High palmitic oil compositions and uses thereof

Abstract

Provided herein are algal oil compositions, including high palmitic and high palmitic-oleic-palmitic (POP) containing algal oil compositions. Further provided herein are methods of producing said compositions and applications thereof in personal and home care products.

Description

HIGH PALMITIC OIL COMPOSITIONS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international application claims benefit to U.S. Provisional Application No. 63 / 549,843, filed on February 5, 2024, which is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a sequence listing Sequence Listing XML which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on February 3, 2025, is named 009919_00098_WO_SL.xml and is 142,039 bytes in size. BACKGROUND

[0003] Oleaginous microorganisms have the ability to convert carbon substrates into oils, including triacylglycerols (TAG) or lipids, and accumulate these oils intracellularly. Some microorganisms can accumulate lipids in amounts of up to 89% dry weight. Thus, oleaginous microorganisms, including microalgae, bacteria, fungi, and yeasts, can serve as an ideal source for biobased oil production. Genetic and non-genetic modification techniques can allow for the production of oils with particular fatty acid profiles.

[0004] Oils containing significant amounts of palmitic acid are valuable across many industries, including foods, personal care, home care, cosmetics, and biofuel. However, the primary oil responsible for providing high levels of palmitic acid is palm oil derived from oil palms (Elaeis guineensis). Because palm oil is semisolid at room temperature, they are routinely fractioned into a liquid fraction (palm olein) and a solid fraction (palm stearin). It is generally known that palm olein contains higher levels of oleic and linoleic acids compared to palm oil. The demand for palm oil and the development of this industry has led to a significant reduction of biodiversity in tropical locations where palms are grown. Thus, a need exists for sustainable sources of palm oil. INCORPORATION BY REFERENCE

[0005] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.SUMMARY

[0006] In some aspects, the present disclosure provides an oil comprising a TAG component, wherein the TAG component has a fatty acid content comprising at least 30% or more of palmitic (C16:0) acid.

[0007] In some aspects, the present disclosure provides an oil, obtained through non- genetic engineering means, comprising TAG components having a fatty acid content comprising 30% or more of palmitic (C16:0) acid, 45% or more of oleic (C18:1) acid and 8% or less of linoleic (C18:2) acid.

[0008] In some aspects, the present disclosure provides an oil derived from a microalgal cell comprising a TAG component, wherein the TAG component has a fatty acid content comprising 30% or more of (C16:0) acid, wherein the TAG component has a low linoleic content.

[0009] In some aspects, the present disclosures provide an oil derived from a microalgal cell, wherein the oil provides an alternative to palm oil-derived olein.

[0010] In some aspects, the present disclosure provides an oil derived from a microalgal cell comprising a TAG component, wherein the TAG component has at least 30% C16:0 fatty acids and at least 40% C18:1 fatty acids, and wherein the oil is produced through classical strain improvement of a microalgal cell.

[0011] In some aspects, the present disclosure provides an oil derived from a microalgal cell comprising a TAG component, wherein the TAG component has a fatty acid content comprising 30% or more C16:0 fatty acid, wherein the oil further comprises less than 1% tripalmitin.

[0012] In some aspects, the present disclosure provides an oil comprising a TAG component having a fatty acid content comprising at least 30% C16:0 fatty acids, less than about 1% diacylglycerols, and less than about 1% monoacylglycerols.

[0013] In some aspects, the present disclosure provides an oil that further comprises sterols, such as ergosterol.

[0014] In some aspects, the present disclosure provides an oil, obtained through non- genetic engineering means, comprising TAG components having a fatty acid content comprising 49% or more of palmitic (C16:0) acid, about 36% or less of oleic (C18:1) acid, and 8% or less of linoleic (C18:2) acid.

[0015] In some aspects, the present disclosure provides an oil obtained through non- genetic engineering means, comprising TAG components having a POP (palmitic-oleic-palmitic) TAG population comprising 45% or more of all TAG species, while PPP (tripalmitin) comprises less than 2.7%.

[0016] In some aspects, the present disclosure provides a combination of screening modalities for Prototheca moriformis, including mutagenesis with agents including UV light (254 nM) and 4-nitroquinoline-1-oxide (4-NQO), followed by repeated cycles of growth in a lipid production medium at temperatures up to about 38°C followed by a brief (up to about 4 min) incubation in near lethal temperatures (up to about 68 °C ). After incubation at lethal temperatures, the cells are allowed to recover for about 1 to 5 days (e.g., up to about 3 days) in a low glucose (i.e., a media containing up to 5 g / L) growth media, followed by plating the cells to growth medium containing plates. Individual clones are then selected for growth in a lipid production medium, followed by screening for fatty acid profile of their resulting lipid (oil). The fatty acid profile is screened by generating fatty acid methyl esters (FAMEs), interrogating those FAMEs by gas chromatography utilizing flame ionization detection (GC / FID), and analyzing the triacylglycerol (TAG) composition of the oils by Liquid- Chromatography / Time of Flight-Mass Spectrometry (LC / TOF-MS) equipped with an Atmospheric Pressure Chemical Ionization (APCI) source. Using such a method, a person of skill in the art can produce a microalgal cell that can make an oil with the desired TAG composition.

[0017] In some aspects, the present disclosure provides a means to produce valuable structured fats through the biotransformation of a sugar, wherein the fats have a similar composition or components to the palm mid fraction, but in the absence of the capital equipment, exhaustive fractionation, and wasteful processes.

[0018] In one embodiment, an isolated microalgal cell is provided such as a Prototheca sp. cell obtained through classical microalgal cell improvement, wherein the microalgal cell produces an oil characterized as comprising: (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%, and (b) a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

[0019] In another embodiment, the isolated microalgal cell has a POP content that is at least about 45%, or at least about 50% or at least about 55%, or at least about 60%, and a PPP content that is less than about 2.7% of the TAG component.

[0020] In another embodiment, the microalgal oil comprising the TAG component, has a TAG component of (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or at least 23%, or at least 29%, or at least 32%, or at least 35%, or at least 45%, and (b) a tripalmitin (PPP) content less than 0.3% of the TAG component, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG, and wherein the microalgal oil is produced by a microalgal Prototheca cell obtained through classical strain improvement.

[0021] In one aspect, the microalgal oils produced by the microalgal cells described are from a cell that has had no exogenous nucleic acid introduced. In another aspect, the oil- producing microalgal cell obtained by the classical means described herein can be further recombinantly engineered with an exogenous nucleic acid that is not native to the parent microalgal cell that produced the strain.

[0022] In a further embodiment, the microalgal oil has a TAG component that comprises: (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 45%, and (b) a tripalmitin (PPP) content of less than about 2.7% of the TAG. Alternatively, a contemplated microalgal oil can have (a) the POP content of at least 35% and the PPP content of less than about 1.5%, or (b) the POP content of at least 45% and the PPP content of less than about 2.7%. The microalgal oils described above and herein can further comprise at least about 30% C16:0 fatty acids, or at least about 35% C16:0 fatty acids, or at least about 40% C16:0 fatty acids, or at least about 45% C16:0 fatty acids, or at least about 50% C16:0 fatty acids.

[0023] Also contemplated is a method of obtaining a microalgal cell that produces the oil of any of claims 4 to 8 comprising the steps of: (a) mutagenizing microalgal cells with at least one or more agents, which promote errors in DNA excision repair known (e.g., ultraviolet light (e.g., 254 nm), 4-nitroquinoline 1-oxide (4-NQO), methyl methanesulfonate (MMS), Ni, Ar, or polyaromatic hydrocarbons while culturing the microalgal cells at about 28 ºC to about 38 ºC in a vegetative or a lipid production media; (b) enriching the mutagenized microalgal cells by exposing the mutagenized cells to physical conditions (e.g., an elevated temperature of about 50 to about 68°C or by using density fractionation on sucrose or percoll gradients to isolate specific subpopulations based on density) or a chemical compound (e.g., herbicides, fungicides, or inhibitors of lipid biosynthesis, such as tebutam or cofenstrole); (c) growing the mutagenized, enriched microalgal cells in a lipid production medium; and (d) harvesting the microalgal oil from the microalgal cells and interrogating the microalgal oil using a FAME analysis and a triacylglyceride (TAG) analysis to determine the component of the screenedmicroalgal cells; and wherein the TAG component of the screened microalgal cells comprises (e) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%, and (f) a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component. In a preferred embodiment, the method uses a Prototheca sp. cell.

[0024] Another embodiment is a method of obtaining a microalgal oil of a microalgal cell as described herein by: (a) mutagenizing cells with agents that introduce mutations through promoting errors in DNA excision repair known to include, but not limited to, UV light (254 nm), 4-nitroquinoline 1-oxide (4-NQO), methyl methanesulfonate (MMS), Ni, Ar, or polyaromatic hydrocarbons culturing the microalgal cells at about 28 ºC to about 38 ºC in vegetative or lipid production media and (b) Enriching said cell populations using physical change (e.g., elevated temperatures of about 50, about 60 or up to about 68°C or fractionation on sucrose or percoll gradients to isolate specific subpopulations based on density) or chemical compound (e.g., herbicides, fungicides or inhibitors of lipid biosynthesis such as tebutam or cofenstrole) and (c) plating aforementioned mutagenized and enrich populations to solid vegetative medium, (d) growing said individuals in a lipid production medium, (e) harvesting the microalgal cells from said lipid production medium, (f) obtaining the microalgal oil from the cultured microalgal cells and interrogating the oil via FAME analysis as well as TAG analysis.

[0025] This Summary is provided to introduce certain concepts in a simplified form that are further described in the Detailed Description. The intention of this Summary is not to identify key or necessary features of the claimed subject matter, nor is it intended to be used to limit, in any way, the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the TAG component of microalgal oils, microalgal oils, and the microalgae producing them, as well as the methods of obtaining said unpurified oils, are set forth with particularity in the appended claims. A better understanding of the features and advantages of the methods, compositions, and components will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the compositions, components, and methods are utilized, and the accompanying drawings of which:

[0027] FIG.1A and 1B show a sequence alignment of a plastidic 23 S rDNA between a UTEX 1533; SEQ ID NO:30, designated as Prototheca wickerhamii, and a Prototheca moriformis strain, designated UTEX 1435; SEQ ID NO:31.

[0028] FIG. 2A and 2B show an outline of the mutagenesis trait selection, high throughput, and automated screening steps of the improvement process described herein.

[0029] FIG. 3 shows an overview of the workflow and strategy utilized in classical strain improvement for the generation of mutants derived from UTEX 1533 in the current disclosure.

[0030] FIG. 4 shows TIC chromatograms of extracted triacylglycerols derived from classically improved mutant isolates (a-c / CHK100 mutant isolates 1-3) derived from the classically improved base strain CHK100(d).

[0031] FIG. 5 is a mass spectrum of POP / PPO triglyceride present in mutant isolate (a) / CHK100 mutant isolate 1 in FIG.4.

[0032] FIG. 6 is a mass spectrum of POP / PPO triglyceride present in mutant isolate (b) / CHK100 mutant isolate 2 in FIG.4.

[0033] FIG. 7 is a mass spectrum of POP / PPO triglyceride present in mutant isolate (c) / CHK100 mutant isolate 3 in FIG.4.

[0034] FIG. 8 is a mass spectrum of POP / PPO triglyceride present in the CHK100 parental strain in FIG.4.

[0035] FIG.9 is a mass spectrum of POP / PPO triglyceride of red palm oil.

[0036] FIG.10 is a mass spectrum of 1,3-palmitin-2-olein analytical standard.

[0037] FIG.11 is a mass spectrum of 1,2-palmitin-3-olein analytical standard.

[0038] FIG. 12 is a graph illustrating the triglyceride profiles of some of the higher palmitate strains of the present disclosure compared to red palm oil.

[0039] FIG. 13 is a graph illustrating the triglyceride profiles of mutant isolate (b) in FIG.4 compared to red palm oil.

[0040] FIG.14 illustrates a comparison between the levels of palmitic acid (C16:0) and unsaturated fatty acids (sum of C18:1 and C18:2 fatty acids) found in a novel oil of the present disclosure compared to the fatty acid levels found in palm olein.

[0041] FIG. 15 shows the TAG profile of the oil produced by the high-palmitic acid algal strains of the present disclosure compared to the TAG profiles of palm olein.

[0042] FIG. 16 shows FAME (fatty acid methyl ester) profiles of algal train 268U derived mutant BLK08 F04 run in lipid production at 28 °C versus 32 °C.

[0043] FIG. 17 shows TAG profiles of algal train 268U derived mutant BLK08 F04 run in lipid production at 28 °C versus 32 °C with PPO and POP regioisomers indicated.

[0044] FIG. 18 shows levels of PPO and POP regioisomers and PPP in classically improved algal strains of the present disclosure compared with red palm oil and palm olein mid-fraction DETAILED DESCRIPTION

[0045] Provided herein are microalgal oil compositions having high-palmitic acid content, high POP TAG content, and low PPP content, as well as methods of making thereof, formulations, and applications thereof. The microalgal oils having such a ratio of high-palmitic acid content and POP TAG content, and having a low PPP content, can then be a ready source substitute for the fractions obtained from palm oil that involves numerous fractionation steps and many days to obtain the mid-fractions of interest.

[0046] The principal species of palm under global cultivation is Elaeis guineensis or various cultivars and varieties derived from it. One source of palm oil is the palm fruit, which is comprised of a fleshy outer mesocarp that surrounds the hard palm kernel or endocarp. The oil of the palm fruit differs markedly from the oil of the hard palm kernel, termed “palm kernel oil” (“PKO”). PKO is characterized as being high in C12:0 fatty acids (lauric, >45%) and C14:0 (myristic, > 15%), and with relatively low C16:0, palmitic (8%), and C18:1, oleic (15%). The palm oil from the palm fruit is virtually devoid of C12:0 and C14:0 fatty acids, being comprised primarily of palmitic acid (40-46%) and oleic acid (37-45%) and some C18:2 fatty acids (linoleic, 8-12%).

[0047] Palm oil is used ‘as is’ for cooking and other applications, including personal care, but a significant amount of commercial palm oil is fractionated to create more valuable components. (Jin et al., “Characteristics of palm mid-fractions produced from different fractionation paths and their potential usages,” Int. J. Food Prop. 21: 58-69, 2018).

[0048] The first fractionation of refined palm oil, a so-called dry fractionation process because it does not use solvents, yields two fractions: a palm olein fraction and a palm stearin fraction. The palm olein fraction is a liquid at room temperature and is enriched in oleic and linoleic fatty acids; the palm stearin fraction is a solid at room temperature and is highly enriched in palmitate (50-60%).

[0049] Palm olein can be further dry fractionated to a “palm mid-fraction A” (“PMF- A”) component. During the fractionation process, palm olein is cooled to roughly 16°C, with the rate depending on the specific manufacturer and their process. This is followed by a furtherdecrease in temperature to 12-13°C and holding at this temperature for a period of time to allow for further crystallization of the solid PMF-A fraction. The PMF-A fraction is enriched in palmitate, particularly POP (40%). After this fractionation step, the liquid component at these low temperatures is termed “super olein.”

[0050] Palm stearin can be further fractionated to produce a palm mid-fraction B (PMF- B) component employing a wet fractionation technique utilizing acetone (ratios of 1:4-8 wt:wt of solvent:oil) and temperatures ranging from 17-25 °C with holds at the indicated temperature of 8-48 hr. The resulting solid fraction produced by the further fractionation is termed “super stearin,” and enriched in C18:1, while the PMF-B is further enriched in palmitate and the POP fraction (45% POP).

[0051] The PMF-A and PMF-B fractions can be blended and wet fractionated yet again using acetone (ratios of 1:10, wt:wt of solvent:oil) and at a very low temperature (e.g., about 4 °C) combined with a hold time of up to 24 hr, to produce a third, highly enriched POP fraction (60-65% POP), termed PMF-C.

[0052] The resulting high POP fractions obtained by these methods from palm oil are highly valued for their use in the confectionery industry, where they give chocolates their characteristic sharp melt profile (see e.g., BAILEY’S INDUSTRIAL OILS AND FAT PRODUCTS. 2005. 6thEd. Shahidi, Fereidoon, Ed. John Wiley and Sons, Inc., Hoboken, NJ. Volume 4: “Edible oil and fat products: Products and Applications,” 4: 159-173).

[0053] It will not be lost to the average layperson that the processes involved in the generation of these PMF components are indeed quite involved and costly from a capital (cost of equipment), energy (chilling and alternately heating large quantities of oil) and hazardous chemicals (large quantities of acetone) perspective.

[0054] Provided herein are examples of a simpler process, whereby fermentable sugars are converted into triglyceride oils in microalgae with POP compositions equal or superior to those found in PMF derived from a more complicated and more costly process currently employed in the manufacture of these materials.

[0055] Having a more commercially available form of these structured oils for use from a non-GMO organism, given cocoa butter and palm oil availability, will result in economic savings in production and will not be as prone to climate changes or plant viruses and blights.

[0056] Oil compositions provided herein can be produced by a microorganism that is genetically modified or non-genetically modified. Non-genetically modified or non-genetically engineered microorganisms or non-genetically modified organisms (non-GMO) can beproduced using strategies such as those described herein. The non-GMO microorganisms obtained by the methods described herein can be further recombinantly modified if desired.

[0057] Genetic and non-genetic modification techniques can allow for the production of oils having particular phenotypes. While genetic engineering techniques can tend to be more targeted to phenotypes elicited in a host oleaginous microbe. Classical strain improvement or other non-genetic engineering techniques can also be employed to enhance phenotypes. Enhancement of phenotypes can include the elaboration of a particular fatty acid profile (e.g., high-palmitic acid content), yield on carbon, volumetric oil accumulation (e.g., grams oil / L culture), oil productivity (e.g., g oil / L culture day), and oil as a percent dry cell weight (DCW) as a measure of strain performance.

[0058] A further benefit of classical strain improvement techniques, when used as the sole means to alter or improve strain phenotype and performance, can be realized from both a regulatory and business / marketing perspective. From a regulatory perspective, non-genetically engineered microbes may be exempt from regulatory oversight by entities such as the U.S. EPA's Toxic Substances Control Act (TSCA) and the requirement to file a Microbial Commercial Activity Notice (MCAN) when the material is to be used in chemical (non-food) applications. Such dispensation extends to other geographies as well, such as Brazil, for example, where such microbes are exempt from filing a Strain Dossier with the Brazilian regulatory body (the National Technical Commission of Biosafety, Ministry of Science, Technology, Innovation, and Communications or CTNBio) that oversees industrial microbes. The avoidance of such regulatory oversight can save millions of dollars in development costs. From a marketing and consumer branding perspective, the raw materials produced by such non-genetically engineered means can meet GMO-free and organic labeling standards, as well as brands' and consumers' desires for "clean labeling".

[0059] As used herein, the term "classical strain improvement" or “classical strain optimization” refers to methods of random or semi-random mutagenesis of microbes to create non-naturally occurring strains with improved properties. These methods include, but are not limited to, mutagenesis of a population to create genetic variants, random selection or screening of a surviving population to identify an improved strain, and identification of improved strains by assaying fermentation broth for products. Classical strain improvement methods include exposure to UV radiation (e.g., 254 nm), chemical mutagens (e.g., 4-nitroquinoline 1-oxide, which forms bulky quinolone purine adducts but also produces mutagenic superoxide radicals (see e.g., Downes, D. et al., “Characterization of the Mutagenic Spectrum of 4-Nitroquinoline 1-Oxide (4-NQO) in Aspergillus nidulans by Whole Genome Sequencing,” G3, Genes,Genomics and Genetics 1-10, 2014) and / or selective or enrichment agents (see e.g., Tebutam, a microtubule inhibitor and potent inhibitor of fatty acyl-ACP thioesterase, FATA as discussed in Johnen et al., “Inhibition of acyl-ACP thioesterase as site of action of the commercial herbicides cumyluron, oxaziclomefone, bromobutide, methyldymron and tebutam,” Pest Manag Sci.78: 3620-3629, 2022).

[0060] The latter class of agents is deployed after a cell population is mutagenized as a means to enrich or differentiate truly mutagenized cells from those that are not, by looking for phenotypes that lie outside those of the ‘wild type’ or non-mutagenized control population. A person in the art would understand that such control populations, or ‘mock controls’, are always assessed alongside the mutagenized population when using enrichment agents as a means to assess the degree of divergence of the mutagenized population form the control population. Selective or enrichment agents can be both physical (e.g., tolerance to extremes in temperature, the buoyancy of the mutagenized cells in percoll, sucrose, or other gradients, the intensity with which cells take up neutral lipid dyes such as Nile Red or 4,4-difluoro-4-bora- 3a,4a-diaza-s-indacene-BODIPY, and a means to sort highly fluorescent populations from less fluorescent ones) or chemical (e.g., the resistance of mutagenized populations to tolerate inhibition by pharmacological agents, herbicides, salts). Classical strain improvement methods do not include recombinant genetic engineering methods targeted to one or more genomic regions, e.g., via homologous recombination, random insertion, markerless transformation, or CRISPR-Cas9-based technologies.

[0061] As used herein, the term "microbial oil" refers to an oil produced or extracted from a microorganism (microbe), e.g., an oleaginous, single-celled, eukaryotic, or prokaryotic microorganism, including but not limited to, microalgae, yeast, bacteria, and fungi. Preferably, the microorganism is a microalgal organism. More preferably, the microalgal organism is a Prototheca strain and has been classically enhanced via exposure to chemical mutagens, compounds, temperature, and / or UV light.

[0062] As used herein, the term "triacylglycerol", "triglyceride", or "TAG" are synonymous and refer to esters between glycerol and three fatty acids. Generally, fatty acids of TAGs have chain lengths of 6 carbon atoms or more.

[0063] Because there are three possible positions on the glycerol backbone to which fatty acids can esterify, referred to as sn-1, -2, and -3, the number of possible TAG molecules in an oil is equal to the number of fatty acid species present in the oil raised to the 3rdpower. By example, an oil comprised of seven different fatty acids will have 343 possible TAG species.

[0064] Regioisomers of TAGs are triacylglycerol molecules that have the same fatty acid composition and molecular weight but whose fatty acid moieties are arrayed in a different order, e.g., POP contains palmitate at the sn-1 and sn-3 positions and oleate at sn-2, while PPO contains palmitate both at sn-1 and sn-2 and oleate at sn-3. PPP, with palmitate at sn-1, -2, and -3, has no regioisomer.

[0065] As used herein, the term "TAG purity", "molecular purity", or "oil purity" refers to the number of molecular species that make up an oil composition on an absolute basis or present in amounts above a certain threshold. The fewer the number of TAG species in an oil, the greater the "purity" of the oil.

[0066] Properties of oils and their modes of characterization, processing, characteristics, and compositions are well known (see e.g., THE LIPID HANDBOOK. 2007. Gunstone, F.D., Harwood, J.L. and Dijkstra, A.J Eds. CRC Press Taylor and Francis Group).

[0067] As used here, the terms “oil” or “lipid” are synonymous and refer to all the TAGs comprising the oil and lipid, accounting for >98% of the molecules in the lipid or oil; the remaining components are comprised of free fatty acids, sterols, color bodies, metals, and phospholipids. This is largely the composition of oil or lipid as it exists in the microalgal cell prior to being separated from the cell.

[0068] The term “fats” as used here, refers to oils or lipids that are solid at room temperature, about 20-25 °C.

[0069] A microalgal oil can be further categorized based on the degree to which it has been purified and separated from the other cellular debris or non-lipid biomass. This separation can be affected using mechanical means, i.e., by expeller pressing, or by chemical means, i.e., by solvent extraction and bead milling, where solvents include combinations of hydrocarbons such as hexane, ethanol, or propane used alone or separately.

[0070] Solvents can also include aqueous solvents comprised of water and salts such as ammonium sulfate, magnesium sulfate, sodium chloride, etc.

[0071] Once the oil is separated from the non-lipid biomass either by mechanical pressing or solvent extraction, the resulting oil is termed “crude oil.”

[0072] In the case of solvent-extracted oils, the solvent-oil mixture is termed “micellae” and, depending on the solvent type, the solvent is removed, which can be further purified via passage through a centrifuge or filter press to remove “fines” or small particles of non-lipid biomass.

[0073] The composition of the “crude oil” is comprised of TAGs, free fatty acids, sterols, color bodies, metals, and phospholipids.

[0074] Crude oils can be further processed by refining. Refining oil serves to remove free fatty acids and can be achieved by adding NaOH or “caustic” to create soaps, which are removed by centrifugation.

[0075] Refined oils are further processed by degumming, which includes neutralization of residual caustic with citric acid and water washing, followed by centrifugation to remove the precipitated “gums” or phospholipid fraction of the crude oil.

[0076] The refined, degummed oil can next be bleached, using earthen bleaching clays to remove residual free fatty acids, metals including Ca, PO4, Fe, Cl, and K, among others, and color bodies that can affect oil quality and appearance.

[0077] Refined, degummed, and bleached oils are finally purified via deodorization, where steam can be used to strip the oil under a vacuum of the volatile components and off- putting flavors. The final oil is termed an RBD oil (i.e., refined, bleached and deodorized oil).

[0078] In the present disclosure, the fatty acid composition or FAME profile and the TAG composition or profile as determined from an intact cell are equivalent to what is determined from an RBD oil for two reasons. First, the analytical methods used to determine FAME and TAG profiles, whether from whole cells or RBD oil affect a certain purification of the oil away from the cellular debris. Second, the oils within the cells are already highly pure materials, >98% TAGs.

[0079] As used herein, the terms “diacylglycerol”, “diglyceride”, and “DAG” refer to glycerides having two fatty acids esterified to a glycerol molecule.

[0080] As used herein, the terms “monoacylglycerol”, “monoglyceride”, and “MAG” refer to glycerides having one fatty acid esterified to a molecule of glycerol.

[0081] As used herein, the terms "fatty acid profile" or “fatty acid content” refer to a fatty acid composition of an oil, e.g., an oil produced by a cell provided herein or a derivative thereof. Derivatives of an oil produced by a cell provided herein include a refined, bleached, and deodorized oil. Fatty acid profiles can be determined by subjecting an oil to transesterification to generate fatty acid methyl esters (FAMEs) and subsequently quantitating fatty acid type by Gas-Chromatography (GC) equipped with a Flame-Ionization Detector (GC / FID).

[0082] As used herein, the term "sterol profile" refers to a sterol composition of an oil (e.g., an oil produced by a cell provided herein or a derivative thereof). Derivatives of an oil produced by a cell provided herein include a refined, bleached, and deodorized oil.

[0083] As used herein, "oleic content", "oleic acid content", "oleate content", and "olein content" refer to the percentage amount of oleic acid in the fatty acid profile of a substance (e.g., a TAG oil).

[0084] As used herein, the term "C18:1 content" refers to the percentage amount of a C18:1 fatty acid (e.g., oleic acid) in the fatty acid profile of a substance (e.g., a microbial oil).

[0085] As used herein, “linoleic content” and “linoleic acid content” refer to the percentage amount of linoleic acid in the fatty acid profile of a substance (e.g., a TAG oil)

[0086] As used herein, the term “C18:2 content” refers to the percentage amount of a C18:2 fatty acid (e.g., linoleic acid) in the fatty acid profile of a substance (e.g., a microbial oil).

[0087] As used herein, “palmitate content”, “palmitic content”, and “palmitic acid content” refer to the percentage amount of palmitic acid in the fatty acid profile of a substance (e.g., a microalgal oil).

[0088] As used herein, the term “C16:0 content” refers to the percentage amount of C16:0 fatty acid (e.g., palmitic acid) in the fatty acid profile of a substance (e.g., a microbial oil).

[0089] As used herein, “PPP”, “tripalmitin”, “tripalmitate”, and “tripalmitoylglycerol” refer to a triacylglycerol having palmitic acid or palmitate as the only fatty acid on the glycerol backbone. The PPP content or PPP level is generally discussed as a percentage of the TAG component.

[0090] The term “POP” or “palmitic-oleic-palmitic” can refer to a triacylglycerol having palmitic acid or palmitate at the sn-1 and sn-3 positions on the glycerol backbone and oleate at the sn-2 position on the glycerol backbone. The POP content or POP level is generally discussed as a percentage of the TAG component.

[0091] The term “PPO” or “palmitatic-palmitic-oleate” can refer to a triacylglycerol having palmitic acid or palmitate at the sn-1 and sn-2 positions on the glycerol backbone and oleate at the sn-3 position on the glycerol backbone. The PPO content or PPO level is generally discussed as a percentage of the TAG component.

[0092] As used herein, the term “high palmitate” or “high palmitic” can refer to greater than 30% palmitic acid, or greater than 35% palmitic acid, greater than 40% palmitic acid, greater than 45% palmitic acid, greater than 50% palmitic acid, greater than 55% palmitic acid, or greater than 60% palmitic acid.

[0093] As used herein, the term “low linoleic content” or “low linoleic acid content” can refer to lower than 10%, lower than 9%, lower than 8%, lower than 7%, lower than 6%,lower than 5%, lower than 4%, lower than 3%, lower than 2% or lower than 1% linoleic acid of the total fatty acid content.

[0094] As used herein, the term “low MAG” or “low monoacylglycerol” can refer to lower than 5%, lower than 4%, lower than 3%, lower than 2%, or lower than 1% of MAG of the total lipid content of an oil.

[0095] As used herein, the term “low DAG” or “low diacylglycerol” can refer to lower than 5%, lower than 4%, lower than 3%, lower than 2%, or lower than 1% of DAG of the total lipid content of an oil.

[0096] As used herein, "sequence identity" refers to a percentage of identical nucleotide or amino acid residues between two sequences being compared after an optimal alignment of sequences. An optimal alignment of sequences may be produced manually or by means of computer programs that use a sequence alignment algorithm (e.g., Clustal W, T-coffee, COBALT, BestFit, FASTA, BLASTP, BLASTN, and TFastA). Sequence identity can be calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result obtained by 100 to obtain the sequence identity between the two sequences; this calculation can be done for DNA, RNA, and protein sequences.

[0097] As used herein, the term "about" refers to ± 10% of the value provided.

[0098] As used herein, cells are grown in a growth media suitable for the cells being used during classic enrichment and selection strategies. For example, in one embodiment, Prototheca microalgal cells from a specific Prototheca strain can use a vegetative growth media comprising NaH2PO4, K2HPO4, citric acid monohydrate, MgSO47H20, CuCl22H2O, dextrose, (NH4)2SO4, Sigma antifoam 204 at final concentrations of 13.6 mM, 11.4 mM, 5 mM, 0.15 mM, 40 g / L, 7.5 mM and 0.23 g / L, respectively. The vegetative growth media also can comprise vitamins such as thiamine HCl, D-pantothenic acid, biotin, cyanocobalamin, riboflavin, and pyridoxine HCl at final concentrations of 2.96E-06, 2.24E-07, 6.53E-09, 8.30E- 11, 1.33E-08, and 1.27E-08 molar, respectively. The vegetative growth media can further comprise one or more of the following micronutrients: H3BO3, ZnSO4•7H2O, MnSO4•H2O, NaMoO4•2H2O, Ni(NO3)2•6H2O, citric acid monohydrate, CuSO4•5H2O and FeSO47•H2O at final concentrations of 2.96E-05, 1.22E-05M, 1.46E-05, 3.97E-07, 2.75E-07, 1.95E-04, 3.96E- 07, and 5.4E-06 molar, respectively. The Prototheca cells in the log phase of growth in the vegetative medium are then subjected to mutagenesis by means of chemicals or UV light (FIG. 3, Step 1). The exemplified Prototheca cells are then sub-cultured into a lipid production medium where the cells are then subjected to selection / enrichment strategies (FIG.3, Step 2).A suitable lipid production medium can be used to culture the cells. For Prototheca cells, a suitable culture media comprises the same vitamins and micronutrients at the same concentrations as used in a vegetative medium. Macronutrients in the lipid production medium, relative to the vegetative medium, can be adjusted to include 25 mM citric acid monohydrate, 50 g / L dextrose, and 1.5 mM (NH4)2SO4. Microalgal cells can then be plated on a suitable solid vegetative medium containing 0.8% agarose to obtain clonal isolates (FIG. 3, Step 3). Once suitable clonal isolates are obtained, the isolates are interrogated in a suitable lipid production medium in a 96-well format (FIG. 3, Step 4). Using glucose consumption as a surrogate for oil production, high glucose consuming microalgal cell strains can be validated by culturing in a suitable lipid production medium in a tube or shake flask format (FIG.3, Step 5). Clonal isolates that are successfully validated are then subcultured for multiple generations to stabilize mutations (FIG.3, Step 6), followed by purification of clonal isolates (FIG.3, Step 7). The clonal isolates that are stable can undergo subsequent re-interrogation in a lipid production medium (FIG.3, Step 8). Clones deemed to be phenotypically stable (e.g., clones having %cv <5% for profile, glucose consumption, and oil titer) after the second interrogation round are ready for validation in fermentation (FIG. 3, Step 9), while clones that still demonstrate variability in their phenotype (FIG. 3, Step 8), are passaged once more (FIG. 3, Step 6) to generate stable clonal lines. Unless indicated otherwise, microalgal Prototheca cells were grown at about 28 °C with shaking at 200 rpm for cultures grown in tubes or shake flasks while blocks were grown with shaking at 900 rpm.

[0099] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are described herein. Microbial Cells and Oils Produced Therefrom

[0100] An oil provided herein is obtained from a non-genetically modified microorganism (microbe), for example, oleaginous microalgae, yeast, or bacteria. In some embodiments, the non-genetically modified microorganism is a microalgal cell. In some embodiments, the microalgal cell is a non-genetically modified Prototheca sp. strain. The non- genetically modified Prototheca sp. strain can be produced by one or more classical strain improvement strategies described herein.

[0101] In some embodiments, a cell provided herein does not comprise an exogenous gene or exogenous nucleotides that encode for an exogenous protein or gene. For example, a cell provided herein does not comprise an exogenous gene in a lipid biosynthetic pathway. In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles of an endogenous gene in a lipid biosynthetic pathway. In some embodiments, a cell provided herein does not comprise a heterologous insertion within a genomic region that encodes for an endogenous gene in a lipid biosynthetic pathway. Non-limiting examples of genes involved in lipid biosynthesis include acyl-ACP thioesterase (FAT), delta-12 fatty acid desaturase (FAD), ketoacyl-ACP synthase (KAS), stearoyl-ACP desaturase (SAD), lysophosphatidic acid acyltransferase (LPAAT), ketoacyl-CoA reductase (KCR), hydroxyacyl- CoA dehydratase (HACD), and enoyl-CoA reductase (ECR).

[0102] In some embodiments, a cell provided herein does not comprise an exogenous acyl-ACP thioesterase gene. In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles of an endogenous acyl-ACP thioesterase gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within a genomic region that encodes for an endogenous acyl-ACP thioesterase gene. In some embodiments, the endogenous acyl-ACP thioesterase gene is FATA (fatty-acyl-ACP A).

[0103] In some embodiments, a cell provided herein does not comprise an exogenous fatty acid desaturase gene. In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles of an endogenous fatty acid desaturase gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within a genomic region that encodes for an endogenous fatty acid desaturase gene. In some embodiments, the endogenous fatty acid desaturase gene is a delta-12 fatty acid desaturase. In some embodiments, the endogenous fatty acid desaturase gene is FAD2.

[0104] In some embodiments, a cell provided herein does not comprise an exogenous ketoacyl-ACP synthase gene. In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles of an endogenous ketoacyl-ACP synthase gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within a genomic region that encodes for an endogenous ketoacyl-ACP synthase gene. In some embodiments, the endogenous ketoacyl-ACP synthase gene is KASI, KASII, or KASIII.

[0105] In some embodiments, a cell provided herein does not comprise an exogenous stearoyl-ACP desaturase gene. In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles of an endogenous stearoyl-ACP desaturase gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within agenomic region that encodes for an endogenous stearoyl-ACP desaturase gene. In some embodiments, the endogenous stearoyl-ACP desaturase gene is SAD2.

[0106] In some embodiments, a cell provided herein does not comprise an exogenous lysophosphatidic acid acyltransferase gene. In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles of an endogenous lysophosphatidic acid acyltransferase gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within a genomic region that encodes for an endogenous lysophosphatidic acid acyltransferase gene.

[0107] In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles within 1.5 kb of an endogenous V-type proton ATPase catalytic subunit A isoform 1 gene or 6S genomic region. In some embodiments, a cell provided herein does not comprise a heterologous insertion within 1.5 kb of an endogenous V- type proton ATPase catalytic subunit A isoform 1 gene or 6S genomic region.

[0108] In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles within 1.5 kb of an endogenous DAO1B gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within 1.5 kb of an endogenous DAO1B gene.

[0109] In some embodiments, a cell provided herein does not comprise a genetic disruption of one or more alleles within 1.5 kb of an endogenous Thi4 gene. In some embodiments, a cell provided herein does not comprise a heterologous insertion within 1.5 kb of an endogenous Thi4 gene.

[0110] Accordingly, a cell provided herein comprises uninterrupted sequences of endogenous genes or genomic regions, including FAD2, FATA1, KASII, SAD2, V-type proton ATPase catalytic subunit A isoform 1 (6S), DAO1B, and Thi4 described herein.

[0111] In some embodiments, a cell provided herein comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO:1- 26 (shown in Table 11 of the present disclosure). In some embodiments, a cell provided herein comprises any one of SEQ ID NO:1-26 (genomic regions of CHK22 and CHK100). In some embodiments, a cell provided herein comprises SEQ ID NO:1-26 (genomic regions of CHK22 and CHK100).

[0112] In some embodiments, a cell provided herein comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 18, 19, 21, 23, and 25 (genomic regions of CHK22 and CHK100). In some embodiments, a cell provided herein comprises any one of SEQ ID NO:1, 3, 5, 7, 9, 11,13, 15, 18, 19, 21, 23, and 25 (genomic regions of CHK22 and CHK100). In some embodiments, a cell provided herein comprises SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 18, 19, 21, 23, and 25 (genomic regions of CHK22 and CHK100).

[0113] In some embodiments, an oil provided herein is produced by microalgae. In some embodiments, the microalgae is a species of a genus selected from the group consisting of: Chlorella sp., Pseudochlorella sp., Heterochlorella sp., Prototheca sp., Arthrospira sp., E uglena sp., Nannochloropsis sp., Phaeodactylum sp., Chlamydomonas sp., Scenedesmus sp., Ostreococcus sp., Selenastrum sp., Haematococcus sp., Nitzschia, Dunaliella, Navicula sp., Trebouxia sp., Pseudotrebouxia sp., Vavicula sp., Bracteococcus sp., Gomophonema sp., Watanabea, sp., Botryococcus sp., Tetraselmis sp., and Isochyris sp. In some embodiments, the microalgae is Prototheca sp. In some embodiments, the microalgae are a P. moriformis strain. In some embodiments, the microalgae are a P. wickerhammii strain.

[0114] In some embodiments, the microalgal cell described herein for use in manufacturing microbial oil is derived from a UTEX 1435 base strain. In some embodiments, a cell provided herein can be derived from a UTEX 1533 base strain. In some embodiments, a cell provided herein is derived from a base strain having a 23S ribosomal DNA sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:30 or SEQ ID NO:31. In some embodiments, a cell provided herein has a 23S ribosomal DNA sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO:30 or SEQ ID NO:31.

[0115] In some embodiments, an oil provided herein can be produced by oleaginous yeast. In some embodiments, the oleaginous yeast is a species of a genus selected from the group consisting of: Candida sp., Cryptococcus sp., Debaromyces sp., Endomycopsis sp., Geotrichum sp., Hyphopichia sp., Lipomyces sp., Pichia, sp., Rodosporidium sp., Rhodotorula sp., Sporobolomyces sp., Starmerella sp., Torulaspora, sp., Trichosporon sp., Wickerhamomyces sp., Yarrowia sp., and Zygoascus sp.

[0116] In some embodiments, an oil provided herein is obtained from or produced by oleaginous bacteria. In some embodiments, the oleaginous bacteria is a species selected from the group consisting of: Flavimonas oryzihabitans, Pseudomonas aeruginosa, Morococcus sp., Rhodobacter sphaeroides, Rhodococcus opacus, Rhodococcus erythropolis, Streptomyces jeddahensis, Ochrobactrum sp., Arthrobacter sp., Nocardia sp., Mycobacteria sp., Gordonia sp., Catenisphaera sp., and Dietzia sp.

[0117] Further provided herein are bioreactors comprising a non-naturally occurring microorganism. For example, these bioreactors comprise an oleaginous, non-naturally occurring microorganism and an oil produced by the microorganism.

[0118] While in many embodiments, an oil provided herein is obtained from a non- genetically modified microorganism or a classically-improved microorganism, in other embodiments, an oil provided herein is obtained from a genetically modified microorganism, for example, oleaginous microalgae, yeast, or bacteria. In some embodiments, an oil provided herein is obtained from a classically-improved microorganism that is then genetically modified to produce a genetically modified microorganism. In some embodiments, the genetically modified microorganism is a genetically modified Prototheca sp. strain. Classical Strain Improvement.

[0119] “Classical strain improvement” and “classical microalgal strain improvement” or “classical microalgal cell improvement” are the terms used herein to characterize the strategy of selecting for organisms having desired phenotypes, e.g., high palmitic oil production. Classical strain improvement (also called "mutation breeding") involves exposing organisms to chemicals or radiation to generate mutants with desirable traits. These classical strain improvement methods introduce random or semi-random mutations, which can thereby allow the selection of strains exhibiting desirable traits as a result of random mutagenesis. Several iterations of mutagenesis and selection can be performed with one or more mutagens to obtain a strain having desirable phenotypes. Ultraviolet (UV) light can be used to introduce random mutations within a microorganism's nuclear genome. Chemical mutagens include compounds that inhibit or disrupt the biosynthetic processes of a microorganism, e.g., antibiotics, antifungals, or carcinogens. Non-limiting examples of chemical mutagens include ICR-191, ethyl methanesulfonate (EMS), and 4-nitroquinoline- 1-oxide (4-NQO). Non-limiting examples of chemical mutagens include acridine mutagens, amino acid analogs, fatty acid biosynthesis inhibitors, cholesterol biosynthetic inhibitors, mTOR inhibitors, and membrane solubilizing agents. Combinations of chemical mutagens can also be used simultaneously (or serially) to induce mutagenesis. Following mutagenesis, selective or enrichment agents can be used to select or enrich for strains of interest. Non-limiting examples of enrichment agents include L-canavanine, cerulenin, triparanol, clomiphene, clomiphene citrate, clotrimazole, terfenadine, fluphenazine, AZD8055, BASF 13-338, cafenstrole, clomiphene, PF-042110, and phenethyl alcohol, as well as the use of a combination of enrichment agents.

[0120] By “an isolated cell” is meant a microalgal cell or other microorganism as set forth herein that has been isolated from its native environment and grown and cloned such that all the clones are the same.

[0121] Methods provided herein include classical strain improvement methods to improve strain productivity, carbon yield, and palmitic acid content. The glucose consumption rate of the microbial cells can be a highly predictive indicator of lipid titer and is known in the art. As such, the glucose consumption rate can be used as an enrichment tool in the mutant selection process. The methods provided herein can include one or more of determining a total lipid titer of the cell, determining a fatty acid profile of the oil, or determining a C16:0 (e.g., palmitic acid) content of the oil. Further, the methods provided herein can include assaying cell media to determine glucose consumption rate, total lipid titer, a fatty acid profile, a C16:0 (e.g., palmitic acid) content, and TAG profile. High Palmitic Oils of the Disclosure.

[0122] The high palmitic oils of the microbial strains, e.g., microalgal strains, described herein are obtained from isolated clonal cells that have been manipulated using classical strain improvement strategies as discussed herein. Exemplary strains are Prototheca microalgal strains such as P. wickerhamii or P. moriformis.

[0123] The complexity and physical properties of an oil can be evaluated by the fatty acid and the TAG profile of the TAG component of a microalgal oil. The fatty acid profile is a measure of fatty acid composition and can be determined by subjecting an oil to transesterification to generate fatty acid methyl esters (FAMEs) and subsequently quantitating fatty acid type by Gas-Chromatography equipped with a Flame-Ionization Detector (GC / FID). Accordingly, fatty acid content can be determined by GC / FID. Since TAGs comprise three fatty acids arrayed along the glycerol backbone in the TAG molecule, the number of possible distinct regioisomers of TAGs can be defined by the number of fatty acid species in the oil raised to the third power. The TAG profile provides relative amounts of various TAG species in an oil, which can be determined by subjecting the oil to TAG fractionation using Liquid- Chromatography / Time of Flight-Mass Spectrometry (LC / TOF-MS) equipped with an Atmospheric Pressure Chemical Ionization (APCI) source.

[0124] In some embodiments, an oil provided herein has a high palmitic acid content. For example, the fatty acid content of the TAG component of an oil provided herein can be high in palmitic acid, comparable to the palmitic acid content in palm olein derived from palm oil.

[0125] In some embodiments, an oil provided herein has a high palmitic acid content. For example, the fatty acid content of the TAG component of an oil provided herein can be high in palmitic acid, comparable to the palmitic acid content in palm olein derived from palm oil.

[0126] In some embodiments, an oil provided herein comprises saturated fatty acid content of at least 30%, at least 40%, at least 50%, or at least 60%. For example, an oil provided herein has a C16:0 content of at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, or more.

[0127] In some embodiments, an oil provided herein has a fatty acid content comprising at least 30%, at least 40%, at least 50%, or at least 60%, or more of C16:0 fatty acid. In some embodiments, the C16:0 fatty acid comprises palmitic acid.

[0128] In some embodiments, an oil provided herein has a fatty acid content of the TAG component comprising at least 40%, at least 45%, or at least 50%, or more of a C18:1 fatty acid. In some embodiments, the C18:1 fatty acid comprises oleic acid.

[0129] In some embodiments, the oil provided herein has a fatty acid content of the TAG component comprising less than 8%, less than 7%, less than 6%, less than 5%, or less of a C18:2 fatty acid. In some embodiments, the C18:2 fatty acid comprises linoleic acid.

[0130] In some embodiments, an oil provided herein comprises less than 5%, less than 4%, less than 3%, less than 2%, less than 1 %, less than 0.5%, less than 0.1%, or less tripalmitin.

[0131] In some embodiments, an oil provided herein comprises polyunsaturated fatty acids. In some embodiments, the polyunsaturated fatty acid comprises C18:2. In some embodiments, the oil has a C18:2 content from about 3% to about 9%, from about 4% to about 8%, or from about 5% to about8%. In some embodiments, an oil provided herein can have a C18:2 content of less than 8%, less than 7%, less than 6%, or lower.

[0132] In some embodiments, the fatty acid content of the TAG component can comprise from about 35% to about 60% C16:0 fatty acids.

[0133] In addition to the fatty acid profile, an oil can be further evaluated by the sterol profile or composition. Sterol composition can be determined by mass spectrometry, forexample, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (MS / MS), and coupled liquid and gas chromatography with subsequent flame ionization detection (LC-GC-FID). The concentration of the different sterols present in an oil can be expressed as mg sterol / 100 grams of oil. Non- limiting examples of sterols include ergosterol, ergost-8(14)-en-3-ol, (3β), 5.Xi.-Ergost-7-en- 3β-, 9,19-cyclolanost-24-en-3-ol, (3B), and 9,19-cyclolanostan-3-ol, 24-methylene-,(3B). In some embodiments, ergosterol may be present in an amount of at least about 30mg / 100 g oil. In some embodiments, 5.Xi.-Ergost-7-en-3β-ol may be present in an amount of at least about 25 mg / 100 g oil. In some embodiments, 9,19-Cyclolanost-24-en-3-ol, (3B)- may be present in an amount of at least 15 mg / 100 g oil. In some embodiments, 19-cyclolanostan-3-ol, 24- methylene-,(3B) may be present in an amount of at least about 25 mg / 100g oil.

[0134] In some embodiments, an oil provided herein can comprise less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less of monoacylglycerols (MAG).

[0135] In some embodiments, an oil provided herein can comprise less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less of diacylglycerols (DAG).

[0136] The microalgae-derived oil of the present disclosure can be useful as an alternative to palm oil in a variety of applications that utilize palmitic acid. A microalgae- derived oil of the present disclosure can deliver palmitic acid like palm olein and palm mid- fraction, the palm mid-fraction is valued for its high levels of the structuring fat, POP. However, the microalgae-derived oil of the present disclosure can also be advantageous over palm oil and its various fractions due to the reduced C18:2 fatty acid levels. The microalgae- derived oil of the present disclosure can comprise a lower level of C18:2 in comparison to palm oil. Because C18:2 fatty acids can be prone to oxidation, an oil comprising high levels of C18:2 can be more prone to oxidative degradation. Therefore, the oil of the present disclosure can be advantageous in food and nutrition product production that require long-shelf lives. The microalgae-derived oil of the present disclosure can also be advantageous over palm oil due to the reduced levels of MAG and DAG. MAG and DAG can be more sensitive to oxidation than TAG. MAG and DAG can also act as precursors for the formation of glycidyl esters, harmful process contaminants, during the thermal process of refining vegetable oils and fats.

[0137] For at least these reasons, the oil of the present disclosure can be advantageous in the production of food and nutrition products requiring the highest level of food safety.Food, Nutrition and Home and Personal Care Applications

[0138] The oils described herein can be used or formulated with one or more excipients for a variety of food applications, including but not limited to, food products, e.g., food-grade oils, cooking oil, frying oils, coatings, salad dressings, spreads, frozen desserts, pharmaceuticals, nutritional supplements, nutraceuticals, meal replacements, infant formulas, beverages, flavoring agents, and food additives.

[0139] Further, the oils of the present disclosure may be used in personal and home care as alternatives to palm oils and palm oil fractions which are commonly used as feedstock to prepare surfactants and derivatives. EXAMPLES Example 1. Generation of a Classically Improved Microalgal Strain Capable of Producing a Triglyceride Oil that is Highly Enriched in Palmitic Acid. Strain Acquisition, Isolation, and Characterization

[0140] A Prototheca wickerhamii strain (UTEX 1533) was obtained from the University of Texas at Austin Culture Collection of Algae (UTEX). Analysis of the 23S ribosomal DNA (rDNA) sequence of UTEX 1533 (SEQ ID NO:30) suggests that UTEX 1533 is very closely related to a Prototheca moriformis strain, UTEX 1435 (SEQ ID NO:31). The 23S rDNA sequence of UTEX 1533 has 100% sequence identity with the published 23S rDNA sequence from UTEX 1435 (FIG.1). The UTEX 1533 strain acquired from UTEX was verified as axenic through repeated passages on solid media containing various antibiotic and antifungal agents, followed by passage on antibiotic-free solid media to confirm a single cell type and a homogeneous morphology. This parent strain was given the designation “Strain 0” and is also referred to herein as CHK22. First Round of Mutagenesis.

[0141] The classical strain improvement of CHK22 was undertaken to improve its productivity, yield on carbon, and to increase its palmitic acid content. The strain lineage of mutants generated in the examples are shown in FIG.2A and 2B, while an overview of the process flow for mutagenesis, trait selection, and high-throughput, automated screening steps of the improvement process is outlined in FIG. 3. As shown in FIG. 2A and 2B, algal cells were cultured in a vegetative growth medium where indicated, comprised of the following components including macronutrients comprised of NaH2PO4, K2HPO4, citric acid monohydrate, MgSO4•7H2O, CuCl2•2H2O, dextrose, (NH4)2SO4, Sigma antifoam 204, at final concentrations of 13.6 mM, 11.4 mM, 5 mM, 0.15 mM, 40 g / L, 7.5 mM and 0.23 g / L,respectively. Vitamins were present as follows in the vegetative growth media: Thiamine HCl, D-pantothenic Acid, Biotin, Cyanocobalamin, Riboflavin, and Pyridoxine HCL at final concentrations of 2.96E-06, 2.24E-07, 6.53E-09, 8.30E-11, 1.33E-08 and 1.27E-08 molar, respectively. Micronutrients were present in the vegetative growth media used as follows: H3BO3, ZnSO4•7H2O, MnSO4•H2O, NaMoO4•2H2O, Ni(NO3)2•6H2O, citric acid monohydrate, CuSO4•5H2O and FeSO4•7H2O at final concentrations of 2.96E-05, 1.22E-05M, 1.46E-05, 3.97E-07, 2.75E-07, 1.95E-04, 3.96E-07, and 5.4E-06 molar, respectively. Cells in the log phase of growth in the vegetative medium, were subjected to mutagenesis by means of chemicals or UV light (FIG.3, Step 1). Cells were then sub-cultured into the lipid production medium, where they were subjected to selection / enrichment strategies (FIG.3, Step 2).

[0142] The lipid production medium was comprised of the same vitamins and micronutrients at the same concentrations as used in the vegetative medium. Macronutrients in the lipid production medium, relative to the vegetative medium, were adjusted to include 25 mM citric acid monohydrate, 50 g / L dextrose, and 1.5 mM (NH4)2SO4. The microalgal strains were plated to a solid medium vegetative medium containing 0.8% agarose to obtain clonal isolates (FIG.3, Step 3), followed by the interrogation of these isolates in the lipid production medium in a 96-well format (FIG.3, Step 4).

[0143] Using glucose consumption as a surrogate for oil production, high glucose- consuming strains can be validated in a lipid production medium in a tube or shake flask format (FIG. 3, Step 5). Isolates that successfully were validated were sub-cultured for multiple generations to stabilize mutations (FIG.3, Step 6), followed by purification of clonal isolates (FIG.3, Step 7) and subsequent re-interrogation in the lipid production medium (FIG.3, Step 8). Clones deemed to be phenotypically stable (% cv <5% for profile, glucose consumption and oil titer) were then validated via fermentation (FIG. 3, Step 9), while clones that still exhibited variability in (FIG.3, Step 8) were passaged once more (FIG.3, Step 6) to generate stable lines. Unless indicated otherwise, cells were grown at about 28 °C with shaking at 200 rpm for cultures grown in tubes or shake flasks, while blocks were grown with shaking at 900 rpm.

[0144] As detailed in FIG. 3, CHK22 was chemically mutagenized with 44 μM 4- nitroquinoline 1-oxide (4-NQO) or subjected to sham mutagenesis with the addition of only the mutagen solvent, dimethyl sulfoxide (DMSO). The mutagen was inactivated with the addition of sodium thiosulfate which was then removed with repeated washing in with water. The cells were then allowed to recover in limited a sugar growth media. The limited sugargrowth media is the same as the vegetative media, but contains 5 g / L glucose. The mutagenized and the mock-mutagenized populations were then each independently cultured in the lipid production media at 38 °C for five days. The optimal lipid production temperature for CHK22 is typically between 28-32 °C, so the higher-than-optimal temperature was applied as a stressor, with the goal of providing an advantage to those mutants with a predisposition toward exhibiting higher palmitate levels. A similar phenomenon has been observed in both fungal model systems and in some commercially relevant seed oil-producing higher plant species following chronic exposure to higher temperatures during growth or cultivation, respectively. At the end of the 5-day, 38 °C lipid culture, the cells were collected and then exposed next to 65 °C for 4 min. Based on range-finding experiments conducted in the same format, exposure to temperatures in this range for this duration of time typically kills >99% of the cell population. After heat exposure, the cells were recovered in limited sugar growth media for three days and then diluted and plated. Mutant clones were then picked and assessed for glucose consumption rate and fatty acid profile in a 72 hrs, 96-well block-based lipid production assay (FIG. 2A). The first three rows in Table 1 depict values for glucose consumption rate and fatty acid profile for the CHK22 parental strain, tested in triplicate. The last three columns in Table 1 depict CHK22 mutants. As shown in Table 1, CHK22 mutant 1 exhibited nearly 8% more palmitate than the CHK22 parent, albeit with a significant reduction in glucose consumption rate, a hallmark of lower lipid titer. Table 1: Fatty acid profiles of select CHK22 mutant strains exhibiting higher glucose consumption in a 96-well block lipid assay and increased palmitate levels.

[0145] As shown in FIG.2A, mutant strains were grown in lipid production medium (0.5 mL) with shaking (900 rpm) for 72 hours at 28 °C at which point glucose consumption was measured. Isolates with high glucose consumption levels were further interrogated for fatty acid composition with lead strains shown in Table 1.

[0146] To further adapt the CHK22 mutant 1 to its altered fatty acid composition and improve its lipid productivity, CHK22 mutant 1 was serially passaged in the absence of any selection and then plated to solid media to yield single colonies. These sub-clones were screened in a lipid production assay to assess the phenotypic stability of the lineage (e.g., %cv <5%) for both glucose consumption rate and fatty acid profile. After one cycle of serial passaging, the mutant was still phenotypically unstable. Several sub-clones that retained the higher palmitate level phenotype, along with increased rates of glucose consumption, were taken through additional rounds of serial passaging and stability assessment. After a total of five rounds of serial passaging and stability assessment, a stable strain, designated CHK100, was identified. CHK100 exhibited about 5% more palmitate and had an oil content, dry cell weight, and lipid titer greater than the CHK22 parental strain (FIG.2A). Table 2: Tube based assays on CHK100, a classically improved derivative of strain CHK22, showing increased C16:0 content and improved lipid titer.

[0147] As shown in FIG. 2A, all strains were run under standard lipid production conditions in which strains were grown in duplicate in 10 mL of lipid production medium. Cultures were grown with shaking (200 rpm) for 121 hours at 28 °C at which point 1 mL of biomass was removed, applied to a polycarbonate filter, washed with an equal volume of Milli Q water, and placed in a tared glass vial at -80 °C for 30 minutes. Vials containing filters and frozen biomass were lyophilized to dryness overnight, their weights recorded, and filters withdried biomass were subjected to direct transesterification followed by GC / FID to quantitate FAMEs. Mutagenesis of CHK100

[0148] CHK100 cells were recovered from a 24-hour lipid production culture and chemically mutagenized for 30 min at 32 °C with 22 μM 4-nitroquinoline 1-oxide (4-NQO) or subjected to sham mutagenesis with the addition of only the mutagen solvent, DMSO. The mutagen was inactivated with the addition of sodium thiosulfate which was then removed with repeated washing with water and media. The cells were then allowed to recover in limited sugar growth media. The mutagenized and the mock-mutagenized populations were then each independently cultured, first in seed media for 24 h at 38 °C and then in lipid production media for three days at 38 °C. At the end of the 3 days in the 38 °C lipid culture, the cells were collected and then exposed next to 65 °C for 1, 2, 3, or 4 min. After heat exposure, the surviving cells were recovered in limited sugar growth media for three days and then diluted and plated. Viable cells were only recovered from the 1 min at 65 °C exposure condition. Mutant clones were then picked and assessed for glucose consumption rate and fatty acid profile in a 72 h, 96-well block-based lipid production assay (FIG. 2A and 2B). Two of the mutants identified (Table 3; highlighted in bold font and underlined) exhibited 4-4.5% more palmitate than the CHK100 parent while maintaining a reasonable glucose consumption rate, which is typically a hallmark of a strain having an acceptable lipid titer. Table 3: Fatty acid profiles of select mutant strains exhibit higher glucose consumption rate (g / L*d, grams per liter per day cultured) in a 96-well block lipid assay and increased palmitate levels.

[0149] CHK100 mutant strains were grown in the lipid production medium (0.5 mL) described above with shaking (900 rpm) for 72 hours at which point glucose consumption was measured. Isolates with high glucose consumption levels were further interrogated for fatty acid composition with lead strains shown in Table 3 here compared to aggregated CHK100 parental control data.

[0150] These mutants were re-screened in a 5-day bioreactor tube-based lipid production assay to quantitatively assess their fatty acid profile, lipid productivity, and triacylglycerol (TAG) distribution, with a particular focus on the level of valuable palmitic- oleic-palmitic (POP)-containing TAGs. POP is valuable as an alternative to traditional POP obtained from palm oil, which requires a great deal of industrial processing to obtain and can be subject to supply issues given climate change.

[0151] As shown in Table 4, one of the mutants, “…Blk07;F02” or “CHK100 mutant isolate 2”, continues to exhibit ~4.5% more palmitate than the CHK100 parental strain. In addition, the lipid titer and oil content are modestly improved compared to the parent. An overview of the lineage history of these classically improved high palmitate strains is illustrated in FIG. 2A, with oleate (C18:1) and palmitate (C16:0) levels reported for each strain in the lineage. The method of mutagenesis utilized at each step is presented above each arrow, and the enrichment strategy utilized in conjunction with each mutagenesis strategy is presentedbelow each arrow. “...Blk07;F02” is otherwise referred to as “CHK mutant isolate 2” in the present disclosure. Table 4: Tube-based assays evaluating CHK100 mutants showing increased C16:0 levels and improved lipid titer. “Dry cell weight” (“DCW”) was determined by applying 1 mL of culture broth to a pre-weighed glass filter sitting atop a sintered glass funnel under vacuum and washing with 5 mL of water. Filters were then lyophilized to dryness and reweighed to determine the DCW of each sample. “Non lipid biomass” (“NLB”) was determined by subtracting the lipid (g / L) from the DCW. “Per cell production” (“PCP”) was determined by dividing the NLB by the lipid titer.

[0152] All strains included in Table 4 were run under standard lipid production conditions in which strains were grown in duplicate in 10 mL of the lipid production medium. Cultures were grown with shaking (200 rpm) for 121 hours at 28 °C at which point 1 mL of biomass was removed, applied to a polycarbonate filter, washed with an equal volume of Milli Q water and placed in a tared glass vial at -80 °C for 30 minutes. Vials containing filters and frozen biomass were lyophilized to dryness overnight, their weights recorded and filters with dried biomass were subjected to direct transesterification followed by GC / FID to quantitate FAMEs. Abbreviations included in Table 4 include: DCW (dried cell weight), NLB (non-lipid biomass), and PCP (per-cell production, defined as [lipid titer (g / L)] / NLB).

[0153] FIG.2B shows the continued progression of the mutant isolate “...Blk07;F02”, also referred to herein as strain “CHK mutant isolate 2.” Serial passaging of the isolate and screening in the lipid assays, as described above, resulted in a stable strain, CHK224, having a palmitate level of 37% in the isolated crude oil as determined by FAME analysis. A separate stability screen resulting from SSID 1447 produced subclone “C06” (FIG. 2B) and the intermediate strain CHK243u, which, upon additional serial passage and interrogation of isolates in lipid production, resulted in subclone “A10.” Subclone A10 is characterized as having a palmitate level of 39% in the isolated oil. Subclone A10, upon another round of passaging and interrogation in lipid production assays (SSID 1515), resulted in a branch in the lineage. One branch in the lineage gave rise to clone “E04’, which, upon additional passaging and stability assays in lipid production medium, resulted in the stable strain, CHK263, which is characterized as having 40% palmitate in the isolated crude oil.

[0154] The second branch point, as illustrated in FIG. 2B from the SSID 1515 clone, gave rise to the unstable strain “sub-clone E07”, which was subsequently designated CHK256u. A lipid stage culture of CHK256u was subsequently exposed to 2.8 µM 4-NQ at 30 °C for 30 minutes and allowed to recover for 24 hrs in fresh vegetative medium at 30 °C. The mutant population of the exposed CHK256u cells was then exposed during culture in lipid production medium to 100 µM tebutam (herbicide) at 35 °C for 72 h. Surviving cells were recovered and subjected to screening. The first clone from the screening of SSID1642 resulted in a mutant, “clone F07”, with a palmitate level of 42% in the isolated crude oil; this clone is designated CHK 268u. CHK 268u was subsequently subjected to serial passage and further interrogation using lipid production assays (SSID 1710). After screening of the SSID 1710cells, from this screen was cloned “Blk08,F04,” which has 49.9% palmitate when cultivated in lipid production medium at 32 °C in the isolated microalgal oil. LCMS Analytical Method

[0155] Triglyceride (TAG) profile analysis was conducted on CHK100 and the CHK100 mutant strains using liquid chromatography-mass spectrometry (LCMS). The TAGs were extracted from P. moriformis dried biomass into a solution of 3:1 toluene / 2-propanol (v / v) by means of a mechanical disruption using ceramic beads and vigorous agitation. The filtered extracts were then injected into an Agilent 1290 Infinity II UHPLC system coupled to a 6470B triple quadrupole mass spectrometer and APCI ionization source according to the parameters described by Co, E.D. et al. (Co, E.D., Koutchekinia, M., Carney, J.R. et al., “Matching the Functionality of Single-Cell Algal Oils with Different Molecular Compositions,” J. Am. Oil Chem. Soc.91: 533–48, 2014 ) and their Total Ion Chromatograms (TIC) are shown in FIG.4. FIG.4 illustrates, through the LCMS data shown, that TAG designations are non-regiospecific. LCMS Determination of Regioisomers

[0156] Chromatographic separation of regioisomers is not achieved by the LCMS method described, however, a qualitative assessment regarding the regiospecificity of the predominant isomer can be made based on the abundance of diacylglycerol (DAG) ions resulting from fragmentation of the TAG. In LC-APCI-MS analyses of TAGs, the loss of the fatty acid at the sn-1 and sn-3 positions are energetically more favorable compared to loss at the sn-2 position, resulting in a greater abundance of the diacylglycerol ions formed from the former fragments. For example, in the case of 1,2-palmitin-3-olein, (PPO), a near 1:1 ratio of [OP]+DAG ion (m / z 577) and [PP]+DAG ion (m / z 551) can be observed as the contribution of the 1-palmitin, 3-olein DAG species is a relatively rare event (also the [OP]+ DAG ion species (m / z 577). In the case of 1,3-palmitin-2-olein (POP) a greater abundance of the [OP]+DAG ion (m / z 577) can be observed, as elimination of palmitate at either sn-1 or sn-3 results in [OP]+DAG ion (m / z 577) while the corresponding [PP]+ DAG ion (m / z 551) can only arise via the rare loss of olein at the sn-2 position. For AAB and ABA type triglycerides, a linear calibration curve between [AA]+ / [AB]+ratios and mixtures of AAB and ABA pure regioisomers with known compositions can be constructed. The method used is described in, e.g., Byrdwell (Byrdwell WC., “The updated bottom-up solution applied to mass spectrometry of soybean oil in a dietary supplement gelcap,” Anal. Bioanal. Chem. 407(17): 5143-60, 2015); using thismethod, the precise composition of each regioisomer can be calculated based on this linear relationship and simplified by means of a two-point calibration curve using only the endpoints of the calibration curve. This can be expressed by the following equation:

[0157] Mass spectra and DAG ion ratios for the higher palmitate-producing microalgal strains obtained using the procedure depicted in FIG. 3, along with the CHK100 control and palm oil, are shown in FIGs. 5-8. FIGs. 9-12 depict the mass spectra and DAG ion ratios of native palm oil, and 1,3-palmitin-2-olein and 1,2-palmitin-3-olein, respectively, purchased from Larodan Research Chemicals. In Table 5, the [OP]+and [PP]+DAG ion abundance and calculated the %POP in the TAG component of the unpurified oil for each sample are shown (including their biological replicates designated rep A and rep B, and instrument injection replicates designated r001 and r002). In FIG.9, the red palm oil analyzed was Nutiva®Nurture Vitality purchased from Berkeley Bowl, Berkeley, California. In FIGs. 10 and 11, the 1,3- palmitin-2-olein and 1,2-palmitin-3-olein analytical standards were both purchased from Larodan Research Chemicals. Table 5: Calculated [OP]+ / [PP]+DAG ion abundance and calculated %POP based on their ratio compared to pure POP and PPO regioisomer standards from palm oil versus triglycerides extracted from various classically improved P. moriformis isolates using the screening method depicted in FIG.2A and 2B.

[0158] The complete triglyceride profile (area %) of each classically improved derivative of CHK100, the parental CHK100 control, and red palm oil are shown in Table 6. Further, as illustrated in Table 6, Sample CHK100 mutant isolate 2 (RepA), has higher POP and lower PPO compared to Red Palm Oil. Tripalmitin levels are also higher in red palm oil.Triglyceride designations for all other TAGs are non-regiospecific. The acronyms used in Table 6 and elsewhere are as follows: LLL linoleic-linoleic-linoleic LLP linoleic-linoleic-palmitic LOL linoleic-oleic-linoleic MLP myristic-linoleic-palmitic MOL myristic-oleic-linoleic MOP myristic-oleic-palmitic OOA oleic-oleic-arachidic OOB oleic-oleic-behenic OOL oleic-oleic-linoleic OOLn oleic-oleic-linolenic OOM oleic-oleic-myristic OOO oleic-oleic-oleic OOP oleic-oleic-palmitic OOS oleic-oleic-stearate PCP palmitic-capric-palmitic PLP palmitic-linoleic-palmitic PLS palmitic-linoleic-stearic PMP palmitic-myristic-palmitic POA palmitic-oleic-arachidic POB palmitic-oleic-behenic POL palmitic-oleic-linoleic POP palmitic-oleic-palmitic POLn palmitic-oleic-linolenic POS palmitic-oleic-stearic PPA palmitic-palmitic-arachidic PPO palmitic-palmitic-oleic PPP palmitic-palmitic-palmitic PPS palmitic-palmitic-stearic PSS palmitic-stearic-stearic SOL palmitic-oleic-linoleic SOS stearic-oleic-stearic SSG stearic-stearic-gondolic (C18:1n-11)SSID Strain / Sample Identifier TAG triacylglycerols or triglyceride and are non-regiospecific unless indicated Table 6: Triglyceride profiles of the higher palmitate strains compared to Nutiva®Nurture Vitality Red Palm Oil.

[0159] TAG designations are non-regiospecific, unless indicated, as is the case with POP and PPO. FIGs.12 and 13 are graphical representations of the same data shown in Table 6. “ECN” in Table 6 denotes the equivalent carbon number. Sample CHK100 mutant isolate 2 Rep A specifically shows a higher POP content compared to palm oil. The red palm oil used as the analytical standard in FIGs.12 and 13 was Nutiva® Nurture Vitality Red Palm Oil. Palm oil has a higher occurrence of PPO compared to the CHK100 control and CHK100 mutant isolates as well as higher tripalmitin.

[0160] Further, FAME analysis was performed on sample CHK100 mutant isolate 2 Rep A (see Table 4) as well as the analytical standard red palm oil (Nutiva® Nurture Vitality Red Palm Oil) and show the palmitate levels of the oil derived from CHK100 mutant isolate 2 of the present disclosure. Table 7: FAME Analysis of Red Palm Oil.Example 2. Fatty acid profile.

[0161] As shown in FIG.14, the fatty acid profile of the CHK100 mutant isolate 2, a high-palmitic acid strain obtained through classical strain optimization of CHK100 using the methods described herein, was analyzed using GC-FID (gas chromatography flame ionization detection). The results reveal levels of both palmitic and unsaturated fatty acids, calculated by adding the concentrations of oleic acid and linoleic acids (abbreviated as 18:1 and 18:2, respectively in FIG. 14) found in the samples. These levels of palmitic acid and unsaturated fatty acids of the microalgal oils described herein were found to be equivalent to those reported in the literature for palm olein (Sampaio et al., (2011) Steam deacidification of palm oil. Food and Bioproducts Processing.89, 383-90). Example 3. Triacylglycerol profile.

[0162] FIG. 15 illustrates the triacylglycerol (TAG) profile of an exemplary high- palmitic acid strain of the present disclosure, obtained through classical strain optimization of CHK100, has been analyzed using LC-MS (liquid chromatography – gas spectrometry). FIG. 18 compares the levels of PPP, PPO regioisomer and POP regioisomer of the various strainsdepicted in FIG.2A and 2B, or which the strain depicted in FIG.15 provides the results for a different strain.

[0163] FIG. 15 shows the levels of 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) in the classically improved algae strain, CHK100 mutant isolate 2, is equivalent to the level of POP level found in palm olein reported in the literature (Sampaio et al., 2011). The analysis also showed that the algae strain contains less than 1% tripalmitin (referred to as PPP in FIG.15). Example 4. Diacylglycerol and Monoacylglycerol profiles.

[0164] Both palm oil and palm fractions contain significant levels of partial glycerides (Sampaio et al., 2011 & Sasue et al., (2015) “Synthesis of palm oil high in diacylglycerol through direct esterification.” The Malaysian J. of Analytical Sciences. 19(1): 222-29), particularly diacylglycerols (DAG) and monoacylglycerols (MAG). According to the literature, palm oil and its fractions typically have about 2-6% diacylglycerols and 2-5% monoacylglycerols (Sasue et al., 2015). The algae oil described herein, with a fatty acid and triacylglycerol (TAG) profile similar to palm olein, contains only trace amounts (e.g., <2%) of the total MAG and DAG combined.

[0165] As documented in existing literature, the presence of partial glycerides such as DAG and MAG can adversely affect the shelf-life stability of products, given their heightened susceptibility to oxidative degradation compared to triacylglycerols (TAG). The high-palmitic oil resulting from the classical strain optimization of CHK100 also contains less than 6% of linoleic acid (expressed as % of total TAG), while palm oil and palm olein contain typically levels of linoleic acid ranging from 9-12%, which also makes palm oil and palm olein more sensitive to oxidative degradation.

[0166] Additionally, the occurrence of DAG in oil has been identified as a precursor for the formation of process contaminants, including glycidyl esters, that generated when one takes a microalgal oil through a deodorization process (Destaillats et al. (2012) Glycidyl esters in refined palm (Elaeis guineensis) oil and related fractions. Part I: Formation mechanism. Food Chemistry, 131: 1391-98).

[0167] Consequently, owing to the reduced MAG and DAG levels, the high-palmitic oil resulting from the classical strain optimization of CHK100 increases the oxidative stability of the oil and is seen to decrease the residual levels of glycidyl esters, making the resulting crude oil from the strain significantly more useful than what can be obtained from existing strains. A more oxidatively stable oil increases to the number of applications for use of the oil in comestibles for human and animal health and nutrition.Example 5. Characterization of High Palmitic Acid Triacylglycerol Oils Produced by Classically Improved Microalgal Strains.

[0168] The sterol compositions of high palmitic refined, bleached, and deodorized (RBD) triacylglycerol oils of the present disclosure were determined using gas chromatography combined with mass spectrometry and flame ionization detection. Samples were prepared using the technique described in the German standardized method F-III for sterols in fats and oils from Aitzetmüller, et al. (1998) (“Analysis of sterol content and composition in fats and oils by capillary-gas liquid chromatography using an internal standard. Comments on the German sterol method,” Fett / Lipid 100: 429-435), which is incorporated herein by reference in its entirety. After addition of an internal standard, the oils were saponified. The unsaponifiable fractions were isolated using solid-phase extraction. Trimethylsilyl (TMS) derivatives of the unsaponifiable fraction were injected on an Agilent 8890 gas chromatograph equipped with a 5977B single quadrupole EI-MS and FID (flame ionization detection) detector. A capillary flow technology (CFT) splitter was used to split the sample between the MS and FID detectors following separation using a DB-5MS Ultra Inert column (60 m length×0.25 mm inner diameter X 0.25 μm film thickness). Mass spectra data, in conjunction with retention time comparison with analytical standards, if available, were used to identify each of the sterol species. Quantification was based on the GC-FID detected response of the sterol TMS ethers compared to that of the internal standard.

[0169] The resulting sterol profiles are shown in Table 8. Amounts are shown as mg / 100 g of the oil and as approximate percentages of total detected sterols. Minor unidentified peaks were not accounted for. Not all sterols were identifiable and are listed as unknown. Ergosterol was the main sterol present in both oils. Various other sterols were present in the RBD oils in significant amounts, including 5.xi.-ergost-7-en-3(3-ol, (3β) and 9,19- cyclolanostan-3-ol, 24-methylene-(3B)-,O-TMS. Table 8: Sterol Profile of RBD Oil Strains - CHK22 and CHK100Example 6: Fatty acid and TAG profile of clone “Blk08,F04”.

[0170] As shown in FIG.16 and Table 9, the fatty acid profile of the “Blk08,F04” high- palmitic acid strain, obtained through classical strain optimization of CHK 268u (FIG. 2B), resulted in a strain producing almost 50% palmitate when grown at 32 °C and 47% when grown at 28 °C for the same length of time.

[0171] Table 9 shows the results of two biological replicates of clone “Blk08,F04” when run in a 5-day tube assay at either 32 or 28 °C. As can be seen, the lipid titer, DCW, and palmitate levels in the obtained oil are higher when grown at 32 °C, but the lipid % and per cell production (PCP;[Lipid g / L] / [DCW-lipid g / L]) are lower.

[0172] FIG.17 shows the TAG profiles of the lipids isolated from the same samples of clone “BLK08,F04” grown at 32 or 28 °C in which the isolate makes 45 or 40% POP, respectively. Table 9: Clone “Blk08,F04” run at 32 or 28 °C.

[0173] Table 10 and FIG.18 show levels of POP and PPO regioisomers and PPP levels in classically improved strains, which are the subject of this disclosure, as shown in FIG.2, as well as commercially available oils, including palm mid fraction (PMF) and red palm oil. The PMF is valued for its high POP content and is a key component in structuring fats generated through multiple stages of fractionation of palm oil fractions. POP, stearate-oleate-stearate (SOS), and palmitic-oleic-stearic (POS) are the principal structuring fats in cocoa butter. PMF and algal fats described herein can be use d as blend stocks along with cocoa butters from different geographies, whose content of the three principle structuring fats can differ, or with other cocoa butter extenders or equivalents used to affect the physical properties of these important structuring fats. As can be seen, clone “BLK08;F04” shows POP levels significantly higher than commercially available palm oil and approximately 70% of what is seen in commercially available palm mid fraction (PMF). Importantly, the levels of PPO, and PPP, which can adversely affect the melt profile and mouthfeel of confections, is markedly reduced in microalgal-derived oils described herein.

[0174] Close inspection of Table 10 and FIG. 18 demonstrate the utility of the microalgal cells of the genus Prototheca for generating fats of superior composition relative to traditional incumbent oils, such as red palm oil. Note that the levels of PPP and PPO, two TAG species which can disrupt and interfere with the melt profile and mouthfeel of important structuring fats, such as cocoa butter, are elevated in palm oil and in the palm mid fraction (PMF, designated as “AAK PMF” in FIG. 18 and below in Table 10) compared to the crudemicroalgal oils of the enhanced strains described herein. Note the inherently elevated levels of both PPO and PPP in red palm oil and PPO in particular in PMF.

[0175] All microalgal oils of the present disclosure show PPP and PPO levels well below those for palm oil (e.g., clone BLK08;FO4 shows PPP levels only 65% that seen in red palm oil and PPO levels at just 16% of what is seen in red palm oil, while at the same time possessing POP levels over 1.9 times higher) and PPO levels between 4.7- and 12.3-fold lower than what is seen in AAK PMF. Table 10. Levels of POP and PPO regioisomers and PPP levels in classically improved strains, which are the subject of this disclosure, and commercially available oils, also shown in FIG.18.The sequences of Table 11 represent exemplary insertion loci in Prototheca that can be used to assess the absence of recombinantly engineered introductions of heterologous genes into the parent strain. Given that certain governments regulate the use of products and labeling of products produced by or from genetically manufactured organisms (GMOs), the developed Prototheca strains obtained in this application can use these exemplary loci to demonstrate that they were not engineered. However, the strains can be recombinantly engineered if so desired. Table 11: shows SEQ ID NOS: 1-26, 30-31 as referenced herein.with reference to a variety of examples, but it should be understood that the features of the embodiments are susceptible to modification, alteration, changes or substitution without departing significantly from the spirit of the invention. One skilled in the art will recognize that numerous variations and modifications may be made to the examples described above without departing from the scope of the present disclosure. EMBODIMENTS

[0177] The following provides various embodiments exemplifying the compositions, methods of making, and uses of the methods and compositions described herein:

[0178] Embodiment [1]. An oil derived from a microalgal cell comprising a triacylglyceride (TAG) component, wherein the TAG component has a fatty acid content of at least 30% C16:0 fatty acids and at least 40% C18:1 fatty acids, and wherein the oil is produced through classical strain improvement of the microalgal cell.

[0179] Embodiment [2]. The oil of Embodiment [1], wherein the fatty acid content of the TAG component comprises at least 35% C16:0 fatty acids.

[0180] Embodiment [3]. The oil of Embodiment [1], wherein the fatty acid content of the TAG component comprises at least 40% C16:0 fatty acids.

[0181] Embodiment [4]. The oil of any one of Embodiments [1] to [3], wherein the fatty acid content of the TAG component further comprises less than 8% C18:2 fatty acids.

[0182] Embodiment [5]. The oil of any one of Embodiments [1] to [4], wherein the microalgal cell is of the genus Prototheca.

[0183] Embodiment [6]. The oil of any one of Embodiments [1] to [5], wherein the fatty acid content of the TAG component comprises at least 45% C18:1 fatty acids.

[0184] Embodiment [7]. The oil of any one of Embodiments [1] to [6], further comprising less than 1% monoacylglycerols (MAG).

[0185] Embodiment [8]. The oil of any one of Embodiments [1] to [7], further comprising less than 1% of diacylglycerols (DAG).

[0186] Embodiment [9]. The oil of any one of Embodiments [1] to [8], further comprising less than 1% tripalmitin.

[0187] Embodiment

[0010] . The oil of any one of Embodiments [1] to [9], wherein the oil comprises ergosterol.

[0188] Embodiment

[0011] . An unpurified oil derived from a Prototheca microalgal cell strain obtained by classical strain improvement, wherein the unpurified oil comprises a triacylglyceride (TAG) component, wherein the TAG component has a fatty acid content comprising 30% or more C16:0 fatty acid and less than 1% tripalmitin.

[0189] Embodiment

[0012] . The oil of Embodiment

[0011] , wherein the oil is produced from classical strain improvement of the microalgal cell.

[0190] Embodiment

[0013] . The oil of any one of Embodiments

[0010] to

[0012] , wherein the fatty acid content of the TAG component further comprises at least 40% C18:1 fatty acids.

[0191] Embodiment

[0014] . The oil of any one of Embodiments

[0010] to

[0013] , wherein the oil further comprises ergosterol.

[0192] Embodiment

[0015] . The oil of any one of Embodiments

[0010] to

[0014] , wherein the microalgal cell does not comprise an exogenous gene.

[0193] Embodiment

[0016] . The oil of any one of Embodiments

[0010] to

[0015] , further comprising less than 1% of monoacylglycerols (MAG).

[0194] Embodiment

[0017] . The oil of any one of Embodiments

[0010] to

[0016] , further comprising less than 1% of diacylglycerols (DAG).

[0195] Embodiment

[0018] . The oil of any one of Embodiments

[0010] to

[0014] , wherein the fatty acid content of the TAG component further comprises less than 8% C18:2 fatty acids.

[0196] Embodiment

[0019] . An unpurified microalgal oil comprising: a triacylglyceride (TAG) component having a fatty acid content comprising at least 30% C16:0 fatty acids, less than about 1% diacylglycerols, and less than about 1% monoacylglycerols.

[0197] Embodiment

[0020] . The oil of any one of Embodiment

[0019] , wherein the oil is derived from a Prototheca microalgal cell.

[0198] Embodiment

[0021] . The oil of any one of Embodiments

[0019] or

[0020] , wherein the oil is derived from an oleaginous microorganism.

[0199] Embodiment

[0022] . The oil of any one of Embodiments

[0019] or

[0021] , wherein the microalgal cell does not comprise an exogenous gene.

[0200] Embodiment [1A]. An isolated microalgal cell, wherein the microalgal cell is a Prototheca sp. cell obtained through classical microalgal cell improvement, wherein the microalgal cell produces an oil characterized as comprising: (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of a triacylglyceride (TAG component), or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%, and (b) a tripalmitin (PPP) content less than 0.3% of the TAG component, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

[0201] Embodiment [2A]. The isolated microalgal cell of Embodiment [1A], wherein the POP content is at least 45% and the PPP content is less than about 2.7% of the TAG component.

[0202] Embodiment [3A]. The isolated microalgal cell of Embodiment [1A] or Embodiment [2A], wherein the microalgal cell oil comprises: (a) the 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least about 50%, and (b) the tripalmitin (PPP) content of less than about 2.7% of the TAG component.

[0203] Embodiment [4A]. A microalgal oil comprising a triacylglyceride (TAG) component, wherein the TAG component comprises: (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or at least 23%, or at least 29%, or atleast 32%, or at least 35%, or at least 45% of the TAG component, (b) a tripalmitin (PPP) content less than 0.3% of the TAG component, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component, and wherein the microalgal oil is produced by a microalgal Prototheca cell obtained through classical strain improvement.

[0204] Embodiment [5A]. The microalgal oil of any one of Embodiments [4A] to [5A] or the microalgal oil produced by the isolated microalgal cell of any one of Embodiments [1A] to [3A], wherein the Prototheca sp. cell has an exogenous nucleic acid introduced recombinantly.

[0205] Embodiment [6A]. The microalgal oil of any one of Embodiments [4A] and [5A], wherein the TAG component comprises: (a) the 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 45%, or at least 50% or at least 55%, or at least 60%, and (b) the tripalmitin (PPP) content of less than about 2.7% of the TAG.

[0206] Embodiment [7A]. The microalgal oil of Embodiment [5A], wherein the microalgal oil has (a) the POP content of at least 35% and the PPP content of less than about 1.5%, or (b) the POP content of at least 45% and the PPP content of less than about 2.7%.

[0207] Embodiment [8A]. The microalgal oil of any one Embodiments [4A] to [7A], wherein the oil further comprises at least about 30% C16:0 fatty acids, or at least about 35% C16:0 fatty acids, or at least about 40% C16:0 fatty acids, or at least about 45% C16:0 fatty acids, or at least about 50% C16:0 fatty acids.

[0208] Embodiment [9A]. A method of obtaining a microalgal cell that produces the oil of any of Embodiments [4A] to [8A], the method comprising the steps of: (a) mutagenizing microalgal cells with at least one or more agents, which promote errors in DNA excision repair known (e.g., ultraviolet light (e.g., 254 nm), 4- nitroquinoline 1-oxide (4-NQO), methyl methanesulfonate (MMS), Ni, Ar, or polyaromatic hydrocarbons while culturing the microalgal cells at about 28 ºC to about 38 ºC in a vegetative or a lipid production media; (b) enriching the mutagenized microalgal cells by exposing the mutagenized cells to physical conditions (e.g., an elevated temperature of about 50 to about 68°C or by using density fractionation on sucrose or percoll gradients to isolate specific subpopulations based on density) or exposure to a chemical compound (e.g., herbicides, fungicides, or inhibitors of lipid biosynthesis, such as tebutam or cofenstrole);(c) growing the mutagenized, enriched microalgal cells in a lipid production medium; (d) harvesting the microalgal oil from the microalgal cells and interrogating the microalgal oil using a FAME analysis and a triacylglyceride (TAG) analysis to determine the component of the screened microalgal cells; and wherein the TAG component of the screened microalgal cells comprises: (i) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%, and (ii) a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

[0209] Embodiment [10A]. The method of Embodiment [9A], wherein the microalgal cell is a Prototheca moriformis.

[0210] Embodiment [11A]. An isolated microalgal cell obtained by the method of Embodiments [9A] to [10A].

[0211] Embodiment [12A]. The microalgal cell of Embodiments [10A] to [11A], wherein the oil of the microalgal cell further comprises at least about 30% C16:0 fatty acids, or at least about 35% C16:0 fatty acids, or at least about 40% C16:0 fatty acids, or at least about 45% C16:0 fatty acids, or at least about 50% C16:0 fatty acids.

[0212] Embodiment [13A]. A method of obtaining a microalgal oil of any of Embodiments [1A] to [3A] comprising the steps of: (a) mutagenizing cells with agents that introduce mutations through promoting errors in DNA excision repair known to include, but not limited to exposure to UV light (254 nm), 4-nitroquinoline 1-oxide (4-NQO), methyl methanesulfonate (MMS), Ni, Ar, or polyaromatic hydrocarbons, culturing the exposed microalgal cells at about 28 ºC to about 38 ºC in vegetative or lipid production media for a sufficient time; (b) enriching said cell populations using physical conditions (e.g., elevated temperatures of about 50, about 60, or up to about 68 °C, or fractionation on sucrose or percoll gradients to isolate specific subpopulations based on density) or chemical compound exposure (e.g., herbicides, fungicides or inhibitors of lipid biosynthesis, such as tebutam or cofenstrole);(c) plating the mutagenized and enriched cell populations on a solid vegetative medium; (d) selecting and growing selected cells in a lipid production medium; (e) harvesting the microalgal cells from said lipid production medium; and (f) obtaining and interrogating a microalgal oil from the selected microalgal cells using FAME analysis and and determining if the selected cells have a 1, 3- dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%; and a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

[0213] Embodiment [14A]. An isolated microalgal cell, wherein the microalgal cell is a Prototheca sp. that produces a microalgal oil, said oil is characterized as comprising: (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 60%, and (b) a tripalmitin (PPP) content of less than about 2% of the triacylglyceride (TAG) component, and the Prototheca sp. producing said microalgal oil was obtained through classical microalgal cell improvement of an isolated Prototheca sp.

[0214] Embodiment [15A]. A microalgal oil comprising a triacylglyceride (TAG) component, wherein the TAG component comprises: (a) a 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 60% of the TAG component, and (b) a tripalmitin (PPP) content of less than about 2% of the TAG component, and wherein the microalgal oil is produced by a microalgal Prototheca sp. cell obtained through a classical microalgal strain improvement method of the isolated Prototheca sp.

Claims

AMENDED CLAIMS received by the International Bureau on 01 August 2025 (01.08.2025)1. An isolated microalgal cell, wherein the microalgal cell is a Prototheca sp. cell obtained through classical microalgal cell improvement, wherein the microalgal cell produces an oil characterized as comprising:(a) a l, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of a TAG (triacylglyceride) component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45% of the TAG component, and(b) a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

2. The isolated microalgal cell of claim 1, wherein the POP content is at least 45% and the PPP content is less than about 2.7% of the TAG component.

3. The isolated microalgal cell of claim 1 or claim 2, wherein the microalgal cell oil comprises:(a) a l, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 50%, and(b) a tripalmitin (PPP) content of less than about 2.7% of the TAG component.

4. A microalgal oil comprising a triacylglyceride (TAG) component, wherein the triacylglyceride (TAG) component comprises:(a) a l, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or at least 23%, or at least 29%, or at least 32%, or at least 35%, or at least 45% of the TAG component,(b) a tripalmitin (PPP) content of less than 0.3% , or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component, and wherein the microalgal oil is produced by a microalgal Prototheca sp. cell obtained through classical strain improvement.

965. The microalgal oil of claim 4 or produced by the isolated microalgal cell of any one of claims 1 to 3, wherein the Prototheca cell has an exogenous nucleic acid introduced recombinantly.

6. The microalgal oil of claim 4 or claim 5 or of the isolated microalgal cell of any of claims 1 to 3, wherein the TAG component comprises:(a) the 1, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 45% of the TAG component, and(b) the tripalmitin (PPP) content of less than about 2.7% of the TAG component.

7. The microalgal oil of claim 5, wherein the microalgal oil has (a) the POP content of at least 35% and the PPP content of less than about 1.5%, or (b) the POP content of at least 45% and the PPP content of less than about 2.7%.

8. The microalgal oil of any one of claims 4 to 7, wherein the oil comprises at least about 30% C16:0 fatty acids, or at least about 35% C16:0 fatty acids, or at least about 40% C16:0 fatty acids, or at least about 45% C16:0 fatty acids, or at least about 50% C16:0 fatty acids.

9. A method of obtaining a microalgal cell that produces a microalgal oil as defined by any of claims 4 to 8 comprising the steps of:(a) mutagenizing microalgal cells with at least one or more agents, which promote errors in DNA excision repair known (e.g., ultraviolet light (e.g., 254 nm), 4- nitroquinoline 1-oxide (4-NQO), methyl methanesulfonate (MMS), Ni, Ar, or polyaromatic hydrocarbons while culturing the microalgal cells at about 28 °C to about 38 °C in a vegetative or a lipid production media;(b) enriching the mutagenized microalgal cells by exposing the mutagenized cells to physical conditions (e.g., an elevated temperature of about 50 to about 68°C, or by using density fractionation on sucrose or percoll gradients to isolate specific subpopulations based on density) or a chemical compound exposure (e.g., herbicides, fungicides, or inhibitors of lipid biosynthesis, such as tebutam or cafenstrole);(c) growing the mutagenized, enriched microalgal cells in a lipid production medium;97(c) harvesting the microalgal oil from the microalgal cells and interrogating the microalgal oil using a FAME analysis and a triacylglyceride (TAG) analysis to determine the component of the screened microalgal cells; wherein the TAG component of the screened microalgal cells comprises:(i) a l, 3-dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%, and(ii) a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

10. The method of claim 9, wherein the microalgal cell is a Prototheca sp.

11. An isolated microalgal cell obtained by the method of claim 9 or 10.

12. A microalgal oil obtained from the microalgal cell of any of claims 1 to 3 and 10, wherein the oil of the microalgal cell further comprises at least about 30% C16:0 fatty acids, or at least about 35% C16:0 fatty acids, or at least about 40% C16:0 fatty acids, or at least about 45% C16:0 fatty acids, or at least about 50% C16:0 fatty acids.

13. A method of obtaining a microalgal cell that produces a microalgal oil of any of claims 1 to 3 comprising the steps of:(a) mutagenizing cells with agents that introduce mutations through promoting errors in DNA excision repair known to include, but not limited to, UV light (254nm), 4- nitroquinoline 1-oxide (4-NQO), methyl methanesulfonate (MMS), Ni, Ar, or polyaromatic hydrocarbons culturing the microalgal cells at about 28 °C to about 38 °C in vegetative or lipid production media and(b) Enriching said cell populations using a physical change (e.g., elevated temperatures of about 50, about 60, or up to about 68 °C, or fractionation on sucrose or percoll gradients to isolate specific subpopulations based on density) or chemical compound (e.g., herbicides, fungicides, or inhibitors of lipid biosynthesis such as tebutam or cafenstrole),98(c) plating the mutagenized and enriched cell populations to a solid vegetative medium,(d) growing selected clones from the plated cells in a lipid production medium,(e) harvesting the selected clones from said lipid production medium, and(f) obtaining and characterizing a microalgal oil from the selected cells using FAME analysis and TAG analysis and determining if the selected cells have a 1, 3- dipalmitoyl-2-oleoyl-glycerol (POP) content of at least 17% of the TAG component, or of at least 23%, or of at least 29%, or of at least 32%, or of at least 35%, or at least 45%; and a tripalmitin (PPP) content less than 0.3%, or less than about 0.6%, or less than about 1.0%, or less than at least 1.2%, or less than about 1.5%, or less than about 2.7% of the TAG component.

14. A biomass obtained from culturing the microalgal cell of any one of claims 1-3, optionally wherein the biomass is washed and / or dried.99

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