Patent Description:
The present invention is defined by the appended claims and provides a non-naturally occurring oil comprising a triacylglyceride (TAG) component and at least <NUM> of ergosterol per <NUM> of the oil, wherein the TAG component has a fatty acid content comprising <NUM>% or more C18:<NUM> fatty acids and <NUM>% or more C18:<NUM> fatty acids, wherein the TAG component further comprises at least <NUM>% triolein (OOO) and wherein the oil is produced from classical strain improvement of a cell.

Formulations comprising the non-naturally occurring oil are disclosed herein.

A method for producing the non-naturally occurring oil is disclosed herein, the method comprising: culturing in a bioreactor an oleaginous, non-naturally occurring microorganism described herein, thereby producing the non-naturally occurring oil.

Provided herein are oil compositions having high oleic acid content. Methods of making the oil, and formulations and applications of the oil are disclosed. Oil compositions provided herein are produced by a microorganism that is produced by classical strain improvement strategies such as those described herein. In turn, these non-naturally occurring microorganisms can produce non-naturally occurring oils provided herein.

Genetic and non-genetic modification techniques can allow for the production of non-naturally occurring 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 elaboration of a particular fatty acid profile (e.g., high oleic acid content), yield on carbon, volumetric oil accumulation (e.g., g 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.

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 U. 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".

As used herein, the term "classical strain improvement" refers to methods of random or semi-random mutagenesis of microbes to create non-naturally occurring strains with improved properties. These methods include, but 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, chemical mutagens, and/or selective or enrichment agents. Classical strain improvement methods do not include recombinant genetic engineering methods targeted to one or more genomic regions, e.g., via homologous recombination.

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.

As used herein, the term "triacylglycerol", "triglyceride", or "TAG" refers to esters between glycerol and three saturated and/or unsaturated fatty acids. Generally, fatty acids of TAGs have chain lengths of <NUM> carbon atoms or more.

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.

As used herein, the term "fatty acid profile" refers 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 equipped with a Flame Ionization Detector (GC/FID).

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.

As used herein, the term "polyol" refers to triglycerols or fatty acid alcohols comprising hydroxyl functional groups. As used herein, the term "polyol derived from a TAG oil" generally refers to a polyol obtained from chemical conversion of a TAG oil, e.g., via epoxidation and ring opening, ozonolysis and reduction, or hydroformylation and reduction.

As used herein, the term "polyurethane", "PU", or "urethane" refers to a class of polymers comprised of carbamate (urethane) linkages formed between a polyol and an isocyanate moiety.

As used herein, the term "oleic content", "oleic acid content", "oleate content", or "olein content" refers the percentage amount of oleic acid in the fatty acid profile of a substance (e.g., a TAG oil). As used herein, the term "C18:<NUM> content" refers the percentage amount of a C18:<NUM> fatty acid (e.g., oleic acid) in the fatty acid profile of a substance (e.g., a microbial oil).

As used herein, the term "high oleic" can refer to greater than <NUM>% oleic acid, greater than <NUM>% oleic acid, greater than <NUM>% oleic acid, greater than <NUM>% oleic acid, greater than <NUM>% oleic acid, greater than <NUM>% oleic acid, or greater than <NUM>% oleic acid.

As used herein, "sequence identity" refers to a percentage of identical 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., ClustalW, 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 <NUM> to obtain the sequence identity between the two sequences.

As used herein, the term "about" refers to ±<NUM>% from the value provided.

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.

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. The non-genetically modified Prototheca sp. strain can be produced by one or more classical strain improvement strategies described herein.

In some embodiments, the cell does not comprise an exogenous gene or exogenous nucleotides that encodes for an exogenous protein or gene. For example, the cell does not comprise an exogenous gene in a lipid biosynthetic pathway. In some embodiments, the cell does not comprise a genetic disruption of one or more alleles of an endogenous gene in a lipid biosynthetic pathway. In some embodiments, the cell 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-<NUM> 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).

In some embodiments, the cell does not comprise an exogenous acyl-ACP thioesterase gene. In some embodiments, the cell does not comprise a genetic disruption of one or more alleles of an endogenous acyl-ACP thioesterase gene. In some embodiments, the cell 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.

In some embodiments, the cell does not comprise an exogenous fatty acid desaturase gene. In some embodiments, the cell does not comprise a genetic disruption of one or more alleles of an endogenous fatty acid desaturase gene. In some embodiments, the cell 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-<NUM> fatty acid desaturase. In some embodiments, the endogenous fatty acid desaturase gene is FAD2.

In some embodiments, the cell does not comprise an exogenous ketoacyl-ACP synthase gene. In some embodiments, the cell does not comprise a genetic disruption of one or more alleles of an endogenous ketoacyl-ACP synthase gene. In some embodiments, the cell 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.

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

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

In some embodiments, the cell does not comprise a genetic disruption of one or more alleles within <NUM> kb of an endogenous V-type proton ATPase catalytic subunit A isoform <NUM> gene or <NUM> genomic region. In some embodiments, the cell does not comprise a heterologous insertion within <NUM> kb of an endogenous V-type proton ATPase catalytic subunit A isoform <NUM> gene or <NUM> genomic region.

In some embodiments, the cell does not comprise a genetic disruption of one or more alleles within <NUM> kb of an endogenous DAO1B gene. In some embodiments, the cell does not comprise a heterologous insertion within <NUM> kb of an endogenous DAO1B gene.

In some embodiments, the cell does not comprise a genetic disruption of one or more alleles within <NUM> kb of an endogenous Thi4 gene. In some embodiments, the cell does not comprise a heterologous insertion within <NUM> kb of an endogenous Thi4 gene.

Accordingly, the cell comprises uninterrupted sequences of endogenous genes or genomic regions, including FAD2, FATA1, KASII, SAD2, V-type proton ATPase catalytic subunit A isoform <NUM> (<NUM>), DAO1B, and Thi4 described herein.

In some embodiments, the cell comprises at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% sequence identity to any one of SEQ ID NO:<NUM>-<NUM>. In some embodiments, the cell comprises any one of SEQ ID NO:<NUM>-<NUM> (genomic regions of CHK22 and CHK80). In some embodiments, the cell comprises SEQ ID NO:<NUM>-<NUM> (genomic regions of CHK22 and CHK80).

In some embodiments, the cell comprises at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% sequence identity to any one of SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> (genomic regions of CHK80). In some embodiments, the cell comprises any one of SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> (genomic regions of CHK80). In some embodiments, the cell comprises SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> (genomic regions of CHK80).

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. , Euglena 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. , Gomphonema sp. , Watanabea, sp. , Botryococcus sp. , Tetraselmis sp. , and Isochrysis sp. In some embodiments, the microalgae is Prototheca sp. In some embodiments, the microalgae is P. moriformis. In some embodiments, the microalgae is P. wickerhamii. In some embodiments, the cell is derived from a UTEX <NUM> base strain. In some embodiments, the cell is derived from a UTEX <NUM> base strain. In some embodiments, the cell is derived from a base strain having a <NUM> ribosomal DNA sequence that is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or <NUM>% identity to SEQ ID NO:<NUM> or SEQ ID NO:<NUM>. In some embodiments, the cell has a <NUM> ribosomal DNA sequence that is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or <NUM>% identity to SEQ ID NO:<NUM> or SEQ ID NO:<NUM>.

In some embodiments, an oil provided herein is 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.

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.

Classical strain improvement strategies can be used to select for organisms having desired phenotypes, e.g., high oleic 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 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 arrive to 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 which inhibit or disrupt biosynthetic processes of a microorganism, e.g., antibiotics, antifungals, or carcinogens. Non-limiting examples of chemical mutagens include ICR-<NUM>, ethyl methanesulfonate (EMS), and <NUM>-nitroquinoline-<NUM>-oxide (<NUM>-NQO). Non-limiting examples of chemical mutagens also 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 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 <NUM>-<NUM>, cafenstrole, clomiphene, PF-<NUM>, and phenethyl alcohol.

Classical strain improvement includes methods to improve strain productivity, carbon yield, and oleic acid content. Glucose consumption rate can be highly predictive indicator of lipid titer. As such, glucose consumption rate can be used as an enrichment tool in the mutant selection process. The methods can further include one or more of determining a total lipid titer of the cell, determining a fatty acid profile of the oil, or determining a C18:<NUM> (e.g., oleic acid) content of the oil. Further, the methods can include assaying cell media to determine glucose consumption rate, total lipid titer, a fatty acid profile, and/or a C18:<NUM> (e.g., oleic acid) content.

The complexity and physical properties of an oil can be evaluated by the fatty acid profile and the TAG profile of the TAG component of an 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 of three fatty acids arrayed along the glycerol backbone in the triglyceride 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.

In some embodiments, an oil provided herein has a high oleic acid content and low saturated fatty acid content. For example, the fatty acid content of the TAG component of an oil provided herein can be high in oleic acid and low in saturated fatty acids.

Non-limiting examples of monounsaturated fatty acids include C10:<NUM>, C12:<NUM>, C14:<NUM>, C16:<NUM>, C17:<NUM>, C18:<NUM>, C18:<NUM>-OH, C20:<NUM>, C22:<NUM>, and C24:<NUM>. In some embodiments, an oil provided herein comprises one or more of C10:<NUM>, C12:<NUM>, C14: <NUM>, C16: <NUM>, C17:<NUM>, C18:<NUM>, C18:<NUM>-OH, C20:<NUM>, C22:<NUM>, or C24:<NUM> fatty acids. In some embodiments, an oil provided herein has a monounsaturated fatty acid content of at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%. For example, an oil provided herein has a C18:<NUM> content of at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or more.

In some embodiments, an oil provided herein has a fatty acid content comprising <NUM>% or more, <NUM>% or more, <NUM>% or more, or <NUM>% or more of C18:<NUM> fatty acids. In some embodiments, the C18:<NUM> fatty acids comprise oleic acid. In some embodiments, the C18:<NUM> fatty acids comprise at least <NUM>% oleic acid. In some embodiments, the C18:<NUM> fatty acids comprise at least <NUM>% oleic acid. In some embodiments, the C18:<NUM> fatty acids comprise at least <NUM>% oleic acid.

An oil provided herein has a C18:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C18:<NUM> content of at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%. For example, an oil provided herein has a C18:<NUM> content of at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or more.

An oil provided herein has an oleic content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has an oleic content of at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%. For example, an oil provided herein has an oleic content of at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or more.

An oil provided herein has a triolein (OOO) content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a triolein content of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%. For example, an oil provided herein has a triolein content of at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or more.

An oil provided herein has a C16:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C16:<NUM> content of greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, or greater than <NUM>%. In some embodiments, an oil provided herein has a C16:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. In some embodiments, this oil has a C16:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C16:<NUM> content of about <NUM>% or about <NUM>%.

An oil provided herein has a C20:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C20:<NUM> content of greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, greater than <NUM>%, or greater than <NUM>%. In some embodiments, an oil provided herein has a C20:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. In some embodiments, this oil has a C20:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C20:<NUM> content of about <NUM>% or about <NUM>%.

Non-limiting examples of saturated fatty acids include C10:<NUM>, C12:<NUM>, C14:<NUM>, C16:<NUM>, C17:<NUM>, C18:<NUM>, C20:<NUM>, C22:<NUM>, and C24:<NUM>. In some embodiments, an oil provided herein comprises one or more of C10:<NUM>, C12:<NUM>, C14:<NUM>, C16:<NUM>, C17:<NUM>, C18:<NUM>, C20:<NUM>, C22:<NUM>, or C24:<NUM> fatty acids. In some embodiments, an oil provided herein has a saturated fatty acid content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. For example, an oil provided herein has a saturated fatty acid content of less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, or less. In some embodiments, this oil has a saturated fatty acid content of greater than <NUM>%. In some embodiments, an oil provided herein has a saturated fatty acid content of about <NUM>%.

An oil provided herein has a C14:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C14:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. In some embodiments, this oil has a C14:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C14:<NUM> content of about <NUM>%.

An oil provided herein has a C16:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C16:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. In some embodiments, this oil has a C16:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C16:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C16:<NUM> content of about <NUM>%.

An oil provided herein has a C18:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C18:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. In some embodiments, this oil has a C18:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C18:<NUM> content of about <NUM>%.

An oil provided herein has a C20:<NUM> content of from <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C20:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. In some embodiments, this oil has a C20:<NUM> content of greater than <NUM>%. In some embodiments, an oil provided herein has a C20:<NUM> content of about <NUM>%.

Non-limiting examples of polyunsaturated fatty acids include C18:<NUM>, C18:<NUM>, C18:<NUM> alpha, C18:<NUM> gamma, and C22:<NUM>. In some embodiments, an oil provided herein comprises one or more of C18:<NUM>, C18:<NUM>, C18:<NUM> alpha, C18:<NUM> gamma, or C22:<NUM> fatty acids. In some embodiments, an oil provided herein has a polyunsaturated fatty acid content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. For example, an oil provided herein has a polyunsaturated fatty acid content of less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, or less. The oil has a polyunsaturated fatty acid content of greater than <NUM>%.

An oil provided herein has a C18:<NUM> content of from <NUM>-<NUM>% or <NUM>-<NUM>%. In some embodiments, an oil provided herein has a C18:<NUM> content of less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>%, or less than <NUM>%. The oil has a C18:<NUM> content of greater than <NUM>%.

In some embodiments, the fatty acid content of a TAG component comprises one or more of C14:<NUM>, C16:<NUM>, C16:<NUM>, C18:<NUM>, C18:<NUM>, C18:<NUM>, C18:<NUM> alpha, C20:<NUM>, or C20:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises one or more of C14:<NUM>, C16:<NUM>, C18:<NUM>, C18:<NUM>, C20:<NUM>, or C20:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises C16:<NUM>, C18:<NUM>, C18:<NUM>, and C18:<NUM> fatty acids. In some embodiments, the C18:<NUM> fatty acids of the TAG component comprises at least <NUM>% of oleic acid.

The fatty acid content of the TAG component can comprise <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>% of C16:<NUM> fatty acids. In some embodiments, the fatty acid content of a TAG component comprises <NUM>% or more of C16:<NUM> fatty acids. In some embodiments, the fatty acid content of a TAG component comprises <NUM>% or more of C16:<NUM> fatty acids. In some embodiments, the fatty acid content of a TAG component comprises more than <NUM>% of C16:<NUM> fatty acids. In some embodiments, the fatty acid content of a TAG component comprises more than <NUM>% of C16:<NUM> fatty acids.

The fatty acid content of the TAG component can comprise <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>% of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises <NUM>% or more of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises <NUM>% or more of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises more than <NUM>% of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises less than <NUM>% of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises less than <NUM>% of C20:<NUM> fatty acids.

The fatty acid content of the TAG component can comprise <NUM>-<NUM>% of C18:<NUM> fatty acids. The fatty acid content of the TAG component comprises <NUM>% or more of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises <NUM>% or more of C18:<NUM> fatty acids. In some embodiments, the fatty acid content of the TAG component comprises more than <NUM>% of C18:<NUM> fatty acids.

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, for example, 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). Concentration of the different sterols present in an oil can be expressed as mg sterol/<NUM> of oil. Non-limiting examples of sterols include brassicasterol; campesterol; stigmasterol; beta-sitosterol (β-sitosterol); ergosterol; ergosta-<NUM>,<NUM>,<NUM>(<NUM>),<NUM>-tetraen-<NUM>-ol,(3β,22E), ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β,22E); ergost-<NUM>(<NUM>)-en-<NUM>-ol, (3β); ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β); <NUM>. -ergost-<NUM>-en-3β-ol; <NUM>,<NUM>-cyclolanost-<NUM>-en-<NUM>-ol, (3β); and <NUM>,<NUM>-cyclolanostan-<NUM>-ol, <NUM>-methylene-, (3β).

In some embodiments, an oil provided herein comprises one or more of ergosterol; ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β); <NUM>. -ergost-<NUM>-en-3β-ol; <NUM>,<NUM>-cyclolanost-<NUM>-en-<NUM>-ol, (3β); and <NUM>,<NUM>-cyclolanostan-<NUM>-ol, <NUM>-methylene-, (3β). In some embodiments, the non-naturally occurring oil comprises one or more of brassicasterol, campesterol, stigmasterol, β-sitosterol, delta-<NUM>-avenasterol, cycloartenol, or ergosterol. In some embodiments, the non-naturally occurring oil comprises brassicasterol, cycloartenol, and ergosterol.

An oil provided herein may comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of ergosterol per <NUM> of the oil. In some embodiments, an oil provided herein comprises more than <NUM>, more than <NUM>, or more than <NUM> of ergosterol per <NUM> of the oil. In some embodiments, an oil provided herein comprises no more than <NUM> of ergosterol per <NUM> of the oil. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% ergosterol on a weight-by-weight basis.

An oil provided herein comprises <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of campesterol per <NUM> of the oil. In some embodiments, an oil provided herein comprises no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, or no more than <NUM> of campesterol per <NUM> of the oil. In some embodiments, an oil provided herein does not comprise campesterol. In some embodiments, an oil provided herein does not contain a detectable level of campesterol. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises about <NUM>% campesterol on a weight-by-weight basis.

An oil provided herein can comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of brassicasterol per <NUM> of the oil. In some embodiments, the non-naturally occurring oil comprises more than <NUM> of brassicasterol per <NUM> of the non-naturally occurring oil. In some embodiments, the non-naturally occurring oil comprises more than <NUM> of brassicasterol per <NUM> of the non-naturally occurring oil. In some embodiments, an oil provided herein does not contain brassicasterol. In some embodiments, an oil provided herein does not contain a detectable level of brassicasterol. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises about <NUM>% brassicasterol on a weight-by-weight basis.

An oil provided herein comprises <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of stigmasterol per <NUM> of the oil. In some embodiments, the non-naturally occurring oil comprises more than <NUM> of stigmasterol per <NUM> of the non-naturally occurring oil. In some embodiments, an oil provided herein comprises no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, or no more than <NUM> of stigmasterol per <NUM> of the oil. In some embodiments, an oil provided herein does not contain stigmasterol. In some embodiments, an oil provided herein does not contain a detectable level of stigmasterol. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises about <NUM>% stigmasterol on a weight-by-weight basis.

An oil provided herein can comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of β-sitosterol per <NUM> of the oil. In some embodiments, the non-naturally occurring oil comprises less than <NUM> of β-sitosterol per <NUM> of the non-naturally occurring oil. In some embodiments, an oil provided herein comprises no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, no more than <NUM>, or no more than <NUM> of β-sitosterol per <NUM> of the oil. In some embodiments, an oil provided herein does not contain β-sitosterol. In some embodiments, an oil provided herein does not contain a detectable level of β-sitosterol. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises about <NUM>% β-sitosterol on a weight-by-weight basis.

An oil provided herein can comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β)- per <NUM> of the oil. In some embodiments, an oil provided herein comprises more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, or more than <NUM> of ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β)-per <NUM> of the oil. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises <NUM>-<NUM>%, <NUM>-<NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β)- on a weight-by-weight basis.

An oil provided herein can comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of <NUM>. -ergost-<NUM>-en-3β-ol, (3β) per <NUM> of the oil. In some embodiments, an oil provided herein comprises more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, or more than <NUM> of <NUM>. -ergost-<NUM>-en-3β-ol, (3β) per <NUM> of the oil. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% <NUM>. -ergost-<NUM>-en-3β-ol, (3β) on a weight-by-weight basis.

An oil provided herein can comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of <NUM>,<NUM>-cyclolanost-<NUM>-en-<NUM>-ol, (3β) per <NUM> of the oil. In some embodiments, an oil provided herein comprises more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, or more than <NUM> of <NUM>,<NUM>-cyclolanost-<NUM>-en-<NUM>-ol, (3β) per <NUM> of the oil. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% <NUM>,<NUM>-cyclolanost-<NUM>-en-<NUM>-ol, (3β) on a weight-by-weight basis.

An oil provided herein can comprise <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> of <NUM>,<NUM>-cyclolanostan-<NUM>-ol, <NUM>-methylene-, (3β) per <NUM> of the oil. In some embodiments, an oil provided herein comprises more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM> more than <NUM>, more than <NUM>, more than <NUM>, more than <NUM>, or more than <NUM> of <NUM>,<NUM>-cyclolanostan-<NUM>-ol, <NUM>-methylene-, (3β) per <NUM> of the oil. In some embodiments, the sterol content (as a percentage of total sterols) of an oil provided herein comprises <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% <NUM>,<NUM>-cyclolanostan-<NUM>-ol, <NUM>-methylene-, (3β) on a weight-by-weight basis. In some embodiments, the non-naturally occurring oil comprises less than <NUM> of cycloartenol per <NUM> of the non-naturally occurring oil.

The oils described herein can be used or formulated with one or more excipients for a variety of applications, including but not limited to, process oils (e.g., for tires), waxes, lubricants, polyols, macrodiols, polyesterdiols, and polyurethane products, e.g., hard foams, soft foams, cast polyurethanes, thermoplastic polyurethanes (TPUs), elastomers, adhesives, coatings, laminates, films, and dispersions. Polyurethane products can be used to construct aerospace, automotive, medical, electronic, building and construction goods; sporting goods or recreational equipment, e.g., skis, snowboards, sidewalls, boating equipment, kayaks; and other consumer goods, e.g., industrial containers, coolers, mattresses, leather goods, apparel, footwear, mannequins, and phone cases. These polyurethane applications can serve as sustainable alternatives to petroleum-based, non-renewable materials, such as acrylonitrile butadiene styrene (ABS), ultra-high molecular weight polyethylene (UHMWPE), or high density polyethylene (HDPE).

Oil provided herein can have improved production efficiency and a TAG composition that is enhanced for improved control of hydroformylation chemistry for generating polyols. These characteristics of microbial oil result in a greater degree of hydroxyl group (-OH) functionality relative to oils with greater TAG heterogeneity (hence, lower purity) and/or diversity (e.g., oilseed or plant derived oils). Thus, polyols produced from hydroformylation of high oleic oils can be preferable in generating polymers, including in instances where physical properties of a polymer can be compromised by molecular impurities, such as non-hydroxylated fatty acids that may be present in oils having a more diverse or heterogeneous TAG profile.

Polyols derived from hydroformylation of a high oleic oil can be particularly useful for producing polyurethane materials. For example, oils provided herein can have relatively low TAG diversity, low fatty acid diversity, and the majority of fatty acids present in the oils may be unsaturated fatty acids. A higher ratio of unsaturated fatty acid to saturated fatty acid can allow for increased chemical reactivity at the double bonds. Oils having low TAG diversity and a high proportion of unsaturated fatty acids can be especially desirable in production of polyurethanes because hydroformylation of such a mixture yields a greater percentage of fatty acids that can participate in crosslinking reactions with isocyanates. Unlike unsaturated fatty acids, saturated fatty acids do not contain carbon-carbon double bounds and cannot participate in crosslinking reactions with isocyanates. Thus, polyols generated from hydroformylation of unsaturated fatty acids from high oleic oil can yield polyurethane materials having superior properties.

The hydroxyl functionality can be introduced into oils provided herein via a chemical conversion of the TAG component. This conversion requires the presence of a double bond on the acyl moiety of the fatty acid, e.g., an olefinic group, which can be accomplished using several different chemistries including, for example:.

While typically carried out in organic solvent, processes that utilize aqueous systems have been developed to improve sustainability of these chemistries. Of the chemistries described above, only hydroformylation results in the preservation of fatty acid length and formation of primary hydroxyl moieties. Primary hydroxyl functionalities are highly desirable due to increased reactivity compared to secondary hydroxyl moieties. Furthermore, only olefinic fatty acids with a double bond that is converted into a site possessing hydroxyl functionality, through epoxidation and ring opening, ozonolysis, or hydroformylation/reduction, can participate in subsequent downstream chemistries, i.e., reaction with an isocyanate moiety to form a urethane linkage or reaction with methyl esters to form polyesters. All other fatty acids, namely, fully saturated fatty acids that do not contain carbon-carbon double bonds, cannot participate in crosslinking reactions with isocyanates. Hence, saturated fatty acids can compromise the structural integrity and degrade performance of the polymer produced therefrom.

The oils described herein can be used or formulated with one or more excipients for a variety of personal care product applications, including but not limited to, cosmetics, creams, face creams, hand creams, balms, lip balms, serums, face serums, body oils, hair oils, soaps, shampoos, conditioners.

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.

A Prototheca wickerhamii strain (UTEX <NUM>) was obtained from the University of Texas at Austin Culture Collection of Algae (UTEX). Analysis of the <NUM> ribosomal DNA (rDNA) sequence of UTEX <NUM> (SEQ ID NO:<NUM>) suggests that UTEX <NUM> is very closely related to a Prototheca moriformis strain, UTEX <NUM> (SEQ ID NO:<NUM>). The <NUM> rDNA sequence of UTEX <NUM> has <NUM>% sequence identity with the published <NUM> rDNA sequence from UTEX <NUM> (<FIG>). The UTEX <NUM> 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 <NUM>" and also known as CHK22.

Classical strain improvement of Strain <NUM> was undertaken in an effort to improve strain productivity, carbon yield, and oleic acid content. First, Strain <NUM> was exposed to UV light to introduce a diverse collection of random mutations within the nuclear genome. To accomplish this, Strain <NUM> cells were grown to mid-log phase in <NUM> of vegetative growth media (M21 seed media) in a <NUM>-mL baffle flask at <NUM>/<NUM> rpm. The cells were then washed in seed media and <NUM>×<NUM><NUM> cells were spread on a <NUM>-mm solid tryptone soy agar (TSA) + <NUM>/L glucose plate + <NUM>% agar and allowed to dry. The cells on the plate were then exposed to <NUM>,<NUM>µJoules of UV radiation (approximately <NUM> sec) using a Stratalinker® <NUM> UV Crosslinker system. Following UV exposure, the cells were scraped off of the solid media surface with a sterile cell scraper and into M21 seed media lacking both sugar and nitrogen. These cells were then washed extensively in the same nitrogen-free and sugar-free media, and then collected in a sterile <NUM>-micron filter sterilization unit by vacuum filtration. Light exposure was kept to a minimum during this process to reduce the frequency of light-dependent repair of UV damage. The cells were washed off of the sterile filter and resuspended in M20 + <NUM>/L glucose pre-seed media formulated with <NUM> L-serine as a nitrogen source, in lieu of the typical ammonium sulfate. Half of the mutagenized cell population was exposed to the acridine mutagen ICR-<NUM> in M20 + <NUM>/L glucose pre-seed media for <NUM> at <NUM> with <NUM> rpm agitation. ICR-<NUM> can introduce both double strand breaks and frame-shift mutations when used as a single agent, and significant chromosomal rearrangements when used in conjunction with other mutagens, including UV radiation.

The culture of Strain <NUM> in M22 pre-seed media formulated with <NUM> L-serine as a nitrogen source was previously shown to have little impact or no impact growth. Nonetheless, amino acid uptake studies in related microalgae have suggested that culture under such conditions stimulates the uptake of L-arginine. After <NUM>-<NUM> of incubation at <NUM>/<NUM> rpm agitation, a portion of the mutagenized and mock-mutagenized cultures were independently plated to M20 + <NUM>/L glucose + <NUM> L-serine + <NUM>µg/mL L-canavanine + <NUM>% agarose solid media. L-canavanine is a toxic analog of L-arginine that disrupts protein synthesis and leads to growth arrest. It is possible, albeit exceedingly rare, (i.e., approximately <NUM> in <NUM><NUM> to <NUM> in <NUM><NUM>) to spontaneously obtain L-canavanine-resistant (L-canR) colonies when plating otherwise wild-type populations of cells onto L-canavanine-containing media in the presence of L-arginine. Based on this possibility, the frequency of L-canR individuals observed in UV-mutagenized versus mock-mutagenized cell populations can serve as a rough measure of the efficiency of a given mutagenesis at increasing the rate of mutation over background. In the case of this UV-mutagenesis campaign, L-canR colonies were obtained at a rate ><NUM>,<NUM> times more frequently in the UV-mutagenized population than in the mock-mutagenized population, indicating a significant mutagenic load.

The mutagenesis, trait selection, high-throughput, and automated screening steps of the improvement process are outlined in <FIG>. Briefly, algal cells in log phase of growth were subjected to mutagenesis by means of chemicals or UV light (<NUM>). Cells were then sub-cultured into lipid production medium where the cells were subjected to selection/enrichment strategies (<NUM>). A non-mutagenized strain was run in parallel with the mutagenized strain until the growth in lipid production medium (as determined by an increase in A750) of the mutant strain exceeded that of the non-mutagenized control, as determined by a statistical analysis. Strains were then plated to solid medium to obtain clonal isolates (<NUM>), followed by the interrogation of these isolates in lipid production medium in a <NUM>-well plate format (<NUM>). Using glucose consumption as a surrogate for oil production, high glucose consuming strains were validated in lipid production medium in a tube or shake flask format (<NUM>). Isolates that were successfully validated were sub-cultured for multiple generations to stabilize mutations (<NUM>), followed by purification of clonal isolates (<NUM>) and subsequent re-interrogation in lipid production medium (<NUM>). Clones deemed to be phenotypically stable were then ready for validation in fermentation (<NUM>), while clones that still show variability in (<NUM>) are passaged once more (<NUM>) to generate stable lines.

Cerulenin is an antifungal antibiotic reported to inhibit fatty acid and sterol biosynthesis. Data from a number of studies suggest that cerulenin can specifically inhibit both the β-ketoacyl-ACP synthase I (KASI) and the β-ketoacyl-ACP synthase II (KASII) enzymes in plants, bacteria, and fungi. KASI catalyzes the two carbon at a time condensation reaction that generates <NUM>-<NUM> carbon long fatty acids. KASII functions in the condensation of palmitoyl-ACP with malonyl-ACP to yield stearoyl-ACP, which is rapidly desaturated to oleate. UV-induced mutants of Strain <NUM> that show resistance to cerulenin could achieve this resistance through mutations that increase KASI/KASII activity, a phenotype that could be highly beneficial for oleate production. In order to isolate cerulenin-resistant Strain <NUM> mutants, the approach illustrated in <FIG> was utilized. Briefly, the UV-mutagenized and mock mutagenized populations described previously were allowed to incubate in <NUM> M20 pre-seed media at <NUM>/<NUM> rpm for an additional <NUM> hours after sampling for the L-canavanine resistance test (<NUM> total). An <NUM>% (v/v) inoculum from each culture was used to initiate a <NUM> seed culture in standard M21 seed media in a <NUM>-mL bioreactor tube. These seed cultures were incubated for <NUM>-<NUM> hour to mid-log phase, after which the cultures were divided and used to perform a <NUM>% inoculation of different wells of a <NUM>-well block containing <NUM> of M22 lipid production media and varying amounts of cerulenin or DMSO as a vehicle control. These lipid cultures were incubated for approximately <NUM> hours. Growth was then assessed by measuring optical density at <NUM> wavelength (OD750) in a Molecular Devices Spectramax M5 plate reader.

While growth of the vast majority of the cells can be negatively impacted by cerulenin, a very small subset of cells with beneficial mutations within the mutant population can be resistant to the antibiotic. To increase the likelihood of isolating these resistant cells, wells in which inhibition of growth was ≥<NUM>% of the growth observed in DMSO-treated controls were collected onto sterile filters, extensively washed in M20 pre-seed media, and then used to initiate another round of M20 pre-seed culture. These cells were then subjected to another round of seed and then lipid culture in the presence of differing amounts of cerulenin or DMSO, as was performed in the first round of enrichment for cerulenin resistance. This cycle was repeated until a "differentiation event" was observed in which the MIC50 of the mutant population is higher than that of the mock mutagenized population. Wells in which statistically significant differences were observed were pooled, collected on sterile filters, washed extensively, and then diluted and plated for single colonies on <NUM>-cm M20+<NUM>/L glucose + <NUM>% agar plates. Clonally isolated mutants from the cerulenin-resistant population constituted a cerulenin-resistant "mutant library".

Using the UV-mutagenized and UV+ICR-<NUM>-mutagenized library of Strain <NUM>, four cycles of enrichment were conducted as described at which time a very small but statistically significant increase in the MIC50 was observed only in the UV-mutagenized mutant population when compared to the mock-mutagenized controls (<FIG>). Cells in these wells were pooled, collected with <NUM>-µm filter sterilization units, washed with media, and then resuspended in media and counted. These cells were then spread onto M20 + glucose + <NUM>% agar solid media to clonally isolate individual mutants.

<FIG> shows a prophetic example of the enrichment strategy used to isolate cerulenin-resistant microalgae. As illustrated in <FIG>, a portion of a <NUM>-deep well block shows wells containing microalgae, with grey circles representing wild type, cerulenin-sensitive a portion of a <NUM>-deep well blocks with wells containing microalgae, with grey circles representing wild type, cerulenin-sensitive cells, and yellow circles representing cerulenin-resistant cells. When resistant cells are collected in the first round of enrichment and then redistributed across the cerulenin concentration gradient in a second round of enrichment, cells with a high tolerance for the cerulenin are present in concentrations that easily out-compete the sensitive cells in the population. These wells should thus have higher levels of growth than the mock-mutagenized populations of cells that have undergone a similar treatment. This "differentiation event" indicates the outgrowth of cerulenin resistance.

<FIG> illustrates an actual enrichment strategy used to isolate cerulenin-resistant microalgae. After the first round of exposure to cerulenin, very little difference is observed between the UV-mutagenized and UV+ICR-<NUM>-mutagenized populations and the mock-mutagenized controls (left panel). After two additional round of drug exposure, the UV-mutagenized population shows slightly higher growth even at the highest cerulenin concentration then the mock mutagenized controls (right panel; red box). Wells containing these more cerulenin resistant cells were pooled, collected, and washed on sterile filters, and diluted and spread on solid media to isolate single colonies.

Colonies arising from individual mutant clones were picked from the solid media plates for screening using a Picolo™ automated colony picking inoculation tool integrated into a TECAN Freedom EVO® <NUM> automated liquid handling system. Each selected colony was used to inoculate both a single well of an archival <NUM>-well solid media M20 + <NUM>/L glucose + <NUM>% agarose storage plate, as well as the corresponding well of a <NUM>-well deep well block containing <NUM> of M20 pre-seed media. Pre-seed cultures were incubated at <NUM>/<NUM> rpm/<NUM>% humidity for <NUM>. An <NUM>% (v/v) inoculum from each culture was then used to initiate a seed culture in <NUM> of standard M21 seed media in corresponding wells of <NUM>-deep well block. These seed cultures were incubated at <NUM>/<NUM> rpm/<NUM>% humidity for <NUM>-<NUM> to mid-log phase, and then the cultures were used to perform a <NUM>% inoculation of corresponding wells of a <NUM>-deep well block containing <NUM> of M22 lipid production media. Lipid cultures were incubated for <NUM> and then samples from each culture were assessed for rate of glucose consumption using an automated glucose assay described in <FIG> to measure residual glucose in each well. Individual clones with glucose consumption rates greater than or equal to the average consumption rate of the Strain <NUM> controls were selected for further analysis. Approximately <NUM>µL of broth from each of the selected lipid cultures were frozen at -<NUM> for approximately <NUM> hour and lyophilized overnight and then submitted for quantitative FAME analysis of fatty acid composition by GC-FID. A number of clones were identified with significant increases in oleate levels and glucose consumption rates equal to or greater than Strain <NUM> (TABLE <NUM>). These primary hits were re-screened in a tube assay to assess oleate production and lipid titer following <NUM>-days in lipid culture (TABLE <NUM>). Three mutants, Strain 0_G07, Strain 0_E05, and Strain 0_E06, had lipid titers that were comparable to the Strain <NUM> parental strain while manifesting <NUM>-<NUM>% more oleate content.

TABLE <NUM> summarizes oleate content of select mutant strains exhibiting higher glucose consumption in a <NUM>-well block lipid assay. Mutant strains (approximately <NUM>,<NUM> isolates) were grown in lipid production medium (<NUM>) with shaking (<NUM> rpm) for <NUM> hours at <NUM> at which point glucose consumption was measured. Isolates with high glucose consumption levels were further interrogated for fatty acid composition with lead strains shown here.

TABLE <NUM> summarizes the performance of select mutant strains in a tube-based assay based on average dry cell weight (Avg DCW), average oil titer (g/L) (Avg Oil), oil titer g/L, % coefficient of variance (Oil %CV), average non lipid biomass (Avg NLB), average per cell production (oil titer/NLB-Avg PCP), and average % oleate content (Avg % Oleate). Mutants described in TABLE <NUM> were re-interrogated in a larger tube-based format in which isolates were grown in duplicate in <NUM> of lipid production medium in a <NUM>-mL bioreactor with shaking (<NUM> rpm) for <NUM> hours at <NUM> at which point <NUM> of biomass was removed, applied to a polycarbonate filter, washed with an equal volume of Milli Q water, and placed in tared glass vial at -<NUM> for <NUM> minutes. Vials containing filters and frozen biomass were lyophilized to dryness overnight, weights were recorded, and filters with dried biomass were subjected to direct transesterification followed by GC/FID to quantitate FAMEs. Mutants highlighted in bold showed both high productivity and high oleate levels.

The phenotypic stability of each of the most promising mutants (highlighted in bold in TABLE <NUM>) was evaluated by repeated sub-culturing in vegetative growth media (M21 + <NUM>/L glucose seed media) until <NUM> or more population doublings had occurred. Cultures were then plated to M20 + <NUM>/L glucose + <NUM>% agarose solid media. Six colonies were picked to a solid media archival storage plate and were subsequently analyzed in duplicate <NUM>-mL lipid production assays conducted in <NUM>-mL bioreactor tubes at <NUM>/<NUM> rpm. One of the strains showed a coefficient of variance (%CV) of less than <NUM>% for oil titer and less than <NUM>% in oleate levels across all six sub-clones. This strain was deemed phenotypically stable and cryo-preserved as strain "Strain <NUM>". The other mutant clones were not phenotypically stable and were reserved for additional rounds of sub-culturing and phenotypic evaluation or subject to additional round of classical strain improvement.

TABLE <NUM> summarizes the results of tube-based assays assessing stability of classically improved high oleic strains. Tube-based assays in which six isolates from each strain were grown in duplicate in <NUM> of lipid production medium. Cultures were grown with shaking (<NUM> rpm) for <NUM> hours at <NUM> at which point <NUM> of biomass was removed, applied to a polycarbonate filter, washed with an equal volume of Milli Q water, and placed in tared glass vial at -<NUM> for <NUM> 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. Strain 0_G07 was determined to be stable and was cryo-preserved as "Strain <NUM>".

Following the cryo-preservation of Strain <NUM>, a series of fermentation development runs were conducted to ascertain which process conditions would yield the highest oleic acid content compared to the parental base strain, Strain <NUM>, which elaborates ca. <NUM>% oleic acid in low cell density fermentations, and ca. <NUM>% oleic acid under an optimized high cell density, high oleic process in a <NUM>-L bioreactor. As illustrated in TABLE <NUM>, feeding a macronutrient bolus (Mg++ and PO<NUM>---) or administering a complete media replacement bolus, had little impact on oleic accumulation. In contrast, increasing the dissolved oxygen (DO) from <NUM> to <NUM>%, further elevated the oleic acid content by roughly <NUM>%. Additional attempts at stressing Strain <NUM> by reducing the run temperature after the base transition from <NUM> to <NUM> showed no additional impact on oleic acid levels.

TABLE <NUM> shows fermentation development runs for Strain <NUM> in comparison with the highest oleic content observed in runs of Strain <NUM> (all at <NUM>-L scale). All runs were carried out at <NUM>-L scale. Nitrogen refers to the total nitrogen used in the process (batched + fed ammonia). DO refers to dissolved oxygen. Mg++ and PO<NUM>--- bolus refers to a bolus (equivalent to the amount of each macronutrient that is batched in the initial vessel seed medium) that is administered just after base transition. Complete media replacement refers to administering a bolus (just after base transition) containing all batched media components with the exception of nitrogen. All broth samples for analysis were applied to a polycarbonate filter, washed with an equal volume of Milli Q water, and placed in a tared glass vial at -<NUM> for <NUM> minutes. Vials containing filters and frozen biomass were lyophilized to dryness overnight, weights were recorded, and filters with dried biomass were subjected to direct transesterification followed by GC/FID to quantitate FAMEs.

The mutant strain, Strain 0_E06 (Strain E06), while not phenotypically stable, consistently achieved higher oleate levels than the mutant lineage that gave rise to Strain <NUM> (TABLE <NUM>). As such, this mutant lineage was selected for further rounds of classical strain improvement, and designated as "Strain K". Following overnight growth in M21 + <NUM>/L glucose seed media to mid-log phase, the E06 mutant was exposed to <NUM> ethyl methanesulfonate (EMS) in <NUM> potassium phosphate buffer (pH <NUM>) for <NUM> at room temperature in dark and with periodic gentle agitation. Mutagenesis was terminated with the addition of a sodium thiosulfate solution to final concentration of <NUM>% (w/v). In parallel, a mock-mutagenized control cell population was taken through all the same manipulations, except for exposure to EMS. Cells were collected using <NUM>-micron filter sterilization units, washed extensively with water, and then washed with and into <NUM> M20+ <NUM>/L glucose pre-seed media. Pre-seed cultures were incubated at <NUM>/<NUM> rpm/<NUM>% humidity for <NUM>. An <NUM>% (v/v) inoculum from each culture was then used to initiate a seed culture in <NUM> of standard M21 + <NUM>/L glucose seed media in <NUM>-mL bioreactor tubes. These seed cultures were incubated at <NUM>/<NUM> rpm/<NUM>% humidity for <NUM>-<NUM> to mid-log phase, and then the cultures were used to perform a <NUM>% inoculation of replicate wells in a <NUM>-deep well block containing <NUM> of M22 lipid production media formulated with <NUM> triparanol and varying concentrations of the membrane fluidizing chemical, phenethyl alcohol, in a range from <NUM>-<NUM>. Triparanol is a <NUM>-dehydroreductase inhibitor that disrupts production of cholesterol in mammalian cells. Triparanol can inhibit phytosterol biosynthesis in some microalgae. These lipid cultures were incubated for approximately <NUM> hours at <NUM>/<NUM> rpm/<NUM>% humidity. Growth was then assessed by measuring optical density at <NUM> wavelength (OD750) in a Molecular Devices Spectramax M5 plate reader. Cells from lipid cultures with <NUM>% or greater inhibition of growth relative to vehicle (DMSO)-treated controls as measured by optical density (<FIG>) were pooled in sterile <NUM>-micron filter sterilization units, washed extensively with fresh M20 + <NUM>/L glucose pre-seed media, and used to initiate a <NUM> M20 + <NUM>/L glucose pre-seed cultures in <NUM>-mL bioreactor tubes. The surviving population was then subjected to a second round of exposure to a combination of <NUM> triparanol and a range of phenethyl alcohol concentrations, ranging from <NUM>-<NUM>, again under lipid production culture conditions. Surviving cells were collected, washed, and recovered into M21 + <NUM>/L glucose seed media, allowed to grow overnight to mid-log phase and then cryopreserved with the addition of dimethyl sulfoxide to <NUM>% (v/v) and flash freezing. This expanded population of rare, surviving mutants was subsequently interrogated for the ability to survive under lipid production conditions in the presence of increasing levels of triparanol.

The cryo-vials were thawed and used to initiate a <NUM> pre-seed culture in bioreactor tube, which was incubated for <NUM> at <NUM>/<NUM> rpm. Two rounds of selection and enrichment with increasing amounts of triparanol were conducted following the strategy outlined in <FIG>. Following the third cycle of selection and enrichment, the mutant population began to significantly differentiate from the mock-mutagenized population when growth was measured during lipid production (<FIG>). This population was collected on sterile filters, diluted, and spread on solid media. The resulting clonal isolates were screened in our automated <NUM>-well block-based lipid production and glucose consumption assay. A number of strains with increased oleate levels were identified among the mutant clones (TABLE <NUM>). Strains validated in repeat assays were then assessed for phenotypic stability. Stable strains were cryo-preserved.

TABLE <NUM> summarizes the oleate content of select mutant strains exhibiting higher glucose consumption in a <NUM>-well block lipid assay. Mutant strains (approximately <NUM>,<NUM> isolates) were grown in lipid production medium (<NUM>) with shaking (<NUM> rpm) for <NUM> hours at <NUM> at which point glucose consumption was measured. Isolates with high glucose consumption levels were further interrogated for fatty acid composition with lead strains shown here.

<FIG> shows growth curves for selection for mutants with greater tolerance to membrane stress. Cells were exposed to a fixed concentration (<NUM>) of triparanol and increasing concentrations of the membrane fluidizer phenethyl alcohol during a <NUM>-day lipid production culture in a <NUM>-deep well block. Significant inhibition of growth occurred at phenethyl alcohol concentrations ><NUM>. Rare surviving cells were recovered by sterile filtration from wells treated with ≥<NUM> phenethyl alcohol, washed with and collected from the filter in M20 + glucose pre-seed media and the cultures used to initiate another round of selection for mutants with greater tolerance of membrane stress.

<FIG> shows growth curves for selection for mutants with greater tolerance to the triparanol. Cells were exposed to increasing amounts of triparanol during a <NUM>-day lipid production culture in a <NUM>-deep well block. Significant inhibition of growth occurred at phenethyl alcohol concentrations ><NUM>. Rare surviving cells presumed to be in wells where <NUM>% inhibition was observed were recovered by sterile filtration, washed with and collected from the filter in M20 + glucose pre-seed media. The cultures were then used to initiate another round of selection. By the third round of selection (right panels), a subtle difference in the growth of the mutant population compared to the mock-mutagenized controls was observed. Cells were collected from the replicated wells treated with <NUM> triparanol (green boxes) and were plated to non-selective media to generate a mutant library for screening.

Lead strains where subsequently evaluated side-by-side in <NUM> lipid production cultures experiment conducted in <NUM>-mL bioreactor tubes. As shown in TABLE <NUM>, the best performing strain, designated as "Strain <NUM>", manifested ><NUM>% more oleate than Strain <NUM>. Strain <NUM> also achieved a <NUM>% higher lipid titer than Strain <NUM>. Based on its performance in low cell density lipid assays in tubes, the performance of Strain <NUM> was then evaluated under high cell density fermentation conditions.

TABLE <NUM> summarizes results of tube based assays on classically improved derivatives of Strain <NUM> showing increased C18:<NUM> content. All strains were run under standard lipid production conditions in which strains were grown in duplicate in <NUM> of lipid production medium. Cultures were grown with shaking (<NUM> rpm) for <NUM> hours at <NUM> at which point <NUM> 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 -<NUM> for <NUM> minutes. Vials containing filters and frozen biomass were lyophilized to dryness overnight, weights were recorded, and filters with dried biomass were subjected to direct transesterification followed by GC/FID to quantitate FAMEs.

A series of development runs utilizing Strain <NUM> were conducted in an effort to explore whether process conditions could further elevate C18:<NUM> levels relative to the ca. <NUM>-<NUM>% oleic obtained in tube and block based assays as described above. The results of these efforts are illustrated in TABLE <NUM> below. Reference runs showing Strain <NUM> and Strain <NUM> are also displayed, illustrating the best high oleic processes for these two strains as well. As shown in the data, the <NUM> nitrogen process that incorporated a Mg++ and PO4--- bolus post base transition, delivered the highest oleic content. At approximately <NUM>% oleic content, a <NUM>% increase in oleate over the previous high oleic strain, Strain <NUM>, was achieved.

TABLE <NUM> summarizes results of fermentation development runs for Strain <NUM> in comparison with the highest oleic content observed for Strain <NUM> and Strain <NUM> (all at <NUM>-L scale). All runs were carried out at <NUM>-L scale. Nitrogen refers to the total nitrogen used in the process (batched + fed ammonia). DO refers to dissolved oxygen. Mg++ and PO4--- bolus refers to a bolus (equivalent to the amount of each macronutrient that is batched in the initial vessel seed medium) that is administered just after base transition. Complete media replacement refers to administering a bolus (just after base transition) containing all batched media components with the exception of nitrogen. All broth samples for analysis were applied to a polycarbonate filter, washed with an equal volume of Milli Q water, and placed in a pre-weighed glass vial at -<NUM> for <NUM> minutes. Vials containing filters and frozen biomass were lyophilized to dryness overnight, weights were recorded, and filters with dried biomass were subjected to direct transesterification followed by GC/FID to quantitate FAMEs.

To further increase the oleate levels achieved in Strain <NUM> by classical strain improvement methods, a number of different cocktails of metabolic inhibitors and herbicides were tested with the goal of increasing genetic diversity in mutant libraries and biasing mutant populations toward higher oleate and/or increased lipid production. To generate a diverse library of mutants, Strain <NUM> was exposed to <NUM> ethyl methane sulfonate for <NUM> hour at room temperature, followed by inactivation of the mutagen and recovery of the cells after culture for <NUM>. The resulting mutant population was subjected to a single round of enrichment with different cocktails of selective agents (<FIG>, Panel A) that impact disparate aspects of fatty acid, triacylglycerol, or sterol production. Two selective agent cocktail-treated mutant cultures exposed to inhibitor mixtures <NUM> and <NUM> (<FIG>, Panels A and B) grew ≥<NUM>-fold more than their paired, mock-treated control populations and were thus considered to have differentiated phenotypically. The mutant libraries derived from these cultures were plated on solid media to isolate individual mutant clones. These clones were initially screened for glucose consumption rates and those showing a statistically significant increase in glucose consumption compared to controls were analyzed for fatty acid profile. One isolate, M05_H03, showed a significant increase in oleate compared to the Strain <NUM> parental strain and was evaluated for phenotypic stability (TABLE <NUM>). In initial stability evaluations, some instability in both profile and glucose consumption was observed. As such, the sub-clone manifesting the highest oleate level, M05_H03. D02, was selected for additional multi-generational sub-culture, and subsequently a re-evaluation of phenotypic stability. However, the sub-clone was shown to be unstable and was designated as "Strain <NUM>".

TABLE <NUM> shows representative fatty acid profiles for sub-clones of a promising Strain <NUM> mutant generated through an additional round of classical strain improvement. All strains were run under standard lipid production conditions in a <NUM>-well block format in which cells are grown in <NUM> of lipid production media. Cultures were grown with shaking (<NUM> rpm) for <NUM> hours at <NUM>, at which point <NUM>µL of biomass was removed, transferred to glass micro-vials, and frozen -<NUM> for <NUM>-<NUM> minutes. The micro-vials containing frozen biomass were lyophilized to dryness overnight and then subjected to direct transesterification followed by GC/FID to quantitate FAMEs. The sub-clone, M05 _H03. C05 (highlighted in bold italics), showed a significant deviation in fatty acid profile from the other sub-clones of the original mutant that was identified. Note that the glucose consumption rate in grams/liter*day (g/L*D) correlated linearly with lipid titer in this assay and also showed significant variability in this mutant clone. Given this phenotypic instability, one of the sub-clones that produced the highest level of oleate, M05_H03. D02 (also highlighted in bold), was selected for additional sub-culture stability re-evaluation.

<FIG> summarizes the classical strain improvement of Strain <NUM> by generation of diverse mutant libraries through exposure to different mixtures of metabolic inhibitors and herbicides. Following mutagenesis with ethyl methane sulfonate (EMS) and recovery, the resulting mutated cell populations were cultured under standard <NUM>-well block lipid production conditions in <NUM> of lipid production media containing different combinations of inhibitors and/or herbicides. Mock-mutagenized cultures taken through all the same manipulations except for exposure to EMS were exposed to the same mixtures of inhibitors and herbicides in parallel. Cultures were grown with shaking (<NUM> rpm) for <NUM> hours at <NUM> after which samples were removed from each well and diluted. Growth was assessed through optical density measurement at <NUM> in a <NUM>-well plate reader (<FIG>, Panel A). Error bars in the bar plot for each inhibitor mixture show the standard error derived from <NUM> replicate wells subjected to the same treatment. Cultures treated with inhibitor mixtures that showed a ≥<NUM>-fold higher level of growth compared to the mock-mutagenized control (yellow arrows; student t-test assuming unequal variances, α=<NUM>) were plated to solid media to generate clonal mutant libraries and screened. Mixtures <NUM> and <NUM> contained different combinations and concentrations of metabolic inhibitors (<FIG>, Panel B) whose reported target activity and hypothesized mechanism of action in the lipid assay are described in the table in <FIG>, Panel C.

As an alternative to stabilizing Strain <NUM>, a new round of mutagenesis was pursued for Strain <NUM> utilizing treatment of cells with the mutagen <NUM>-nitroquinoline-<NUM>-oxide (<NUM>-NQO) for <NUM> minutes at <NUM>. Mutagenized cells were enriched by growing under conditions of limited glucose (<NUM>/L) for three days, then the cells were subjected to fraction over a <NUM>% Percoll/<NUM> NaCl density gradient. Cells recovered from a density zone of <NUM>/mL were plated and assessed for glucose consumption and fatty acid profile. One of these clones was subsequently stabilized and given the strain designation "Strain <NUM>".

A subpopulation of the Strain <NUM> lineage was concurrently subjected to an enrichment strategy employing one round of enrichment in an inhibitor cocktail (Inhibitor Cocktail <NUM> in <FIG>, Panel B) comprised of <NUM> clomiphene citrate, <NUM> terfenadine, <NUM> fluphenazine, and <NUM> triparanol. Cells recovered from the enrichment were plated and subsequently assessed for glucose consumption and fatty acid profile in a <NUM>-hour, <NUM>-well block based format. One of these clones was subsequently stabilized and given the strain designation "Strain <NUM>".

Strain <NUM> was subjected to another round of mutagenesis with increasing concentrations and exposure time to <NUM>-NQO (<NUM> for <NUM> minutes at <NUM>). This population of cells was subsequently subdivided and grown in standard lipid production medium supplemented with a range of cerulenin concentrations (<NUM>-<NUM>). Cells from all concentrations were pooled and fractionated over a <NUM>% Percoll/<NUM> NaCl density gradient. Oil laden cells recovered from a density zone of <NUM>/mL were plated and assessed for glucose consumption and fatty acid profile. One of these clones was subsequently stabilized and given the strain designation "Strain <NUM>".

Strain <NUM> was subsequently mutagenized with <NUM>,<NUM>µJoules of UV radiation. The resulting population of cells were subdivided and grown in standard lipid production medium supplemented with a range of concentrations (<NUM>-<NUM>) of the mTOR (mammalian target of rapamycin) ATP-competitive inhibitor, AZD8055. Cells from all concentrations were pooled and fractionated over a <NUM>% Percoll/<NUM> NaCl density gradient. Oil laden cells recovered from a density zone of <NUM>-<NUM>/mL were plated and assessed for glucose consumption and fatty acid profile. One of these clones was subsequently stabilized and given the strain designation "Strain <NUM>". TABLE <NUM> shows the fatty acid profiles of strains depicted in <FIG> from a <NUM>-day, <NUM>-well block lipid assay.

<FIG> shows an overview of classical strain improvement and provenance of Strain <NUM> by generation of mutants derived from UTEX <NUM> using mutagens and a series of selective agents and enrichment strategies. Inhibitor cocktail <NUM> (Strain <NUM> to Strain <NUM>) comprised of <NUM> BASF <NUM>-<NUM>, <NUM> cafenstrole, <NUM> clomiphene, <NUM> PF-<NUM>, and <NUM> triparanol. Inhibitor cocktail <NUM> (Strain <NUM> to Strain <NUM>) comprised of <NUM> clomiphene citrate, <NUM> terfenadine, <NUM> fluphenazine, and <NUM> triparanol.

TABLE <NUM> shows the fatty acid profiles of various strains described herein.

Strain <NUM> (CHK74) described in Example <NUM> was further subjected to classical strain improvement to produce the strain "CHK80". CHK74 was mutagenized for <NUM> at <NUM> with <NUM> or <NUM> <NUM>-NQO or subjected to sham mutagenesis with the addition of only the mutagen solvent, <NUM>% DMSO. The two mutagenized and the mock-mutagenized populations were each independently cultured in lipid production media supplemented with different concentrations of the inhibitor, clomiphene, along with <NUM>/L Tween-<NUM>, <NUM>/L Tween-<NUM>, & <NUM>/L linoleic acid. The Tween-emulsified linoleic acid was added to enrich for mutants that inherently produce lower levels of C18:<NUM>. After the first cycle of exposure, mutant cells exposed to concentrations of clomiphene where growth was reduced ><NUM>% based on OD750 measurements were collected, pooled, washed free of the inhibitor, and subjected to another round of clomiphene exposure for <NUM> in lipid media with linoleic supplementation, each time increasing the range of clomiphene exposures. By the third cycle of exposure, a resistant population arose from the <NUM> <NUM>-NQO mutant library compared to both the <NUM> <NUM>-NQO mutant population or the mock-mutagenized controls (<FIG>). This population was collected, washed free of inhibitor, expanded in pre-seed media for <NUM> days and plated for single colonies. Mutant clones were then picked and assessed for glucose consumption and fatty acid profile in a <NUM>, <NUM>-well block-based lipid production assay. A mutant identified in the screen was subsequently stabilized and given the strain designation "CHK80".

TABLE <NUM> shows sequence alignments of genes involved in fatty acid biosynthesis in CHK80 and CHK22 (Strain <NUM>; original progenitor strain): FAD2-<NUM>, FAD2-<NUM>, FATA1-<NUM>, FATA1-<NUM>, KASII-<NUM>, KASII-<NUM>, SAD2-<NUM>, and SAD2-<NUM>.

TABLE <NUM> shows sequence alignments of genomic regions of CHK80 and CHK22: <NUM>, DAO1B, FAD2, FATA1, and Thi4.

TABLE <NUM> shows genetic sequences of selectable markers that can be used to generate genetically modified microalgae. The classically improved microalgal strains provided herein do not comprises any of these exogenous sequences. Saccharomyces cerevisiae suc2 (ScSUC2) encodes for sucrose invertase, which allows for selection of strains that grow on sucrose media. Saccharomyces carlbergenesis MEL1 (ScarMEL) encodes for alpha-galactosidase (melibiase), which allows for selection of strains that grow on melibiose media. Arabidopsis thaliana ThiC (AtTHIC) encodes for phosphomethylpyrimidine synthase, an enzyme involved in pyrimidine synthesis in the thiamine biosynthesis pathway.

The sterol and triterpene alcohol compositions of high oleic refined, bleached, and deodorized (RBD) triglyceride oils of the 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 <NPL>. 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 <NUM> gas chromatograph equipped with a 5977B single quadrupole EI-MS and FID detector. A capillary flow technology (CFT) splitter was used to split the sample between the MS and FID detectors following separation using a DB-<NUM> Ultra Inert column (<NUM> length X <NUM> inner diameter X <NUM> 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 FID response of the sterol TMS ethers compared to that of the internal standard.

The resulting sterol profiles are shown in TABLE <NUM>. Amounts are shown as mg/<NUM> 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 high oleic oils in significant amounts, including ergosta-<NUM>,<NUM>-dien-<NUM>-ol, (3β)- and <NUM>. -ergost-<NUM>-en-3β-ol, (3β). Campesterol, stigmasterol, and β-sitosterol were not detected in either oils.

TABLE <NUM> shows fatty acid profiles of a TAG oil produced by strains CHK74 and CHK80. Values are in percentages of total detected fatty acids.

TAG analysis was carried out utilizing Liquid Chromatography/Time of Flight-Mass Spectrometry (LC/TOF-MS), where fractionation of TAG species is carried out on a C18 column followed by interrogation of individual peaks on a TOF LC-MS equipped with an Atmospheric Pressure Chemical Ionization (APCI) source. LC TAG standards from NuChek and samples were run on a Shimadzu Shim-pack XR-ODS III <NUM> micron, <NUM> x <NUM> column and confirmed by MS on an Agilent TOF LC-MS equipped with an APCI ionization source utilizing a method from <NPL>. TABLE <NUM> shows TAG profiles of a TAG oil produced by strains CHK74 and CHK80. Values are in percentages of total detected TAG species.

Claim 1:
An oil comprising a triacylglyceride (TAG) component and at least <NUM> of ergosterol per <NUM> of the oil, wherein the TAG component has a fatty acid content comprising <NUM>% or more C18:<NUM> fatty acids and <NUM>% or more C18:<NUM> fatty acids, wherein the TAG component further comprises at least <NUM>% triolein (OOO) and wherein the oil is produced from classical strain improvement of a cell.