comprises amino acids having the sequence set forth in SEQ ID NO: 55, 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: 55.

[0107] In an embodiment of the above aspect, the microbial cell, preferably a yeast cell, comprises: 1) an exogenous polynucleotide encoding a FATA fatty acylthioesterase and a genetic modification resulting in reduced expression and / or activity of an endogenous Δ12 desaturase gene; 2) an exogenous polynucleotide encoding a fatty acid acyltransferase, preferably a diacylglycerol acyltransferase (DGAT), and a genetic modification resulting in reduced expression and / or activity of an endogenous Δ12 desaturase gene; 3) exogenous polynucleotides encoding a FATA fatty acylthioesterase and a fatty acid acyltransferase, preferably a diacylglycerol acyltransferase (DGAT), and genetic modifications resulting in reduced expression and / or activity of an endogenous Δ12 desaturase gene; 4) an exogenous polynucleotide encoding a FATA fatty acylthioesterase and a genetic modification resulting in reduced expression and / or activity of the endogenous DGA2 gene; 5) an exogenous polynucleotide encoding a fatty acid acyltransferase, preferably diacylglycerol acyltransferase (DGAT), and a genetic modification resulting in reduced expression and / or activity of the endogenous DGA2 gene; 6) exogenous polynucleotides encoding FATA fatty acylthioesterase and fatty acid acyltransferase, preferably diacylglycerol acyltransferase (DGAT), and genetic modifications resulting in reduced expression and / or activity of the endogenous DGA2 gene; 7) an exogenous polynucleotide encoding a FATA fatty acylthioesterase, a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase gene, and a genetic modification that results in reduced expression and / or activity of an endogenous DGA2 gene; 8) an exogenous polynucleotide encoding a fatty acid acyltransferase, preferably a diacylglycerol acyltransferase (DGAT), a genetic modification that results in reduced expression and / or activity of an endogenous Δ12 desaturase gene, and a genetic modification that results in reduced expression and / or activity of an endogenous DGA2 gene; or 9) exogenous polynucleotides encoding a FATA fatty acylthioesterase and a fatty acid acyltransferase, preferably a diacylglycerol acyltransferase (DGAT), genetic modifications that result in reduced expression and / or activity of an endogenous Δ12 desaturase gene, and genetic modifications that result in reduced expression and / or activity of an endogenous DGA2 gene; wherein each polynucleotide is operably linked to one or more promoters capable of directing expression of said polynucleotide in said microbial cell.

[0108] In an embodiment of the above aspect, the microbial cells, preferably yeast cells, comprise or consist of eukaryotic cells, fungal cells, bacterial cells, or algal cells, live microbial cells, dead microbial cells, or any mixture thereof. In embodiments of the above aspects, the microbial cells, preferably yeast cells, are one or more or all of: (i) cells suitable for fermentation; (ii) oleaginous cells; (iii) non-oleaginous cells, preferably non-oleaginous cells derived from oleaginous cells by genetic engineering; and (iv) heterotrophic cells.

[0109] In an embodiment of the above aspect, the microbial cell is a yeast cell. Examples of suitable yeast cells include, but are not limited to, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and any mixture thereof. In an embodiment of the above aspect, the yeast cell is Yarrowia lipolytica.

[0110] In an embodiment of the above aspect, the microbial cells are present in or have been cultured in a medium having less than about 5 g / L, less than about 2 g / L, less than about 1 g / L stearate, or no stearate added.

[0111] In another aspect, the present invention provides a microbial cell extract, preferably a yeast cell extract, comprising a lipid as defined herein or produced from a microbial cell of the present invention.

[0112] In another aspect, the present invention provides a DNA construct or a combination of DNA constructs encoding one or more of the enzymes defined herein, preferably integrated into the genome of a microbial cell, such as a yeast cell.

[0113] In one embodiment, at least one or all DNA constructs in a cell encoding a DGAT comprise nucleotides having a sequence set forth in any one of SEQ ID NOs: 144 to 154, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 144 to 154. In one embodiment, at least one or all DNA constructs encoding a DGAT in a cell encode a sequence set forth in one or more of SEQ ID NOs: 115 to 125 and differ from one of SEQ ID NOs: 144 to 154 only by codon degeneracy.

[0114] In another embodiment, at least one or all of the DNA constructs in the cell encoding the FATA fatty acylthioesterase comprise a sequence set forth in any one of SEQ ID NOs: 84 or 86, a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 84 or 86.

[0115] In another embodiment, at least one or all of the DNA constructs in the cell encoding acyl-CoA synthetase (ACS) comprise a sequence set forth in any one of SEQ ID NOs: 88-89, that is a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to any one or more of SEQ ID NOs: 88-89.

[0116] In another embodiment, at least one or all DNA constructs in the cell encoding lysophosphatidic acid acyltransferase (LPAAT) comprise nucleotides having the sequence set forth in SEQ ID NO:91, or a nucleotide sequence that is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95%, or at least 97% identical to SEQ ID NO:91.

[0117] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) obtaining a microbial cell of the invention; and (b) extracting lipids from the microbial cells, thereby producing extracted lipids. Thus, each of the embodiments of the microbial cells described herein can be used in a process for producing extracted lipids.

[0118] In one embodiment, the process further comprises culturing the microbial cells or treating the cells with an acid prior to step (b). In one embodiment, the process further comprises culturing the microbial cells or treating the cells after culturing but before the extraction step to improve the extraction step, for example treating the cells with an acid before step (b).

[0119] In one embodiment, the cells are cultured in a medium having less than 5 g / L, less than 2 g / L, less than 1 g / L stearate, or in a medium that is stearate-free. Alternatively, the cells are cultured in a medium that contains at least 1 g / L, at least 2 g / L, or at least 5 g / L stearate.

[0120] In one embodiment, the step of extracting lipids comprises exposing the cells to an organic solvent, pressing the cells, or treating the cells with microwave irradiation, sonication, high speed homogenization, high pressure homogenization, bead beating, autoclaving, pyrolysis, or any combination thereof. In one embodiment, the method further comprises modifying or purifying the lipids after extraction.

[0121] In another aspect, the present invention provides a method for culturing microbial cells, the method comprising: (a) obtaining a microbial cell of the invention; and (b) Expanding the number of cells by culturing the cells in an appropriate medium. Thus, each of the microbial cell embodiments described herein can be used in a process for culturing microbial cells.

[0122] In another aspect, the present invention provides a process for producing a lipid-producing microbial cell of the present invention, the process comprising introducing one or more genetic modifications and / or exogenous polynucleotides defined herein into a precursor microbial cell, which process can be used to generate each of the embodiments of the microbial cell of the present invention described herein.

[0123] In one embodiment, the process comprises: (i) generating progeny cells from cells containing the introduced genetic modification and / or exogenous polynucleotide; (ii) mutagenesis of progenitor cell populations; (iii) introducing one or more exogenous polynucleotides, whereby the exogenous polynucleotides are integrated into the genome of the microbial cell, preferably at a predetermined location; (iv) determining the fatty acid composition of the cell or its progeny; and (v) selecting progeny cells containing the lipid of the present invention.

[0124] In another aspect, the present invention provides a composition comprising one or more or all of the lipids of the present invention, the microbial cells of the present invention, or the microbial cell extracts of the present invention. Thus, each embodiment of the microbial cells and extracted lipids described herein can be used to produce a composition. The composition can be used as an ingredient to produce a food or beverage.

[0125] In one embodiment, the composition comprises a food, feed, or beverage ingredient in addition to the lipid of the present invention. The lipid or composition of the present invention can be used in personal care products such as pharmaceuticals, cosmetics, and toiletries.

[0126] In one embodiment, the composition comprises one or more fatty acids, esterified or non-esterified, from a source other than extracted microbial lipids, cells, or extracts.

[0127] In another aspect, the invention provides a food, feed, or beverage comprising 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, and at least one other food, feed, or beverage ingredient. Thus, each of the microbial cell and extracted lipid embodiments described herein can be used to produce the food, feed, or beverage ingredient of the food, feed, or beverage.

[0128] In one embodiment, the food, feed, or drink is devoid of animal-derived ingredients.

[0129] In one embodiment, the food, feed, or beverage ingredient, or food, feed, or beverage of the invention is a meat substitute, fat, oil, or dressing, soup, noodle product, stew, stock, broth, sauce, gravy, pasta sauce, tomato product, dry seasoning mix, bread, bread substitute, pastry, croissant, biscuit, cracker, cake, pizza dough, pie dough, dry bakery mix, bakery dough, donut, mixed ingredient dish, snack, such as a sweet snack, savory food, or savory snack, meat substitute or similar, dairy substitute or similar, or another food.

[0130] In another aspect, the present invention provides a method of producing a food, feed, or beverage, the method comprising 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 at least one other food, feed, or beverage ingredient. Accordingly, each of the microbial cell and extracted lipid embodiments described herein can be used in the method.

[0131] Also provided is the use of 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 to produce a food, feed, or beverage ingredient, or a food, feed, or beverage.

[0132] Any embodiment herein shall apply mutatis mutandis to any other embodiment unless expressly stated otherwise. The present invention is not limited in scope by the specific embodiments described herein, which are for purposes of example only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention as described herein. Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition, group of steps or group of compositions should be understood to encompass one and more (i.e., one or more) of that step, composition, group of steps or group of compositions.

[0133] The invention will now be described by way of the following non-limiting examples and with reference to the accompanying drawings. [Brief explanation of the drawings]

[0134] [Figure 1] Figure 1: Growth curves of S. cerevisiae cultured in YPD medium for up to 7 days. [Figure 2] Figure 2: Schematic diagram for creating a gene construct to introduce an inactivating deletion in a gene of interest, such as microbial FAD2 or URA3. Panel A. DNA synthesis of a 2 kb fragment with a 1,000 bp 5' upstream region and a 1,000 bp 3' downstream region of the gene of interest joined with a SacII site between these two regions. The positions of the restriction 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 compatible with the SacII sites. Construction of a gene construct by inserting a SacII-terminated antibiotic resistance gene cassette into a CA DNA fragment. Not to scale. [Figure 3] Figure 3: Schematic diagram of the construction of gene constructs for introducing gene deletions into microorganisms. Panel A. PCR amplification and ligation of the 5' upstream and 3' downstream regions of the gene of interest to create a 2 kb fragment. Oligonucleotide primers are indicated by small horizontal arrows, and 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) resistance genes using primers compatible with the flanking AsiSI sites. C. Assembly of gene constructs for introduction into microorganisms such as Y. lipolytica. [Figure 4] Figure 4: Schematic structure of a phospholipid. One of the hydroxyls can be replaced with a variety of head groups, such as choline, serine, or inositol. [Figure 5]Figure 5: Schematic of the gene construct for the expression of the candidate PDAT combination in Y. lipolytica. Each PDAT is under the control of the pTEF promoter and the LIP2 gene transcription terminator. [Figure 6] Figure 6: Schematic diagram of cloning vectors for introducing candidate acyltransferase genes into Y. lipolytica. The sequence of pNIz0hyg[dga2] is provided as SEQ ID NO: 173. The sequence of pNIz0ura[fad2] is provided as SEQ ID NO: 174. The sequence of pNIz0nat[lro1] is provided as SEQ ID NO: 175. The sequence of pNIz0hyg[pox2] is provided as SEQ ID NO: 176. [Figure 7] Figure 7: (A) Light micrograph of W29 cells. (B) Transformed W29 cells after lipid accumulation in defined medium (M1).

[0135] Array table key SEQ ID NO: 1. Amino acid sequence of the Y. lipolytica strain W29 FAD2 polypeptide, accession number XP_500707.1.

[0136] SEQ ID NO: 2. Nucleotide sequence of the FAD2 gene of Y. lipolytica strain W29, including the upstream and downstream regions. Nucleotides 1 to 1000 correspond to the 5' upstream sequence, nucleotides 1001 to 2260 correspond to the protein coding region for the Δ12 desaturase, and nucleotides 2261 to 3260 correspond to the 3' downstream region.

[0137] SEQ ID NO: 3. Nucleotide sequence of the hygromycin resistance selectable marker gene (pTEF-Hyg-tLip2). Nucleotides 1 to 417 correspond to the TEF promoter (Müller et al., 1998; accession number AF054508), nucleotides 418 to 1443 correspond to the protein coding region for the hygromycin phosphotransferase (Hph) enzyme, and nucleotides 1444 to 1620 correspond to the polyadenylation region / transcription terminator from the Yersinia lipolytica U6 strain lipase 2 gene from accession number HM486900 (Darvishi et al., 2011). Nucleotides 20 to 53 correspond to a loxP site, and nucleotides 1569 to 1598 correspond to a loxR site.

[0138] SEQ ID NO: 4. Amino acid sequence of hygromycin B phosphotransferase (Hph) encoded by pTEF-Hyg-tLip2.

[0139] SEQ ID NO: 5. Nucleotide sequence of the nourseothricin resistance selectable marker gene (pTEF-Nat1-tLip2) from GGE368 (accession number AIC06992, Calvey et al. (2014)). Nucleotides 1-418 correspond to the TEF promoter, nucleotides 419-988 correspond to the protein coding region of the nourseothricin acetyltransferase (Nat1) enzyme, and nucleotides 989-1165 correspond to the polyadenylation region / transcription terminator of the Lip2 gene. Nucleotides 20-53 correspond to a loxP site, and nucleotides 1114-1143 correspond to a loxR site. 1165 nt.

[0140] SEQ ID NO: 6. Amino acid sequence of nourseothricin acetyltransferase (Nat1) encoded by the pTEF-Nat1-tLip2 gene. SEQ ID NO: 7. Nucleotide sequence of the Y. lipolytica TEF promoter (Muller et al., 1998; accession number AF054508). SEQ ID NOs: 8 to 49. Oligonucleotide primers (see Table 13) SEQ ID NO: 50. Amino acid sequence of Y. lipolytica strain URA3 polypeptide, GenBank accession number Q12724; 286 aa.

[0141] SEQ ID NO:51. Nucleotide sequence of the URA3 gene of Y. lipolytica, including the upstream and downstream regions. Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 1,861 correspond to the protein coding region of orotidine-5'-phosphate decarboxylase, and nucleotides 1,862 to 2,861 correspond to the 3' downstream region.

[0142] SEQ ID NO: 52. Nucleotide sequence of the DGA1 gene (YALI0E32769p) on chromosome E of Y. lipolytica strain W29, accession number CR382131.1, from nucleotides 3885857 to 3889401 (including the upstream and downstream regions of the DGA1 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 2,545 correspond to the protein coding region of the DGA1 polypeptide, and nucleotides 2,546 to 3,545 correspond to the 3' downstream region; 3,545 nt.

[0143] SEQ ID NO: 53. Amino acid sequence of the DGA1 polypeptide from Y. lipolytica strain W29 encoded by the YALI0E32769p gene (GenBank accession no. XP_504700.1; 514 aa).

[0144] SEQ ID NO: 54. Nucleotide sequence of the DGA2 gene (YALI0D07986p) on chromosome D of Y. lipolytica strain W29, accession number CP017556.1, from nucleotides 1025413 to 1028993 (including the upstream and downstream regions of the DGA2 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 2,581 correspond to the protein coding region of the DGA2 polypeptide, and nucleotides 2,582 to 3,581 correspond to the 3' downstream region; 3,581 nt.

[0145] SEQ ID NO: 55. Amino acid sequence of Y. lipolytica strain W29 DGA2 polypeptide, GenBank accession number XP_502557; 526 aa.

[0146] SEQ ID NO: 56. Nucleotide sequence of the LRO1 gene (YALI0E16797p) on chromosome E of Y. lipolytica strain CLIB122, accession number CR382131.1, nucleotides 1989950 to 1993896 (including the upstream and downstream regions of the LRO1 gene). Nucleotides 1 to 1000 correspond to the 5' upstream sequence, nucleotides 1001 to 2947 correspond to the protein-coding region of PDAT, and nucleotides 2948 to 3947 correspond to the 3' downstream region; 3947 nt.

[0147] SEQ ID NO: 57. Amino acid sequence of PDAT from Y. lipolytica strain CLIB122 encoded by LRO1 gene (YALI0E16797p), GenBank accession number XP_504038; 648 aa.

[0148] SEQ ID NO: 58. Nucleotide sequence of the ARE1 gene (YALI0F06578p) on chromosome F of Y. lipolytica strain W29, accession number CP028453.1, nucleotides 957751 to 961382 (including the upstream and downstream regions of the ARE1 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 2,632 correspond to the protein-coding region of ASAT, and nucleotides 2,633 to 3,632 correspond to the 3' downstream region; 3,632 nt.

[0149] SEQ ID NO: 59. Amino acid sequence of ASAT from Y. lipolytica strain W29 encoded by ARE1 gene (YALI0F06578p), GenBank accession number XP_505086; 543 aa.

[0150] SEQ ID NO: 60. Nucleotide sequence of the POX1 gene (YALI0E32835g) on ​​chromosome E of Y. lipolytica strain CLIB122, accession number CR382131.1, nucleotides 3897102 to 3899135 (including the upstream and downstream regions of the POX1 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 3,103 correspond to the protein-coding region of POX1, and nucleotides 3,104 to 4,103 correspond to the 3' downstream region; 4,103 nt.

[0151] SEQ ID NO: 61. Amino acid sequence of POX1 from Yersinia lipolytica strain CLIB122 encoded by YALI0E32835p, GenBank accession number XP_504703.1; 677aa.

[0152] SEQ ID NO: 62. Nucleotide sequence of the POX2 gene (YALI0F10857g) on ​​chromosome F of Y. lipolytica strain CLIB122, accession number CR382132.1, nucleotides 1449289 to 1451391 (including the upstream and downstream regions of the POX2 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 3,103 correspond to the protein-coding region of POX2, and nucleotides 3,104 to 4,103 correspond to the 3' downstream region; 4,103 nt.

[0153] SEQ ID NO: 63. Amino acid sequence of the POX2 gene from Yersinia lipolytica strain CLIB122 encoded by YALI0F10857p, GenBank accession number _XP_505264.1; 700 aa.

[0154] SEQ ID NO: 64. Nucleotide sequence of the POX3 gene (YALI0D24750g) on ​​chromosome D of Y. lipolytica strain CLIB122, accession number CR382130.1, nucleotides 3291579 to 3293681 (including the upstream and downstream regions of the POX3 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 3,103 correspond to the protein-coding region of POX3, and nucleotides 3,104 to 4,103 correspond to the 3' downstream region; 4,103 nt.

[0155] SEQ ID NO: 65. Amino acid sequence of POX3 from Y. lipolytica strain CLIB122 encoded by YALI0D24750p, GenBank accession number XP_503244; 700 aa.

[0156] SEQ ID NO: 66. Nucleotide sequence of the MFE1 gene (YALI0E15378g) on ​​chromosome E of Y. lipolytica strain CLIB122, accession number CR382131.1, nucleotides 1829460 to 1832239 (including the upstream and downstream regions of the MFE1 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 3,706 correspond to the protein-coding region of PDAT, and nucleotides 3,706 to 4,706 correspond to the 3' downstream region; 4,706 nt.

[0157] SEQ ID NO: 67. Amino acid sequence of MFE1 from Y. lipolytica strain CLIB122, encoded by YALI0E15378p, GenBank accession number XP_503980. 901 aa.

[0158] SEQ ID NO: 68. Nucleotide sequence of the PEX10 gene (YALI0C01023g) on ​​chromosome C of Y. lipolytica strain CLIB122, accession number CR382129.1, nucleotides 139718 to 140851 (including the upstream and downstream regions of the PEX10 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 2,134 correspond to the protein-coding region of PEX10, and nucleotides 2,135 to 3,134 correspond to the 3' downstream region; 3,134 nt.

[0159] SEQ ID NO: 69. Amino acid sequence of PEX10 from Y. lipolytica strain CLIB122 encoded by YALI0C01023p, GenBank accession number XP_501311; 377aa.

[0160] SEQ ID NO: 70. Nucleotide sequence of the SNF1 gene (YALI0D02101g) on ​​chromosome D from Y. lipolytica strain CLIB122, accession number CR382130.1, nucleotides 236133 to 237872 (including the upstream and downstream regions of the SNF1 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 2,740 correspond to the protein-coding region of SNF1, and nucleotides 2,741 to 3,740 correspond to the 3' downstream region; 3,740 nt.

[0161] SEQ ID NO: 71. Amino acid sequence of SNF1 from Y. lipolytica strain CLIB122 encoded by YALI0D02101p, GenBank accession number XP_502312; 579 aa.

[0162] SEQ ID NO: 72. Nucleotide sequence of the SPO14 gene (YALI0E18898g) on ​​chromosome E of Y. lipolytica strain CLIB122, accession number CR382131.1, nucleotides 2251884 to 2257373 (including the upstream and downstream regions of the SPO14 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 6,490 correspond to the protein-coding region of SPO14, and nucleotides 6,491 to 7,490 correspond to the 3' downstream region; 7,490 nt.

[0163] SEQ ID NO: 73. Amino acid sequence of SPO14 from Y. lipolytica strain CLIB122 encoded by YALI0E18898p, GenBank accession number XP_504124; 1829 aa.

[0164] SEQ ID NO: 74. Nucleotide sequence of the OPI1 gene (YALI0C14784g) on ​​chromosome E of Y. lipolytica strain CLIB122, accession number CR382129.1, nucleotides 2251884 to 237872 (including the upstream and downstream regions of the OPI1 gene). Nucleotides 1 to 1,000 correspond to the 5' upstream sequence, nucleotides 1,001 to 2,863 correspond to the protein-coding region of OPI1, and nucleotides 2,864 to 3,863 correspond to the 3' downstream region; 3,863 nt.

[0165] SEQ ID NO: 75. Amino acid sequence of OPI1 from Y. lipolytica strain CLIB122 encoded by YALI0C14784p, GenBank accession number XP_501843; 620 aa.

[0166] SEQ ID NO: 76. Nucleotide sequence of the S. cerevisiae POX1 gene (YGL205W; chrVII:108158-110404), including upstream and downstream regions. Nucleotides 1-1,000 correspond to the 5' upstream sequence, nucleotides 1,001-3,247 correspond to the protein coding region for acyl-CoA oxidase, and nucleotides 3,248-4,247 correspond to the 3' downstream region.

[0167] SEQ ID NO: 77. Amino acid sequence of the POX1 gene product of S. cerevisiae strain S288C (accession number NP_011310.1); 748 aa.

[0168] SEQ ID NO: 78. Nucleotide sequence of the FBAIN promoter from Y. lipolytica (U.S. Patent No. 8,815,566); 973 nt. Translation initiation ATG is at nucleotides 803-805, intron is at nucleotides 866-967.

[0169] SEQ ID NO: 79. Nucleotide sequence of the modified Y. lipolytica FBAIN gene promoter (FBAINm); 927 nt. FBAINm was generated by deleting the region corresponding to nucleotides 805-857 of the FBAIN promoter (SEQ ID NO: 78). The promoter was also modified to remove the wild-type ATG from FBAIN and provide an optional translation consensus sequence (CACA; Gasmi et al., 2011) immediately upstream of the ATG start codon, nucleotides 925-927.

[0170] SEQ ID NO: 80. Nucleotide sequence of the GPD gene promoter of Y. lipolytica (U.S. Patent No. 8,815,566); 971 nt. The translation initiation codon ATG is at nucleotides 969 to 971.

[0171] SEQ ID NO: 81. Amino acid sequence of DGAT1 from M. tetraphylla (macadamia), NCBI accession number KT736302.1; 535 aa.

[0172] SEQ ID NO: 82. Nucleotide sequence of the protein coding region cloned into pAT117 encoding MtDGAT1 from M. tetraphylla, flanked by BsaI sites; 1634 nt.

[0173] SEQ ID NO: 83. Amino acid sequence of GmFATA1 from G. mangostana (mangosteen), NCBI accession number U92876.1; 352 aa.

[0174] SEQ ID NO: 84. Nucleotide sequence of the protein coding region cloned into pAT066 encoding the FATA1 protein from Z. mangostana, flanked by BsaI sites. The start codon ATG is at nucleotides 9-11 and the stop codon is at nucleotides 1068-107; 1081 nt.

[0175] SEQ ID NO: 85. Amino acid sequence of GmFATA2 from Z. mangostana (mangosteen), NCBI accession number U92877.1; 355 aa.

[0176] SEQ ID NO: 86. Nucleotide sequence of the protein coding region cloned into pAT067 encoding FATA2 from Z. mangostana. The start codon ATG is at nucleotides 9-11 and the stop codon is at nucleotides 1077-1079; 1090 nt.

[0177] SEQ ID NO: 87. Amino acid sequence of PcACS-X1 from P. chlororaphis, NCBI accession number BAD90933; 545 aa.

[0178] SEQ ID NO: 88. Nucleotide sequence of the protein coding region cloned into pAT136 (PcACS-X1) from P. chlororaphis, flanked by BsaI sites. The start codon ATG is at nucleotides 12-14, and the stop codon is at nucleotides 1647-1649; 1660 nt.

[0179] SEQ ID NO: 89. Nucleotide sequence of the protein coding region cloned into pAT138 (PcACS-X2) from P. chlororaphis, flanked by BsaI sites; 1660 nt.

[0180] SEQ ID NO: 90. Amino acid sequence of MaLPAAT from Mortierella alpina, NCBI accession number: 314 aa.

[0181] SEQ ID NO: 91. Nucleotide sequence of the protein coding region of MtLPAAT from M. alpina; 945 nt.

[0182] SEQ ID NO: 92. Nucleotide sequence of the NotI DNA fragment of pAT207 used to transform Y. lipolytica cells. Nucleotides 1-8 and 9096-9103, NotI restriction enzyme site; nucleotides 9-308, 5' portion of the Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 1903-2261, 3960-4318, and 6595-6967, pTEF promoter; nucleotides 1644-1902, 3483-3959, and nucleotides 2269–3330, the protein-coding region of GmFATA1; nucleotides 4329–5966, the protein-coding region of PcACS-X1; nucleotides 6968–8575, the protein-coding region of MtDGAT1; nucleotides 3331–3482, 5971–6117, and 8580–8696, the lip2 transcription terminator; and nucleotides 8701–9095, the 3' portion of the Y. lipolytica zeta sequence.

[0183] SEQ ID NO: 93. Nucleotide sequence of the NotI DNA fragment of pAT208 used to transform Y. lipolytica cells. Nucleotides 1-8 and 9096-9103, NotI restriction enzyme site; nucleotides 9-308, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 1903-2261, 3960-4318, and 6595-6967, pTEF promoter. Nucleotides 1644 to 1902, 3483 to 3959, and 6118 to 6594, enhancer sequence; nucleotides 2269 to 3330, protein coding region of GmFATA1; nucleotides 4329 to 5966, protein coding region of PcACS-X2; nucleotides 6968 to 8575, protein coding region of MtDGAT1; 3331 to 3482, 5971 to 6117, and 8580 to 8696, lip2 transcription terminator; and nucleotides 8701 to 9095, 3' portion of the Y. lipolytica zeta sequence.

[0184] SEQ ID NO: 94. Nucleotide sequence of the NotI DNA fragment of pAT209 used to transform Y. lipolytica cells. Nucleotides 1-8 and 9105-9112, NotI restriction enzyme site; nucleotides 9-308, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, containing the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 1903-2261, 3969-4327, and 6604-6976, pTEF promoter. Nucleotides 1644 to 1902, 3492 to 3968, and 6127 to 6603, enhancer sequence; nucleotides 2269 to 3330, protein coding region of GmFATA2; nucleotides 4338 to 5975, protein coding region of PcACS-X1; nucleotides 6977 to 8584, protein coding region of MtDGAT1; 3340 to 3491, 5980 to 6126, and 8589 to 8705, lip2 transcription terminator; and nucleotides 8710 to 9104, 3' portion of the Y. lipolytica zeta sequence.

[0185] SEQ ID NO: 95. Nucleotide sequence of the NotI DNA fragment of pAT210 used to transform Y. lipolytica cells. Nucleotides 1-8 and 9105-9112, NotI restriction enzyme site; nucleotides 9-308, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 1903-2261, 3969-4327, and 6604-6976, pTEF promoter. Nucleotides 1644 to 1902, 3492 to 3968, and 6127 to 6603, enhancer sequence; nucleotides 2269 to 3330, protein coding region of GmFATA2; nucleotides 4338 to 5975, protein coding region of PcACS-X2; nucleotides 6977 to 8584, protein coding region of MtDGAT1; 3340 to 3491, 5980 to 6126, and 8589 to 8705, lip2 transcription terminator; and nucleotides 8710 to 9104, 3' portion of the Y. lipolytica zeta sequence.

[0186] SEQ ID NO: 96. Nucleotide sequence of the NotI DNA fragment of pAT211 used to transform Y. lipolytica cells. Nucleotides 1-8 and 8410-8417, NotI restriction enzyme site; nucleotides 9-308, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 1903-2261, 3960-4318, and 5909-6267, pTEF promoter. Nucleotides 1644 to 1902, 3483 to 3959, and 5432 to 5908, enhancer sequence; nucleotides 2269 to 3330, protein coding region of GmFATA1; nucleotides 4333 to 5280, protein coding region of MaLPAAT; nucleotides 6282 to 7889, protein coding region of MtDGAT1; 3331 to 3482, 5281 to 5431, and 7890 to 8010, lip2 transcription terminator; and nucleotides 8015 to 8409, 3' portion of the Y. lipolytica zeta sequence.

[0187] SEQ ID NO: 97. Nucleotide sequence of the NotI DNA fragment of pAT212 used to transform W. lipolytica cells. Nucleotides 1-8 and 8419-8426, NotI restriction enzyme site; nucleotides 9-308, 5' portion of W. lipolytica zeta sequence; nucleotides 365-1643, W. lipolytica URA3 gene in reverse orientation containing the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 1903-2261, 3960-4318, and 5909-6267, pTEF promoter; nucleotides 1644-1902, 3483-3959, and and 5432 to 5908, enhancer sequence; nucleotides 2272 to 3339, protein coding region of GmFATA2; nucleotides 4342 to 5289, protein coding region of MaLPAAT; nucleotides 6291 to 7898, protein coding region of MtDGAT1; nucleotides 3340 to 3491, 5290 to 5490, and 7899 to 8019, lip2 transcription terminator; and nucleotides 8024 to 8418, 3' portion of the Y. lipolytica zeta sequence.

[0188] SEQ ID NO: 98. Oligonucleotide primer at001 SEQ ID NO: 99. Oligonucleotide primer at002. SEQ ID NO: 100. Oligonucleotide primer at085. SEQ ID NO: 101. Oligonucleotide primer at086. SEQ ID NO: 102. Oligonucleotide primer at274. SEQ ID NO: 103. Oligonucleotide primer at275. SEQ ID NO: 104. Oligonucleotide primer at297. SEQ ID NO: 105. Oligonucleotide primer at298. SEQ ID NO: 106. Oligonucleotide primer at299. SEQ ID NO: 107. Oligonucleotide primer at300.

[0189] SEQ ID NO: 108. Nucleotide sequence of the NotI DNA fragment of pAT091 used to transform Y. lipolytica cells. Nucleotides 1-8 and 4648-4655, NotI restriction enzyme sites; nucleotides 9-311, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 2080-2479, pTEF promoter; nucleotides 1644-2079, enhancer sequence; nucleotides 2487-4127, protein coding region of PcACS-X1; nucleotides 4128-4248, lip2 transcription terminator; and nucleotides 4254-4647, 3' portion of Y. lipolytica zeta sequence.

[0190] SEQ ID NO: 109. Nucleotide sequence of the NotI DNA fragment of pAT108 used to transform Y. lipolytica cells. Nucleotides 1-8 and 4078-4085, NotI restriction enzyme sites; nucleotides 9-311, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 2080-2479, pTEF promoter; nucleotides 1644-2079, enhancer sequence; nucleotides 2487-3557, protein coding region of GmFATA2; 3558-3682, lip2 transcription terminator; and nucleotides 3683-4077, 3' portion of Y. lipolytica zeta sequence.

[0191] SEQ ID NO: 110. Nucleotide sequence of the NotI DNA fragment of pAT135 used to transform Y. lipolytica cells. Nucleotides 1-8 and 4622-4629, NotI restriction enzyme sites; nucleotides 9-311, 5' portion of Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 2080-2479, pTEF promoter; nucleotides 1644-2079, enhancer sequence; nucleotides 2494-4101, protein coding region of MtDGAT1; nucleotides 4102-4226, lip2 transcription terminator; and nucleotides 4227-4621, 3' portion of Y. lipolytica zeta sequence.

[0192] SEQ ID NO: 111. Nucleotide sequence of the NotI DNA fragment of pAT213 used to transform Y. lipolytica cells. Nucleotides 1 to 8 and 6714 to 6721, NotI restriction enzyme site; nucleotides 9 to 311, 5' portion of the Y. lipolytica zeta sequence; nucleotides 365 to 1643, the Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398 to 1621; nucleotides 2080 to 2479 and 4146 to 4546, pTEF promoter; nucleotides 1644 to 2079 and 3710 to 4145, enhancer sequence; nucleotides 2487 to 3557, protein coding region of GmFATA2; nucleotides 4556 to 6193, protein coding region of PcACS-X1; 3558 to 3709 and 6194 to 6318, lip2 transcription terminator; and nucleotides 6319 to 6713, 3' portion of the Y. lipolytica zeta sequence.

[0193] SEQ ID NO: 112. Nucleotide sequence of the NotI DNA fragment of pAT214 used to transform Y. lipolytica cells. Nucleotides 1 to 8 and 6688 to 6695, NotI restriction enzyme site; nucleotides 9 to 311, 5' portion of the Y. lipolytica zeta sequence; nucleotides 365 to 1643, the Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398 to 1621; nucleotides 2080 to 2479 and 4146 to 4546, pTEF promoter; nucleotides 1644 to 2079 and 3710 to 4145, enhancer sequence; nucleotides 2487 to 3557, protein coding region of GmFATA2; nucleotides 4560 to 6167, protein coding region of MtDGAT1; 3558 to 3709 and 6168 to 6292, lip2 transcription terminator; and nucleotides 6293 to 6687, 3' portion of the Y. lipolytica zeta sequence.

[0194] SEQ ID NO: 113. Nucleotide sequence of the NotI DNA fragment of pAT215 used to transform Y. lipolytica cells. Nucleotides 1 to 8 and 7258 to 7265, NotI restriction enzyme site; nucleotides 9 to 311, 5' portion of the Y. lipolytica zeta sequence; nucleotides 365 to 1643, Y. lipolytica URA3 gene in reverse orientation, including the promoter of the URA3 gene at nucleotides 1398 to 1621; nucleotides 2080 to 2479 and 4716 to 5116, pTEF promoter; nucleotides 1644 to 2079 and 4280 to 4715, enhancer sequence; nucleotides 2487 to 4127, protein coding region of PcACS-X1; nucleotides 5130 to 6737, protein coding region of MtDGAT1; 4128 to 4279 and 6738 to 6862, lip2 transcription terminator; and nucleotides 6863 to 7257, 3' portion of the Y. lipolytica zeta sequence.

[0195] SEQ ID NO: 114. Nucleotide sequence of the NotI DNA fragment of pAT216 used to transform Y. lipolytica cells. Nucleotides 1-8 and 9260-9267, NotI restriction enzyme site; nucleotides 9-311, 5' portion of the Y. lipolytica zeta sequence; nucleotides 365-1643, Y. lipolytica URA3 gene in reverse orientation containing the promoter of the URA3 gene at nucleotides 1398-1621; nucleotides 2080-2479, 4146-4545, and 6781-7181, pTEF promoter; nucleotides 1644-2079, 3710-4145, and and 6345 to 6780, enhancer sequence; nucleotides 2487 to 3557, protein coding region of GmFATA2; nucleotides 4556 to 6193, protein coding region of PcACS-X1; nucleotides 7195 to 8739, protein coding region of YIDGA1; nucleotides 3558 to 3709, 6194 to 6344, and 8740 to 8864, lip2 transcription terminator; and nucleotides 8865 to 9259, 3' part of the Y. lipolytica zeta sequence.

[0196] SEQ ID NO:115—Amino acid sequence of TcDGAT1 from T. cacao. SEQ ID NO: 116—Amino acid sequence of TcDGAT2 from tea cocoa. SEQ ID NO: 117—Amino acid sequence of TcDGAT3 from tea cocoa. SEQ ID NO: 118—Amino acid sequence of TcDGAT4 from tea cocoa. SEQ ID NO: 119—Amino acid sequence of TcDGAT5 from tea cocoa. SEQ ID NO:120—Amino acid sequence of TcDGAT6 from tea cocoa. SEQ ID NO: 121—Amino acid sequence of TcDGAT7 from tea cocoa. SEQ ID NO: 122—Amino acid sequence of TcDGAT8 from tea cocoa. SEQ ID NO: 123—Amino acid sequence of TcDGAT9 from tea cocoa. SEQ ID NO:124—Amino acid sequence of TcDGAT10 from tea cocoa. SEQ ID NO:125—Amino acid sequence of TcDGAT11 from tea cocoa. SEQ ID NO: 126—Amino acid sequence of TcGPAT1 from tea cocoa. SEQ ID NO: 127—Amino acid sequence of TcGPAT2 from tea cocoa. SEQ ID NO: 128—Amino acid sequence of TcGPAT3 from tea cocoa. SEQ ID NO: 129—Amino acid sequence of TcGPAT4 from tea cacao. SEQ ID NO: 130—Amino acid sequence of TcGPAT5 from tea cocoa. SEQ ID NO: 131—Amino acid sequence of TcGPAT6 from tea cocoa. SEQ ID NO: 132—Amino acid sequence of TcGPAT7 from tea cacao. SEQ ID NO: 133—Amino acid sequence of TcGPAT8 from tea cacao. SEQ ID NO: 134—Amino acid sequence of TcGPAT9 from tea cacao. SEQ ID NO: 135—Amino acid sequence of TcGPAT10 from tea cocoa. SEQ ID NO: 136—Amino acid sequence of TcGPAT11 from tea cocoa. SEQ ID NO: 137—Amino acid sequence of TcGPAT12 from tea cocoa. SEQ ID NO: 138—Amino acid sequence of TcGPAT13 from tea cocoa. SEQ ID NO: 139—Amino acid sequence of TcPDAT1 from tea cacao. SEQ ID NO:140—Amino acid sequence of TcPDAT2 from tea cocoa. SEQ ID NO: 141—Amino acid sequence of TcPDAT4 from tea cocoa. SEQ ID NO:142—Amino acid sequence of TcPDAT5 from tea cacao. SEQ ID NO:143—Amino acid sequence of TcPDAT6 from tea cocoa.

[0197] SEQ ID NO:144—Codon-optimized open reading frame encoding TcDGAT1 from tea cacao. SEQ ID NO:145—Codon-optimized open reading frame encoding TcDGAT2 from tea cacao. SEQ ID NO:146—Codon-optimized open reading frame encoding TcDGAT3 from tea cacao. SEQ ID NO:147—Codon-optimized open reading frame encoding TcDGAT4 from tea cacao. SEQ ID NO:148—Codon-optimized open reading frame encoding TcDGAT5 from tea cacao. SEQ ID NO:149—Codon-optimized open reading frame encoding TcDGAT6 from tea cacao. SEQ ID NO:150—Codon-optimized open reading frame encoding TcDGAT7 from tea cacao. SEQ ID NO:151—Codon-optimized open reading frame encoding TcDGAT8 of tea cacao. SEQ ID NO:152—Codon-optimized open reading frame encoding TcDGAT9 from tea cacao. SEQ ID NO:153—Codon-optimized open reading frame encoding TcDGAT10 from tea cacao. SEQ ID NO:154—Codon-optimized open reading frame encoding TcDGAT11 from tea cacao. SEQ ID NO:155—Codon-optimized open reading frame encoding TcGPAT1 from tea cacao. SEQ ID NO:156—Codon-optimized open reading frame encoding TcGPAT2 from tea cacao. SEQ ID NO:157—Codon-optimized open reading frame encoding TcGPAT3 from tea cacao. SEQ ID NO:158—Codon-optimized open reading frame encoding TcGPAT4 from tea cacao. SEQ ID NO:159—Codon-optimized open reading frame encoding TcGPAT5 from tea cacao. SEQ ID NO:160—Codon-optimized open reading frame encoding TcGPAT6 from tea cacao. SEQ ID NO:161—Codon-optimized open reading frame encoding TcGPAT7 from tea cacao. SEQ ID NO:162—Codon-optimized open reading frame encoding TcGPAT8 from tea cacao. SEQ ID NO:163—Codon-optimized open reading frame encoding TcGPAT9 from tea cacao. SEQ ID NO:164—Codon-optimized open reading frame encoding TcGPAT10 from tea cacao. SEQ ID NO:165—Codon-optimized open reading frame encoding TcGPAT11 from tea cacao. SEQ ID NO:166—Codon-optimized open reading frame encoding TcGPAT12 from tea cacao. SEQ ID NO:167—Codon-optimized open reading frame encoding TcGPAT13 from tea cacao. SEQ ID NO:168—Codon-optimized open reading frame encoding TcPDAT1 from tea cacao. SEQ ID NO:169—Codon-optimized open reading frame encoding TcPDAT2 from tea cacao. SEQ ID NO:170—Codon-optimized open reading frame encoding TcPDAT4 from tea cacao. SEQ ID NO:171—Codon-optimized open reading frame encoding TcPDAT5 from tea cacao. SEQ ID NO:172—Codon-optimized open reading frame encoding TcPDAT6 from tea cacao.

[0198] SEQ ID NO: 173 - Nucleotide sequence of the cloning vector pNIz0hyg[dga2] used for insertion of genes for transformation of Y. lipolytica cells. Nucleotides 416-423 and 6134-6141, NotI restriction enzyme sites; nucleotides 424-736, 5' portion of the Y. lipolytica zeta sequence; nucleotides 737-742 and 5733-5738, BsaI restriction enzyme site; nucleotides 743-1742, 5' upstream sequence from the Y. lipolytica DGA2 gene; nucleotides 1743-1755, SfiI restriction enzyme site; nucleotides 1784-1811 and 4699-4732, loxPsym recombination sites; nucleotides 1829-2245 and 3460-385 8, pTEF promoter; nucleotides 2246 to 3271, protein coding region for hygromycin B phosphotransferase (HygR); nucleotides 3859 to 4581, protein coding region for fuGFP polypeptide; nucleotides 3272 to 3453 and 4582 to 4698, lip2 polyadenylation / transcription terminator; nucleotides 4733 to 5732, 3' downstream sequence from the Y. lipolytica DGA2 gene; nucleotides 5739 to 6141, 3' portion of the Y. lipolytica zeta sequence; 8397 nt.

[0199] SEQ ID NO: 174 - Nucleotide sequence of the cloning vector pNIz0ura[fad2] used for insertion of genes for transformation of Y. lipolytica cells. Nucleotides 416-423 and 5737-5744, NotI restriction enzyme sites; nucleotides 424-736, 5' portion of the Y. lipolytica zeta sequence; nucleotides 737-742 and 5320-5325, BsaI restriction enzyme sites; nucleotides 743-1742, 5' upstream sequence from the Y. lipolytica FAD2 gene; nucleotides 1743-1755, SfiI restriction enzyme site; nucleotides 1784-1817 and 4286-4319, loxPsym recombination sites; nucleotides 1818-3037, URA3 selection marker gene (reverse orientation), containing nucleotides 1999 to 2859, the complement of the protein coding region of URA3; nucleotides 3038 to 3445, pTEF promoter; nucleotides 3446 to 4168, the protein coding region for the fuGFP polypeptide; nucleotides 4169 to 4285, lip2 polyadenylation / transcription terminator; nucleotides 4320 to 5319, 3' downstream sequence from the Y. lipolytica FAD2 gene; nucleotides 5326 to 5728, 3' portion of the Y. lipolytica zeta sequence; 7984 nt.

[0200] SEQ ID NO: 175—Nucleotide sequence of the cloning vector pNIz0nat[Iro1] used for insertion of genes for transformation of Y. lipolytica cells. Nucleotides 416-423 and 5679-5686, NotI restriction enzyme sites; nucleotides 424-736, 5' portion of Y. lipolytica zeta sequence; nucleotides 737-742 and 5320-5325, BsaI restriction enzyme site; nucleotides 743-1742, 5' upstream sequence from the Y. lipolytica FAD2 gene; nucleotides 1743-1755, SfiI restriction enzyme site; nucleotides 1784-1817 and 4244-4277, loxPsym recombination site; nucleotides 1818-3037, URA3 selectable marker gene (reverse orientation), containing nucleotides 1999-2859, the complement of the protein coding region of URA3; nucleotide 1883 ~2246 and 3005-3403, pTEF promoter; nucleotides 3404-4126, protein coding region for fuGFP polypeptide; nucleotides 2817-2998 and 4127-4243, lip2 polyadenylation / transcription terminator; nucleotides 4278-5277, 3' downstream sequence from the FAD2 gene of Y. lipolytica; nucleotides 5284-5678, 3' portion of Y. lipolytica zeta sequence. 7942 nt.

[0201] SEQ ID NO: 176 - Nucleotide sequence of the cloning vector pNIz0hyg[pox2] used for insertion of genes for transformation of Y. lipolytica cells. Nucleotides 416-423 and 6134-6141, NotI restriction enzyme sites; nucleotides 424-736, 5' portion of the Y. lipolytica zeta sequence; nucleotides 737-742 and 5733-5738, BsaI restriction enzyme site; nucleotides 743-1742, 5' upstream sequence from the Y. lipolytica POX2 gene; nucleotides 1743-1755, SfiI restriction enzyme site; nucleotides 1784-1811 and 4699-4732, loxPsym recombination sites; nucleotides 1882-2245 and 3460-3858, p TEF promoter; nucleotides 2246 to 3271, protein coding region for hygromycin B phosphotransferase (HygR; SEQ ID NO: 4); nucleotides 3859 to 4581, protein coding region for fuGFP polypeptide; nucleotides 3272 to 3453 and 4582 to 4698, lip2 polyadenylation / transcription terminator; nucleotides 4733 to 5732, 3' downstream sequence from the Y. lipolytica POX2 gene; nucleotides 5739 to 6141, 3' portion of the Y. lipolytica zeta sequence; 8397 nt. DETAILED DESCRIPTION OF THE INVENTION

[0202] Detailed Description of the Invention Abbreviation Acetyl-CoA and malonyl-CoA: acetyl-coenzyme A and malonyl-coenzyme A, ACCase: acetyl-CoA carboxylase; FAS: fatty acid synthase complex; KASII: ketoacyl-ACP synthase II (EC2.3.1.41); PAP: PA phosphorylase (EC 3.1.3.4), G3P: glycerol-3-phosphate; LPA: lysophosphatidic acid; PA: phosphatidic acid; MAG: monoacylglycerol DAG: diacylglycerol; TAG: Triacylglycerol, Acyl-CoA: acyl-coenzyme A, PC: phosphatidylcholine; GPAT: glycerol-3-phosphate acyltransferase; LPAAT: lysophosphatidic acid acyltransferase (EC2.3.1.51), LPCAT: acyl-CoA:lysophosphatidylcholine acyltransferase, or synonyms 1-acylglycerophosphocholine O-acyltransferase, acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23); CPT: CDP-choline:diacylglycerol choline phosphotransferase, or synonyms 1-alkyl-2-acetylglycerol choline phosphotransferase, alkylacylglycerol choline phosphotransferase, choline phosphotransferase, phosphorylcholine-glyceride transferase (EC 2.7.8.2); PDCT: phosphatidylcholine:diacylglycerolcholinephosphotransferase; PLC: phospholipase C (EC3.1.4.3); PLD: phospholipase D, choline phosphatase, lecithinase D, lipophosphodiesterase II (EC3.1.4.4), PDAT: phospholipid:diacylglycerol acyltransferase, or synonymously phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase (EC 2.3.1.158); FAD2: fatty acid Δ12-desaturase, FAD3, fatty acid Δ15-desaturase; UDP-Gal: uridine diphosphate galactose.

[0203] General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein shall be construed 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).

[0204] 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 and explained in such publications as 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), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editor), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editor), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editor), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editor) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0205] 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 explicitly endorsing both meanings or either meaning. As used herein, unless otherwise specified, the term "about" refers to ±10%, more preferably ±5%, more preferably ±1% of the specified value. Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, steps, or groups of elements, integers, or steps.

[0206] Selected Definitions As used herein, "lipid" refers to any of a class of organic compounds that are or contain fatty acids, whether esterified or unesterified, or their derivatives, that are insoluble in water but soluble in organic solvents, such as chloroform. As used herein, lipids include nonpolar lipids, such as triacylglycerols (TAGs), diacylglycerols (DAGs), and monoacylglycerols (MAGs), as well as waxes, wax esters, and sterol esters, and polar lipids, such as free fatty acids (FFAs), phospholipids, galactolipids, ceramides, and other sphingolipids. Lipids are composed primarily of carbon (C), hydrogen (H), and some oxygen (O), and may also contain phosphorus (P), nitrogen (N), and sulfur (S). As used herein, the term "extracted lipid" refers to a lipid composition extracted from microbial cells. The extracted lipids may be relatively crude compositions, such as those obtained by lysing cells, or may be more purified compositions in which most, if not all, of one or more of the following components have been removed: water, nucleic acids, proteins, and carbohydrates from the cells. Exemplary purification methods are described below. In one embodiment, the extracted lipids comprise at least about 10% (w / w), at least about 20% (w / w), at least about 30% (w / w), at least about 40% (w / w), at least about 50% (w / w), at least about 60% (w / w), at least about 70% (w / w), at least about 80% (w / w), at least about 90% (w / w), or at least about 95% (w / w) of lipid by weight of the composition. In one embodiment, the extracted lipids comprise between about 10% and 95% lipid by weight, e.g., between about 10% and about 50% or between about 50% and 95% lipid by weight. Lipids may be solid or liquid at room temperature (25°C), or a mixture of the two; if liquid, they are considered oils, and if solid, they are considered fats. In one embodiment, the extracted lipids of the present invention are not mixed with other lipids produced from other sources, such as animal lipids. Alternatively, the extracted lipids may be blended with other lipids.

[0207] As used herein, the term "polar lipid" refers to an amphipathic lipid molecule having a hydrophilic head group and one or more hydrophobic hydrocarbon tails, e.g., one or more fatty acyl groups, including phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol), galactolipids, cephalins, sphingolipids (sphingomyelin and glycosphingolipids), phosphatidic acid, cardiolipin, and glycoglycerolipids. The hydrophilic head group is a polar group that is charged or uncharged at physiological pH, conferring amphipathic properties to these lipids. Polar lipids have the ability to ionize and / or form hydrogen bonds with water molecules via their hydrophilic head groups, thereby increasing their aqueous solubility compared to nonpolar lipids. Phospholipids are composed of fatty acid, glycerol, and phosphoric acid as their main structural units, and may contain amino alcohols. They function primarily as structural lipids within cells and are thought to play an important role in the structure of membranes in plants, microorganisms, and animals. Polar lipids exhibit bipolarity due to their chemical structure, and are soluble or partially soluble in both polar and nonpolar solvents.

[0208] As used herein, the term "phospholipid" refers to an amphipathic molecule containing a fatty acid with a hydrocarbon chain hydrophilic head group and one (in the case of lysophosphatidic acid) or two hydrophobic tails, with a glycerol backbone esterified to the phosphate-containing "head" group and the fatty acid providing the hydrophobic tail. The phosphate group can be covalently attached to simple organic molecules such as choline, ethanolamine, or serine. Because the head group is charged at neutral pH, phospholipids are polar lipids and have some solubility in solvents such as chloroform as well as ethanol. Phospholipids are important components of all cell membranes. Their amphipathic properties allow them to form lipid bilayers. Well-known phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin.

[0209] As used herein, the term "nonpolar lipid" refers to a lipid that has one or more hydrocarbon groups, e.g., a hydrophobic "tail" of one or more acyl chains, but is not an amphipathic molecule and lacks the hydrophilic head group of polar lipids. The fatty acids of nonpolar lipids are in esterified form, and free (unesterified) fatty acids are considered polar lipids herein. Examples of esterified forms include, but are not limited to, triacylglycerol (TAG), diacylglycerol (DAG), and monoacylglycerol (MAG). Nonpolar lipids also include sterol esters and wax esters. Nonpolar lipids are also known as "neutral lipids" because they are essentially uncharged at physiological pH. Nonpolar lipids, such as TAG and sterol esters, do not form monolayers (monolayers) at air-water or oil-water interfaces and are therefore essentially insoluble in aqueous solutions. In contrast, polar lipids can form monolayers at air-water or oil-water interfaces and can form micelles or bilayers in the aqueous phase. Non-polar lipids can be liquid at room temperature, or solid 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.Extracted lipids that are liquid at room temperature are referred to herein as "oil", while extracted lipids that are solid at room temperature are referred to herein as "fat".

[0210] 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 may be 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 two to six carbon-carbon double bonds. Fatty acids may be free fatty acids (FFA) or esterified to glycerol or glycerol phosphate molecules, CoA molecules, or other head groups known in the art, preferably esterified as part of a polar lipid such as a phospholipid.

[0211] 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. In one example, total fatty acid content includes the total saturated fatty acid content of saturated fatty acids (SFAs) and the total monounsaturated fatty acid content of monounsaturated fatty acids (MUFAs). 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 relative 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, which uses the addition of known amounts of unique fatty acid standards as quantitative standards. 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. Therefore, TFA represents the weight of the fatty acid alone, not the weight of the fatty acid within the lipid and its attached moieties.

[0212] "Saturated fatty acids" do not contain double bonds or other functional groups along the acyl chain. The term "saturated" refers to hydrogen atoms in every carbon (excluding the carboxylic acid [-COOH] group) containing as many hydrogen atoms as possible. Examples of saturated fatty acids in the lipids of the present invention include stearic acid (C18:0), palmitic acid (C16:0), myristic acid (C14:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0).

[0213] "Unsaturated fatty acids" have a similar structure to saturated fatty acids, except that there are one or more alkene functional groups along the chain, each replacing a single-bonded "-CH-CH-" portion of the chain with a double-bonded "-CH=CH-" portion (i.e., a carbon double-bonded to another carbon). The two adjacent carbon atoms in the chain, attached on either side of the double bond, can be in the cis or trans configuration, although the cis configuration is preferred.

[0214] As used herein, the term "monounsaturated fatty acid" refers to a fatty acid containing 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).

[0215] As used herein, the term "polyunsaturated fatty acid" or "PUFA" refers to a fatty acid whose carbon chain contains at least 12 carbon atoms and at least two alkene groups (carbon-carbon double bonds). Typically, the number of carbon atoms in the carbon chain of a fatty acid refers to an unbranched carbon chain. Unless otherwise specified, if the carbon chain is branched, the number of carbon atoms does not include the carbon atoms of the side groups. The polar lipids of the present invention, such as 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), dihomogammalinolenic 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), gamma-linolenic acid (GLA, C18:3Δ6,9,12; ω6), and linoleic acid (LA, C18:2Δ9,12; ω6). In some embodiments, the polar lipids of the present invention, such as the extracts or cells of the present invention, contain at least one ω3 fatty acid having an unsaturation (carbon-carbon double bond) in the third carbon-carbon bond from the methyl end of the fatty acid. In some embodiments, the polar lipids of the present invention, such as the extracts or cells of the present invention, do not contain specific ω3 fatty acids, such as one or more of C16:3ω3, ALA, EPA, and DHA, or do not contain any ω3 fatty acids. Examples of ω3 fatty acids include, but are not limited to, α-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, C22:6Δ4,7,10,13,16,19; 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 contain one or more or all of the following omega-3 fatty acids: C16:3 omega-3, EPA, and DHA.

[0216] As used herein, the term "L / S-SFA ratio" refers to the total amount of saturated fatty acids having 18 or more carbons (e.g., stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0)) divided by the total amount of saturated fatty acids having 16 or fewer carbons (e.g., palmitic acid (C16:0), myristic acid (C14:0), arachidic acid (C12:0)).

[0217] As used herein, "C12:0" refers to lauric acid. As used herein, "C14:0" refers to myristic acid. As used herein, "C15:0" refers to n-pentadecanoic acid. As used herein, "C16:0" refers to palmitic acid. As used herein, "C17:Δ1" refers to heptadecenoic acid. As used herein, "C16:1Δ9" refers to palmitoleic acid or -hexadec-9-enoic acid. As used herein, "C18:0" refers to stearic acid. As used herein, "C18:1Δ9" (sometimes abbreviated as "C18:1") refers to oleic acid. As used herein, "C18:1Δ11" refers to vaccenic acid. As used herein, "C20:0" refers to eicosanoic acid, also known as arachidic acid. As used herein, "C20:1" refers to eisocosenoic acid. As used herein, "C22:0" refers to docosanoic acid, also known as behenic acid. As used herein, "C22:1" refers to erucic acid. As used herein, "C24:0" refers to tetracosanoic acid, also known as lignoceric acid.

[0218] A "triacylglyceride" or "TAG" is a glyceride in which glycerol is esterified with three fatty acids, either the same (e.g., triolein) or, more commonly, different. All three fatty acids may be different, or two may be the same and the third may be different. The Kennedy pathway for TAG synthesis, as described below, produces DAG, followed by the esterification of the third acyl group to the glycerol backbone by the activity of diglyceride acyltransferase (DGAT). TAG is a type of nonpolar lipid. The three esterified acyl groups within a TAG molecule are referred to as esterified at the sn-1, sn-2, and sn-3 positions, referring to their positions within the glycerol backbone of the TAG molecule. While 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 distinct acyltransferase enzymes catalyze the esterification.

[0219] "Diacylglyceride" or "DAG" refers to a glyceride in which glycerol is esterified with two fatty acids, either the same or preferably different. As used herein, DAG does not include phosphorylated glycerolipid molecules such as PA or PC, since DAG contains hydroxyl groups at the sn-1, 3, or sn-2 positions. In the Kennedy pathway for 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 the first reaction catalyzed by glycerol-3-phosphate acyltransferase (GPAT) to generate LysoPA, followed by a second acylation at the sn-2 position catalyzed by lysophosphatidic acid acyltransferase (LPAAT) to generate phosphatidic acid (PA). This intermediate is then dephosphorylated by PAP to generate DAG.

[0220] As used herein, an "oil" is a composition that contains primarily lipids and is liquid at room temperature.

[0221] As used herein, an "oleaginous" cell or microorganism is one that can store at least 20% lipid, e.g., 20% to 70% lipid, of its cell mass on a dry weight basis. As known in the art, lipid content can depend on culture conditions. 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 understood that the microorganism is considered an oleaginous cell, even if lipid accumulation is less than 20% 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 it 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 produce a derived microorganism include one or more genetic modifications that result in reduced synthesis and / or accumulation of TAG.

[0222] 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 the appropriate conditions.

[0223] As used herein, "fermentation" refers to a metabolic process that causes chemical changes in organic molecules through the action of enzymes within cells under conditions in which oxygen is deficient or the oxygen level is reduced compared to air.

[0224] As used herein, the term "volatile solvent" refers to a gaseous liquid that can be easily evaporated and used to disrupt and disperse microbial cells, and thus may be present in the extracted lipids of the present invention. Examples of volatile solvents that may be present in the extracted lipids of the present invention include, but are not limited to, hexane and other alkanes, chloroform, ether, methanol, ethanol, propanol, and mixtures of any one or more of these.

[0225] As used herein, the term "corresponding extracted microbial lipid derived from a corresponding microorganism lacking at least one genetic modification" refers to lipid produced under the same culture conditions as lipid produced from a cell having at least one genetic modification.

[0226] Fatty acid biosynthesis The present invention relates to the genetic modification of microbial cells to increase the production of saturated fatty acids, particularly saturated fatty acids having a length of 18 carbons or more. Details of the genes that can be modified, either by expression of an exogenous polynucleotide or by modification of an endogenous gene, are discussed below.

[0227] As used herein, the term "fatty acyl acyltransferase" refers to a protein that can transfer an acyl group from acyl-CoA, PC, or acyl-ACP, preferably acyl-CoA or PC, onto a substrate molecule to which the acyl group is transferred, covalently linking the acyl group to the substrate molecule to form an ester bond. Preferred substrate molecules are 3-phosphoglycerol, lysophosphatidic acid, or diacylglycerol, producing MAG, DAG, or TAG, respectively. These acyltransferases include DGAT, PDAT, MGAT, GPAT, LPAAT, and LPCAT.

[0228] Diacylglycerol acyltransferase (DGAT) As used herein, the term "diacylglycerol acyltransferase" (DGAT), also known as acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20), transfers a fatty acyl group from acyl-CoA to a DAG substrate to produce TAG. Thus, the term "diacylglycerol acyltransferase activity" refers to the transfer of an acyl group from acyl-CoA to DAG to produce TAG. Although DGATs can also possess monoacylglycerol acyltransferase (MGAT) activity, they primarily function as DGATs; i.e., when enzyme activity is expressed in nmol of product / min / mg of protein, they have higher catalytic activity as DGATs than as MGATs (see, e.g., Yen et al., 2005). DGATs use an acyl-CoA substrate as the acyl donor and transfer it to the sn-3 position of DAG to produce TAG. The enzyme functions naturally in the endoplasmic reticulum (ER) of cells, or in the case of soluble DGAT3, in the cytoplasm.

[0229] There are three known types of DGATs, designated DGAT1, DGAT2, and DGAT3. The diversity and relationships of DGAT species have been reviewed by Lung and Weselake (2006) and Turchetto-Zolet et al. (2011 and 2016). DGAT1 polypeptides are membrane proteins, often with 10 transmembrane domains, but can also have only 6–9 transmembrane domains. They share sequence homology with sterol:acyl-CoA acyltransferases (ACATs; EC 2.3.1.26). Both enzymes belong to a large family of membrane-bound O-acyltransferase (MBOAT) proteins. The DGAT2 polypeptide, first identified in Mortierella rammanniana (Lardizabal et al., 2001), is also a membrane protein but has one or two transmembrane domains, whereas the DGAT3 polypeptide typically lacks a transmembrane domain and is thought to be soluble in the cytoplasm. DGAT1 polypeptides from plant, animal, and microbial sources typically contain 510–550 amino acid residues, while DGAT2 polypeptides from plant and animal sources typically contain approximately 310–330 residues. DGAT2 is thought to be the primary enzyme responsible for the synthesis of TAG from DAG in most microbial cells, including the yeast Saccharomyces cerevisiae. The DGAT2 polypeptide is related in amino acid sequence to acyl-CoA:monoacylglycerol acyltransferase (MGAT, EC 2.3.1.22) and acyl-CoA wax-alcohol acyltransferase (AWAT, EC 2.3.1.75), Turchetto-Zolet et al. (2011).

[0230] Plant DGAT1 and DGAT2 appear to have non-redundant functions in triacylglycerol biosynthesis. DGAT1 is the primary enzyme responsible for TAG synthesis during seed development. Examples of DGAT1 polypeptides include those encoded by the YALI0D07986g gene (also known as the DGA2 gene, SEQ ID NO: 55) in Yarrowia lipolytica, Mortierella alpina (AQX34626.1), Aspergillus fumigatus (XP_755172.1), Arabidopsis thaliana (CAB44774.1), Ricinus communis (AAR11479.1), Vernicia fordii (ABC94472.1), Vernonia galamensis (ABV21945.1 and ABV21946.1), Euonymus alatus (AAV31083.1), Nannochloropsis oceanica (Zienkiewicz et al., 2017), Saccharomyces cerevisiae (Zulu et al., 2017), Caenorhabditis elegans (AAF82410.1), Rattus norvegicus (NP_445889.1), and Homo sapiens (NP_036211.2), as well as variants and / or mutants thereof. Examples of DGAT2 polypeptides include proteins encoded by DGAT2 genes from Arabidopsis thaliana (NP_566952.1), Ricinus communis (AAY16324.1), Taurine curcas (ABC94474.1), Mortierella ramanniana (AAK84179.1), Homo sapiens (Q96PD7.2; Q58HT5.1), Bos taurus (Q70VZ8.1), and Mus musculus (AAK84175.1), as well as variants and / or mutants thereof.The amino acid sequences of DGAT1 and DGAT2 show little homology (Turchetto-Zolet et al., 2011, Turchetto-Zolet et al., 2016). For example, compared to Arabidopsis DGAT1, Arabidopsis DGAT2 has a preference for polyunsaturated linoleoyl-CoA and linolenoyl-CoA as acyl donors over monounsaturated oleoyl-CoA.

[0231] Examples of DGAT3 polypeptides include proteins encoded by the DGAT3 gene from peanut (Arachis hypogaea, Saha, et al., 2006), as well as variants and / or mutants thereof. The DGAT has little or no detectable MGAT activity, for example, less than 300 pmol / min / mg protein, preferably less than 200 pmol / min / mg protein, and more preferably less than 100 pmol / min / mg protein.

[0232] In one embodiment, the exogenous polynucleotide encoding a DGAT comprises one or more of the following: i) a nucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 53, 55, or 115 to 125; ii) a nucleotide encoding a polypeptide comprising an amino acid sequence that is at least 30% identical to any one or more of SEQ ID NOs: 53, 55, or 115 to 125; iii) a nucleotide having a sequence set forth in any one of SEQ ID NOs: 52, 54, or 144 to 154; iv) a nucleotide having a sequence that is at least 30% identical to one or more of SEQ ID NOs: 52, 54, or 144-154; or v) A polynucleotide that hybridizes to any one or more of i) to iv) under stringent conditions.

[0233] In one embodiment, the exogenous polynucleotide of the invention encoding DGAT1 comprises one or more of the following: i) a nucleotide encoding a polypeptide whose sequence comprises the amino acid sequence set forth in SEQ ID NO: 55, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical to SEQ ID NO: 55; ii) nucleotides whose sequence is at least 30% identical, at least 40% identical, or at least 95% identical to i); and iii) A polynucleotide that hybridizes to either or both of i) and ii) under stringent conditions.

[0234] In one embodiment, the exogenous polynucleotide of the invention encoding DGAT2 comprises one or more of the following: i) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 53, or a biologically active fragment thereof, or a polypeptide whose amino acid sequence is at least 30% identical, at least 40% identical, or at least 95% identical to SEQ ID NO: 53; ii) nucleotides whose sequence is at least 30% identical to i); and iii) A polynucleotide that hybridizes to either or both of i) and ii) under stringent conditions.

[0235] In one embodiment, the DGAT does not comprise an amino acid having the sequence provided in SEQ ID NO:115 or SEQ ID NO:116.

[0236] The TcDGAT1 polypeptide (SEQ ID NO:115) is a member of the DGAT1 class (PLN02401) and is predicted by software available at www.cbs.dtu.dk / services / TMHMM / to have nine transmembrane domains. It contains an MBOAT domain (pfam03062) at amino acids 266-489. The TcDGAT2 polypeptide (SEQ ID NO:116) is a member of the DGAT2 class (PLN02783) and is predicted by software to have two transmembrane domains. It contains a DAGAT domain (pfam03982) at amino acids 74-318. TcDGATs 3-8 all share homology with wax ester synthase (WES, pfam03007), which is also an acyltransferase. TcDGAT3 (SEQ ID NO:117) is predicted to have no transmembrane domains but contains a WES domain at amino acids 64-268. TcDGAT4 (SEQ ID NO: 118) is predicted to have one transmembrane domain and contains a WES domain at amino acids 128-276. TcDGAT5 (SEQ ID NO: 119) is predicted to have one transmembrane domain and contains a WES domain at amino acids 146-303. TcDGAT6 (SEQ ID NO: 120) is predicted to have one transmembrane domain and contains a WES domain at amino acids 102-291. TcDGAT7 (SEQ ID NO: 121) is predicted to have no transmembrane domain and does not have an identified WES domain. TcDGAT8 (SEQ ID NO: 122) is predicted to have one transmembrane domain and contains a WES condensation domain (cd19533) at amino acids 36-177. TcDGAT9-11 all share homology with yeast LRO1 (PLN02517) of the PDAT family. TcDGAT9 (SEQ ID NO: 123) is predicted to have one transmembrane domain and contains an LCAT domain (pfam02450) at amino acids 134 to 630. TcDGAT10 (SEQ ID NO: 124) is predicted to have no transmembrane domain but contains an LCAT domain (pfam02450) at amino acids 148 to 651. TcDGAT11 (SEQ ID NO: 125) is predicted to have one transmembrane domain and contains an LCAT domain (pfam02450) at amino acids 150 to 645.

[0237] As used herein, a "DGA1 polypeptide" is a polypeptide having at least 30% sequence identity along the entire length of SEQ ID NO: 53, the amino acid sequence of the Y. lipolytica DGA1 polypeptide. As used herein, a functional DGA1 polypeptide is a DGA1 polypeptide that can generate TAG from DAG and acyl-CoA. Numerous DGA1 polypeptides have been reported, and sequences are available in databases, such as accession number NC_001147.6 (794076..795332, complement), Saccharomyces cerevisiae chromosome XV, gene YOR245C; accession number KABA2_02S14982, Kazachstania barnettii; accession number NC_030983.1, chromosome VIII (552596..553852, complement), Saccharomyces eubayanus; and accession number NC_005784.3, chromosome III (594117..595502, complement), Eremothecium gossypii. As used herein, a "Yarrowia DGA1 polypeptide" is a polypeptide having at least 95% sequence identity along the entire length of SEQ ID NO: 53 and having DGAT activity. In addition to SEQ ID NO: 53, exemplary Yarrowia DGA1 polypeptide sequences include accession numbers: QNQ00885.1 (513 / 514 identical); KAG5365696.1 (496 / 514 identical), and KAG5357621.1 (494 / 514 identical).

[0238] As used herein, a "DGA2 polypeptide" is a polypeptide having at least 30% sequence identity along the entire length of SEQ ID NO: 55, the amino acid sequence of the Yarrowia lipolytica DGA2 polypeptide. As used herein, a functional DGA2 polypeptide is a DGA2 polypeptide capable of producing TAG from DAG and acyl-CoA. Numerous DGA2 polypeptides have been reported, and their sequences are available in databases. As used herein, a "Yarrowia DGA2 polypeptide" is a polypeptide having at least 95% sequence identity along the entire length of SEQ ID NO: 55 and having DGAT activity. In addition to SEQ ID NO: 55, exemplary Yarrowia DGA2 polypeptide sequences include the following accession numbers: RDW42020.1 (525 / 526 identical); RDW42020.1 (525 / 526 identical); KAG5361387.1 (495 / 526 identical); and KAG5358063.1 (494 / 526 identical).

[0239] Phospholipid:diacylglycerol acyltransferase (PDAT) As used herein, the term "phospholipid:diacylglycerol acyltransferase" (PDAT; EC 2.3.1.158) or its synonym "phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase" transfers an acyl group from a phospholipid, typically from the sn-2 position of PC to the sn-3 position of DAG, to produce TAG and lysophosphocholine (LPC). This reaction differs from DGAT in that it uses a phospholipid as the acyl donor. Increasing expression of a PDAT, such as PDAT1, which can be exogenous or endogenous to the cells of the present invention, increases the production of TAG from PC. The enzyme LPCAT reacylates LPC to produce more PC, allowing PDAT to continue producing TAG. Several forms of PDAT exist in plant cells, including PDAT1, PDAT2, or PDAT3 (Ghosal et al., 2007).

[0240] In one embodiment, the exogenous polynucleotide encoding PDAT comprises one or more of the following: i) a nucleotide encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 57 and 139 to 143; ii) a nucleotide encoding a polypeptide comprising an amino acid sequence that is at least 30% identical to any one or more of SEQ ID NOs: 57 or 139 to 143; iii) a nucleotide having a sequence set forth in any one of SEQ ID NOs: 56 and 168 to 172; iv) a nucleotide having a sequence that is at least 30% identical to one or more of SEQ ID NOs: 56 or 168-172; or v) A polynucleotide that hybridizes to any one or more of i) to iv) under stringent conditions.

[0241] However, any PDAT-encoding gene can be used. Homologs and naturally occurring variants of PDAT from microbial or plant, fungal, or algal species can be readily identified and used in the present invention. In one embodiment, the homolog or variant is at least 95% identical, preferably at least 99% identical, to the amino acid sequence of the listed SEQ ID NO: or accession number. PDAT can be exogenous or endogenous to the microorganism of the present invention.

[0242] Monoacylglycerol acyltransferase (MGAT) As used herein, the term "monoacylglycerol acyltransferase" or "MGAT" refers to a protein that transfers a fatty acyl group from acyl-CoA to a MAG substrate, such as sn-2 MAG, to produce DAG. Thus, the term "monoacylglycerol acyltransferase activity" refers at least to the transfer of an acyl group from acyl-CoA to MAG to produce DAG. As used herein, the term "MGAT" includes enzymes that act on sn-1 / 3 MAG and / or sn-2 MAG substrates to produce sn-1,3 DAG and / or sn-1,2 / 2,3-DAG, respectively. In preferred embodiments, the MGAT has a preference for sn-2 MAG substrates over sn-1 MAG, or uses substantially only sn-2 MAG as a substrate. As used herein, MGAT does not include enzymes that preferentially transfer an acyl group to LysoPA over MAG; such enzymes are known as LPAATs. That is, MGAT preferentially uses non-phosphorylated monoacyl substrates, even if its catalytic activity towards LysoPA is low. Preferred MGATs do not have detectable activity in the acylation of LysoPA. Although MGATs may also have DGAT function, they primarily function as MGATs, i.e., when enzyme activity is expressed in moles of product / minute / mg protein, they have higher catalytic activity as MGATs than DGATs (see also Yene et al. 2002). There are three known classes of MGATs, called MGAT1, MGAT2, and MGAT3, respectively. Examples of MGAT1, MGAT2, and MGAT3 polypeptides are described in WO2013 / 096993.

[0243] sn-glycerol-3-phosphate acyltransferase (GPAT) A key component in glycerolipid synthesis from fatty acids esterified to ACP or CoA is the enzyme sn-glycerol-3-phosphate acyltransferase (GPAT), a polypeptide involved in the biosynthesis of nonpolar lipids. This enzyme catalyzes the following reaction: G3P + fatty acyl-ACP or -CoA → LPA + free -ACP or -CoA. There are at least three distinct types of GPAT enzymes: a soluble form located in the plastid stroma that uses acyl-ACP as the natural acyl substrate, and two membrane-bound forms located in the ER and mitochondria that use acyl-CoA and acyl-ACP as the natural acyl donors, respectively (Chen et al., 2011).

[0244] As used herein, the term "glycerol-3-phosphate acyltransferase" (GPAT; EC 2.3.1.15), and its synonym "glycerol-3-phosphate O-acyltransferase," refer to a protein that acylates glycerol-3-phosphate (G-3) to produce LysoPA and / or MAG, the latter product being produced when the GPAT also possesses phosphatase activity toward LysoPA. If the GPAT is an ER-type GPAT (also called "acyl-CoA:sn-glycerol-3-phosphate 1-O-acyltransferase" or "microsomal GPAT"), the transferred acyl group is either from acyl-CoA or, if the GPAT is a plastid-type GPAT, from acyl-ACP. Thus, the term "glycerol-3-phosphate acyltransferase activity" refers to the acylation of G-3-P to produce LysoPA and / or MAG. The term "GPAT" encompasses enzymes that acylate G-3-P to produce sn-1LPA and / or sn-2LPA. Preferably, the GPAT that can be overexpressed in cells is a membrane-bound GPAT that functions in the ER of the cell, more preferably GPAT9.

[0245] The GPAT family is large, and all known members contain two conserved domains: the plsC acyltransferase domain (PF01553) and the HAD-like hydrolase superfamily domain (PF12710) and their variants. Additionally, at least in Arabidopsis, all GPATs of the subclasses GPAT4-GPAT8 contain an N-terminal region homologous to a phosphoserine phosphatase domain (PF00702). GPATs that produce MAG as a product can be identified by the presence of such a homologous region.

[0246] Arabidopsis homologs of GPAT4 (accession number NP_171667.1) and GPAT6 (NP_181346.1) include AAF02784.1 (Arabidopsis thaliana), AAL32544.1 (Arabidopsis thaliana), AAP03413.1 (rice (Oryza sativa)), ABK25381.1 (Picea sitchensis), and ACN34546.1 (maize (Zea mays)). mays), BAF00762.1 (Arabidopsis thaliana), BAH00933.1 (rice), EAY84189.1 (rice), EAY98245.1 (rice), EAZ21484.1 (rice), EEC71826.1 (rice), EEC76137.1 (rice), EEE59882.1 (rice), EFJ08963.1 (Selaginella moellendorffii), EFJ11200.1 (Selaginella moellendorffii), NP_001044839.1 (rice), NP_001045668.1 (rice), NP_001147442.1 (maize), NP_001149307.1 (maize), NP_001168351.1 (maize), AFH02724.1 (Brassica napus), NP191950.2 (Arabidopsis thaliana), XP_001765001.1 (Physcomitrella patens), XP_001769671.1 (Physcomitrella patens), (Vitis vinifera), XP_002275348.1 (Vitis vinifera), XP_002276032.1 (Vitis vinifera), XP_002279091.1 (Vitis vinifera), XP_002309124.1 (Cottonwood), XP_002309276.1 (Cottonwood), XP_002322752.1 (Cottonwood), XP_002323563.1 (Cottonwood), XP_002439887.1 (Sorghum bicolor), XP_002458786.1 (sorghum), XP_002463916.1 (sorghum), XP_002464630.1 (sorghum), XP_002511873.1 (castor), XP_002517438.1 (castor), XP_002520171.These include ACT32032.1 (castor bean), ACT32032.1 (Vernicia fordii), NP_001051189.1 (rice), AFH02725.1 (Brassica napus), XP_002320138.1 (cottonwood), XP_002451377.1 (sorghum), XP_002531350.1 (castor bean), and XP_002889361.1 (Arabidopsis lyrata).

[0247] Soluble plastid GPATs have been purified and the genes encoding them have been cloned from several plant species, e.g., soybean (Pisum sativum, accession number: P30706.1), spinach (Spinacia oleracea, accession number: Q43869.1), pumpkin (Cucurbita moschate, accession number: P10349.1), cucumber (Cucumis sativus, accession number: Q39639.1), and Arabidopsis thaliana (accession number: Q43307.2).

[0248] In one embodiment, the exogenous polynucleotide encoding a GPAT comprises one or more of the following: i) a nucleotide encoding a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 126 to 138; ii) a nucleotide encoding a polypeptide comprising an amino acid sequence that is at least 30% identical to any one or more of SEQ ID NOs: 126 to 138; iii) a nucleotide having a sequence set forth in any one of SEQ ID NOs: 155 to 167; iv) a nucleotide having a sequence that is at least 30% identical to one or more of SEQ ID NOs: 155 to 167; or v) A polynucleotide that hybridizes to any one or more of i) to iv) under stringent conditions.

[0249] 1-acyl-sn-glycerophosphate acyltransferase (LPAAT) As used herein, the term "lysophosphatidic acid acyltransferase" (LPAAT; EC 2.3.1.51) and its synonyms "1-acyl-glycerol-3-phosphate acyltransferase," "acyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase," and "1-acylglycerol-3-phosphate O-acyltransferase" refer to a protein that acylates lysophosphatidic acid (LPA) to produce phosphatidic acid (PA). The transferred acyl group is from acyl-CoA when the LPAAT is an ER-type LPAAT or from acyl-ACP when the LPAAT is a plastid-type LPAAT. Thus, the term "lysophosphatidic acid acyltransferase activity" refers to the acylation of LPA to produce PA.

[0250] Acyl-ACP thioesterase As used herein, the term "acyl-ACP thioesterase" refers to an enzyme that hydrolyzes the thioester bond of acyl-acyl carrier protein (ACP) substrates (EC 3.1.2.14). These are soluble enzymes targeted to plastids and encoded by nuclear genes in plants and other eukaryotes. Acyl-ACP thioesterase cleaves the acyl group from ACP, thereby terminating the acyl chain elongation activity of fatty acid synthase (FAS) in plastids where fatty acid synthesis occurs. This final step releases free fatty acids within the plastid and converts them to acyl-CoA thioesters that are subsequently transported from the plastid to the cytoplasm and endoplasmic reticulum to generate TAGs, other neutral lipids, and phospholipids. Fatty acyl-ACP thioesterases are classified into two distinct but closely related classes based on amino acid sequence (Martins-Noguerol et al. 2020) and activity: FATB acyl-ACP thioesterases, which have specificity primarily for C16 and shorter SFAs, and FATA acyl-ACP thioesterases, which are more active toward C18-ACP substrates than C16-ACP substrates, particularly toward MUFA-ACP substrates such as oleoyl-ACP, but may also have activity toward stearoyl-ACP (Jones et al., 1995; Salas and Ohlrogge, 2002). Hawkins and Kridl (1998) reported that both FATA and FATB class enzymes have limited activity toward stearoyl-ACP, but neither exhibits a substrate preference for oleoyl-ACP. However, a more recent report (Ghosh et al., 2007) identified FATB thioesterases from mahua (Madhuca latifolia, accession number AAX51637) and Jatropha curcas (Dani et al., 2011; accession number ACT09366) that have specificity for stearoyl-ACP over oleoyl- and palmitoyl-ACP.As used herein, a "FATA polypeptide" or "FATA thioesterase" refers to an acyl-ACP thioesterase of the FATA class, and a "FATB polypeptide" or "FATB thioesterase" refers to an acyl-ACP thioesterase of the FATB class.

[0251] Overexpression of FATA polypeptide in transgenic plants increases stearic acid levels in seed oil in some cases but not in others (Hawkins and Kridl, 1998).

[0252] Bhattacharjee et al. (2011) identified a FATA thioesterase from mango (Mangifera indica) with a relative substrate specificity of 100:35:1.8 for oleoyl-ACP, stearoyl-ACP, and palmitoyl-ACP, respectively. As described herein, we selected two related FATA acyl-ACP thioesterases, designated GarmFATA1 (accession number U92876) and GarmFATA2 (Hawkins and Kridl, 1998), from the tropical plant mangosteen (Garcinia mangostana). Both proteins contain an N-terminal transit peptide sequence (TPS) that functions to target the protein to plastids. The former polypeptide contains 352 amino acids, including the TPS, while the latter polypeptide contains 355 amino acids, including the TPS. The amino acid sequences of GarmFATA1 and GarmFATA2 are 73% identical along the entire length of GarmFATA1. GarmFATA1 exhibited greatest activity toward C18:1-ACP, was approximately 7-fold less active toward C18:0-ACP, was again less active toward C16:0-ACP, and exhibited relative activities of 100:15:6 toward oleoyl, stearoyl-ACP, and palmitoyl-ACP, respectively. In contrast, GarmFATA2 had 50-fold less activity toward C18:0-ACP compared to C18:1-ACP. Thus, both enzymes had their primary activity toward the MUFA, C18:1-ACP. The amino acid sequences of GarmFATA1 and GarmFATA2 are provided herein as SEQ ID NO:83 and SEQ ID NO:85.

[0253] 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, which is 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. Accordingly, desaturases can generally be classified into three groups. In one embodiment, the desaturase is a front-end desaturase.

[0254] 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 in 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).

[0255] As used herein, "Δ12-desaturase" refers to a protein capable of carrying out a desaturase reaction to introduce a carbon-carbon double bond one carbon-carbon bond from the carboxyl terminus of a fatty acid substrate. Δ12-desaturases typically convert 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 called phospholipid-dependent Δ12-desaturases, while the latter subclass is called acyl-CoA-dependent Δ12-desaturases. Plant and fungal Δ12-desaturases generally belong to the former subclass, while animal Δ12-desaturases generally belong to the latter subclass, with the exception of Δ12-desaturases from lower animals such as C. elegans, such as the Δ12-desaturase encoded by the gene cloned from an insect by Zhou et al. (2008). Many other Δ12-desaturase sequences can be easily identified by searching sequence databases. Numerous desaturase-encoding genes have been isolated from fungal sources. U.S. Patent No. 7,211,656 describes a Δ12-desaturase from Saprolegnia diclina.WO2009016202 is a microbial fungus that can be isolated from Helobdella robusta, Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosiga brevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola, Naegleria gruben, Nectria haematococca, Nematostella vectensis, Phycomyces blakesleeanus, Trichoderma reesei, and other fungi. WO 2005 / 012316 describes 12-desaturases from Thalassiosira pseudonana and other fungi. WO 2003 / 099216 describes genes encoding fungal 12-desaturases isolated from Neurospora crassa, Aspergillus nidulans, Botrytis cinerea, and Mortierella alpina.

[0256] Other genes In addition to manipulating the expression of the above enzymes, the production of saturated fatty acids in the lipids of microbial cells can be enhanced by genetic modifications that regulate the expression of one or more endogenous genes involved in microbial fatty acid biosynthesis, catabolism, and regulation. Exemplary such microbial genes are listed in Table 1.

[0257] In some embodiments, the genetic modification that increases the production of saturated fatty acids in lipids results in increased expression and / or activity of one or more genes in Table 1. In some embodiments, the genetic modification results in increased expression and / or activity of fatty acid synthesis genes (see Table 1 for examples). In some embodiments, the genetic modification results in increased expression and / or activity of phospholipid synthesis genes (see Table 1 for examples). In some embodiments, the genetic modification results in increased expression and / or activity of lipid synthesis regulatory genes (see Table 1 for examples). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0258] In some embodiments, the genetic modification that increases the production of saturated fatty acids in lipids reduces or disrupts the expression and / or activity of one or more genes in Table 1. In some embodiments, the genetic modification reduces or disrupts the expression and / or activity of lipid catabolic genes. (See Table 1 for examples.)

[0259] Phospholipid synthesis in microorganisms As major structural components of biological membranes, phospholipids play important roles in cell morphology and organelle function, and some also function as secondary messengers. Phospholipids are amphipathic molecules with a phosphate head group and a glycerol backbone esterified to two fatty acids (Figure 4). Because the head groups are charged at neutral pH, they are polar lipids, exhibiting some solubility in solvents such as chloroform as well as ethanol. 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 (C18:0) (Carman and Gil-Soo, 2011). The major phospholipids in S. cerevisiae whole-cell extracts 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 primarily of PE, PG, and CL, and these phospholipids contain primarily the fatty acids 16:0, 16:1, and 18:1Δ11 (De Siervo, 1969). E. coli and many other bacteria lack PC.

[0260] The enzymes and corresponding genes involved in microbial phospholipid synthesis are listed in Table 1. The enzymes and genes involved in yeast phospholipid synthesis have been extensively characterized in S. cerevisiae (Carman and Zeimetz, 1996). Phospholipid specific synthesis 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 acyltransferases and lysophospholipid acyltransferases (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 in all other yeasts. In one reaction, CDP-DAG donates its phosphatidyl moiety to inositol to generate PI in a reaction catalyzed by PI synthase (Nikawa and Yamashita, 1984). The inositol used in this reaction can be 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 for the synthesis of PI can also be utilized from exogenously supplied inositol in the medium by inositol permease.CDP-DAG can also donate its phosphatidyl moiety to glycerol-3-phosphate to generate phosphatidylglycerophosphate (PGP) in a reaction catalyzed by PGP synthase (Chang et al., 1998a). PGP is then dephosphorylated by PGP phosphatase to generate PG (Osman et al., 2010). Cardiolipin (CL) synthase catalyzes the reaction of PG with another molecule of CDP-DAG to generate CL (Chang et al., 1998b). The final enzyme that utilizes CDP-DAG is PS synthase (Letts et al., 1983), which catalyzes the production 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, in which the first methylation reaction is catalyzed by PE methyltransferase, and the last two methylation reactions are catalyzed by phospholipid methyltransferase (Kodaki and Yamashita 1987).

[0261] PE and PC can also be synthesized from exogenously supplied ethanolamine and choline via the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway. Exogenously supplied ethanolamine and choline are phosphorylated with ATP by ethanolamine kinase and choline kinase to generate phosphoethanolamine and phosphocholine, respectively (Kim et al., 1999; Hosaka et al., 1989). These intermediates are then activated with CTP to generate CDP-ethanolamine and CDP-choline, respectively, via 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 reactions with DAG to generate 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 synthetase enzyme. DAG, used in the synthesis of PE and PC via the Kennedy pathway, is derived from PA by 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 to synthesize the neutral lipid TAG by acyltransferase enzymes encoded by DGA1 and LRO1. Furthermore, additional acyltransferase enzymes involved in the synthesis of ergosterol esters may also acylate DAG to generate TAG.

[0262] The Kennedy pathway plays a crucial role in the synthesis of PE and PC when enzymes in the CDP-DAG pathway are dysfunctional or defective (Carman and Henry, 1999; Greenberg and Lopes, 1996). For example, mutants defective in the three-step methylation of PE require supplemental choline for growth and synthesize PC via the CDP-choline branch of the Kennedy pathway. Mutants defective in PS or PE synthesis 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 subsequently methylated to produce 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, once 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 exclusively through the CDP-DAG pathway. However, unlike CDP-DAG pathway mutants, Kennedy pathway mutants do not exhibit auxotrophy and have an essentially normal complement of phospholipids.

[0263] Evidence supports that the CDP-DAG pathway is primarily responsible for the synthesis of PE and PC when cells are grown in the absence of ethanolamine and choline (Carman and Henry 1989). However, the Kennedy pathway may contribute to the synthesis of PE and PC when their precursors are not supplemented in the medium. For example, PC synthesized via the CDP-DAG pathway is always hydrolyzed to choline and PA by phospholipase D. Choline is then converted back to PC via the CDP-choline branch of the Kennedy pathway, and PA is converted to other phospholipids via the intermediates CDP-DAG and DAG.

[0264] The details presented above about phospholipid synthesis in S. cerevisiae and the genes and enzymes involved were found to also apply to the oleaginous yeast Yarrowia lipolytica. Another common yeast, S. pombe, uses a PL biosynthetic pathway 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, meaning it cannot generate L-myo-inositol 3-phosphate from the 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 in S. cerevisiae, as evidenced by the observation that expression of Pichia pastoris inositol-3-phosphate synthase in S. pombe converts this natural inositol auxotroph into inositol prototrophy.

[0265] Phospholipids in Escherichia coli and other Gram-negative bacteria are used to construct the inner and outer membranes. E. coli possesses only three major phospholipid species in its membrane: PE, which accounts for the majority (75%) of phospholipids, and PG and CL, which account for the remaining 15–20% and 5–10%, respectively. Bacterial phospholipid synthesis begins with the acylation of glycerol 3-phosphate (G3P), producing lysophosphatidic acid (lysoPA). This detergent-like intermediate undergoes a second acylation to generate phosphatidic acid (PA), an important 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 PS synthase and PS decarboxylase (Psd)-mediated PE synthesis. PG is produced 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.

[0266] microbial cells A wide variety of different microbial cells can be used in the present invention. In one embodiment, microbial cells exist as unicellular organisms, although such cells may aggregate. Examples of microbial cells of the present invention include bacterial cells and fungal cells, such as eukaryotic cells and algal cells. Eukaryotic microorganisms are preferred over bacterial (prokaryotic) microorganisms. As used herein, the terms "microbial cell," "microbe," and "microorganism" mean the same thing.

[0267] In one embodiment, the microbial cells are suitable for fermentation, although they can also be cultured under ambient oxygen concentrations. In another embodiment, the microbial cells are derived from oleaginous cells, preferably oleaginous eukaryotic microorganisms, or precursor oleaginous microorganisms, such as precursor eukaryotic oleaginous microorganisms. In another embodiment, the microbial cells are heterotrophic cells, preferably heterotrophic eukaryotic microorganisms. Preferably, the microbial cells have at least two of these characteristics, and more preferably, are characterized by all of these characteristics.

[0268] 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, such as Saccharomyces cerevisiae, Yarrowia, such as Yarrowia lipolytica, Pichia, such as Pichia pastoris, Candida, such as Candida rugosa, Aspergillus, such as Aspergillus niger, Cryptococcus, such as Cryptococcus curvatus, Rhodosporidium, such as Rhodosporidium toruloides, and Rhodotorula, such as Rhodotorula glutinis. glutinis, and the Trichosporon genus, e.g., Trichosporon fermentans.

[0269] 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, the genus Cunninghamella, such as Cunninghamella echinulate, the genus Mortierella, such as Mortierella isabellina or Mortierella alpina (a fungus), the genus Mucorales, such as Mucorales fungi, and the genus Trichoderma, such as Trichoderma harzianum.

[0270] In one embodiment, the cell is a bacterial cell. Examples of bacterial cells useful in the present invention include Acinetobacter, such as Acinetobacter baylyi, Alcanivorax, such as Alcanivorax borkumensis, Gordonia, such as DG, Mycobacterium, such as Mycobacterium tuberculosis, Nocardia, such as Nocardia globerula, Rhodococcus, such as Rhodococcus opacus, and Streptomyces, such as Streptomyces coelicolor.

[0271] In one embodiment, the cell is an algal cell, such as a microalgae or Bacillariophyceae cell. Examples of algal cells useful in the present invention include, but are not limited to, the genus Prototheca, e.g., Prototheca moriformis, the genus Thraustochytrium, and the genus Chlorella. Chlorella protothecoides, Chlorella vulgaris or Chlorella ellipsoidea, the genus Schizochytrium, for example Schizochytrium strain FCC-1324, the genus Dunaliella, the genus Haematococcus, for example Haematococcus pluvialis, the genus Neochloris, for example Neochloris oleabundans, for example strain UTEX#1185, the genus Pseudochlorococcum, the genus Scenedesmus, for example Scenedesmus oblicus obliquus, the genus Tetraselmis, such as Tetraselmis chui or Tetraselmis tetrathele, the genus Chaetoceros, for example Chaetoceros calcitrans, Chaetoceros gracilis or Chaetoceros muelleri, the genus Nitzschia, for example Nitzschia cf. pusilla,pusilla, the genus Phaeodactylum, for example Phaeodactylum tricornutum, the genus Skeletonema, for example strain CS252, the genus Thalassiosira, for example Thalassiosira pseudonana, the genus Crypthecodinium, for example Crypthecodinium cohnii, the genus Isochrysis, for example Isochrysis zhangjiangensis, the genus Nannochloropsis, for example Nannochloropsis oculata oculata, for example strain NCTU-3, Pavlova species, for example Pavlova salina, Rhodomonas species, and Thalassiosira species, for example Thalassiosira weissflogii.

[0272] Polypeptides The terms "polypeptide" and "protein" are generally used interchangeably. A polypeptide or class of polypeptides can be defined by the degree of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a higher % identity to one reference amino acid sequence than 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) using parameters of a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 100 amino acids in length, 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 in length, 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 class of polypeptides may have the same enzymatic activity as the reference polypeptide, or may have a different activity from 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.

[0273] The polynucleotides defined herein can encode biologically active fragments of enzymes such as acyltransferases or thioesterases. As used herein, a "biologically active" fragment is a portion of a polypeptide defined herein that retains a defined activity of the full-length reference polypeptide, e.g., acyltransferase or thioesterase 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.

[0274] It will be understood that with respect to a defined polypeptide or enzyme, % identity figures higher than those provided herein encompass preferred embodiments. Thus, taking into account minimum % identity figures, where applicable, it is preferred that the polypeptide / enzyme 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% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.1% or more, more preferably 99.2% or more, more preferably 99.3% or more, 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 named 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 the designated SEQ ID NO.

[0275] 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.

[0276] Mutant (modified) 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 / modified DNA can be readily screened using the techniques described herein to determine whether they have, for example, acyltransferase or thioesterase activity.

[0277] In designing amino acid sequence variants, the location of the mutation site and the nature of the mutation depend on the property to be altered. Mutation sites can be altered individually or sequentially. For example, (1) substituting conservative amino acid selections first, followed by more radical selections depending on the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the identified site.

[0278] Amino acid sequence deletions generally range from about 1 to 15 contiguous residues, more preferably about 1 to 10 residues, and typically about 1 to 5 contiguous residues.

[0279] Substitutional variants involve removing at least one amino acid residue in a polypeptide molecule and inserting another residue in its place. Sites of primary interest for substitutional mutagenesis include those that are not conserved in naturally occurring proteins, such as acyltransferases and thioesterases. 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 "Exemplary Substitutions."

[0280] In preferred embodiments, mutant / variant polypeptides have only one or two or three or four or fewer conservative amino acid changes compared to the naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 2. As will be appreciated by those skilled in the art, such minor changes can reasonably be expected not to alter the activity of the polypeptide when expressed in a recombinant cell. [Table 2]

[0281] 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. They may be double- or single-stranded DNA or RNA, of genomic or synthetic origin, and may be combined with carbohydrates, lipids, proteins, or other substances to perform a specific activity as defined herein. The term "polynucleotide" is used interchangeably herein with the term "nucleic acid molecule."

[0282] In one embodiment, the polynucleotide is not naturally occurring. Examples of non-naturally occurring polynucleotides include, but are not limited to, those that have been codon-optimized for expression in a microbial cell, e.g., mutated using the methods described herein, and polynucleotides in which an open reading frame encoding a protein is operably linked to a promoter to which it is not naturally associated, i.e., a promoter that is heterologous with respect to the open reading frame.

[0283] As used herein, "chimeric DNA" or "chimeric gene construct" or the like refers to any DNA molecule that is not a native DNA molecule in its native location, also referred to herein as a "DNA construct." Typically, chimeric DNA or chimeric genes contain regulatory and transcriptional or protein-coding sequences that are not found operably linked in nature, i.e., are heterologous to one another. Thus, chimeric DNA or chimeric genes can 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.

[0284] An "endogenous gene" refers to a native gene in its natural location within the genome of an organism. As used herein, "recombinant nucleic acid molecule," "recombinant polynucleotide," or variations thereof refer to a nucleic acid molecule constructed or modified by recombinant DNA technology. The terms "foreign polynucleotide," "exogenous polynucleotide," "heterologous polynucleotide," and the like refer to any nucleic acid introduced into the genome of a cell by experimental manipulation. A foreign gene or exogenous gene can be a gene inserted into a non-native organism, a native gene introduced into a new location in its native host, or a chimeric gene. A "transgene" is a gene introduced into a genome by a transformation procedure. The terms "genetic modification," "gene variation," "transgenic," and variations thereof include the introduction of a gene into a cell by transformation or transduction, the mutation of a gene in a cell, the deletion of a gene, and the alteration or modulation of gene control through genetic changes in the genome of a cell or organism or its progeny. As used herein, a "genomic region" refers to a location within the genome where a transgene or group of transgenes (also referred to herein as a cluster) has been inserted into a cell or its ancestor. Such regions include only nucleotides incorporated by human intervention, such as the methods described herein.

[0285] The term "exogenous" in the context of 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 also contain 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 they reside, as well as polynucleotides that are produced in such cells or cell-free systems and then purified and removed from at least the other components. An exogenous polynucleotide (nucleic acid) may be a contiguous stretch of nucleotides found in nature, or two or more contiguous stretches of nucleotides from different sources (natural and / or synthetic) may be joined to produce a single polynucleotide. Typically, such chimeric polynucleotides comprise 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.

[0286] It will be understood that with respect to defined polynucleotides, % identity figures higher than those provided above encompass preferred embodiments. That is, taking into account minimum % identity figures, where applicable, it is preferred that the polynucleotide comprise 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 designated 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 the designated SEQ ID NO.

[0287] A polynucleotide may have one or more mutations, which are deletions, insertions, or substitutions of nucleotide residues, when compared to a naturally occurring molecule. Polynucleotides having mutations compared to a reference sequence can be either naturally occurring (i.e., isolated from a natural source) or synthetic (e.g., by subjecting the nucleic acid to site-directed mutagenesis or DNA shuffling, as described above). Thus, it is clear that polynucleotides can be derived from natural sources or recombinantly. Preferred polynucleotides are those having coding regions that are codon-optimized for translation in microbial cells, as is known in the art.

[0288] Recombinant vector Recombinant expression can be used to produce the genetically modified microorganisms of the present invention. Recombinant vectors contain heterologous polynucleotide sequences, i.e., polynucleotide sequences not naturally found adjacent to a polynucleotide molecule as defined herein, preferably derived from a species other than the one 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 allow for easy selection, amplification, and transformation of the expression cassette in prokaryotic cells, such as pYES-derived vectors, pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. Suitable yeast expression vectors include the pPIC series of vectors, Yeast Integrating Plasmids (YIp), Yeast Replicating Plasmids (YRp), Yeast Centromeric Plasmids (YCp), and Yeast Episomal Plasmids (YEp). Additional nucleic acid sequences include an origin of replication that provides 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 facilitate transformation of microbial cells. A recombinant vector may comprise two or more polynucleotides as defined herein, for example a combination of three, four, five, or six polynucleotides as defined herein, preferably a chimeric gene construct as described herein, each polynucleotide operably linked to an expression control sequence operable in the cell.

[0289] 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 between a transcriptional regulatory element (promoter) and 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, promoter transcriptional regulatory elements operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically adjacent or located in close proximity to the coding sequence whose transcription they enhance. For example, introns within a 5'UTR sequence or introns near the 5' end of a protein-coding region may contain transcriptional enhancers, such as the FBAIN promoter region, that confer increased expression levels.

[0290] 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 on cells expressing the marker gene, thus allowing such transformed cells to be distinguished from cells lacking the marker. A selectable marker gene confers a trait that can be "selected" for 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 by observing or testing, i.e., "screening," for example, β-glucuronidase, luciferase, GFP, or other enzyme activity that is not present in non-transformed cells. The marker gene and the nucleotide sequence of interest need not be linked. The actual choice of marker is not critical, as long as the marker is functional (i.e., selective) in combination with the cells of choice.

[0291] Examples of selectable markers are those that confer antibiotic resistance, such as hygromycin, nourseothricin, ampicillin, erythromycin, chloramphenicol, or tetracycline resistance, preferably hygromycin or kanamycin resistance.

[0292] The recombinant yeast of the present invention can contain a reporter gene encoding either a galactosidase or a selectable growth marker.

[0293] A "galactosidase" can be any enzyme capable of cleaving terminal galactose residues from various 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 Escherichia coli) LacZ. In another embodiment, the galactosidase is a β-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-chloroindolyl-β-D-galactopyranoside), which produces a deep blue product upon cleavage; ONPG (o-nitrophenyl galactoside), which produces a water-soluble yellow pigment with an absorbance maximum at approximately 420 nm upon cleavage; and CPRG (chlorophenol red-β-D-galactopyranoside), which produces a water-soluble red product upon cleavage that can be measured spectrophotometrically. α-Galactosidase activity can be detected using substrates for the enzyme, such as o-nitrophenyl-D-galactopyranoside, which produces an indigo dye upon cleavage, or chlorophenol red-α-D-galactopyranoside, which produces a red, water-soluble product upon cleavage that can be measured spectrophotometrically. Kits for detecting galactosidase expression in yeast are commercially available, such as the Thermo Scientific β-galactosidase (LacZ) Expression Kit.

[0294] Preferably, the selectable growth marker is a nutritional marker or an antibiotic resistance marker.

[0295] 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 renders the host inviable, a so-called essential gene, can be used as a selectable marker if a functional gene is provided, for example, on a plasmid, as has been demonstrated for PGK1 in pgk1 yeast strains. Suitable essential genes can be found in the Stanford Genome Database (SGD) (http: / / db.yeastgenome.org). An essential gene product (e.g., PDI1, PSE1, PGK1, or FBA1) that, when deleted or inactivated, does not result in an auxotrophic (biosynthetic) requirement can be used as a selectable marker on a plasmid in yeast host cells that, in the absence of the plasmid, are unable to produce the gene product to improve plasmid stability without the drawback of having to culture the cells under specific selective conditions. "Auxotrophic (biosynthetic) requirement" includes a deficiency that can be compensated for by additions to or modifications to the growth medium.

[0296] Expression An expression vector can direct gene expression in a microbial cell. As used herein, an expression vector is a vector capable of transforming a host cell and resulting in the expression of one or more specific polynucleotide molecules. Expression vectors useful in the present invention contain regulatory sequences, such as transcriptional control sequences, translational control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and control the expression of the polynucleotide molecules of the present invention. In particular, polynucleotides or vectors useful in the present invention contain transcriptional control sequences. Transcriptional control sequences are sequences that control the initiation, elongation, and termination of transcription. Particularly important transcriptional control sequences are sequences that control transcription initiation, such as promoter sequences and enhancer sequences. Suitable transcriptional control sequences include any transcriptional control sequence that can function in at least one of the recombinant cells of the present invention. The choice of regulatory sequence to be used will depend on the target microbial cell. A variety of such transcriptional control sequences are known to those skilled in the art.

[0297] Yeast cells are typically transformed by chemical methods (e.g., Rose et al., 1990, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Kawai et al., 2010). Typically, cells are treated with lithium acetate for approximately 10 min. 4 A transformation efficiency of colony-forming units (transformed cells) / μg DNA is achieved. 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 bullet method, in which DNA-coated metal particles are fired 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.

[0298] 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 gene due to differences in copy number and other factors. Therefore, it is recommended to screen two or more independent clonal isolates for each transformed receptor to maximize the chances of identifying a suitable receptor-ligand pair during screening. Different clonal isolates can be screened individually or combined into a single well for screening. The latter option is particularly convenient when nutritional rather than colorimetric reporters are used.

[0299] A "constitutive promoter" refers to a promoter that directs expression of an operably linked transcriptional sequence in a cell without the need for induction by specific growth conditions. Examples of constitutive promoters useful in the yeast cells of the present invention include, but are not limited to, the 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). 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 can be a naturally occurring molecule or a variant thereof containing, for example, one, two, or three nucleotide substitutions that do not eliminate (and preferably do not enhance) promoter function.

[0300] Recombinant DNA technology can be used to improve expression of transformed polynucleotide molecules, for example, by manipulating the copy number of polynucleotide molecules in host cells, the efficiency with which these 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 polynucleotide molecules 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 mRNA, substitution or modification of transcriptional control signals (e.g., promoters, operators, enhancers), substitution or modification of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of the polynucleotide molecule to correspond to the codon usage of the host cell, and deletion of sequences that destabilize the transcript.

[0301] 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; Becker et al., 1991).

[0302] In one embodiment, integration of a gene of interest into a specific chromosomal site in a microbial cell occurs via homologous recombination. According to this embodiment, an integration cassette containing a module (internal module) containing at least one marker gene and / or a gene to be integrated is flanked on both sides by DNA fragments homologous to the DNA fragments at the ends of the target integration site (recombination-inducing sequences). After transforming a microbial cell with the cassette using an appropriate method, the chromosomal region between the two sites in the genome corresponding to the recombination sequences of the integration cassette can be replaced with the internal module by homologous recombination between the recombination-inducing sequences (Orr-Weaver et al., 1981).

[0303] In one embodiment, the integration cassette for the integration of a gene of interest into a microbial cell comprises a heterologous gene under the control of a suitable promoter, together with a selectable marker flanked by recombination-inducing sequences for integration of the heterologous gene into the chromosome of the microbial cell. In one embodiment, the heterologous gene comprises any of the fatty acid biosynthetic genes described herein.

[0304] If deletion of an endogenous gene is desired, the integration cassette can contain a selectable marker (containing no other heterologous gene sequences) flanked by DNA fragments 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 nucleases or RNA-guided nucleases) are described below. 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 recombination sequence can be freely selected depending on the desired integration site suitable for the desired application.

[0305] In another embodiment, integration of the gene into the chromosome of the microbial cell can occur via random integration (Kooistra et al., 2004). Furthermore, in one embodiment, the specific introduced marker gene is removed from the genome using techniques known to those skilled in the art. For example, the loss of the URA3 marker can be achieved by plating URA3-containing cells on FOA (5-fluoroorotic acid)-containing medium and selecting FOA-resistant colonies (Boeke et al., 1984).

[0306] Exogenous nucleic acid molecules contained within the microbial cells of the present disclosure can be maintained in any form within the cell. For example, exogenous nucleic acid molecules 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 (e.g., plasmids, mitochondrial genomes) may further contain selectable markers that ensure the presence of such genetic elements in daughter cells. Furthermore, microbial cells can be stably transformed or transiently transformed. Furthermore, the microbial cells described herein can contain a single copy or multiple copies of a particular exogenous nucleic acid molecule, as described above.

[0307] Genome editing using site-specific nucleases Genome editing uses artificial nucleases composed of a sequence-specific DNA-binding domain fused to a nonspecific DNA-cleaving module. These chimeric nucleases induce targeted DNA double-strand breaks that stimulate the cell's endogenous cellular DNA repair mechanisms to repair the induced breaks, allowing for efficient and precise genetic modifications (including deletions, mutations, and insertions). Such mechanisms include error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR).

[0308] In the presence of a donor plasmid with extended homology arms, HDR can result in the introduction of single or multiple transgenes that correct or replace existing genes. In the absence of a donor plasmid, NHEJ-mediated repair generates targeted small insertion or deletion mutations that cause gene disruption.

[0309] Artificial nucleases useful in the methods of the present invention include zinc finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs).

[0310] Typically, the gene encoding the nuclease is delivered to cells by plasmid DNA, viral vectors, or in vitro transcribed mRNA. The use of fluorescent surrogate reporter vectors also allows for enrichment of ZFN- and TALEN-modified cells. As an alternative to ZFN gene delivery systems, cells can be contacted with purified ZFN proteins, which can cross the cell membrane and induce endogenous gene disruption.

[0311] Zinc finger nucleases (ZFNs) contain a DNA-binding domain and a DNA-cleavage domain, where the DNA-binding domain consists of at least one zinc finger and is operably linked to the DNA-cleavage domain, and the zinc finger DNA-binding domain is located at the N-terminus of the protein, and the DNA-cleavage domain is located at the C-terminus of the protein.

[0312] ZFN requires 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 cells. Generally, ZFN with more than three zinc fingers will have incrementally greater specificity with each additional zinc finger.

[0313] The zinc finger domain can be derived from any class or type of zinc finger. In certain embodiments, the zinc finger domain comprises a Cis2His2 type of zinc finger, which is most commonly represented, for example, by the zinc finger transcription factors TFIIIA or Sp1. In a preferred embodiment, the zinc finger domain comprises three Cis2His2 type zinc fingers. The DNA recognition and / or binding specificity of ZFNs can be altered to achieve targeted genetic 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).

[0314] ZFN DNA cleavage domains are derived from a class of nonspecific DNA cleavage domains, such as the DNA cleavage domains of type II restriction enzymes such as FokI (Kim et al., 1996). Other useful endonucleases include, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI.

[0315] 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 within the host microbial cell DNA. These recognition sites are oriented in opposite directions and separated by approximately 6 bp of DNA. ZFNs are then generated by designing and engineering a combination of zinc fingers that specifically bind to DNA at the target locus and linking the zinc fingers to a DNA-cleavage domain.

[0316] ZFN activity can be improved by using transient hypothermic culture conditions to increase nuclease expression levels (Doyon et al., 2010) and by co-delivery of DNA end-processing enzymes and site-specific nucleases (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).

[0317] Transcription activator-like (TAL) effector nucleases (TALENs) contain a TAL effector DNA-binding domain and an endonuclease domain.

[0318] TAL effectors are proteins from plant pathogenic bacteria that are injected into plant cells by pathogens, where they translocate to the nucleus and function as transcription factors to turn on specific plant genes. The primary amino acid sequence of a TAL effector determines the nucleotide sequence to which it binds. Therefore, the target site of a TAL effector can be predicted, and TAL effectors can be engineered and generated to bind to specific nucleotide sequences.

[0319] A sequence encoding a nuclease or portion of a nuclease, typically a nonspecific cleavage domain from a type II restriction endonuclease such as FokI, is fused to a nucleic acid sequence encoding a TAL effector (Kim et al., 1996). Other useful endonucleases include, for example, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. The fact that some endonucleases (e.g., FokI) function only as dimers can be exploited to enhance the target specificity of TAL effectors. For example, in some cases, each FokI monomer can be fused to a TAL effector sequence that recognizes a different DNA target sequence; only when the two recognition sites are in close proximity will the inactive monomers bind to generate a functional enzyme. By requiring DNA binding for nuclease activation, highly site-specific restriction enzymes can be created.

[0320] Sequence-specific TALEN can recognize specific sequences in the preselected target nucleotide sequence present in cells.Therefore, in some embodiments, target nucleotide sequence can be scanned for nuclease recognition site, and specific nuclease can be selected based on target sequence.In other cases, TALEN can be engineered to target specific cellular sequence.

[0321] Genome editing using a programmable RNA-guided DNA endonuclease 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 genetic changes. In bacteria, the CRISPR system provides adaptive immunity against invading foreign DNA through RNA-guided DNA cleavage.

[0322] The CRISPR system relies on CRISPR RNA (crRNA) and a trans-activating 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 recognition of the crRNA-tracrRNA target. CRISPR RNA pairs with tracrRNA to form two RNA structures that guide the Cas9 endonuclease to the complementary DNA site and cleave it.

[0323] They are a distinct class of interspersed short sequence repeats (SSRs) first recognized in Escherichia coli (Ishino et al., 1987; Nakata et al., 1989). Similar interspersed SSRs have also 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).

[0324] CRISPR loci differ from other SSRs by the structure of their repeats, termed short regularly interspaced repeats (SRSRs) (Janssen et al., 2002; Mojica et al., 2000). These repeats are short elements that occur in clusters and are regularly spaced by unique intervening sequences of a consistent length (Mojica et al., 2000). While the repeats are highly conserved among strains, the number of interspersed repeats and the sequence of the spacer regions vary from strain to strain (van Embden et al., 2000).

[0325] Common structural features of CRISPR loci have been described by Jansen et al. (2002) as (i) the presence of multiple short direct repeats with little or no sequence variation within a given locus, (ii) the presence of non-repeated spacer sequences between similarly sized repeats, (iii) the presence of a common leader sequence of several hundred base pairs in most species with multiple CRISPR loci, (iv) the absence of long open reading frames within the locus, and (v) the presence of one or more cas genes.

[0326] CRISPRs are typically short, 24-40 bp partial palindromic sequences containing internal and terminal inverted repeats of up to 11 bp. While isolated elements have been detected, they are generally arranged in clusters of repeat units (up to approximately 20 or more per genome) spaced by unique intervening 20-58 bp sequences. CRISPRs are generally uniform within a given genome, with most of them being identical. However, there are examples of heterogeneity, for example in Archaea (Mojica et al., 2000).

[0327] As used herein, the term "cas gene" generally refers to one or more cas genes associated with, adjacent to, or nearby linked to adjacent CRISPR loci. A comprehensive review of the Cas protein family is provided in Haft et al. (2005). The number of cas genes at a particular CRISPR locus varies between species.

[0328] cell culture Effective culture conditions are known to those skilled in the art and include, but are not limited to, appropriate media, bioreactors, temperature, pH, and oxygen conditions that allow lipid production. A suitable media refers to any medium in which cells can be cultured to produce lipids as defined herein. Such media typically include aqueous media having 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. Culturing can be carried out at temperatures, pH, and oxygen content appropriate for the recombinant cells. Such culture conditions are within the expertise of those skilled in the art.

[0329] lipid extraction Lipid extraction from microbial cells of the present invention uses methods similar to those known in the art for extracting lipids from oleaginous microorganisms, as described, for example, in Patel et al. (2018). In one embodiment, 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 pulverized, but also in a wet state. The solvent dissolves the intracellular lipids, 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 for disrupting the cellular integrity of microbial cells. Other pretreatment methods may include treatment with acids such as sulfuric acid, microwave irradiation, high-speed homogenization, high-pressure homogenization, bead beating, autoclaving, and pyrolysis. For example, treatment with 2% sulfuric acid is performed at 60°C for 5 minutes. The organic solvent can then be separated from the nonpolar lipids (e.g., by distillation). This second separation step yields the nonpolar lipids from the cells, and conventional steam recovery can be used to obtain reusable solvent.

[0330] 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 pulverized. The solvent dissolves the lipids in the biomass, and the solution is then separated from the biomass by mechanical action (e.g., by the process described above). This separation step can also be performed by filtration (e.g., using a filter press or similar device) or centrifugation. 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 conventional steam recovery can be used to obtain reusable solvent.

[0331] The lipid extracted from the microbial cells of the present invention can be subjected to conventional oil processing operations. As used herein, the term "refined," when used in reference to the lipid of the present invention, typically means that the extracted lipid has been subjected to one or more processing steps to increase the purity of the lipid component. For example, the refining step may include one or more or all of the following: degumming, deodorizing, bleaching, drying, and / or fractionation of the extracted oil. However, the term "refined" as used herein does not include transesterification or other processes that alter the fatty acid composition of the lipid or oil 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 refined lipid is essentially the same as that of the unrefined lipid.

[0332] degumming Degumming is the initial stage in the purification of liquid lipids (oils), and its primary goal is to separate the majority of phospholipids, which may be present at approximately 1-2% of the total extracted lipids, from the oil. Adding approximately 2% water, typically containing phosphoric acid, to crude oil at 70-80°C separates most 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 accomplished by adding concentrated phosphoric acid to the crude extracted lipids to convert non-hydratable phospholipids to a hydratable form and chelate any trace metals present. The gum is separated from the oil by centrifugation. Recovered gums containing omega-6 fatty acids other than LA alone are also encompassed by this invention.

[0333] Alkali Refining Alkali refining, sometimes called neutralization, is a refining process that treats lipids in the form of oil. It is usually performed after degumming and before bleaching. After degumming, the oil is treated with a sufficient amount of alkaline solution to titrate all fatty acids and phosphates and remove any soaps that have formed. Suitable alkaline substances 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 extraction into a soap-soluble solvent, and the neutralized oil is washed with water. If necessary, excess alkali in the oil can be neutralized with a suitable acid, such as hydrochloric acid or sulfuric acid.

[0334] bleaching Bleaching is a refining process in which 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, using nitrogen or steam or operating under vacuum. This step in oil processing is designed to remove unwanted pigments; the process also removes oxidation products, trace metals, sulfur compounds, and small amounts of soap.

[0335] deodorant Deodorization is the treatment of fats and oils at high temperatures (200-260°C) and low pressures (0.1-1 mmHg). This is typically accomplished by introducing steam into the oil at a rate of approximately 0.1 ml / min per 100 ml of oil. After approximately 30 minutes of sparging, the oil is cooled under vacuum. Typically, the oil is transferred to glass containers, flushed with argon, and then refrigerated for storage. This treatment improves the oil's color and removes most of the volatile and odorous compounds, including residual free fatty acids, monoacylglycerols, and oxidation products.

[0336] Esterification and transesterification As used herein, "esterification" refers to a chemical reaction that produces at least one fatty acid ester through an esterification reaction between a fatty acid and an alcohol. A fatty acid ester (FAE) is a type of ester resulting from the combination of a fatty acid and an alcohol. When the alcohol component is glycerol, the fatty acid ester produced can include a monoglyceride, a diglyceride, or a triglyceride.

[0337] As used herein, "transesterification" refers to the process of exchanging fatty acids within and between TAGs (transesterification) or transferring a fatty acid to another alcohol to produce an ester. This may involve first releasing the fatty acid from the TAG as a free fatty acid, or it may involve directly producing a fatty acid ester, preferably a fatty acid methyl or ethyl ester. In the transesterification of TAGs with alcohols such as methanol or ethanol, the alkyl group of the alcohol forms an ester bond with an acyl group (including SCFAs) of the TAG.

[0338] Foods, Feeds, Beverages, and Compositions The present invention includes lipids and compositions that can be used as ingredients in foods or beverages for human consumption or feed for animal consumption, preferably at least foods for human consumption, or foods or beverages that can be used to prepare foods or beverages. The compositions can also be added to foods, beverages, or feeds to improve one or more of the texture, appearance, aroma, and / or flavor of the foods, beverages, or feeds. Lipids can be used to improve dough consistency or workability, or to enhance the consistency, texture, or taste of foods when warmed above room temperature. For purposes of the present invention, a food, beverage, or feed is a preparation for human or animal consumption that, when ingested, (a) serves to nourish, build, or provide energy to tissues, and / or (b) maintain, restore, or support proper nutritional status or metabolic function.

[0339] Suitable foods / feeds include meat substitutes, soup bases, stew bases, snack foods, bouillon powders, bouillon cubes, flavor packs, or frozen meals. Meat substitutes can be formulated as, for example, hot dogs, hamburgers, ground meat, sausages, steaks, fillets, roasts, breasts, thighs, wings, meatballs, meatloaf, bacon, strips, fingers, nuggets, cutlets, or cubes.

[0340] The lipids of the present invention can be used as a food ingredient as fats or oils, and in dressings, or in products such as butter, powdered butter, margarine, mayonnaise, or salad dressings. They can be used in soups, such as canned or instant soups, noodle bowl or noodle cup products, stews, stocks, soups, canned vegetables, dried vegetables, etc. They can be used in sauces and gravies, pasta sauces, tomato products, dried seasoning mixes, seasoning cubes, etc. They can be used in bakery products, such as bread, bread substitutes, pastries, croissants, biscuits, savory biscuits, crackers, cakes, pizza dough, pie pastry, dried bakery mixes, bakery dough, etc. Specific examples include, for example, muffins (e.g., English muffins), crackers (e.g., saltine crackers, baked crackers, graham crackers, etc.), rolls (e.g., soft rolls, dinner rolls, crescent rolls), biscuits (e.g., buttermilk biscuits, cobbler biscuits), pie dough, bread (e.g., focaccia, bruschetta, sourdough bread, soda bread, breadsticks, cornbread), pizza dough, and bagels. Sweet dough can be used to make brownies, cookies, muffins, turnovers, donuts, cakes, pastries, pies, scones, etc. It can be used in mixed dishes, such as frozen and canned foods. For example, it can be used in sweet snacks, savory foods, or savory snacks such as potato chips, crisps, nuts, tortilla tostadas, pretzels, cheese snacks, corn snacks, potato snacks, instant popcorn, microwaveable popcorn, pork rinds, nuts, crackers, cracker snacks, breakfast cereals, meats, cured meats, lunch / breakfast meats, and peanut butter.It can be used in dairy substitutes and analogs, such as ice cream, ice cream desserts, frozen yogurt, cow's milk, raw / pasteurized milk, full-fat raw / pasteurized milk, semi-skimmed raw / pasteurized milk, long-life / UHT milk, full-fat long-life / UHT milk, semi-skimmed long-life / UHT milk, non-fat long-life / UHT milk, goat's milk, condensed / unsweetened milk, plain condensed / unsweetened milk, flavored condensed milk, functional condensed milk, other condensed milk, flavored milk drinks, dairy-only flavored milk drinks, soy milk, sour milk drinks, cultured milk drinks, coffee creamers / whiteners, milk powders, flavored powdered milk drinks, creamers, yogurt, plain / natural yogurt, flavored yogurt, fruit yogurt, probiotic yogurt, yogurt drinks, and other dairy-based desserts. It can also be used in other foods, such as breakfast cereals, cereal flakes, muesli, children's breakfast cereals, and hot cereals.

[0341] The food, beverage, or feed of the present invention comprises, for example, the extracted lipid of the present invention, the microbial cell of the present invention, the microbial cell extract, or the composition of the present invention. The food may be in either solid or liquid form. Furthermore, the composition 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 by normal individuals or individuals with special needs, such as those suffering from metabolic disorders.

[0342] Examples of suitable nutritional ingredients 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, coconut oil, borage oil, fungal oil, blackcurrant oil, soybean oil, monoglycerides, and diglycerides. Examples of such carbohydrates include, but are not limited to, glucose, edible lactose, and hydrolyzed starch. Furthermore, examples of proteins that can be used in the nutritional compositions of the present invention include, but are not limited to, soy protein, electrodialyzed whey, electrodialyzed skim milk, milk whey, or hydrolysates of these proteins.

[0343] With regard to vitamins and minerals, the following may be added to the food, drink, or feed of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, vitamins A, E, D, C, and B complex. Other such vitamins and minerals may also be added.

[0344] Additional ingredients include food-grade oils, such as canola oil, corn oil, sunflower oil, soybean oil, olive oil, coconut oil, seasonings, such as 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).

[0345] The foods and feeds described herein can contain natural coloring agents, such as turmeric or beet juice, or artificial coloring agents, such as azo dyes, triphenylmethane, xanthene, quinine, indigoid, titanium dioxide, Red No. 3, Red No. 40, Blue No. 1, or Yellow No. 5.

[0346] The foods and feeds described herein can include meat shelf life extenders, such as carbon monoxide, nitrites, sodium metabisulfite, bombal, vitamin E, rosemary extract, green tea extract, catechin, and other antioxidants.

[0347] The ingredients utilized in the foods, beverages, or feeds of the present invention may be of semi-purified or purified origin, by which is meant materials prepared by purification of natural materials or de novo synthesis.

[0348] In one embodiment, the food, drink, or feed does not contain any animal-derived ingredients. That is, in a preferred embodiment, at least some of the ingredients are plant materials or plant-derived materials. In some embodiments, the food, drink, or feed may be soy-free, wheat-free, yeast-free, MSG-free, and / or protein hydrolysate-free, and may have a meaty flavor, a very flavorful taste, and may be free of or have reduced levels of off-flavors or off-flavors.

[0349] Additionally, the compositions of the present invention can be used to modify the taste, texture, appearance, mouthfeel, and / or aroma profile of other foods (e.g., meat replicas, meat substitutes, tofu, mock duck, or other gluten-based vegetable products, textured vegetable proteins such as textured soy protein, pork, fish, lamb, or poultry products such as chicken or turkey products) and can be applied to other foods before or during cooking.

[0350] In some embodiments, the compositions described herein include ingredients necessary to induce a Maillard reaction upon heating the composition. For example, the compositions may include one or both of: (i) a sugar, sugar alcohol, sugar acid, or sugar derivative; and (ii) an amino acid or derivative thereof.

[0351] 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.

[0352] 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.

[0353] The composition can also include one or more flavor precursors, including oils (e.g., vegetable oils), free fatty acids, alpha-hydroxy acids, dicarboxylic acids, nucleosides, nucleotides, vitamins, peptides, protein hydrolysates, extracts, phospholipids, lecithins, and organic molecules.

[0354] 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 composition can be sprinkled or spread on the food before or during cooking.

[0355] The foods, beverages, and feeds described herein can be evaluated for texture, appearance, mouthfeel, flavor, and aroma using trained panelists. Evaluation can include seeing, feeling, chewing, smelling, and tasting the food, beverage, or feed product to determine its 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 and quality of the food, or specific quality attributes such as meatiness, texture, and flavor.

[0356] 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 measurements. In various embodiments, the olfactometer can be used to evaluate odor concentration, odor threshold, odor threshold exceedance compared to a reference gas, hedonic scale score to determine the degree of appreciation, or relative odor intensity.

[0357] In some embodiments, GCMS can be used to evaluate the identified volatile chemicals.For example, people can evaluate the smell experience of the chemicals that cause specific peaks.This information can be used to further improve the flavor and aroma compound profile that is produced by the composition of the present invention.

[0358] Characteristic flavor and aroma components are primarily produced during the cooking process by chemically reactive molecules, such as amino acids, fats, and sugars, contained in plants and meat. Thus, in some embodiments, food, beverages, or feeds are tested for their similarity to meat during or after cooking. In some embodiments, human ratings, human evaluations, olfactometer measurements, or GC-MS measurements, or a combination thereof, are used to create an olfactory map of the food or feed. Similarly, olfactory maps can be created for foods, beverages, or feeds, such as meat imitation products. These maps can be compared to assess how similar the cooked food or feed is to meat.

[0359] Personal Care Products The lipids of the present invention can be used in personal care products such as pharmaceuticals, cosmetics, toiletries, etc. Examples include aftershaves, baby lotions, oils, powders and creams, baby shampoos, base coats and undercoats, bath capsules, bath oils, tablets and salts, bath soaps and detergents, beard softeners, blushers, body and hand preparations, bubble baths, cleaning products, colognes and toilet waters, cuticle softeners, dentifrices, deodorants, depilatories, douches, dressings, eye lotions, eye makeup preparations, eye makeup removers, eye shadows, eyebrow pencils, eyeliners, face and neck preparations, face powders, feminine hygiene deodorants, foot powders and sprays, foundations, fragrances, hair bleaches, hair color sprays, hair color preparations, hair conditioners, hair dyes, etc. and color, colored hair lighteners, hair preparations, hair rinses, hair shampoos, hair sprays, hair tints, hair wave sets, hair grooming aids, indoor tanning products, leg and body paints, lipsticks, lozenges, makeup bases, makeup fixatives, makeup products, nail polish, mascara, moisturizers, mouthwashes and breath fresheners, nail creams and lotions, nail extensions, nail polish and enamel, night skin care products, oral care products, oral hygiene products, paste masks, perfumes, personal hygiene products, pre-shave lotions, lipsticks, sachets, shampoos, shaving creams, shaving preparations, shaving soaps, skin care preparations, skin fresheners, tanning gels, creams and liquids, tanning products, toothpastes, tooth gels, teeth whitening products, and tonics. [Example]

[0360] Example 1. Materials and Methods Culture medium 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). This medium was supplemented with uracil as needed.

[0361] Large-scale culture media Unless otherwise specified, the medium used to prepare seed cultures for large-scale cultivation (≥2 L) was a defined medium (DM-Glu) with a basal medium (BM) containing 10.64 g / L potassium dihydrogen orthophosphate (KH2PO4), 4.0 g / L diammonium hydrogen orthophosphate ((NH4)2HPO4), and 1.7 g / L citric acid (monohydrate). These components were dissolved in approximately 70% of the required amount of water purified by reverse osmosis, adjusted to pH 6.0 with 2 M NaOH, and made up to volume with 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 of 660 g / L glucose (autoclaved) to a final concentration of 20 g / L, 10 ml / L of 1 M magnesium sulfate heptahydrate (autoclaved), 10 ml / L of trace metals solution (see below, filter sterilized), 10 ml / L of 15 g / L thiamine hydrochloride (filter sterilized), and 3 ml / L of 10% (v / v) Sigma antifoam 204 (autoclaved).

[0362] Fermentation medium (FM) for 2 L and 10 L cultures also used BM as the base medium. The required amount was added to the bioreactor, sterilized at 121 °C for a 60-minute 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) to a final concentration of 80 g / L, 5 ml / L of 1 M magnesium sulfate heptahydrate (autoclaved), 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). The glucose, magnesium, trace metals solution, and thiamine solution were mixed and added together to the bioreactor. After the medium was formulated, the pH was checked and typically found to be slightly below 6.0. Ammonia solution was added to the medium using a pH controller to bring the pH to 6.0.

[0363] Small-scale (50 ml) cultures and larger cultures (>2 L) were also grown in a defined medium (DM-Glyc-LowN) containing 8% (w / v) glycerol and a lower nitrogen content to induce greater TAG synthesis. This medium was identical to DM-Gluc, except that glucose was replaced with 80 g / L glycerol (final concentration) as the carbon source and the (NH4)2HPO4 content was reduced to 0.5 g / L. For larger cultures, seed cultures were grown for 24–48 h in SD-Ura medium, supplemented with uracil and optional amino acids as needed. A sample of the seed culture was centrifuged, and the cells were used to inoculate larger cultures. These cultures were incubated for 48–96 h, and the pH was maintained at 6.0 unless otherwise noted.

[0364] The alternative media were the same as DM-Glyc-LowN but contained 8% (w / v) glucose or glycerol as the carbon source, (NH)HPO was replaced with (NH)SO to a final concentration of 0.5 g / L to obtain low nitrogen levels, yeast extract was added at 1 g / L, and MgSO and citric acid were omitted. These media are referred to herein as DM-Gluc-LowN-LowMg and DM-Glyc-LowN-LowMg, respectively.

[0365] The trace metals solution (TM) used in the above media contained the following per liter: 2.0 g CuSO4·5H2O, 0.08 g NaI, 3.0 g MnSO4·H2O, 0.2 g NaMoO4·2H2O, 0.02 g H3BO3, 0.5 g CoCl2·6H2O, 7.0 g ZnCl2, 22.0 g FeSO4·7H2O, 0.50 g CaSO4·2H2O, and 1 ml sulfuric acid. The reagents were added in the order listed. The addition of sulfuric acid dissolved the calcium sulfate. The trace metals solution was filter-sterilized using a 0.2 μm filter and stored in a bottle wrapped in aluminum foil at 2–8°C.

[0366] One pH control reagent was a phosphoric acid solution (10% w / v) prepared by adding 118 mL of 85% H3PO4 to 882 mL of purified water. This solution was sterilized by autoclaving.

[0367] The other was an ammonia solution (10% v / v) prepared by adding 330 ml of 30% ammonia solution to 670 ml of purified water. This solution was considered self-sterilizing. An antifoam solution was prepared by mixing 100 ml of Sigma Antifoam 204 with 900 ml of purified water to provide a 10% concentration. The mixture was sterilized by autoclaving.

[0368] 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 autoclaving.

[0369] Microbial strains and cloning vectors S. cerevisiae strains INVSc1 (ThermoFisher, catalog no. C81000) and D5A (ATCC 200062) were used as host strains for experiments on lipid production, including phospholipids. When testing various lipid-modifying genes in yeast by transgene addition, the pYES2 plasmid was used as the base vector for gene transfer. INVSc1 and pYES2 were obtained from Invitrogen (catalog 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 contained unique HindIII and XhoI restriction enzyme sites, which were used to insert DNA fragments encoding various proteins, as described herein. The pYES2 expression vector contained the URA3 gene as a selectable marker for introduction into Ura- yeast strains, a 2μ origin of replication for high copy maintenance, and an inducible Gal1 promoter for expression of protein-coding regions in yeast. This plasmid also contained an ampicillin resistance gene for selection in E. coli during cloning experiments.

[0370] Several strains of Yarrowia lipolytica were obtained from the American Type Culture Collection (Manassas, VA, USA): strain JM23 (ATCC90812) with the genotype leu2-35 lys5-12 ura3-18 xpr2::LYS5B, strain IFP29 (ATCC20460) with the genotype leu2-35 lys5-12 ura3-18 xpr2::LYS5B, and wild-type strain W29 (Casaregola et al., 2000). E. coli DH5α and BL21 strains were obtained from ThermoFisher Scientific (catalog no. 18265017, EC0114).

[0371] Growth of S. cerevisiae and S. 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 at 29°C for 24 hours. For experiments, the inoculum culture was diluted to an optical density at 600 nm (OD600) of 0.1 in growth medium with volumes ranging from 50 to 2000 ml. Cultures were grown in polypropylene tubes for 10 ml cultures or in glass flasks for larger volumes, with a volume at least 5 times larger than the culture volume. The vessels were sealed with 3M Micropore surgical tape (Cat. No. 1530-1) and incubated in a shaker at 200 rpm for aeration at a set temperature of 29°C unless otherwise specified.

[0372] When SD-Ura medium was used, a carbon source such as 2% glycerol or raffinose (w / v) (MP Chemicals, USA, catalog number 4010022) was used. The culture was incubated overnight at 28°C with shaking for aeration. The inoculum culture was diluted with 10 ml of SD-Ura medium containing 2% (w / v) glycerol or raffinose, or other volume as specified, to obtain an initial OD600 of 0.1. The culture in a 50 ml tube or 250 ml flask was incubated in a shaker at 28°C and 200 rpm for aeration. The OD600 was checked at 15- or 30-minute time intervals. When the OD600 reached 0.3, exogenous compounds were added as potential substrates (if present) along with 2% galactose, if necessary, to induce the transgene from the GAL1 promoter.

[0373] For transformants such as pYES2 derivatives, large-scale cultures of S. cerevisiae cells were grown in 3 L volumes. These were inoculated from glycerol stocks. The seed cultures were grown in 10 ml of SD-Ura medium containing 2% (w / v) raffinose for up to 48 hours. The cells were transferred to 3 L of SD-Ura medium containing 2% (w / v) raffinose until an OD600 of 0.1 was reached and grown at 28°C with shaking at 200 rpm. The OD600 was checked at 15- and 30-minute intervals. When the OD600 reached 0.3, galactose was added to a final concentration of 2% (w / v) to induce the transgene. If desired, sodium butyrate was added to the culture to a final concentration of 2 mg / ml, or sodium stearate was added to a final concentration of 0.5 mg / L, unless otherwise noted. The flasks were then loosely closed with sterile aluminum foil. Cultures were grown in an incubator for 48 hours, after which the cells were harvested by centrifugation.

[0374] Cultures of E. coli were grown from glycerol stocks in 5 ml of LB medium for 24 hours to obtain inoculum. Cultures were diluted to an OD of 0.1 with LB medium in polypropylene tubes or glass flasks and incubated at 37°C and 200 rpm in a shaker for aeration unless otherwise specified.

[0375] Providing lipid substrates to cells For substrate feeding experiments, both yeast and bacterial inoculum cultures were diluted to an OD of 0.1 in their respective growth media containing 1% Tergitol (Sigma-Aldrich catalog no. NP40S) and incubated with shaking for a set period of time (typically 2 hours). A lipid substrate, such as a fatty acid, oil, or oil hydrolysate, was then added to the medium, and the cultures were further incubated for different periods of time. Fatty acid substrates were either dissolved in ethanol and added to the cultures at a final concentration of 0.5 mg / ml, or sodium salts of fatty acids were added as aqueous solutions.

[0376] When compounds were added to the medium as potential carbon sources or substrates (feeding assays), the following compounds were obtained from Sigma-Aldrich: ethanolamine (catalog no. 110167), choline chloride (C7017), myo-inositol (13011), butyric acid (B103500), sodium butyrate (B5887), tributyrin (W222305), or palmitic acid (76119).

[0377] 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 starting medium volume was 1 L. The initial temperature setpoint was 31 °C and remained unchanged throughout the process. The temperature controller configuration was: min: -100%; max: 100%; XP: 4%; TI: 300 s; TD: 75 s; Dead: 0.0%; cascade control using a dissolved oxygen controller; min agitation speed: 500 rpm; max agitation speed: 1200 rpm; pH control setpoint: 6.0; pH controller configuration: min: -100%, max: 100%, XP: 30%, TI: 30 s, TD: 0 s; Dead: 0.2% (equivalent to 0.02 pH units). The acid and base used for automatic pH control were 10% H3PO4 and 10% ammonia solution.

[0378] The initial dissolved oxygen setpoint was 30%. The dissolved oxygen (DO) electrode was calibrated after sterilization and after the media 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. Saturation was established with a stirrer speed of 500 rpm and an air flow rate of 0.5 L / min. Cascade control used a stirrer in a two-stage cascade followed by gas mixing to obtain oxygen enrichment of the air stream. Because yeast cells tend to float on foam, oxygen enrichment was used to reduce the air flow rate, thereby reducing foaming that could adversely affect the process. The air flow rate was constant at 0.5 L / min, with a minimum oxygen enrichment rate of 0% and a maximum oxygen enrichment rate of 50%. The dissolved oxygen controller configuration was set as follows: Dead: 0%, Min: 0% (510 rpm), Max: 100% (1425 rpm), XP: 90%, TI: 50 seconds, TD: 0 seconds.

[0379] For foam control, 10% Sigma antifoam 204 was used, and automatic chemical foam control was achieved by 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, and sensitivity: 04.

[0380] The target inoculation OD600 was 0.20, which was calculated based on the starting volume of basal medium using a secondary seed culture. In fed-batch mode, feeding of the feed solution was initiated 14 hours after inoculation at a feed flow rate of 20 ml / h. At the completion of each process, the vessels were drained and the cells were harvested by centrifugation.

[0381] 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, with the following differences: To calibrate the dissolved oxygen (DO) electrode, 0% saturation was established using pure nitrogen at a stirrer speed of 100 rpm and a nitrogen flow rate of 1 L / min. Saturation was established by setting the stirrer speed at 500 rpm and the air flow rate at 3 L / min. For cascade control, the air flow rate was constant at 3.0 L / min. The dissolved oxygen controller configuration was set as follows: HTime Stirrer: 0 min, Dead: 0.5%, Min: 34% at 510 rpm, Max: 95% at 1425 rpm, XP: 150%, TI: 100 s, TD: 0 s, HTime GasMix: 0 min, Dead: 0.5%, Min: 0% (no supplemental oxygen), Max: 50%, XP: 5%, TI: 200 s, TD: 0 s.

[0382] For 2 L fermentations, the target inoculum OD was 0.20 when using a secondary seed culture. In fed-batch mode, feeding with the feed solution began 14 hours after inoculation at 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, then cooled to 31°C before harvesting the cells by centrifugation.

[0383] Seed culture for large-scale culture 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 Erlenmeyer flask with a vented cap and plastic baffles. This was incubated at 28°C for 24 ± 2 hours 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 Erlenmeyer flask with a vented cap and plastic baffles to a starting OD600 of 0.04. The second seed culture was incubated at 28°C for 16 ± 2 hours with shaking at 200 rpm. The OD600 was measured at the end of incubation. This culture was used to inoculate large-scale fermentations.

[0384] Cell harvesting, washing, and freeze-drying Cells from small-scale cultures were harvested, for example, by centrifugation in a 50 ml tube at 4600 g for 15 minutes, and washed twice with 10 ml of MilliQ water, followed by a final wash with 1 ml of MilliQ water. For the final wash, the cell suspension was transferred to a pre-weighed 2 ml Eppendorf tube and centrifuged to measure dry cell weight. The cell pellet was lyophilized (VirTis benchtop freeze-dryer, SP Scientific) and then weighed for lipid extraction. When lipid substrates such as ARA, DGLA, γ-linolenic acid (GLA), butyrate, or palmitate 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 any remaining substrate from the outside of the cells. The pellet was then lyophilized 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, the absence of oil stains on the cell walls was confirmed by microscopic observation after staining with Bodipy. After the final wash, the pellet was transferred to a pre-weighed 2 ml Eppendorf tube, lyophilized, and weighed for lipid extraction.

[0385] Lipid extraction from yeast cells Total cellular lipids were extracted from yeast cells, such as S. cerevisiae or Y. lipolytica, using a modified method from Bligh and Dyer (1959). Approximately 50 mg of lyophilized cells were homogenized in 0.6 ml of a chloroform / methanol (2 / 1, v / v) mixture containing 0.5 g of zirconium oxide beads (catalog no. ZROB05, Next Advance, Inc., USA) in a 2 ml Eppendorf tube using a Bullet Blender Blue (Next Advance, Inc., USA) at speed 6 for 5 minutes. The mixture was then sonicated in a water bath for 5 minutes, and 0.3 ml of 0.1 M KCl was added. The mixture was shaken for 10 minutes and centrifuged at 10,000 g for 5 minutes. The lower organic phase containing lipids was transferred to a glass vial, and the upper layer containing cell debris was mixed with 0.4 ml of chloroform and centrifuged for 20 minutes to extract the remaining lipids. The lower phase was collected and combined with the first extract in a glass vial. The solvent was evaporated from the lipid samples under a stream of nitrogen gas, and the extracted lipids were resuspended in a measured volume of chloroform. If necessary, the lipid samples were stored at -20°C until further analysis.

[0386] Lipid extraction from larger biomass To extract total lipids from larger biomasses, a different cell homogenization method was used, using a larger volume of solvent unless otherwise stated. Approximately 1.5 g of lyophilized cells distributed into six 50 ml Cellstar polypropylene tubes (6 × Tube A) (catalog no. 227261, Greiner bio-one) was homogenized for 3 min with 9 ml of 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 continued for an additional 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 vigorously vortexed for 1 min. 6 ml of chloroform was added to each tube, and the mixture was 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 minutes. 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 minutes. 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.

[0387] Lipid fractionation by thin-layer chromatography To separate different types of lipids, such as TAGs, DAGs, free fatty acids, and polar lipids, e.g., phospholipids (PLs), total lipids were fractionated on thin-layer chromatography (TLC) plates (silica gel 60, catalog no. 1.05626.0001, MERCK, Darmstadt, Germany) using a solvent system of hexane:diethyl ether:acetic acid (70 / 30 / 1 v / v / v). Samples of lipid standards, such as 18-6A (Nu-Chek Prep Inc, USA), containing TAGs, DAGs, FFAs, and MAGs, were run in adjacent lanes to identify the various lipid spots. To distinguish between various TAGs containing short-chain fatty acids (SCFAs), a standard containing triheptadecanoin (Nu-Chek USA, Catalog No. T-155), a triglyceride mixture C2–C10 (TAG 6:0), triacetin (TAG 12:0), tricaprylin (TAG 18:0), and tridecanoin (TAG 30:0) (Sigma-Aldrich, Catalog No. 17810-1AMP-S) were run in adjacent lanes to identify TAG lipid spots. After chromatography, the plate was sprayed with a solution of primulin (Cat. No. 206865, Sigma, Taufkirchen, Germany) 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 containing the lipids was scraped off from each spot and transferred to a tube. The lipid fraction was extracted from the silica for derivatization using either methylation, propylation, or butylation.

[0388] Large-scale fractionation of PL and AG from total lipids PL and TAG were fractionated from approximately 100 mg of total lipids by loading the lipids onto the 18 cm line of each of eight TLC plates (silica gel 60; catalog number 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). Aliquots of lipid standards (18-6A; NuChek Inc., USA) containing TAG, DAG, FFA, and MAG were analyzed in parallel to aid in the identification of lipid bands. 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 a Falcon tube. The lipid / silica sample was extracted with a mixture of 6 ml chloroform and 3 ml methanol, mixed vigorously for 5 minutes, and then 3 ml water was added and mixed for another 5 minutes. After centrifugation at 3,000 g for 5 minutes, the lower organic phase was transferred to a new tube. After centrifugation at 3,000 rcf for 5 minutes, the lower phase was transferred to a Falcon tube. The upper phase was mixed with 5 ml of chloroform for 5 minutes to extract any 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, catalog no. CINY-02) to remove any particulates. The fatty acid composition and quantity of each PL and TAG fraction were determined by FAME preparation and GC analysis. Such preparations were used, for example, to separate different polar lipid classes, such as PC, PE, PI, and PS, or in Maillard reactions for aroma testing or to detect volatile compounds as reaction products.

[0389] Derivatization of lipids to fatty acid methyl esters (FAMEs) For GC analysis, fatty acid methyl esters (FAMEs) were prepared from total extracted lipids or purified TAG or PL fractions by treatment with 0.7 ml of 1 N methanolic HCl (Sigma-Aldrich, Cat. No. 90964) in 2 ml glass vials with PTFE-lined screw caps at 80°C for 2 h. 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 and then used as an internal standard for quantification. After cooling the vials, 0.3 ml of 0.9% NaCl (w / v) and 0.1 ml of hexane were added, and the mixture was vortex-mixed for 5 min. The mixture was centrifuged at 1700 x g for 5 min, and the upper hexane phase containing FAMEs was analyzed by GC.

[0390] 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 i.d., 0.25 μm film thickness), a 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) at an oven temperature of 150 °C. The column temperature was programmed as follows: 150 °C for 1 min, ramped 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 to 240 °C, and the detector temperature was set to 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, Nu-Chek Inc., USA). Peaks were integrated using Agilent Technologies ChemStation software (Rev. B.04.03 (16), Palo Alto, California, USA) based on the response of known amounts of the external standard GLC-411 (Nu-Chek) and the internal standard C17:0-ME. The resulting data represent the fatty acid composition on a weight basis, representing the percentage (wt%) of each fatty acid in 100% total fatty acid content. These weight-based percentages can be easily converted to molar percentages (mol%) based on the known molecular weights of each fatty acid.

[0391] 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 vortex mixed for 5 min and centrifuged at 1700 g for 5 min. The upper organic phase was collected for GC analysis.

[0392] Derivatization of lipids 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 a 1N HCl / ethanol solution at 80°C for 2 hours. After the sample was cooled to room temperature, 100 μl of hexane was added to the mixture. The mixture was vortex mixed for 5 minutes, centrifuged at 1700 g for 5 minutes, 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, but the rest was treated similarly.

[0393] Peak body by GC-MS The identity of unknown or uncertain peaks in the GC-FID chromatogram was confirmed by gas chromatography-mass spectrometry (GC-MS). Samples were analyzed by GC-MS operating in electron ionization mode at 70 eV to confirm peak identity and identify any extraneous peaks that may correspond to possible contamination, degradation products, or reagent signals. A Shimadzu GC-MS QP2010 Plus (Shimadzu Corporation, Japan) system connected to an HTX-Pal liquid autosampler was used with the following parameters: a 1 or 2 μl injection volume 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. The 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 achieved using a Stabilwax or Stabilwax-DA (Restek / Shimadzu) capillary column (30 m × 0.25 mm diameter, 0.25 μm film thickness) using He as the carrier gas at 30 cm / s. Mass spectral correlation was performed using the NIST library, retention indices, and matching retention times of available standards. Identified SCFAs were set as present if the signal-to-noise ratio was greater than 10:1. Instrument and procedural blanks were run for quality control purposes.

[0394] recombinant DNA technology Derivatives of pYES2 with single genes inserted for testing in S. cerevisiae were generated by inserting the protein-coding region into the unique HindIII and XhoI sites or other restriction enzyme sites within the plasmid as needed using standard cloning methods. The E. coli strain DH5α was used for cloning, plasmid propagation, and DNA preparation according to standard methods.

[0395] The Golden Gate (GG) method (Larroude et al., 2018) was used to create multigene constructs for testing in S. cerevisiae or Y. lipolytica because it allows for the rapid and efficient combinatorial assembly of multiple expression cassettes within a single vector. GG DNA segments and donor vectors (also known as L0 vectors) were obtained from Addgene, USA, according to Celinska et al. (2017) and Larroude et al. (2018). The DNA segments included promoters (GGE146, GGE151, and GGE294), terminators (GGE014, GGE015, GGE080, GGE020, and GGE021), and the backbone assembly vector (destination vector) was GGE114.

[0396] 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 at Twist Bioscience or GeneArt (ThermoFisher, USA) or in-house. Internal BsaI restriction enzyme sites were avoided in the codon-optimized nucleotide sequences of the protein-coding regions because BsaI sites were used in the GG assembly method. NotI restriction enzyme sites were also avoided within the nucleotide sequences because NotI was used to linearize the gene constructs for transformation of Y. lipolytica. When inserting one, two, or three genes into a single vector, individual components were designed according to Table 3, with 4-nucleotide overhangs immediately 5' of each translation initiation codon (ATG) and 3' of the translation termination codon, with each 4-nucleotide overhang depending on the component's location within the backbone vector GGE114. An external BsaI site with an appropriate 4-nt overhang was added to the 5' end of each DNA strand. The 4-nt overhang sequence used between consecutive components can be varied, as known in the art, using any 4-nucleotide sequence at each site to allow ligation between consecutive components.

[0397] To avoid false positives when performing GG reactions using the GG backbone vector GGE114 with an ampicillin selectable marker gene, the protein coding region was synthesized in a cloning vector with a kanamycin selectable marker gene. Escherichia coli DH5α strain was used for cloning and plasmid propagation according to standard methods. Antibiotics were used appropriately to select for transformed cells; for example, ampicillin was added at 100 μg / mL for the selection of constructs with an ampicillin selectable marker gene.

[0398] 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). In addition to the bacterial replicon, the GGE114 vector contains a ZETA sequence and a URA3 selectable marker gene for more efficient integration into the Y. lipolytica genome. This combination was used to reduce the number of fragments to be assembled into the backbone vector pYES2, which contains a 2µ origin for high-copy maintenance. In this case, the RFP was located between the URA3 marker gene and ZETAdown. In the presence of the BsaI enzyme, the RFP was released, allowing the insertion of one, two, or three transcription units (TUs; promoter-protein-coding region-terminator). [Table 3]

[0399] The GG assembly reaction mix contained equimolar amounts (50 ng) of a GG backbone vector such as GGE114 and other DNA components (donor vector) in a final volume of 7.5 μl by adding 0.75 μl of 10x T4 ligase buffer, 0.75 μl of 10x BSA (bovine serum albumin), 0.75 μl of BsaI HF-V2 (NEB), and 0.5 μl of T4 ligase (NEB). The reaction mixture was incubated at 37°C for 3 minutes, followed by 25 cycles of 16°C for 4 minutes, and then one cycle of 50°C for 5 minutes and 80°C for 5 minutes. A 2-3 μl sample was transformed into competent cells of E. coli DH5α using standard methods. Colonies lacking RFP were confirmed to contain the desired gene insert by colony PCR using appropriate primers and verified by restriction digestion. Glycerol stocks were made and stored at -80°C.

[0400] Transformation of S. cerevisiae A rapid method was used to introduce pYES2-based gene constructs into S. cerevisiae without the use of competent cells. A loopful of S. cerevisiae cells was scraped from a new plate and resuspended in 100 μl of transformation buffer (Sigma-Aldrich, catalog no. T0809). Approximately 1 μg of plasmid DNA containing 10 μl of 10 mg / ml salmon testis DNA, boiled for 5 minutes before use, was added to the cell suspension along with 600 μl of plating buffer (Sigma-Aldrich, catalog no. P8966) and mixed thoroughly. The mixture was incubated at room temperature in a rotor wheel at the lowest speed for 16 hours. The mixture was then heat-shocked at 42°C for 15 minutes, centrifuged at 3500 rpm for 3 minutes, and the cell pellet was resuspended in 200 μl of sterile water. For selection of transformants, aliquots of up to 100 μl were plated onto synthetic dropout selection medium lacking uracil (SD-URA, Sigma-Aldrich, catalog no. Y1501). Plates were incubated at 28°C for 3 days or until colonies appeared. Two or more colonies from each plate were picked and tested for the presence of the gene construct by colony PCR to identify transformants.

[0401] Transformation of Y. lipolytica for integration of expression cassettes 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 or other desired recipient strain, prepared using the Frozen-EZ Yeast Transformation II Kit (Zymo Research, California, USA). Briefly, 5 μl (2 μg) of the NotI-digested, linearized expression vector was mixed with 50 μl of competent cells and 500 μl of the kit's EZ3 solution and mixed thoroughly. A negative control transformation contained competent cells without any DNA from the gene construct. The mixture was incubated at 28°C for 2 hours, and then 100 μl was spread onto SD-Ura plates or other selective plates, such as antibiotic-containing media, as needed. The plates were incubated at 28°C for 2 days. When the recipient strain was an auxotroph lacking a functional URA3 gene, only transformants that received the vector carrying the URA gene grew on these plates. W. lipolytica transformation yielded many colonies. Ura + Alternatively, antibiotic-resistant colonies were picked from the selection plates and confirmed to be transformed by colony PCR for a phenotype corresponding to the introduced gene construct and the desired genetic modification.

[0402] Gene expression analysis Transgene expression was analyzed using a DNase RQ1 kit (Promega catalog no. M6101) and Qiagen columns (Qiagen RNAse-free DNAse) to purify RNA from cells, and oligo-dT primers (200–500 ng), dNTPs (10 mM), Superscript III reverse transcriptase, and 0.1 M DTT for reverse transcription using standard methods.

[0403] Example 2. Extraction of lipids from microorganisms Lipid extraction from yeasts, such as S. cerevisiae and Y. lipolytica, is more challenging due to the rigid cell walls of these microorganisms. Various methods for cell disruption and lipid extraction from yeast have been described in the literature, including mechanical, enzymatic, chemical, osmotic shock, and microwave cell disruption (Hein and Hayen, 2012). Chisti and Moo-Young (1986) reviewed mechanical, chemical, and enzymatic methods of microbial cell disruption, as well as cell lysis by osmotic shock. Hegel et al. (2011) described lipid extraction from yeast using supercritical carbon dioxide. Peter et al. (2017) reported cell disruption and homogenization of Schizosaccharomyces pombe cells in a water / methanol solvent mixture using zirconium oxide beads, a bullet blender, and a water bath sonicator.

[0404] To carry out the studies described in the following examples, a simple, high-throughput, small-scale lipid extraction method was desired. Therefore, we tested several methods and variations for extracting lipids from S. cerevisiae and Y. lipolytica, particularly several methods for cell wall disruption and homogenization of cellular material with organic solvents to extract lipids and test extraction efficiency.

[0405] Experiment 1 - Extraction of lipids from S. cerevisiae In initial experiments aimed at testing the efficiency of lipid extraction from yeast cells using sonication for cell disruption in the presence of KCl solution or methanol, S. cerevisiae INVSc1 strain was grown for 3 days in 5 ml of YPD medium. Cells were harvested by centrifugation, washed with water, and lyophilized as described in Example 1. Approximately 25 mg of identical dried cell pellets in 2 ml tubes were processed in four ways:

[0406] 1A. Homogenization in KCl solution, lipid extraction using chloroform / methanol. 1B. Homogenization in KCl solution, sonication for 5 min, lipid extraction using chloroform / methanol. 2A. Homogenization in methanol, lipid extraction using chloroform / methanol / KCl. 2B. Homogenization in methanol, sonication for 5 min, lipid extraction using chloroform / methanol / KCl.

[0407] In Method 1A, 0.3 ml of 1 M KCl was added to the tube and mixed at speed 8 for 3 minutes using zirconium beads (catalog number ZROB05, Next Advance, Inc., USA) and a Bullet Blender Blue (Next Advance, Inc., USA). Subsequently, 0.4 ml of methanol and 0.8 ml of chloroform were added to disrupt the cells. The mixture was shaken for 5 minutes and centrifuged at 10,000 g for 5 minutes. The lower phase containing lipids was transferred to a glass vial. The only difference in Method 1B was an additional step of sonicating the mixture for 5 minutes using a water bath sonicator (Bransonic M2800H-E, Branson Ultrasonic Corporation, USA) after the addition of methanol but before the addition of chloroform. In Method 2A, 0.3 ml of methanol was added to the tube containing the yeast cells and zirconium beads, homogenized using a Bullet Blender, and then 0.3 ml of 1 M KCl, 0.1 ml of methanol, and 0.8 ml of chloroform were added. The mixture was shaken and centrifuged, and the lower phase was collected as before. Method 2B was the same as 2A, except that cells were disrupted in a bullet blender followed by sonication.

[0408] For each sample, the solvent was evaporated from the lipid sample under a stream of nitrogen gas, and the extracted lipids were dissolved in a measured amount of chloroform. To measure the amount of extracted lipids in each sample, a measured aliquot of the lipids in chloroform was transferred to a GC vial with a screw cap lined with PTFE. After evaporating the chloroform under nitrogen gas, a known amount of triheptadecanoin (Nu-Chek Prep, Inc., Cat. No. T-155, Waterville, MN, USA) was added to the vial. The fatty acids in each lipid sample were converted to FAMEs and measured by GC as described in Example 1. The peak areas were integrated and compared with a known amount of heptadecanoin to calculate the weight of fatty acids in the extracted lipids.

[0409] As a control to measure the total amount of lipids present in the cells before extraction, all lipids in duplicate cell pellets were converted to FAMEs by direct methylation with methanolic HCl and triheptadecanoin and analyzed by GC. The average of the two controls gave a total fatty acid content that was considered 100% of the cellular fatty acid content. Comparison of the total fatty acid content in the extracted lipids with the cellular fatty acid content provided the extraction efficiency of the four methods tested.

[0410] Table 4 provides data from this experiment. Of the four methods tested in this experiment, method 2B provided the most efficient lipid extraction from lyophilized S. cerevisiae cells, yielding 62.4% of the total cellular fatty acid content. Method 2B involved cell disruption in methanol using zirconium beads and a bullet blender, followed by sonication. Meanwhile, method 1B, which involved cell disruption by homogenization in a KCl solution and sonication, yielded a lipid extraction efficiency of 26.2%. Methods 1A and 2A did not use sonication, resulting in lower lipid extraction efficiencies.

[0411] Experiment 2 Another experiment was conducted to estimate lipid extraction efficiency using larger cell samples and compare it with the method of disrupting cells in a chloroform / methanol (2 / 1, v / v) mixture. Approximately 47 mg of dried cell pellet and 0.5 g of zirconium beads were transferred to a 2 ml Eppendorf tube. In Method 3A, lipid extraction efficiency was tested using sonication. To do this, 0.4 ml of methanol was added to the tube, and the mixture was sonicated in a water bath at 40 °C for 10 min. Next, 0.3 ml of 1 M KCl and 0.8 ml of chloroform were added to the tube, and the mixture was vortex-mixed for 5 min, followed by centrifugation at 10,000 g for 5 min. The lower phase was collected in a glass vial. A second extraction was performed by adding 0.8 ml of chloroform, vortex-mixing the mixture for 5 min, and then centrifuging to collect the lower phase, which was then combined with the first extract in the glass vial. Method 3B was the same as Method 3B, except that both zirconium beads and a bullet blender were used at speed 8 for 5 minutes, followed by 10 minutes of sonication to disrupt cells in 0.4 ml of methanol. Method 4 tested cell disruption in a mixture of chloroform / methanol (2 / 1, v / v) instead of methanol. Extracted lipids were processed and quantified as in Experiment 1. As in Experiment 1, total fatty acid content was obtained by direct methylation of fatty acids in cell samples and was considered 100%.

[0412] The data are shown in Table 4. When cells were disrupted in methanol by sonication (Method 3A), 27% of the total lipid content was extracted from the cells. When cells were disrupted in methanol using a bullet blender followed by sonication, 46.4% of the total lipids were obtained. On the other hand, when cells were disrupted in a mixture of chloroform / methanol (2 / 1, v / v) followed by sonication, the lowest level of lipids was extracted from S. cerevisiae.

[0413] Experiment 3 In a third experiment, we compared the efficiency of lipid extraction from S. cerevisiae cells using glass beads, zirconium beads, or metal balls to homogenize cells in methanol and using a bead beater or vortex mixer. Cells were obtained from 10 ml cultures as in the previous experiment, and the same cell pellets were processed. Glass beads, zirconium beads, or metal balls were added to the tube and mixed by vortex or using a bullet blender as follows:

[0414] Method 5: 0.3 ml of methanol, 0.5 g of glass beads (catalog number G8772, Sigma), and two 1 mm metal balls were added to the tube containing the cell pellet and vortex mixed for 10 minutes using a Vibramax.

[0415] Method 6: 0.3 ml of methanol, 0.5 g of zirconium beads (catalog number ZROB05, Next Advance, Inc., USA), and two 1 mm metal balls were added to the second tube containing the cells and vortex mixed for 10 minutes.

[0416] Method 7: 0.3 ml of methanol and 0.5 g of zirconium beads were added to the third tube containing the cell pellet and vortex mixed for 10 minutes.

[0417] Method 8: 0.3 ml of methanol and 0.5 g of zirconium beads were added to the fourth tube containing the cell pellet and shaken at 25 rpm / s for 3 min in a TissueLyser II (Qiagen Inc., Germantown, MD, USA).

[0418] After homogenization, 0.4 ml of 1 M KCl, 0.1 ml of methanol, and 0.8 ml of chloroform were added to each tube, and the mixtures were vortexed for an additional 5 minutes. The mixtures were centrifuged at 10,000 g for 5 minutes, and the lower chloroform phase was transferred to a glass vial. As in the previous experiment, the extracted lipid samples were dried, and the fatty acids were converted to FAMEs and quantified by GC.

[0419] During the lipid extraction process, cell debris accumulated at the interface after centrifugation of the mixture. To determine the total lipid content by measuring the lipids remaining in the cell debris, the cell debris was dried in a freeze dryer. A known amount of triheptadecanoin was added, and the fatty acids were converted to FAMEs by incubation at 80°C for 2 hours using 0.7 ml of methanolic HCl. FAMEs were quantified by GC as before.

[0420] The data are shown in Table 4. The most efficient extraction was Method 8, which used zirconium beads with a bead beater, extracting 66.5% of the total fatty acid content. Methods 5-7 extracted less lipid than Method 8 (Table 4). The efficiency of lipid extraction using a bead beater (Method 8) was similar to Methods 2B and 3B, which involve cell disruption using a bullet blender and sonication. The fatty acid composition of the lipids remaining in the cell debris after the initial extraction was the same as that of the extracted lipids. [Table 4]

[0421] Experiment 4. Extraction of Lipids from Yersinia lipolytica For many analyses in Y. lipolytica, where the primary goal was to determine the cellular fatty acid composition and maximum extraction efficiency was not necessary, we decided to routinely use a simpler method that was suitable for high sample throughput yet provided sufficient extracted lipids. This conclusion was based on the observation made in the above experiments that the fatty acid composition of the extracted lipids was identical to that of the residual lipids remaining in the cell debris (Table 4) and thus representative of the cellular fatty acid content. Briefly, in this method, described in Example 1, the dried cell pellet was homogenized and disrupted using a bullet blender in a chloroform / methanol (2 / 1, v / v) solution containing zirconium beads, followed by sonication in a water bath sonicator and mixing for 20 minutes. After the addition of KCl solution, the mixture was vortex-mixed for 10 minutes and centrifuged to separate the phases. The lower phase was collected. The lipids remaining in the upper phase were extracted using another volume of chloroform, and the extracts were combined and dried.

[0422] Another experiment was conducted to determine whether heat treatment of W. lipolytica cells at 105°C for 5 minutes immediately after harvesting the culture by centrifugation could kill the cells and inactivate all intracellular or extracellular lipases. Furthermore, cell aliquots were either lyophilized or not lyophilized before lipid extraction. When lipid extracts from wild-type W29 cells were analyzed by TLC, it was observed that cells that had been heat-treated without drying produced less free fatty acids (FFA) than cells that had been dried, suggesting that heat treatment was effective. Heat treatment itself did not affect the lipid or fatty acid content of the cells. Lipid extraction from undried cells was at least as efficient as that from freeze-dried cells.

[0423] Example 3. Content and composition of lipids derived from microorganisms We wanted to determine the content and composition of total lipid content from various microorganisms, including triacylglycerols (TAGs) and polar lipids, as a baseline, and then genetically modify the microorganisms to alter lipid content and composition.

[0424] Microbial growth and lipid extraction Five widely used strains of three different species were selected to determine the amount and fatty acid composition of total lipids, including polar lipids and TAGs, within microbial cells during the growth cycle. These were E. coli strains DH5α and BL21, the oleaginous wild-type Y. lipolytica strain W29, and S. cerevisiae strains INVSc1 and D5A. These species were selected due to the availability of genetic tools and processes for genetic engineering and the depth of information available on lipid synthesis and metabolism in these species. Strain D5A was selected as an oleaginous strain of S. cerevisiae (He et al., 2018). These microorganisms were cultured for up to 7 days, with samples removed and analyzed at various time points. Inoculum cultures were prepared by growing cells overnight in LB medium for E. coli or YPD or SD+Ura medium for yeast. Samples of these cultures were diluted with 200 ml of the same growth medium in 1 L bottles to an initial OD600 of 0.1. The mouth of each bottle was covered with microporous tape, and the cultures were aerated by shaking. E. coli cells were incubated at 37°C and 250 rpm in a shaker. Yeast cells were grown in YPD medium containing 2% glucose as a carbon source and incubated at 28°C with shaking at 200 rpm. 10 ml samples were taken from each culture at 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days. Cells were harvested from the cultures by centrifugation at 3,400 g for 10 minutes and washed twice with 3 ml of deionized water each time and once with 1.5 ml of deionized water. Cells were transferred to pre-weighed 2 ml tubes and lyophilized for 24 hours. The tubes were then reweighed, and the dry cell weight was calculated before lipid extraction.

[0425] As described in Example 1, total cellular lipids were extracted using a bullet blender in the presence of zirconium beads with 0.6 ml of chloroform / methanol (2 / 1, v / v) as the extraction solvent, followed by sonication in a 40°C water bath. The homogenate was mixed with 0.3 ml of 0.1 M KCl for 10 minutes, and the mixture was then centrifuged at 10,000 g for 5 minutes. The lower phase containing lipids was transferred to a glass vial. The remaining lipids were extracted from the upper phase containing cell debris with 0.6 ml of chloroform for 20 minutes, and the mixture was centrifuged and the lower phase collected as before. The solvent was evaporated from the combined lower phases under a stream of nitrogen gas, and the extracted lipids were resuspended in a predetermined volume of chloroform. Aliquots of lipids extracted from 20 mg of dry cell weight were fractionated on TLC plates using a mixture of hexane / diethyl ether / acetic acid (70 / 30 / 1, v / v / v) to separate TAG lipids and polar lipids as described in Example 1. Again, as described in Example 1, the fatty acid composition of lipids and polar lipid spots from the TAG was determined by GC of FAMEs generated from the lipids.

[0426] First experiments with S. cerevisiae In the first experiment, lipids were extracted from cultured cells of S. cerevisiae strain INVSc1 after growth for 1, 2, 3, or 4 days in YPD and SD+Ura media. Data, including the extracted lipid yield as a percentage of dry cell weight (dcw), are shown in Table 5. The recovery efficiency of TAGs and polar lipids in the TLC fractions was not measured. It was noted that the amount of TAGs produced by INVSc1 cells was low when grown in YPD medium but high when grown in SD+Ura medium. Polar lipid yields ranged from 0.63% to 1.15% on a dry cell weight basis, but this method was not optimized for efficient extraction. Regarding fatty acid composition, C16:1Δ9 was the most abundant fatty acid in both fractions, accounting for 47–67%. Oleic acid (C18:1Δ9) and palmitic acid (C16:0) were the other major fatty acids present, as were low levels of stearic acid (C18:0); linoleic acid (LA, C18:2Δ9,12) was absent. These data were consistent with published reports (e.g., Itoh and Kaneko, 1974; Stukey et al., 1989; Kamisaka et al., 2015) reporting the presence of 40–55% C16:1, 30–35% C18:1Δ9, and small amounts of C16:0 and C18:0. These four fatty acids constitute nearly all of the fatty acid content of many wild-type S. cerevisiae strains. Wild-type strains such as INVSc1 contain only one fatty acid desaturase, a Δ9-desaturase encoded by the OLE1 gene that produces the monounsaturated palmitoleic and oleic acids ( Stuckey et al., 1989 ).

[0427] Like S. cerevisiae, wild-type fission yeast S. pombe is unable to synthesize LA and other polyunsaturated fatty acids (Ratledge and Evans 1989; Holic et al. 2012). In contrast, other wild-type yeasts, such as S. kluyveri and K. lactis, possess Δ12- and Δ15-desaturases and can produce LA and ALA.

[0428] Fatty acid composition in Escherichia coli, Y. lipolytica, and S. cerevisiae after growth for up to 7 days Experiments were performed with E. coli, S. cerevisiae, and Y. lipolytica. Cultures were sampled daily for up to 7 days. Growth curves for the two S. cerevisiae strains are shown in Figure 1, which shows OD600 and dry cell weight over 7 days of culture. The amount and fatty acid composition of lipids were determined for both the polar lipid and TAG fractions of each strain at each time point. Data are presented in Table 6 for the two E. coli strains, Table 7 for Y. lipolytica W29, and Table 8 for S. cerevisiae INVSc1 and D5A strains. The identity of the fatty acid C15:0 (pentadecanoic acid) was confirmed by GC-MS.

[0429] The fatty acid composition of polar lipids from E. coli BL21 strain was similar to that reported by Kanemasa et al. (1967) and by Marr and Ingraham (1962) for other wild-type E. coli strains. As with many other bacteria, polar lipids from E. coli contain four types of fatty acids: straight-chain saturated fatty acids including C12:0, C14:0, C15:0, and C16:0; straight-chain monounsaturated fatty acids including C16:1Δ9 (cis-palmitoleic acid) and C18:1Δ11 (cis-vaccenic acid); branched-chain fatty acids; and C17:0c. * (cis-9,10-methylenehexadecenoic acid) and C19:0c * Cyclopropane fatty acids, including cis-11,12-methyleneoctadecenoic acid (Hildebrand and Law, 1964), were reported in E. coli BL21 by Oldham et al. (2001). All unsaturated fatty acids found in wild-type E. coli are monoenoic in the cis conformation, except for oleic acid (Cronan and Vagelos, 1972). All four fatty acids were observed in lipids extracted from BL21, consisting of approximately 31–36% C18:1Δ11 and approximately 7–10% C16:1Δ9, as well as 30–35% saturated fatty acid C16:0 (palmitic acid), 10–20% cyclopropane fatty acid C17:0c, and 10–20% C18:1Δ11. * , and 1-5% C19:0c *These latter two fatty acids are unique to bacterial lipids and are rarely found in animal fats or yeast lipids. They are produced from the corresponding monoenes C16:1Δ9 and C18:1Δ11 by the activity of cyclopropane fatty acid synthase (CPFAS). Another difference observed in animal fats is that polyunsaturated fatty acids such as LA are absent from wild-type E. coli lipids, whereas this was observed in BL21 and DH5α. Furthermore, oleic acid (C18:1Δ9) was not observed in E. coli polar lipids, but is present at significant levels in animal and plant lipids.

[0430] The DH5α strain exhibited a significantly different fatty acid composition from BL21 in terms of the amounts of several fatty acids in its polar lipids: significantly less C18:1Δ11 (approximately 3–8%), less C16:1Δ9, but more C16:0, and significantly more C15:0 and cyclopropane fatty acids. In DH5α, palmitic acid accounted for nearly half of the total fatty acid content, which was reported to be located almost exclusively at the sn-1 position of phospholipids (Cronan and Vagelos, 1972). Hildebrand and Law (1964) reported the presence of cyclopropane fatty acids in E. coli, and these were also observed here in DH5α. As shown in Table 9, pentadecanoic acid, nonadecanoic acid (C19:0), and cyclopropane fatty acids were observed in the polar lipid fraction. The reduced levels of C16:1 and C18:1Δ11 in DH5α compared to BL21 were accompanied by increased amounts of C14:0, C15:0, C16:0, and cyclopropane fatty acids. In both E. coli strains, stearic acid (C18:0) was present in the lipids at less than 2%. The maximum amount of polar lipids was observed at approximately 2.7% DCW on day 2 of culture.

[0431] The fatty acid composition of Y. lipolytica (Table 7) differed significantly from that of E. coli and S. cerevisiae. A broader range of fatty acids was observed in Y. lipolytica lipids, including polyunsaturated fatty acids such as LA and longer-chain saturated fatty acids with 20, 22, or 24 carbon atoms, C20:0, C22:0, and C24:0 (VLC-SFA), all of which were present in the TAG fraction. C24:0 was present in the TAG fraction at levels between 3% and 9% at most time points. The peak for this fatty acid in the GC chromatogram coincided with that of a C24:0 standard, and GC-MS confirmed that the main component of the peak was C24:0. While C24:0 was commonly present, C20:0 and C22:0 were either absent by weight or present at low levels (<0.5%) of the total fatty acid content in the polar lipid fraction. Although Y. lipolytica is an oleaginous microorganism, the growth conditions in this experiment using rich YPD medium were not favorable for high-level TAG production, resulting in TAG production of less than approximately 1% on a dry cell weight basis. TAG continued to accumulate at low levels over 7 days. The highest level of polar lipids was observed on day 2 of culture. Palmitic acid, palmitoleic acid, oleic acid, and linoleic acid were the major fatty acids in Y. lipolytica. Polar lipids also contained low levels of short-, medium-, and long-chain saturated and monounsaturated fatty acids, along with odd-chain fatty acids such as pentadecanoic acid and heptadecenoic acid (Table 7). The main peak of pentadecanoic acid was also confirmed by GC-MS. The fatty acid composition was similar to that reported by Carsanba et al. (2020). However, we were unaware of any previous reports of the presence of C24:0 in Y. lipolytica TAG at the levels observed here.

[0432] The polar lipid and TAG fractions of Y. lipolytica showed significant differences in the amounts of some fatty acids. In general, polar lipids contained higher levels of LA and palmitoleic acid (C16:1) than TAG, while TAG was enriched in palmitic acid, stearic acid, and lignoceric acid (C24:0). Notably, TAG contained significantly higher levels of the saturated fatty acid stearic acid (approximately 4–12%) compared with less than 1% in polar lipids, as well as higher amounts of saturated C20, C22, and C24 fatty acids. Y. lipolytica polar lipids were easily distinguishable from E. coli lipids; for example, the former possessed C18:1Δ9 (oleic acid) rather than C18:1Δ11 (vaccenic acid) as the predominant monounsaturated fatty acid. As noted above, E. coli lipids lacked oleic acid.

[0433] The polar lipid and TAG fractions of S. cerevisiae INVSc1 and D5A mainly contained four fatty acids: the monounsaturated palmitoleic acid (C16:1Δ9) and oleic acid (C18:1Δ9), and the saturated palmitic acid (C16:0) and stearic acid (C18:0). These data were consistent with a published report (He et al., 20...

Claims

1. An extracted microbial lipid comprising a total fatty acid content comprising a total saturated fatty acid content of saturated fatty acids (SFA) and a total monounsaturated fatty acid content of monounsaturated fatty acids (MUFA), wherein at least a portion of the total fatty acid content comprising at least a portion of SFA and at least a portion of MUFA is esterified in the form of triacylglycerol (TAG), and thus the extracted microbial lipid has a total TAG content, and, herein, (i) The total SFA content of the extracted microbial lipid comprises stearic acid (C18:0), palmitic acid (C16:0), myristic acid (C14:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0), and thus at least 50% by weight of the fatty acids of the total fatty acid content of the extracted microbial lipid are SFA, (ii) 20% to 85% by weight of the total fatty acid content is stearic acid, (iii) The total MUFA content of the extracted microbial lipid comprises oleic acid (C18:1Δ9) and palmitoleic acid (C16:1Δ9), and optionally C16:1Δ7 and / or C17:1, (iv) The total fatty acid (TFA) content of the extracted microbial lipid is lacking in polyunsaturated fatty acids (PUFA) or comprises a PUFA content comprising linoleic acid (C18:2Δ9,12), wherein the PUFA content is less than 5% by weight of the total fatty acid content, (v) The extracted microbial lipid either comprises polar lipids including phospholipids or is lacking in polar lipids, (vi) The extracted microbial lipid is solid at 25 °C, And optionally, (vii) The extracted microbial lipid is obtained from microbial cells, wherein the microbial cells are yeast cells, and optionally, the yeast cells are Yarrowia lipolytica, the microbial lipid. Claim 2: (i) The TFA content of the lipid and / or the TAG content, or both, is less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.2%, or 0.2% to 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2% by weight of linoleic acid (LA), or LA is essentially absent from the TFA content of the lipid and / or the TAG content; (ii) The TFA content of the lipid and / or the TAG content, or both, is less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.2%, or 0.2% to 5%, 0.2% to 4%, 0.2% to 3%, 0.2% to 2%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2% by weight of PUFA, or PUFA is essentially absent from the TFA content of the lipid and / or the TAG content; and / or, (iii) The TFA content of the lipid and / or the TFA content of the TAG contains at least 10%, at least 20%, at least 30%, 10% to 50%, 10% to 40%, 10% to 35%, 10% to 30%, or 20% to 35% by weight of oleic acid, The extracted microbial lipid according to claim 1. Claim 3: The lipid is (i) contains one or more of a higher total SFA content, C18:0 content, C20:0 content, and C22:0 content in the TFA content and / or TAG content of the lipid as compared to the corresponding extracted microbial lipid obtained from the corresponding microorganism lacking at least one genetic modification; (ii) extracted from a microorganism containing at least one genetic modification, and the L / S-SFA ratio of the TFA content or TAG content or both of said lipid is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or about 3 to about 10-fold greater compared to the corresponding extracted microbial lipid obtained from a corresponding microorganism lacking the at least one genetic modification; (iii) extracted from a microorganism containing at least one genetic modification, and containing polar lipids including phospholipids, wherein at least two, preferably three, or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) in said extracted lipid have a greater amount of SFA than the corresponding extracted lipid obtained from a corresponding microorganism lacking the at least one genetic modification; or (iv) (a) an exogenous polynucleotide encoding FATA fatty acyl thioesterase, (b) at least one exogenous polynucleotide encoding at least one fatty acid acyltransferase, preferably at least diacylglycerol acyltransferase (DGAT), and (c) a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase, preferably a genetic modification of the endogenous gene encoding Δ12 desaturase, more preferably a null mutation of the endogenous gene encoding Δ12 desaturase, most preferably a null mutation of the FAD2 gene, extracted from a microorganism containing a genetic modification comprising where each polynucleotide is operably linked to one or more promoters capable of directing the expression of the polynucleotide within said microbial cell, The extracted microbial lipid according to claim 1.

4. The TFA content and / or TAG content of the lipid contains 30 wt% to 90 wt%, 30 wt% to 80 wt%, 30 wt% to 70 wt%, 30 wt% to 60 wt%, or 30 wt% to 50 wt% of SFA, Optionally, when the total SFA content is at least 60 wt% of the TFA content of the lipid and / or the TFA content of the TAG, the stearic acid content is at least 40 wt%, The extracted microbial lipid according to claim 1.

5. The ratio (L / S-SFA ratio) of the total saturated fatty acids containing 18 or more carbons to the total saturated fatty acids containing 16 or fewer carbons in the TFA content of the lipid and / or the TAG content of the lipid and / or both is at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or about 3 to about 10; and / or, The content of C20:0 fatty acid is at least about 1 wt%, at least about 1.5 wt%, at least about 2 wt%, at least about 2.5 wt%, or at least about 3 wt% of the TFA content of the extracted lipid and / or the TAG content of the extracted lipid and / or both, the content of C22:0 fatty acid is at least about 1 wt%, at least about 1.5 wt%, at least about 2 wt%, at least about 2.5 wt%, at least about 3 wt%, or at least about 3.5 wt%, and / or the content of C24:0 fatty acid is at least about 1 wt%, at least about 1.5 wt%, at least about 2 wt%, at least about 2.5 wt%, at least about 3 wt%, at least about 3.5 wt%, or at least 4 wt%, optionally, C20:0, C22:0, and C24:0 fatty acids contain at least 95%, at least 97%, or at least 99 wt% of the TFA content of the extracted lipid and / or the TFA content of the lipid TAG and / or both, and these are at least 20 or more carbon atoms, The extracted microbial lipid according to claim 1. **Claim 6**: The microbial lipid contains polar lipids, and the TFA content of the polar lipids has one or more characteristics defined in claims 2 to 5 for the TFA content or TAG content of the extracted lipid. The extracted microbial lipid according to claim 1. **Claim 7** The TAG is at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, or 100% by weight of the total lipid content of the extracted lipid. The extracted microbial lipid according to claim 1. **Claim 8** A microbial cell having at least one genetic modification, Optionally, the microbial cell is Yarrowia lipolytica, where the microbial cell lacks at least one genetic modification and, when compared to the corresponding microbial cell cultured under the same conditions, in each case: (i) an increase in the total fatty acid (TFA) content of the microbial cell, or the TFA content of TAG in the microbial cell, or both, an increase in the saturated fatty acid (SFA) content, an increase in the SFA content having at least 18 carbons, an increase in the stearic acid content, an increase in the C20:0 and C22:0 fatty acid contents, an increase in the C24:0 fatty acid content, an increase in the L / S-SFA ratio, or any combination thereof, and (ii) an increase in triacylglycerol (TAG) production or accumulation, or both, Optionally, the TFA content of the cell, or the TAG content of the cell, or both, has one or more characteristics defined in claim 2 for the TFA content of the extracted lipid or the TAG, Optionally, the TFA content of said cells, the TAG content of said cells, or the polar lipids of said cells, or any combination thereof, lacks at least one genetic modification and has an increased L / S-SFA when compared to corresponding microbial cells cultured under the same conditions, preferably where the L / S-SFA ratio is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or about 3 to about 10-fold greater than in the TFA content, TAG content, or polar lipids of corresponding microbial cells lacking at least one genetic modification and cultured under the same conditions. **Claim 9** The microbial cell is (i) a) FAT A fatty acyl thioesterase, b) at least one fatty acid acyltransferase, preferably at least diacylglycerol acyltransferase (DGAT), or c) at least one genetic modification that is an exogenous polynucleotide encoding FAT A and at least one fatty acid acyltransferase, preferably at least DGAT, where each polynucleotide is operably linked to one or more promoters capable of directing the expression of the polynucleotide in said microbial cell; (ii) a genetic modification that results in a decrease in the expression and / or activity of an endogenous Δ12 desaturase, preferably a genetic modification of an endogenous gene encoding Δ12 desaturase, more preferably a null genetic modification of an endogenous gene encoding Δ12 desaturase, optionally where the genetic modification is a mutation in the gene encoding endogenous Δ12 desaturase, preferably a null mutation of the FAD2 gene; (iii) a genetic modification that results in a decrease in the expression and / or activity or both of an endogenous gene encoding DGAT, preferably the DGA2 gene, that has a preference for PUFA-CoA over stearoyl-CoA, or has a preference for palmitoyl-CoA over stearoyl-CoA, or both; and / or, (iv) having the following, 1) an exogenous polynucleotide encoding FATA fatty acyl thioesterase and a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase gene, 2) an exogenous polynucleotide encoding a fatty acid acyl transferase, preferably diacylglycerol acyl transferase (DGAT), and a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase gene, 3) an exogenous polynucleotide encoding FATA fatty acyl thioesterase and a fatty acid acyl transferase, preferably diacylglycerol acyl transferase (DGAT), and a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase gene, 4) an exogenous polynucleotide encoding FATA fatty acyl thioesterase and a genetic modification that results in a decrease in the expression and / or activity of the endogenous DGA2 gene, 5) an exogenous polynucleotide encoding a fatty acid acyl transferase, preferably diacylglycerol acyl transferase (DGAT), and a genetic modification that results in a decrease in the expression and / or activity of the endogenous DGA2 gene, 6) an exogenous polynucleotide encoding FATA fatty acyl thioesterase and a fatty acid acyl transferase, preferably diacylglycerol acyl transferase (DGAT), and a genetic modification that results in a decrease in the expression and / or activity of the endogenous DGA2 gene, 7) An exogenous polynucleotide encoding FATA fatty acyl thioesterase, a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase gene, and a genetic modification that results in a decrease in the expression and / or activity of the endogenous DGA2 gene, 8) An exogenous polynucleotide encoding a fatty acid acyltransferase, preferably a diacylglycerol acyltransferase (DGAT), a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase gene, and a genetic modification that results in a decrease in the expression and / or activity of the endogenous DGA2 gene, or 9) An exogenous polynucleotide encoding FATA fatty acyl thioesterase and a fatty acid acyltransferase, preferably a diacylglycerol acyltransferase (DGAT), a genetic modification that results in a decrease in the expression and / or activity of the endogenous Δ12 desaturase gene, and a genetic modification that results in a decrease in the expression and / or activity of the endogenous DGA2 gene, wherein each polynucleotide is operably linked to one or more promoters capable of directing the expression of the polynucleotide in the microbial cell, The microbial cell according to claim 8.

10. At least one acyltransferase, preferably at least DGAT, has at least equivalent or higher activity with respect to a steryl-CoA molecule as a substrate compared to palmitoyl-CoA, Optionally, the DGAT comprises an amino acid having the sequence set forth in SEQ ID NO: 81 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: 81, Optionally, the DGAT is yeast DGAI, Optionally, the DGAI comprises an amino acid having the sequence set forth in SEQ ID NO: 53 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: 53, The microbial cell according to claim 9.

11. The microbial cell according to claim 9, wherein the FATA contains an amino acid having an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to the sequence set forth in SEQ ID NO: 83 or SEQ ID NO: 85, or one or both of SEQ ID NO: 83 and SEQ ID NO:

85.

12. A microbial cell extract containing the lipid according to claim 1 or produced from the microbial cell according to claim 8.

13. (a) a step of obtaining the microbial cell according to claim 8, and (b) a process for producing the extracted lipid, comprising a step of extracting the lipid from the microbial cell, Optionally, the process further comprises a step of culturing the microbial cell or a step of treating the cell with an acid before step (b). Optionally, the cell is cultured in a medium having less than 5 g / l, less than 2 g / l, less than 1 g / l of stearate or in the absence of stearic acid.

14. A composition comprising one or more or all of the lipid according to claim 1 or the microbial cell according to claim 8.

15. A food, feed, or beverage comprising one or more or all of the lipid according to claim 1 or the microbial cell according to claim 8 and at least one other food, feed, or beverage ingredient, Optionally, the food, feed, or beverage is a meat substitute.

15.

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