NOVEL ACYLTRANSFERASES, VARIANT THIOESTERASES, AND USES THEREOF

Abstract

Recombinant nucleic acids and vector constructs encoding acyltransferases and variant thioesterases, and the acyltransferases and variant thioesterases encoded by the nucleic acids are provided. The acyltransferases and variant thioesterases are useful in fatty acid synthesis and triacylglycerol production. Host cells that express the recombinant nucleic acids as well as methods of cultivating the host cells, methods of producing oils from the host cells are provided. The recombinant host cells and the oils produced therefrom have altered fatty acid profiles and/or triacylglycerols with altered regiospecificity.

Claims

1. A recombinant vector construct or a host cell comprising nucleic acids that encodes a protein having acyltransferase activity, wherein the amino acid sequence of the acyltransferase has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to an acyltransferase of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, or 196.

2. The recombinant of claim 1, wherein the amino acid sequence of the protein comprises: a. at least 96.3% identity to an acyltransferase of clade 1 of Table 5; b. at least 93.9% identity to an acyltransferase of clade 2 of Table 5; c. at least 86.5% identity to an acyltransferase of clade 3 of Table 5; or d. at least 78.5% identity to an acyltransferase of clade 4 of Table 5.

3.-7. (canceled)

8. The nucleic acids of claim 1, wherein the nucleic acids encoding the acyltransferase are codon-optimized for expression in Prototheca or Chlorella, and wherein the coding sequence contains the most or second most preferred codon of Table 1 or Table 2 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca or Chlorella than a non-codon optimized sequence.

9.-10. (canceled)

11. The host cell of claim 8, wherein the cell is a microalgal cell, microbial cell or a plant cell, and wherein the fatty acid profile or the sn-2 profile of the host cell is altered by the expression of the nucleic acids.

12. The host cell of claim 11, wherein the microalgal cell is a Prototheca cell or a Chlorella cell.

13. The host cell of claim 12, wherein the cell is a Prototheca moriformis cell.

14. The recombinant vector construct or a host cell of claim 1, wherein the acyl transferase is a lysophosphatidic acid acyltransferase (LPAAT), glycerol phosphate acyltransferase (GPAT), diacyl glycerol acyltransferase (DGAT), lysophosphatidylcholine acyltransferase (LPCAT), or phospholipase A2 (PLA2).

15. The recombinant vector construct or a host cell of claim 14, wherein the acyl transferase is lysophosphatidic acid acyltransferase (LPAAT).

16. A method of cultivating a host cell, the host cell comprising recombinant nucleic acids encoding a protein having acyltransferase activity, wherein the amino acid sequence of the acyltransferase has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to an acyltransferase of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, or 196.

17. The method of claim 16, wherein the amino acid sequence of the protein comprises: a. at least 96.3% identity to an acyltransferase of clade 1 of Table 5; b. at least 93.9% identity to an acyltransferase of clade 2 of Table 5; c. at least 86.5% identity to an acyltransferase of clade 3 of Table 5; or d. at least 78.5% identity to an acyltransferase of clade 4 of Table 5.

18.-22. (canceled)

23. The method of claim 16, wherein the nucleic acids encoding the acyltransferase are codon-optimized for expression in Prototheca or Chlorella, and wherein the coding sequence contains the most or second most preferred codon of Table 1 or Table 2 for at least 60% of the codons of the coding sequence, such that the codon-optimized sequence is more efficiently translated in Prototheca or Chlorella than a non-codon optimized sequence.

24.-25. (canceled)

26. The method of claim 23, wherein the cell is a microalgal cell, microbial cell or a plant cell.

27. The method of claim 26, wherein the microalgal cell is a Prototheca cell or a Chlorella cell.

28. The method of claim 27, wherein the cell is a Prototheca moriformis cell.

29. The method of claim 16, wherein the acyl transferase is a lysophosphatidic acid acyltransferase (LPAAT), glycerol phosphate acyltransferase (GPAT), diacyl glycerol acyltransferase (DGAT), lysophosphatidylcholine acyltransferase (LPCAT), or phospholipase A2 (PLA2).

30. The method of claim 29, wherein the acyltransferase is lysophosphatidic acid acyltransferase (LPAAT).

31. A method of producing a triglyceride oil in a host cell, the host cell comprising recombinant nucleic acids encoding a protein having acyltransferase activity, wherein the amino acid sequence of the acyltransferase has at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to an acyltransferase of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, or 196.

32. The method of claim 31, wherein the amino acid sequence of the protein comprises: a. at least 96.3% identity to an acyltransferase of clade 1 of Table 5; b. at least 93.9% identity to an acyltransferase of clade 2 of Table 5; c. at least 86.5% identity to an acyltransferase of clade 3 of Table 5; or d. at least 78.5% identity to an acyltransferase of clade 4 of Table 5.

33.-38. (canceled)

39. The method of claim 31, wherein the microalgal cell is a Prototheca cell or a Chlorella cell.

40. The method of claim 39, wherein the cell is a Prototheca moriformis cell.

41.-141. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1. TAG profiles of S7815 versus the S6573 parent. TAGs in brackets co-elute with the peak of the main TAG, but are present in trace amounts, and do not contribute significantly to the area. M=myristate (C14:0), P=palmitate (C16:0), Po=palmitoleate (C16:1), Ma=margaric (C17:0), S=stearate (C18:0), 0=oleate (C18:1), L=linoleate (C18:2), Ln=linolenate (C18:3 ), A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0). Sat-Sat-Sat=trisaturates. See Example 5.

[0039] FIG. 2. TAG profiles of lipids from fermentations of S7815 versus S6573. TAGs in brackets co-elute with the peak of the main TAG, but are present in trace amounts, and do not contribute significantly to the area. M=myristate (C14:0), P=palmitate (C16:0), S=stearate (C18:0), 0=oleate (C18:1), L=linoleate (C18:2), Ln=linolenate (C18:3 ), A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0). Sat-Sat-Sat=trisaturates. See Example 5.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0040] An allele refers to a copy of a gene where an organism has multiple similar or identical gene copies, even if on the same chromosome. An allele may encode the same or similar protein.

[0041] An oil, cell oil or cell fat shall mean a predominantly triglyceride oil obtained from an organism, where the oil has not undergone blending with another natural or synthetic oil, or fractionation so as to substantially alter the fatty acid profile of the triglyceride. In connection with an oil comprising triglycerides of a particular regiospecificity, the cell oil or cell fat has not been subjected to interesterification or other synthetic process to obtain that regiospecific triglyceride profile, rather the regiospecificity is produced naturally, by a cell or population of cells. For a cell oil produced by a cell, the sterol profile of oil is generally determined by the sterols produced by the cell, not by artificial reconstitution of the oil by adding sterols in order to mimic the cell oil. In connection with a cell oil or cell fat, and as used generally throughout the present disclosure, the terms oil, and fat are used interchangeably, except where otherwise noted. Thus, an oil or a fat can be liquid, solid, or partially solid at room temperature, depending on the makeup of the substance and other conditions. Here, the term fractionation means removing material from the oil in a way that changes its fatty acid profile relative to the profile produced by the organism, however accomplished. The terms oil, cell oil and cell fat encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching, deodorized, and/or degumming, which does not substantially change its triglyceride profile. A cell oil can also be a noninteresterified cell oil, which means that the cell oil has not undergone a process in which fatty acids have been redistributed in their acyl linkages to glycerol and remain essentially in the same configuration as when recovered from the organism.

[0042] As used herein, an oil is said to be enriched in one or more particular fatty acids if there is at least a 10% increase in the mass of that fatty acid in the oil relative to the non-enriched oil. For example, in the case of a cell expressing a heterologous FatB gene described herein, the oil produced by the cell is said to be enriched in, e.g., C8 and C16 fatty acids if the mass of these fatty acids in the oil is at least 10% greater than in oil produced by a cell of the same type that does not express the heterologous FatB gene (e.g., wild type oil).

[0043] Exogenous gene shall mean a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a transgene. A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.

[0044] FADc, also referred to as FAD2 or FAD is a gene encoding a delta-12 fatty acid desaturase. SAD is a gene encoding a stearoyl ACP desaturase, a delta-9 fatty acid desaturase. The desaturases desaturates a fatty acyl chain to create a double bond. SAD converts stearic acid, C18:0 to oleic acid, C18:1 and FAD converts oleic acid, C18:1 to linoleic acid, C18:2.

[0045] Fatty acids shall mean free fatty acids, fatty acid salts, or fatty acyl moieties in a glycerolipid. It will be understood that fatty acyl groups of glycerolipids can be described in terms of the carboxylic acid or anion of a carboxylic acid that is produced when the triglyceride is hydrolyzed or saponified.

[0046] Fixed carbon source is a molecule(s) containing carbon, typically an organic molecule that is present at ambient temperature and pressure in solid or liquid form in a culture media that can be utilized by a microorganism cultured therein. Accordingly, carbon dioxide is not a fixed carbon source. Typical fixed carbon source include sucrose, glucose, fructose and other well-known monosaccharides, disaccharides and polysaccharides.

[0047] In operable linkage is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.

[0048] Microalgae are eukaryotic microbial organisms that contain a chloroplast or other plastid, and optionally that is capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae also include mixotrophic organisms that can perform photosynthesis and metabolize one or more fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca.

[0049] As used with respect to nucleic acids, the term isolated refers to a nucleic acid that is free of at least one other component that is typically present with the naturally occurring nucleic acid. Thus, a naturally occurring nucleic acid is isolated if it has been purified away from at least one other component that occurs naturally with the nucleic acid.

[0050] In connection with fatty acid length, mid-chain shall mean C8 to C16 fatty acids.

[0051] In connection with a recombinant cell, the term knockdown refers to a gene that has been partially suppressed (e.g., by about 1-95%) in terms of the production or activity of a protein encoded by the gene. Inhibitory RNA technology to down-regulate or knockdown expression of a gene are well known. These techniques include dsRNA, hairpin RNA, antisense RNA, interfering RNA (RNAi) and others.

[0052] Also, in connection with a recombinant cell, the term knockout refers to a gene that has been completely or nearly completely (e.g., >95%) suppressed in terms of the production or activity of a protein encoded by the gene. Knockouts can be prepared by ablating the gene by homologous recombination of a nucleic acid sequence into a coding sequence, gene deletion, mutation or other method. When homologous recombination is performed, the nucleic acid that is inserted (knocked-in) can be a sequence that encodes an exogenous gene of interest or a sequence that does not encode for a gene of interest. The ablation by homologous recombination can be performed in one, two or more alleles of the gene of interest.

[0053] An oleaginous cell is a cell capable of producing at least 20% lipid by dry cell weight, naturally or through recombinant or classical strain improvement. An oleaginous microbe or oleaginous microorganism is a microbe, including a microalga that is oleaginous (especially eukaryotic microalgae that store lipid). An oleaginous cell also encompasses a cell that has had some or all of its lipid or other content removed, and both live and dead cells.

[0054] An ordered oil or ordered fat is one that forms crystals that are primarily of a given polymorphic structure. For example, an ordered oil or ordered fat can have crystals that are greater than 50%, 60%, 70%, 80%, or 90% of the 0 or (3 polymorphic form.

[0055] In connection with a cell oil, a profile is the distribution of particular species or triglycerides or fatty acyl groups within the oil. A fatty acid profile is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID), as in Example 1. The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME-GC-FID measurement approximate weight percentages of the fatty acids. A sn-2 profile is the distribution of fatty acids found at the sn-2 position of the triacylglycerides in the oil. A regiospecific profile is the distribution of triglycerides with reference to the positioning of acyl group attachment to the glycerol backbone without reference to stereospecificity. In other words, a regiospecific profile describes acyl group attachment at sn-1/3 vs. sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate) and SOP (stearate-oleate-palmitate) are treated identically. A stereospecific profile describes the attachment of acyl groups at sn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such as SOP and POS are to be considered equivalent. A TAG profile is the distribution of fatty acids found in the triglycerides with reference to connection to the glycerol backbone, but without reference to the regiospecific nature of the connections. Thus, in a TAG profile, the percent of SSO in the oil is the sum of SSO and SOS, while in a regiospecific profile, the percent of SSO is calculated without inclusion of SOS species in the oil. In contrast to the weight percentages of the FAME-GC-FID analysis, triglyceride percentages are typically given as mole percentages; that is the percent of a given TAG molecule in a TAG mixture.

[0056] The term percent sequence identity, in the context of two or more amino acid or nucleic acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted using the NCBI BLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. For example, to compare two nucleic acid sequences, one may use blastn with the BLAST 2 Sequences tool Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: 2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For a pairwise comparison of two amino acid sequences, one may use the BLAST 2 Sequences tool Version 2.0.12 (Apr. 21, 2000) with blastp set, for example, at the following default parameters: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap x drop-off 50; Expect: 10; Word Size: 3; Filter: on.

[0057] Recombinant is a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi), hairpin RNA or dsRNA that reduce the levels of active gene product in a cell. A recombinant nucleic acid is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, using chemical synthesis, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a recombinant protein is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid. A recombinant protein will have a different pattern of glycosylation than the protein isolated from the wild-type organism.

[0058] The genes can be used in a variety of genetic constructs including plasmids or other vectors for expression or recombination in a host cell. The genes can be codon optimized for expression in a target host cell. The proteins produced by the genes can be used in vivo or in purified form.

[0059] For example, the gene can be prepared in an expression vector comprising an operably linked promoter and 5UTR. Where a plastidic cell is used as the host, a suitably active plastid targeting peptide can be fused to the FATB gene, as in the examples below. Generally, for the newly identified FATB genes, there are roughly 50 amino acids at the N-terminal that constitute a plastid transit peptide, which are responsible for transporting the enzyme to the chloroplast. In the examples below, this transit peptide is replaced with a 38 amino acid sequence that is effective in the Prototheca moriformis host cell for transporting the enzyme to the plastids of those cells. Thus, the invention contemplates deletions and fusion proteins in order to optimize enzyme activity in a given host cell. For example, a transit peptide from the host or related species may be used instead of that of the newly discovered plant genes described here.

[0060] A selectable marker gene may be included in the vector to assist in isolating a transformed cell. Examples of selectable markers useful in microlagae include sucrose invertase antibiotic resistance genes and other genes useful as selectable markers. The S. carlbergensis MEL1 gene (conferring the ability to grow on melibiose), A. thaliana THIC gene (conferring the ability to grow in media free of thiamine, Saccharomyces sucrose invertase (conferring the ability to grow on sucrose) are disclosed in the Examples. Other known selectable markers are useful and within the ambit of a skilled artisan.

[0061] The terms triglyceride, triacylglyceride and TAG are used interchangeably as is known in the art.

II. Embodiments of the Invention

[0062] Illustrative embodiments of the present invention feature oleaginous cells that produce altered fatty acid profiles and/or altered regiospecific distribution of fatty acids in glycerolipids, and products produced from the cells. Examples of oleaginous cells include microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae and, where applicable, oil producing cells of higher plants including but not limited to commercial oilseed crops such as soy, corn, rapeseed/canola, cotton, flax, sunflower, safflower and peanut. Other specific examples of cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae and methods of cultivation are also provided in co-owned applications WO2008/151149, WO2010/063031, WO2010/063032, WO2011/150410, WO2011/150411, WO2012/061647, WO2012/061647, WO2012/106560, and WO2013/158938, WO2014/120829, WO2014/151904, WO2015/051319, WO2016/007862, WO2016/014968, WO2016/044779, WO2016/164495, all of which are incorporated by reference, including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs. The oleaginous cells can be, for example, capable of producing 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, or about 90% oil by cell weight, 5%. Optionally, the oils produced can be low in highly unsaturated fatty acids such as DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA. The above-mentioned publications also disclose methods for cultivating such cells and extracting oil, especially from microalgal cells; such methods are applicable to the cells disclosed herein and incorporated by reference for these teachings. When microalgal cells are used they can be cultivated autotrophically (unless an obligate heterotroph) or in the dark using a sugar (e.g., glucose, fructose and/or sucrose) In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock. Alternately, or in addition, the cells can metabolize xylose from cellulosic feedstocks. For example, the cells can be genetically engineered to express one or more xylose metabolism genes such as those encoding an active xylose transporter, a xylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase and a xylose reductase. See WO2012/154626, GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE, published Nov. 15, 2012, including disclosure of genetically engineered Prototheca strains that utilize xylose.

[0063] The host cells expressing the acyltransferases or the variant B. napus thioesterases or the variant G. mangostana thioesterase may, optionally, be cultivated in a bioreactor/fermenter. For example, heterotrophic oleaginous microalgal cells can be cultivated on a sugar-containing nutrient broth. Optionally, cultivation can proceed in two stages: a seed stage and a lipid-production stage. In the seed stage, the number of cells is increased from a starter culture. Thus, the seed stage(s) typically includes a nutrient rich, nitrogen replete, media designed to encourage rapid cell division. After the seed stage(s), the cells may be fed sugar under nutrient-limiting (e.g. nitrogen sparse) conditions so that the sugar will be converted into triglycerides. As used herein, standard lipid production conditions are disclosed here. In one embodiment, the culture conditions are nitrogen limiting. Sugar and other nutrients can be added during the fermentation but no additional nitrogen is added. The cells will consume all or nearly all of the nitrogen present, but no additional nitrogen is provided. For example, the rate of cell division in the lipid-production stage can be decreased by 50%, 80%, or more relative to the seed stage. Additionally, variation in the media between the seed stage and the lipid-production stage can induce the recombinant cell to express different lipid-synthesis genes and thereby alter the triglycerides being produced. For example, as discussed below, nitrogen and/or pH sensitive promoters can be placed in front of endogenous or exogenous genes. This is especially useful when an oil is to be produced in the lipid-production phase that does not support optimal growth of the cells in the seed stage.

[0064] The oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes. As a result, some embodiments feature cell oils that were not obtainable from a non-plant or non-seed oil, or not obtainable at all.

[0065] The oleaginous cells, including microalgal cells, can be improved via classical strain improvement techniques such as UV and/or chemical mutagenesis followed by screening or selection under environmental conditions, including selection on a chemical or biochemical toxin. For example the cells can be selected on a fatty acid synthesis inhibitor, a sugar metabolism inhibitor, or an herbicide. As a result of the selection, strains can be obtained with increased yield on sugar, increased oil production (e.g., as a percent of cell volume, dry weight, or liter of cell culture), or improved fatty acid or TAG profile. Co-owned application PCT/US2016/025023 filed on 31 Mar. 2016, herein incorporated by reference, describes methods for classically mutagenizing oleaginous cells.

[0066] The cells can be selected on one or more of 1,2-Cyclohexanedione; 19-Norethindone acetate; 2,2-dichloropropionic acid; 2,4,5-trichlorophenoxyacetic acid; 2,4,5-trichlorophenoxyacetic acid, methyl ester; 2,4-dichlorophenoxyacetic acid; 2,4-dichlorophenoxyacetic acid, butyl ester; 2,4-dichlorophenoxyacetic acid, isooctyl ester; 2,4-dichlorophenoxyacetic acid, methyl ester; 2,4-dichlorophenoxybutyric acid; 2,4-dichlorophenoxybutyric acid, methyl ester; 2,6-dichlorobenzonitrile; 2-deoxyglucose; 5-Tetradecyloxy-w-furoic acid; A-922500; acetochlor; alachlor; ametryn; amphotericin; atrazine; benfluralin; bensulide; bentazon; bromacil; bromoxynil; Cafenstrole; carbonyl cyanide m-chlorophenyl hydrazone (CCCP); carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP); cerulenin; chlorpropham; chlorsulfuron; clofibric acid; clopyralid; colchicine; cycloate; cyclohexamide; C75; DACTHAL (dimethyl tetrachloroterephthalate); dicamb a; dichloroprop ((R)-2-(2,4-dichlorophenoxy)propanoic acid); Diflufenican; dihyrojasmonic acid, methyl ester; diquat; diuron; dimethylsulfoxide; Epigallocatechin gallate (EGCG); endothall; ethalfluralin; ethanol; ethofumesate; Fenoxaprop-p-ethyl; Fluazifop-p-Butyl; fluometuron; fomasefen; foramsulfuron; gibberellic acid; glufosinate ammonium; glyphosate; haloxyfop; hexazinone; imazaquin; isoxaben; Lipase inhibitor THL (()-Tetrahydrolipstatin); malonic acid; MCPA (2-methyl-4-chlorophenoxyacetic acid); MCPB (4-(4-chloro-o-tolyloxy)butyric acid); mesotrione; methyl dihydroj asmonate; metolachlor; metribuzin; Mildronate; molinate; naptalam; norharman; orlistat; oxadiazon; oxyfluorfen; paraquat; pendimethalin; pentachlorophenol; PF-04620110; phenethyl alcohol; phenmedipham; picloram; Platencin; Platensimycin; prometon; prometryn; pronamide; propachlor; propanil; propazine; pyrazon; Quizalofop-p-ethyl; s-ethyl dipropylthiocarbamate (EPTC); s,s,s-tributylphosphorotrithioate; salicylhydroxamic acid; sesamol; siduron; sodium methane arsenate; simazine; T-863 (DGAT inhibitor); tebuthiuron; terbacil; thiobencarb; tralkoxydim; triallate; triclopyr; triclosan; trifluralin; and vulpinic acid and others.

[0067] The oleaginous cells produce a storage oil, which is primarily triacylglyceride and may be stored in storage bodies of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. The raw oil may comprise sterols produced by the cells. Patent applications WO2008/151149, WO2010/063031, WO2010/063032, WO2011/150410, WO2011/150411, WO2012/061647, WO2012/061647, WO2012/106560, WO2013/158938, WO2014/120829, WO2014/151904, WO2015/051319, WO2016/007862, WO2016/014968, WO2016/044779, and WO2016/164495 disclose heterotrophic cultivation and oil isolation techniques for oleaginous microalgae. For example, oil may be obtained by providing or cultivating, drying and pressing the cells. The oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939. The raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. Even after such processing, the oil may retain a sterol profile characteristic of the source. Sterol profiles of microalga and the microalgal cell oils are disclosed below. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, drilling fluids, as animal feed, for human nutrition, or for fertilizer.

[0068] In an embodiment of the invention nucleic acids that encode novel acyl transferases are provided. The novel acyltransferases are useful in altering the fatty acid profile and/or altering the regiospecific profile of an oil produced by a host cell. The nucleic acids of the invention may contain control sequences upstream and downstream in operable linkage with the gene of interest. These control sequences include promoters, targeting sequences, untranslated sequences and other control elements. Nucleic acids of the invention encode acyltransferases that function in type II fatty acid synthesis. The acyltransferase genes are isolated from higher plants and can be expressed in a wide variety of host cells. The acyltransferases include lysophosphatidic acid acyltransferase (LPAAT), glycerol phosphate acyltransferase (GPAT), diacyl glycerol acyltransferase (DGAT), lysophosphatidylcholine acyltransferase (LPCAT), or phospholipase A2 (PLA2). and other lipid biosynthetic pathway genes as discussed herein. The acyltransferases of the invention are shown in Table 5. In one embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 96.3%, 98%, or 99% identity to an acyltransferase of clade 1 of Table 5. In another embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 93.9%, 98%, or 99% identity to an acyltransferase of clade 2 of Table 5. In one embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 86.5%, 90%, 95%, 98%, or 99% identity to an acyltransferase of clade 3 of Table 5. In one embodiment, the acyltransferases of the invention have acyltransferase activity and the amino acid sequence comprises at least 78.5%, 80%, 85%, 90%, 95%, 98%, or 99% identity to an acyltransferase of clade 4 of Table 5. The acyltransferases when expressed increase the SOS, POP, POS, SLS, PLO, and/or PLO content DCW in host cells and the oils recovered from the host cells. The acyltransferases when expressed in host cells decreases the sat-sat-sat content of the oil by DCW. The acyltransferases when expressed in host cells increases the sat-unsat-sat/sat-sat-sat ratio of the oil by DCW.

[0069] In an embodiment of the invention nucleic acids that encode variant Brassica napus thiosterases (FATA) are provided. The novel thioesterases are useful in altering the fatty acid profile of an oil produced by a host cell. The variant Brassica napus thiosterases prefer to hydrolyze long chain fatty acyl groups from the acyl carrier protein. The nucleic acids of the invention may contain control sequences upstream and downstream in operable linkage with the gene of interest. These control sequences include promoters, targeting sequences, untranslated sequences and other control elements. Nucleic acids of the invention encode thiosterases that function in type II fatty acid synthesis. The thioesterase genes, isolated from higher plants, are altered to create variant thioesterases that have certain amino acids that have been altered from the wild type enzyme. Due to the altered amino acid(s), the substrate specificity of the thioesterase is altered. The variant thioesterases can be expressed in a wide variety of host cells. The nucleic acids encode the variant thioesterases having amino acid sequences that are 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 165, 166, 167, or 198 and comprise one or more of amino acid variants D124A, D209A, D127A or D212A. The variant BnOTE enzymes increased C18:0 content by DCW, decreased C18:1 content by DCW, and decreased C18:2 content by DCW in host cells and the oils recovered from the host cells.

[0070] In an embodiment of the invention nucleic acids that encode variant Garcinia mangostana thiosterases (FATA) are provided. The novel thioesterases are useful in altering the fatty acid profile of an oil produced by a host cell. The variant Garcinia mangostana thiosterases prefer to hydrolyze long chain fatty acyl groups from the acyl carrier protein. The nucleic acids of the invention may contain control sequences upstream and downstream in operable linkage with the gene of interest. These control sequences include promoters, targeting sequences, untranslated sequences and other control elements. Nucleic acids of the invention encode thiosterases that function in type II fatty acid synthesis. The thioesterase genes, isolated from higher plants, are altered to create variant thioesterases that have certain amino acids that have been altered from the wild type enzyme. Due to the altered amino acid(s), the substrate specificity of the thioesterase is altered. The variant thioesterases can be expressed in a wide variety of host cells. The nucleic acids encode the variant thioesterases having amino acid sequences that are 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NOs: 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 and comprise one or more of amino acid variants L91F, L91K, L91S, G96A, G96T, G96V, G108A, G108V, S111A, S111V T156F, T156A, T156K, T156V, or V193A. The variant GmFATA enzymes increased C18:0 content by DCW, decreased C18:1 content by DCW, and decreased C18:2 content by DCW in host cells and the oils recovered from the host cells.

[0071] The nucleic acids of the invention can be codon optimized for expression in a target host cell (e.g., using the codon usage tables of Tables 1a, 1b, 2a, and 2b. For example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codons used can be the most preferred codon according to Tables 1a, 1b, 2a, and 2b. Alternately, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codons used can be the first or second most preferred codon according to Tables 1a, 1b, 2a, and 2b. Preferred codons for Prototheca strains and for Chlorella protothecoides are shown below in Tables 1a and 1b, respectively.

TABLE-US-00001 TABLE 1a Preferred codon usage in Prototheca strains. Ala GCG 345 (0.36) Asn AAT 8 (0.04) GCA 66 (0.07) AAC 201 (0.96) GCT 101 (0.11) GCC 442 (0.46) Pro CCG 161 (0.29) CCA 49 (0.09) Cys TGT 12 (0.10) CCT 71 (0.13) TGC 105 (0.90) CCC 267 (0.49) Asp GAT 43 (0.12) Gln CAG 226 (0.82) GAC 316 (0.88) CAA 48 (0.18) Glu GAG 377 (0.96) Arg AGG 33 (0.06) GAA 14 (0.04) AGA 14 (0.02) CGG 102 (0.18) Phe TTT 89 (0.29) CGA 49 (0.08) TTC 216 (0.71) CGT 51 (0.09) CGC 331 (0.57) Gly GGG 92 (0.12) GGA 56 (0.07) Ser AGT 16 (0.03) GGT 76 (0.10) AGC 123 (0.22) GGC 559 (0.71) TCG 152 (0.28) TCA 31 (0.06) His CAT 42 (0.21) TCT 55 (0.10) CAC 154 (0.79) TCC 173 (0.31) Ile ATA 4 (0.01) Thr ACG 184 (0.38) ATT 30 (0.08) ACA 24 (0.05) ATC 338 (0.91) ACT 21 (0.05) ACC 249 (0.52) Lys AAG 284 (0.98) AAA 7 (0.02) Val GTG 308 (0.50) GTA 9 (0.01) Leu TTG 26 (0.04) GTT 35 (0.06) TTA 3 (0.00) GTC 262 (0.43) CTG 447 (0.61) CTA 20 (0.03) Trp TGG 107 (1.00) CTT 45 (0.06) CTC 190 (0.26) Tyr TAT 10 (0.05) TAC 180 (0.95) Met ATG 191 (1.00) Stop TGA/TAG/TAA

TABLE-US-00002 TABLE 1b Preferred codon usage in Chlorella protothecoides. TTC (Phe) TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met) AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu)

TABLE-US-00003 TABLE 2a Codon usage for Cuphea wrightii UUU F 0.48 19.5 ( 52) UCU S 0.21 19.5 ( 52) UAU Y 0.45 6.4 ( 17) UGU C 0.41 10.5 ( 28) UUC F 0.52 21.3 ( 57) UCC S 0.26 23.6 ( 63) UAC Y 0.55 7.9 ( 21) UGC C 0.59 15.0 ( 40) UUA L 0.07 5.2 ( 14) UCA S 0.18 16.8 ( 45) UAA * 0.33 0.7 ( 2) UGA * 0.33 0.7 ( 2) UUG L 0.19 14.6 ( 39) UCG S 0.11 9.7 ( 26) UAG * 0.33 0.7 ( 2) UGG W 1.00 15.4 ( 41) CUU L 0.27 21.0 ( 56) CCU P 0.48 21.7 ( 58) CAU H 0.60 11.2 ( 30) CGU R 0.09 5.6 ( 15) CUC L 0.22 17.2 ( 46) CCC P 0.16 7.1 ( 19) CAC H 0.40 7.5 ( 20) CGC R 0.13 7.9 ( 21) CUA L 0.13 10.1 ( 27) CCA P 0.21 9.7 ( 26) CAA Q 0.31 8.6 ( 23) CGA R 0.11 6.7 ( 18) CUG L 0.12 9.7 ( 26) CCG P 0.16 7.1 ( 19) CAG Q 0.69 19.5 ( 52) CGG R 0.16 9.4 ( 25) AUU I 0.44 22.8 ( 61) ACU T 0.33 16.8 ( 45) AAU N 0.66 31.4 ( 84) AGU S 0.18 16.1 ( 43) AUC I 0.29 15.4 ( 41) ACC T 0.27 13.9 ( 37) AAC N 0.34 16.5 ( 44) AGC S 0.07 6.0 ( 16) AUA I 0.27 13.9 ( 37) ACA T 0.26 13.5 ( 36) AAA K 0.42 21.0 ( 56) AGA R 0.24 14.2 ( 38) AUG M 1.00 28.1 ( 75) ACG T 0.14 7.1 ( 19) AAG K 0.58 29.2 ( 78) AGG R 0.27 16.1 ( 43) GUU V 0.28 19.8 ( 53) GCU A 0.35 31.4 ( 84) GAU D 0.63 35.9 ( 96) GGU G 0.29 26.6 ( 71) GUC V 0.21 15.0 ( 40) GCC A 0.20 18.0 ( 48) GAC D 0.37 21.0 ( 56) GGC G 0.20 18.0 ( 48) GUA V 0.14 10.1 ( 27) GCA A 0.33 29.6 ( 79) GAA E 0.41 18.3 ( 49) GGA G 0.35 31.4 ( 84) GUG V 0.36 25.1 ( 67) GCG A 0.11 9.7 ( 26) GAG E 0.59 26.2 ( 70) GGG G 0.16 14.2 ( 38)

TABLE-US-00004 TABLE 2b Codon usage for Arabidopsis UUU F 0.51 21.8 (678320) UCU S 0.28 25.2 (782818) UAU Y 0.52 14.6 (455089) UGU C 0.60 10.5 (327640) UUC F 0.49 20.7 (642407) UCC S 0.13 11.2 (348173) UAC Y 0.48 13.7 (427132) UGC C 0.40 7.2 (222769) UUA L 0.14 12.7 (394867) UCA S 0.20 18.3 (568570) UAA * 0.36 0.9 ( 29405) UGA * 0.44 1.2 ( 36260) UUG L 0.22 20.9 (649150) UCG S 0.10 9.3 (290158) UAG * 0.20 0.5 ( 16417) UGG W 1.00 12.5 (388049) CUU L 0.26 24.1 (750114) CCU P 0.38 18.7 (580962) CAU H 0.61 13.8 (428694) CGU R 0.17 9.0 (280392) CUC L 0.17 16.1 (500524) CCC P 0.11 5.3 (165252) CAC H 0.39 8.7 (271155) CGC R 0.07 3.8 (117543) CUA L 0.11 9.9 (307000) CCA P 0.33 16.1 (502101) CAA Q 0.56 19.4 (604800) CGA R 0.12 6.3 (195736) CUG L 0.11 9.8 (305822) CCG P 0.18 8.6 (268115) CAG Q 0.44 15.2 (473809) CGG R 0.09 4.9 (151572) AUU I 0.41 21.5 (668227) ACU T 0.34 17.5 (544807) AAU N 0.52 22.3 (693344) AGU S 0.16 14.0 (435738) AUC I 0.35 18.5 (576287) ACC T 0.20 10.3 (321640) AAC N 0.48 20.9 (650826) AGC S 0.13 11.3 (352568) AUA I 0.24 12.6 (391867) ACA T 0.31 15.7 (487161) AAA K 0.49 30.8 (957374) AGA R 0.35 19.0 (589788) AUG M 1.00 24.5 (762852) ACG T 0.15 7.7 (240652) AAG K 0.51 32.7 (1016176) AGG R 0.20 11.0 (340922) GUU V 0.40 27.2 (847061) GCU A 0.43 28.3 (880808) GAU D 0.68 36.6 (1139637) GGU G 0.34 22.2 (689891) GUC V 0.19 12.8 (397008) GCC A 0.16 10.3 (321500) GAC D 0.32 17.2 (535668) GGC G 0.14 9.2 (284681) GUA V 0.15 9.9 (308605) GCA A 0.27 17.5 (543180) GAA E 0.52 34.3 (1068012) GGA G 0.37 24.2 (751489) GUG V 0.26 17.4 (539873) GCG A 0.14 9.0 (280804) GAG E 0.48 32.2 (1002594) GGG G 0.16 10.2 (316620)

[0072] The cell oils of this invention can be distinguished from conventional vegetable or animal triacylglycerol sources in that the sterol profile will be indicative of the host organism as distinguishable from the conventional source. Conventional sources of oil include soy, corn, sunflower, safflower, palm, palm kernel, coconut, cottonseed, canola, rape, peanut, olive, flax, tallow, lard, cocoa, shea, mango, sal, illipe, kokum, and allanblackia.

[0073] The oils provided herein are not vegetable oils. Vegetable oils are oils extracted from plants and plant seeds. Vegetable oils can be distinguished from the non-plant oils provided herein on the basis of their oil content. A variety of methods for analyzing the oil content can be employed to determine the source of the oil or whether adulteration of an oil provided herein with an oil of a different (e.g. plant) origin has occurred. The determination can be made on the basis of one or a combination of the analytical methods. These tests include but are not limited to analysis of one or more of free fatty acids, fatty acid profile, total triacylglycerol content, diacylglycerol content, peroxide values, spectroscopic properties (e.g. UV absorption), sterol profile, sterol degradation products, antioxidants (e.g. tocopherols), pigments (e.g. chlorophyll), d13C values and sensory analysis (e.g. taste, odor, and mouth feel). Many such tests have been standardized for commercial oils such as the Codex Alimentarius standards for edible fats and oils.

[0074] Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, b-sitosterol, and stigamsterol are common plant sterols, with b-sitosterol being a principle plant sterol. For example, b-sitosterol was found to be in greatest abundance in an analysis of certain seed oils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74% in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Cell and Molecular Biology 5:71-79, 2006).

[0075] The sterol profile of a microalgal oil is distinct from the sterol profile of oils obtained from higher plants or animals. Oil isolated from Prototheca moriformis strain UTEX1435 were separately clarified (CL), refined and bleached (RB), or refined, bleached and deodorized (RBD) and were tested for sterol content according to the procedure described in JAOCS vol. 60, no. 8, Aug. 1983. Results of the analysis are shown Table 3 below (units in mg/100 g):

TABLE-US-00005 TABLE 3 (units in mg/100 g) Refined, Refined & bleached, & Sterol Crude Clarified bleached deodorized 1 Ergosterol 384 398 293 302 (56%) (55%) (50%) (50%) 2 5,22-cholestadien-24- 14.6 18.8 14 15.2 methyl-3-ol (2.1%) (2.6%) (2.4%) (2.5%) (Brassicasterol) 3 24-methylcholest-5- 10.7 11.9 10.9 10.8 en-3-ol (Campesterol or (1.6%) (1.6%) (1.8%) (1.8%) 22,23- dihydrobrassicasterol) 4 5,22-cholestadien-24- 57.7 59.2 46.8 49.9 ethyl-3-ol (Stigmasterol (8.4%) (8.2%) (7.9%) (8.3%) or poriferasterol) 5 24-ethylcholest-5-en- 9.64 9.92 9.26 10.2 3-ol (-Sitosterol or (1.4%) (1.4%) (1.6%) (1.7%) clionasterol) 6 Other sterols 209 221 216 213 Total sterols 685.64 718.82 589.96 601.1

[0076] These results show three striking features. First, ergosterol was found to be the most abundant of all the sterols, accounting for about 50% or more of the total sterols. The amount of ergosterol is greater than that of campesterol, -sitosterol, and stigmasterol combined. Ergosterol is steroid commonly found in fungus and not commonly found in plants, and its presence particularly in significant amounts serves as a useful marker for non-plant oils. Secondly, the oil was found to contain brassicasterol. With the exception of rapeseed oil, brassicasterol is not commonly found in plant based oils. Thirdly, less than 2% -sitosterol was found to be present. -sitosterol is a prominent plant sterol not commonly found in microalgae, and its presence particularly in significant amounts serves as a useful marker for oils of plant origin. In summary, Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of -sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol: -sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.

[0077] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% -sitosterol. In other embodiments the oil is free from -sitosterol.

[0078] In some embodiments, the oil is free from one or more of -sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from -sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.

[0079] In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-ethylcholest-5-en-3-ol. In some embodiments, the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% clionasterol.

[0080] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-methylcholest-5-en-3-ol. In some embodiments, the 24-methylcholest-5-en-3-ol is 22, 23-dihydrobrassicasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% 22,23-dihydrobrassicasterol.

[0081] In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 5,22-cholestadien-24-ethyl-3-ol. In some embodiments, the 5, 22-cholestadien-24-ethyl-3-ol is poriferasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% poriferasterol.

[0082] In some embodiments, the oil content of an oil provided herein contains ergosterol or brassicasterol or a combination of the two. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 40% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of a combination of ergosterol and brassicasterol.

[0083] In some embodiments, the oil content contains, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, or 5% brassicasterol. In some embodiments, the oil content contains, as a percentage of total sterols less than 10%, 9%, 8%, 7%, 6%, or 5% brassicasterol.

[0084] In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.

[0085] In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% -sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% -sitosterol. In some embodiments, the oil content further comprises brassicasterol.

[0086] Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes. Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols. The sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols b-sitosterol and stigmasterol. In contrast, the sterol profiles of non-plant organisms contain greater percentages of C27 and C28 sterols. For example the sterols in fungi and in many microalgae are principally C28 sterols. The sterol profile and particularly the striking predominance of C29 sterols over C28 sterols in plants has been exploited for determining the proportion of plant and marine matter in soil samples (Huang, Wen-Yen, Meinschein W. G., Sterols as ecological indicators; Geochimica et Cosmochimia Acta. Vol 43. pp 739-745).

[0087] In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than b-sitosterol and stigmasterol. In some embodiments of the microalgal oils, C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.

[0088] In some embodiments the microalgal oils provided herein contain C28 sterols in excess of C29 sterols. In some embodiments of the microalgal oils, C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95% by weight of the total sterol content. In some embodiments the C28 sterol is ergosterol. In some embodiments the C28 sterol is brassicasterol.

[0089] Where a fatty acid profile of a triglyceride (also referred to as a triacylglyceride or TAG) cell oil is given here, it will be understood that this refers to a nonfractionated sample of the storage oil extracted from the cell analyzed under conditions in which phospholipids have been removed or with an analysis method that is substantially insensitive to the fatty acids of the phospholipids (e.g. using chromatography and mass spectrometry). The oil may be subjected to an RBD process to remove phospholipids, free fatty acids and odors yet have only minor or negligible changes to the fatty acid profile of the triglycerides in the oil. Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the TAGs in the cell. Examples 1 and 2 below give analytical methods for determining TAG fatty acid composition and regiospecific structure.

[0090] Broadly categorized, certain embodiments of the invention include (i) recombinant oleaginous cells that comprise an ablation of one or two or all alleles of an endogenous polynucleotide, including polynucleotides encoding lysophosphatidic acid acyltransferase (LPAAT) or (ii) cells that produce oils having low concentrations of polyunsaturated fatty acids, including cells that are auxotrophic for unsaturated fatty acids; (iii) cells producing oils having high concentrations of particular fatty acids due to expression of one or more exogenous genes encoding enzymes that transfer fatty acids to glycerol or a glycerol ester; (iv) cells producing regiospecific oils, (v) genetic constructs or cells encoding a an LPAAT, a lysophosphatidylcholine acyltransferase (LPCAT), a phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), diacylglycerol cholinephosphotransferase (DAG-CPT) or fatty acyl elongase (FAE), (vi) cells producing low levels of saturated fatty acids and/or high levels of C18:1, C18:2, C18:3, C20:1 or C22:1, (vii) and other inventions related to producing cell oils with altered profiles. The embodiments also encompass the oils made by such cells, the residual biomass from such cells after oil extraction, oleochemicals, fuels and food products made from the oils and methods of cultivating the cells.

[0091] In any of the embodiments below, the cells used are optionally cells having a type II fatty acid biosynthetic pathway such as plant cells, yeast cells, microalgal cells including heterotrophic or obligate heterotrophic microalgal cells, including cells classified as Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae, or cells engineered to have a type II fatty acid biosynthetic pathway using the tools of synthetic biology (i.e., transplanting the genetic machinery for a type II fatty acid biosynthesis into an organism lacking such a pathway). Use of a host cell with a type II pathway avoids the potential for non-interaction between an exogenous acyl-ACP thioesterase or other ACP-binding enzyme and the multienzyme complex of type I cellular machinery. In specific embodiments, the cell is of the species Prototheca moriformis, Prototheca krugani, Prototheca stagnora or Prototheca zopfii or has a 23 S rRNA sequence with at least 65, 70, 75, 80, 85, 90 or 95% nucleotide identity SEQ ID NO: 25. By cultivating in the dark or using an obligate heterotroph, the cell oil produced can be low in chlorophyll or other colorants. For example, the cell oil can have less than 100, 50, 10, 5, 1, 0.0.5 ppm of chlorophyll without substantial purification.

[0092] The stable carbon isotope value 613C is an expression of the ratio of .sup.13C/.sup.12C relative to a standard (e.g. PDB, carbonite of fossil skeleton of Belemnite americana from Peedee formation of South Carolina). The stable carbon isotope value 13C (.sup.0/.sub.00) of the oils can be related to the 13C value of the feedstock used. In some embodiments the oils are derived from oleaginous organisms heterotrophically grown on sugar derived from a C4 plant such as corn or sugarcane. In some embodiments the 613C (.sup.0/.sub.00) of the oil is from 10 to 17 .sup.0/.sub.00 or from 13 to 16.sup.0/.sub.00.

[0093] In specific embodiments and examples discussed below, one or more fatty acid synthesis genes (e.g., encoding an acyl-ACP thioesterase, a keto-acyl ACP synthase, an LPAAT, an LPCAT, a PDCT, a DAG-CPT, an FAE a stearoyl ACP desaturase, or others described herein) is incorporated into a microalga. It has been found that for certain microalga, a plant fatty acid synthesis gene product is functional in the absence of the corresponding plant acyl carrier protein (ACP), even when the gene product is an enzyme, such as an acyl-ACP thioesterase, that requires binding of ACP to function. Thus, optionally, the microalgal cells can utilize such genes to make a desired oil without co-expression of the plant ACP gene.

[0094] For the various embodiments of recombinant cells comprising exogenous genes or combinations of genes, it is contemplated that substitution of those genes with genes having 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% nucleic acid sequence identity can give similar results, as can substitution of genes encoding proteins having 60%, 70%, 80%, 85%, 90%, 91% 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 100% amino acid sequence identity. Nucleic acids encoding the acyltransferases encode acyltransferases that have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to the acyltransferase disclosed in clade 1, clade 2, clade 3 or clade 4 of Table 5. Likewise, for novel regulatory elements, it is contemplated that substitution of those nucleic acids with nucleic acids having 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid can be efficacious. In the various embodiments, it will be understood that sequences that are not necessary for function (e.g. FLAG tags or inserted restriction sites) can often be omitted in use or ignored in comparing genes, proteins and variants.

[0095] The novel genes and gene combinations reported here can be used in higher plants using techniques that are well known in the art. For example, the use of exogenous lipid metabolism genes in higher plants is described in U.S. Pat. Nos. 6,028,247; 5,850,022; 5,639,790; 5,455,167; 5,512,482; and 5,298,421 disclose higher plants with exogenous acyl-ACP thioesterases. WO2009129582 and WO1995027791 disclose cloning of LPAAT in plants. FAD2 ablation and/or down regulation in higher plants is taught in WO 2013112578, and WO2008/006171. SAD ablation and/or down regulation in higher plants is taught in WO 2013112578, and WO 2008006171.

[0096] The expression of the novel acyltransferases is shown in Examples 4, 5, 6 and 7. The expression of Cuphea paucipetala or Cuphea ignea LPATs markedly increased the C8:0 and C10:0 fraction of the cell oil. Additionally, the expression of Cuphea paucipetala or Cuphea ignea LPAATs markedly increased the incorporation of C8:0 and C10:0 fatty acids in the sn-2 position of the TAG. This is disclosed in Example 4.

[0097] The expression of LPAT genes in host cells increased C18:2 levels and elevated the sat-unsat-sat/sat-sat-sat, (e.g., SOS/SSS) ratio of the cell oil. For example, the expression of Theobroma cacoa LPAT2 drives the transfer of unsaturated fatty acids toward the sn-2 position and reduces the incorporation of saturated fatty acids at sn-2.

[0098] The novel LPAATs, GPATs, DGATs, LPCATs, and PLA2 with specificity for mid-chain fatty acids are disclosed. In Example 7, expression of LPAATs and DGATs are disclosed.

[0099] When an acyltransferase of the invention is expressed in a host cell, one or more additional exogenous genes can concomitantly be expressed. An embodiment of this invention provides host cells that express a recombinant acyltransferase and concomitantly express one or more additional recombinant genes. The one or more additional genes include invertase, fatty acyl-ACP thioesterase (FATA, FATB), melibiase, ketoacyl synthase (KASI, KASII, KASIII, KASIV), antibiotic selective markers, tags such as FLAG, and THIC. In Examples 4, 5, 6, and 7, the co-expression of nucleic acids that encode LPAATs co-expressed with one or more exogenous genes that encode invertase, fatty acyl-ACP thioesterase, melibiase, ketoacyl synthase, THIC are disclosed.

[0100] When an acyltransferase of the invention is expressed in a host cell, an endogenous gene of the host call can concomitantly be ablated or downregulated, thereby eliminating or decreasing the expression of the gene of the host cell. This can be accomplished by using homologous recombination techniques or other RNA inhibitory technologies. The ablated or downregulated gene can be any gene in the host cell. The ablated or downregulated endogenous gene can be stearoyl ACP desaturase, fatty acyl desaturase, fatty acyl-ACP thioesterase (FATA or FATB), ketoacyl synthase (KASI, KASII, KASIII or KAS IV), or an acyltransferase (LPAAT, DGAT, GPAT, LPCAT). When an endogenous is ablated, one, two or more alleles of the endogenous can be ablated. In Example 5, the expression of a Brassica LPAAT, while concomitantly ablating an endogenous stearoyl ACP desaturase is disclosed. In Example 6, LPAATs, GPATs, DGATs, LPCATs and PLA2s with specificity for mid-chain fatty acids were expressed, while ablating a gene encoding stearoyl ACP desaturase. In Example 7 the down regulation of an endogenous FAD2 and a hairpin RNA is disclosed. In co-owned PCT/US2016/026265, applicants disclosed concomitant ablation of an endogenous LPAAT and expression of an exogenous LPAAT.

[0101] In one embodiment, the expression of the acyl transferases alters the fatty acid profile and/or the sn-2 profile of the oil produced by the host organism. The fatty acid profiles and the sn-2 profiles that result from the expression of various acyltransferases are disclosed in Tables 6, 7, 10, 11, 12, 13, 16, 17, 18, 19, 20, 22, 23, and 24. The invention provides host cells with altered fatty acid profiles and altered sn-2 profiles according to Tables 6, 7, 10, 11, 12, 13, 16, 17, 18, 19, 20, 22, 23, and 24.

[0102] As described in PCT/US2016/026265, co-owned by applicant, transcript profiling was used to discover promoters that modulate expression in response to low nitrogen conditions. The promoters are useful to selectively express various genes and to alter the fatty acid composition of microbial oils. In accordance with an embodiment, there are non-natural constructs comprising a heterologous promoter and a gene, wherein the promoter comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any of the promoters of SEQ ID NOs: 1-18 and the gene is differentially expressed under low vs. high nitrogen conditions. In particular, the Prototheca moriformis AMT02 (SEQ ID NO: 18) and AMT03 promoter (SEQ ID NO: 18) are useful promoters for controlling the expression of an exogenous gene. For example, the promoters can be placed in front of a FAD2 gene in a linoleic acid auxotroph to produce an oil with less than 5, 4, 3, 2, or 1% linoleic acid after culturing first under high nitrogen conditions, then next culturing under low nitrogen conditions. Additional promoters, in particulare Prototheca and Chlorella promoters are described in the sequences and descriptions in this application. For example, the Prototheca HXT1, SAD, LDH1 and other Prototheca promoters are described in Examples 6, 7, 8, and 9. Additionally, the Chlorella SAD, ACT and other Chlorella promoters are described in Examples 6, 7, 8, and 9.

[0103] In embodiments of the present invention, oleaginous cells expressing one or more of the genes encoding acyltransferases and/or variant FATA can produce an oil with at least 20, 40, 60 or 70% of C8, C10, C12, C14, C16, or C18 fatty acids.

[0104] The invention also provides host cells expressing one or more of the genes encoding acyltransferases and/or variant FATA can produce an oil enriched is oils that are sat-unsat-sat. Oils of this type include SOS, POP, POS, SLS, PLO, PLO. The sat-unsat-sat oils comprise at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cell oil by dry cell weight.

[0105] The invention also provides host cells expressing one or more of the genes encoding acyltransferases and/or variant FATA can produce an oil that is decreased in tri-saturated oils, sat-sat-sat. Oils of this type include PPP, PSS, PPS, SSS, SPS, and PSP. The sat-sat-sat oils comprise less than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the cell oil by molar fraction or dry cell weight.

[0106] The host cells of the invention can produce 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, or about 90% oil by cell weight, 5%. Optionally, the oils produced can be low in DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA.

[0107] In other embodiments of the invention, there is a process for producing an oil, triglyceride, fatty acid, or derivative of any of these, comprising transforming a cell with any of the nucleic acids discussed herein. In another embodiment, the transformed cell is cultivated to produce an oil and, optionally, the oil is extracted. Oil extracted in this way can be used to produce food, oleochemicals or other products.

[0108] The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, etc). The oils, triglycerides, fatty acids from the oils may be subjected to CH activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation, methylation, dimerization, thiolation, metathesis, hydro-alkylation, lactonization, or other chemical processes.

[0109] After extracting the oil, a residual biomass may be left, which may have use as a fuel, as an animal feed, or as an ingredient in paper, plastic, or other product. For example, residual biomass from heterotrophic algae can be used in such products.

EXAMPLES

Example 1: Fatty Acid Analysis by Fatty Acid Methyl Ester Detection

[0110] Lipid samples were prepared from dried biomass. 20-40 mg of dried biomass was resuspended in 2 mL of 5% H.sub.2SO.sub.4 in MeOH, and 200 ul of toluene containing an appropriate amount of a suitable internal standard (C19:0) was added. The mixture was sonicated briefly to disperse the biomass, then heated at 70-75 C. for 3.5 hours. 2 mL of heptane was added to extract the fatty acid methyl esters, followed by addition of 2 mL of 6% K.sub.2CO.sub.3 (aq) to neutralize the acid. The mixture was agitated vigorously, and a portion of the upper layer was transferred to a vial containing Na.sub.2SO.sub.4 (anhydrous) for gas chromatography analysis using standard FAME GC/FID (fatty acid methyl ester gas chromatography flame ionization detection) methods. Fatty acid profiles reported below were determined by this method.

Example 2: Analysis of Regiospecific Profile

[0111] LC/MS TAG distribution analyses were carried out using a Shimadzu Nexera ultra high performance liquid chromatography system that included a SIL-30AC autosampler, two LC-30AD pumps, a DGU-20A5 in-line degasser, and a CTO-20A column oven, coupled to a Shimadzu LCMS 8030 triple quadrupole mass spectrometer equipped with an APCI source. Data was acquired using a Q3 scan of m/z 350-1050 at a scan speed of 1428 u/sec in positive ion mode with the CID gas (argon) pressure set to 230 kPa. The APCI, desolvation line, and heat block temperatures were set to 300, 250, and 200 C., respectively, the flow rates of the nebulizing and drying gases were 3.0 L/min and 5.0 L/min, respectively, and the interface voltage was 4500 V. Oil samples were dissolved in dichloromethane-methanol (1:1) to a concentration of 5 mg/mL, and 0.8 L of sample was injected onto Shimadzu Shim-pack XR-ODS III (2.2 m, 2.0200 mm) maintained at 30 C. A linear gradient from 30% dichloromethane-2-propanol (1:1)/acetonitrile to 51% dichloromethane-2-propanol (1:1)/acetonitrile over 27 minutes at 0.48 mL/min was used for chromatographic separations.

Example 3: Cultivation of Microalgae

Standard Lipid Production Conditions:

[0112] Cells scraped from a source plate with toothpicks were used to inoculate pre-seed cultures of 0.5 mL EB03, 0.5% glucose, 1DAS2 cultures in 96-well blocks. Pre-seed cultures were grown for 70-75 h at 28 C., 900 rpm in a Multitron shaker. 40 L of pre-seed cultures were used to inoculate seed cultures of 0.46 mL H29, 4% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1DAS2 (8% inoculum), and grown for 24-28 h at 28 C., 900 rpm in a Multitron shaker. 40 L of seed cultures were used to inoculate lipid production cultures of 0.46 mL H43, 6% glucose, 25 mM citrate pH 5, 1DAS2 (8% inoculum), and grown for 70-75 h at 28 C., 900 rpm in a Multitron shaker. Fatty acid profiles and lipid titer analyses were performed as disclosed in Examples 1 and 2.

50 mL Shake Flask Format

[0113] Cells scraped from a source plate with inoculation loops, or cell cultures from cryovials were used to inoculate pre-seed cultures of 10 mL EB03, 0.5% glucose, 1DAS2 cultures in 50 mL bioreactor tubes. Pre-seed cultures were grown for 70-75 h at 28 C., 200 rpm in a Kuhner shaker. 0.8 mL of pre-seed cultures were used to inoculate seed cultures of 10 mL H29, 4% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1DAS2 (8% inoculum), and grown for 24-28 h at 28 C., 200 rpm in a Kuhner shaker. 100 L of seed cultures were used to inoculate lipid production cultures of 49.9 mL H43, 6% glucose, 25 mM citrate pH 5 or 100 mM PIPES pH 7.3, 1DAS2 (0.2% inoculum), and grown for 118-122 h at 28 C., 200 rpm in a Kuhner shaker. Fatty acid profiles and lipid titer analyses were performed as disclosed in Examples 1 and 2.

EB03

[0114]

TABLE-US-00006 Dry chemicals Component Concentration (g/L) K2HPO4 3 Sodium Phosphate Dibasic Heptahydrate 5.66 (Na2HPO4 7H2O) citric acid monohydrate 1.2 ammonium sulfate 1 MgSO4 7H2O 0.23 CaCl2 2H2O 0.03 Stock solutions Component Concentration (mL/L) 100X C-Trace (3) 10 Antifoam Sigma 204 0.225

H29

[0115]

TABLE-US-00007 Dry chemicals Final Component Concentration (g/L) K.sub.2HPO.sub.4 (Potassium phosphate 0.25 dibasic anhydrous) NaH.sub.2PO.sub.4 (Sodium phosphate 0.18 monobasic) MgSO.sub.47H.sub.2O (Magnesium 0.24 sulfate heptahydrate) Citric acid monohydrate 0.25 Stock solutions Component Concentration (mL/L) 0.017M stock CaCl.sub.22H.sub.2O 10 0.151M (NH.sub.4).sub.2SO.sub.4 52.2 100X C-Trace (2) 10 Antifoam Sigma 204 0.225

H43

[0116]

TABLE-US-00008 Dry chemicals Final Component Concentration (g/L) K2HPO4 0.25 NaH2PO4 0.18 MgSO4 7H2O 0.24 Citric acid H2O 0.25 Stock solutions Component Concentration (mL/L) 0.017M stock CaCl2 2H2O 10 100X C-Trace (2) 10 Antifoam Sigma 204 0.225 0.151M (NH4)2SO4 12.5

1000DAS2

[0117]

TABLE-US-00009 Dry chemicals Final Component Concentration (g/L) Thiamine-HCl 0.67 d-Biotin 0.010 Cyanocobalimin (vit B-12) 0.008 Calcium Pantothenate 0.02 PABA (p-aminobenzoic acid) 0.04

100C-Trace(2)

[0118]

TABLE-US-00010 Dry chemicals Final Component Concentration (g/L) CuSO45H2O 0.011 CoC126H2O 0.081 H3BO3 0.33 ZnSO47H2O 1.4 MnSO4H2O 0.81 Na2MoO42H2O 0.039 FeSO47H2O 0.11 NiCl26H2O 0.013 Citric Acid Monohydrate 3.0

100C-Trace (3)

[0119]

TABLE-US-00011 Dry chemicals Final Component Concentration (g/L) CuSO45H2O 0.011 H3BO3 0.33 ZnSO47H2O 1.4 MnSO4H2O 0.81 Na2MoO42H2O 0.039 FeSO47H2O 0.11 MCl26H2O 0.013 Citric Acid Monohydrate 3.0

Example 4: Identification of Novel LPAAT Genes from Sequenced Transcriptomes and Engineering Sn-2 Tag Regiospecificity in Utex1435 by Expression of Heterologous LPAAT Genes from Cuphea Paucipetala, Cuphea Ignea, Cuphea Painteri, and Cuphea Hookeriana

[0120] Lysophosphatidic acyltransferase (LPAAT) genes from plant seeds were cloned and expressed in the transgenic strain, S6511, derived from UTEX 1435 (P. moriformis). Expression of the heterologous LPAATs increases C8:0 and C10:0 fatty acid levels and dramatically increases incorporation of C8:0 and C10:0 fatty acids at the sn-2 position of triacylglycerols (TAGs) in transgenic strains.

[0121] TAGs are synthesized from various chain length acyl-CoAs and glycerol-3-phosphate by consecutive action of three ER-resident enzymes of the Kennedy pathwayglycerol phosphate acyltransferase (GPAT), LPAAT, and diacylglycerol acyltransferase (DGAT). Substrate specificities of these acyltransferases are known to determine the fatty acid composition of the resulting TAGs. LPAAT acylates the sn-2 hydroxyl group of lysophosphatidic acid (LPA) to form phosphatidic acid (PA), a precursor to TAG. In co-owned applications WO2013/158938, WO2015/051139, and PCT/US2016/026265 we demonstrated expression of LPAAT from Cocos nucifera (CnLPAAT, accession no. AAC49119; Knutzon et al., 1995).

[0122] Strain S6511 expresses the acyl-ACP thioesterase (FATB2) gene from Cuphea hookeriana (ChFATB2), leading to C8:0 and C10:0 fatty acid accumulation of ca. 14% and 28%, respectively. Strain S6511 is a strain made according to the methods disclosed in co-owned WO2010/063031 and WO2010/063032, herein incorporated by reference. Briefly, S6511 is a strain that express sucrose invertase and a C. hookeriana FATB2. The construct pSZ3101:6S::CrTUB2-ScSUC2-CvNR_a:PmAMT03-CpSAD1tp_trimmed:ChFATB2-CvNR_d::6S was engineered into S3150, a strain classically mutagenized to increase lipid yield. We identified novel C8:0- and C10:0-specific LPAATs from seeds exhibiting high levels of C8:0 and C10:0 fatty acids. After we identified and cloned LPAATs we expressed the LPAAT genes in S6511.

Method for Identification of LPAATs

[0123] Seeds were obtained from species exhibiting elevated levels of midchain and other specialized fatty acids (Table 4).

TABLE-US-00012 TABLE 4 Fatty acid profiles of mature seeds. The percentage of each fatty acid making up the seed oil is shown; abundant and unusual fatty acid species are indicated in bold. C18:1 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 (petroselinate) S01_Cc Cinnamomum 0.4 54.7 39.0 1.6 0.7 0.1 2.9 camphora S02_Uc Umbellularia 0.9 28.8 63.0 2.3 0.4 0.1 3.4 californica S03_Ld Limnanthes 0.0 0.0 0.0 0.4 0.7 0.4 2.7 douglasii S04_Chs Cuphea 0.2 6.5 83.7 5.1 1.1 0.1 0.0 hyssopifolia S05_Ccr Cuphea 1.6 8.1 59.2 15.2 3.9 0.6 0.0 carthagenensis S06_Cpr Cuphea 2.0 11.5 61.3 10.8 2.7 0.5 0.0 parsonsia S07_Cg Cuphia 7.1 85.1 1.7 0.3 1.0 0.2 0.0 glossostoma S08_Cht Cuphea 3.5 44.3 40.0 4.3 1.2 0.3 2.2 heterophylla S11_Dc Daucus 0.0 0.0 0.0 0.1 5.9 0.8 11.5 65.9 carrota S14_Cw Cuphea 0.5 20.2 62.5 5.8 2.2 0.3 2.7 wrightii S15_Bj Brassica 0.0 0.0 0.0 0.1 3.2 0.7 12.1 juncea S16_Br Brassica 0.0 0.0 0.0 0.1 2.8 1.0 16.0 rapa nipposinica S17_Ca Cuphea 90.8 2.7 0.0 0.1 1.2 0.1 1.8 avigera var. pulcherrima S18_Ch Cuphea 64.7 29.7 0.1 0.2 1.3 0.1 1.9 hookeriana S19_Cpal Cuphea 28.9 0.8 1.3 55.1 6.2 0.2 3.0 palustris S20_Cpai Cuphea 67.0 20.8 0.1 0.2 2.6 0.3 3.1 painteri S21_Cpau Cuphea 1.5 91.0 1.2 0.7 1.5 0.2 1.1 paucipetala S22_Chook Cuphea 62.8 31.9 0.2 0.2 1.0 0.1 2.1 hookeriana S23_Cglut Cuphea 5.2 29.9 46.4 3.9 1.9 0.4 0.0 glutinosa S24_Caequ Cuphea 27.1 0.0 1.4 57.4 6.0 0.2 3.2 aequipetala S25_Ccalc Cuphea 8.0 20.4 46.8 7.6 3.2 0.6 3.7 calcarata S26_Chook Cuphea 70.4 23.1 0.1 0.2 1.5 0.2 2.5 hookeriana S27_Cproc Cuphea 0.9 86.3 0.0 1.6 2.2 0.4 3.2 procumbens S28_Cignea Cuphea 3.1 84.9 0.7 0.3 2.6 0.2 2.9 ignea S35_Ccras Cuphea 1.3 87.7 1.3 0.4 2.0 0.5 3.3 crassiflora S36_Ckoe Cuphea 0.0 87.4 1.4 0.8 2.2 0.4 2.3 koehneana S37_Clept Cuphea 1.3 86.1 1.3 0.4 2.2 0.5 3.1 leptopoda S40_Clop Cuphea 0.5 82.3 2.4 1.6 3.0 0.6 3.9 lophostoma S41_Sal Sassafras 4.3 65.2 22.8 0.9 0.8 5.1 0.0 albidum db C22: C22: C22:2n9, C18:2 C20:0 C20:1 C22:0 1n17 1n9 17 C22:2n6 S01_Cc 0.6 0.0 S02_Uc 0.6 0.0 S03_Ld 1.5 1.5 59.9 0.3 2.8 17.4 9.3 0.5 S04_Chs 1.7 0.1 S05_Ccr 5.4 0.2 S06_Cpr 5.2 0.1 S07_Cg 2.1 0.1 S08_Cht 3.6 0.1 S11_Dc 13.0 0.5 0.3 0.3 S14_Cw 4.7 S15_Bj 19.2 0.5 6.3 0.8 38.9 1.3 S16_Br 16.8 0.7 8.3 1.0 40.1 0.8 S17_Ca 2.8 S18_Ch 2.0 S19_Cpal 3.4 S20_Cpai 4.5 S21_Cpau 2.1 S22_Chook 1.2 S23_Cglut 8.1 S24_Caequ 3.8 S25_Ccalc 8.5 S26_Chook 1.8 S27_Cproc 3.3 S28_Cignea 4.4 S35_Ccras 2.7 S36_Ckoe 4.5 S37_Clept 4.1 S40_Clop 4.9 S41_Sal 0.6

[0124] Briefly, RNA was extracted from dried plant seeds and submitted for paired-end sequencing using the Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using the Trinity software package. LPAAT-containing cDNA contigs were identified by mining transcriptomes for sequences with homology to a known LPAAT that was previously identified in-house, CuPSR23 LPAAT2-1 (seeWO2013/158938), using BLAST. For some sequences, a high-confidence, full-length transcript was assembled using Trinity. The resulting amino acid sequences of all new LPAATs were subjected to phylogenetic analyses using previously known, full-length LPAAT sequences (available via NCBI) as well as sequences of previously known LPAATs whose sequences were derived at Solazyme. The analysis showed that the amino acid sequences of the newly discovered LPPAATs were not similar to previously known LPAATs. Table 5 shows the clade analysis in which the novel LPAATs were clustered according to a neighbor joining algorithm. These were found to form 4 clades as listed in Table 5.

TABLE-US-00013 TABLE 5 Clade Analysis of LPAATs Percent amino acid Amino Acid identity Clade SEQ ID Nos. to members No. in Clade Full Genus Species Function of clade 1 S15 BjLPAAT1d Brassica juncea 96.3 S15 BjLPAAT1c Brassica juncea S15 BjLPAAT1a Brassica juncea S15 BjLPAAT1b Brassica juncea 2 CuPSR23LPAAT2-1 Cuphea PSR23 Prefer C8/ 93.9 S40 ClopLPAAT1 Cuphea lophostoma C10 sn-2 S21 CpauLPAAT1 Cuphea paucipetala S37 CleptLPAAT1 Cuphea leptopoda S27 CprocLPAAT1b Cuphea procumbens S27 CprocLPAAT1 Cuphea procumbens S04 ChsLPAAT2 Cuphea hyssopifolia S28 CigneaLPAAT1 Cuphea ignea S05 CcrLPAAT2a Cuphea carthagenensis S06 CprLPAAT1 Cuphea parsonsia S05 CcrLPAAT2b Cuphea carthagenensis S17 CaLPAAT3 Cuphea avigera var. pulcherrima S26 ChookLPAAT1 Cuphea hookeriana S20 CpaiLPAAT1 Cuphea painteri S04 ChsLPAAT1 Cuphea hyssopifolia S25 Ccalc1a Cuphea calcarata S25 Ccalc1b Cuphea calcarata S14 CwLPAAT1 Cuphea wrightii S08 ChtLPAAT1a Cuphea heterophylla S08 ChtLPAAT1b Cuphea heterophylla S36 CkoeLPAAT2 Cuphea koehneana S02 UcLPAAT1b Umbellularia californica S02 UcLPAAT1a Umbellularia californica S01 CcLPAAT1a Cinnamomum camphora S01 CcLPAAT1b Cinnamomum camphora S41 SaILPAAT1 Sassafras albidum db 3 S14 CwLPAAT2a Cuphea wrightii C18:2 86.5 S14 CwLPAAT2b Cuphea wrightii S25 CcalcLPAAT2 Cuphea calcarata S19 CpaILPAAT1 Cuphea palustris S22 ChookLPAAT3b Cuphea hookeriana S17 CaLPAAT1 Cuphea avigera var. pulcherrima S22 ChookLPAAT3a Cuphea hookeriana CuPSR23LPAAT3-1 Cuphea PSR23 S27 CprocLPAAT2b Cuphea procumbens S27 CprocLPAAT2a Cuphea procumbens S18 ChLPAAT2a Cuphea hookeriana S24 CaequLPAAT1d Cuphea aequipetala S24 CaequLPAAT1b Cuphea aequipetala S24 CaequLPAAT1a Cuphea aequipetala S24 CaequLPAAT1c Cuphea aequipetala S23 CglutLPAAT1a Cuphea glutinosa S23 CglutLPAAT1b Cuphea glutinosa S26 ChookLPAAT2b Cuphea hookeriana S07 CgLPAAT1c Cuphia glossostoma S07 CgLPAAT1b Cuphia glossostoma S07 CgLPAAT1a Cuphia glossostoma S28 CigneaLPAAT2 Cuphea ignea S36 CkoeLPAAT1 Cuphea koehneana S35 CcrasLPAAT1a Cuphea crassiflora S35 CcrasLPAAT1c Cuphea crassiflora S35 CcrasLPAAT1b Cuphea crassiflora S35 CcrasLPAAT1d Cuphea crassiflora 4 Gh LPAAT2B Garcinia hombroriana Reduced 78.5 Gi LPAAT2B-1 Garcinia indica trisaturates, Gh LPAAT2A Garcinia hombroriana increase Gi LPAAT2A Garcinia indica unsaturates Gh LPAAT2C Garcinia hombroriana at Sn-2 Gi LPAAT2C-2 Garcinia indica position S03 LdLPAAT1 Limnanthes douglasii S11 DcLPAAT1 Daucus carrota (carrot) S11 DcLPAAT2 Daucus carrota (carrot) S11 DcLPAAT2 Daucus carrota (truncated) (carrot)

Functionality of LPAATs in P. Moriformis

[0125] To increase the levels of C8:0 and C10:0 fatty acids in strain S6511, as well as to test the functionality of the newly identified LPAATs, we identified midchain-specific LPAATs from the transcriptomes of species exhibiting high levels of C8:0 and C10:0 fatty acids in their oil seeds and introduced the genes into S56511. LPAATs that co-clustered with CuPSR23 LPAAT2-1, specifically CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1, were selected for synthesis and testing. CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1 were synthesized in a codon-optimized form to reflect UTEX 1435 codon usage. Transgenic strains were generated via transformation of the strain S6511 with a construct encoding one of the four LPAAT genes. The construct pSZ3840 encoding CpauLPAAT1 is shown as an example, but identical methods were used to generate each of the remaining three constructs. Construct pSZ3840 can be written as pLOOP::PmHXT1-ScarMEL1-CvNR:PmAMT3-CpauLPAAT1-CvNR::pLOOP. The sequence of the transforming DNA is provided in FIG. 2 (pSZ3840). The relevant restriction sites in the construct from 5-3, BspQI, KpnI, SpeI, XhoI, EcoRI, SpeI, XhoI, SacI, BspQI, respectively, are indicated in lowercase, bold, and underlined. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Bold lowercase sequences at the 5 and 3 end of the construct represent genomic DNA from UTEX 1435 that target integration to the pLOOP locus via homologous recombination. Proceeding in the 5 to 3 direction, the selection cassette has the P. moriformis HXT1 promoter driving expression of the Saccharomyces carlsbergensis MEL1 (conferring the ability to grow on melibiose) and the Chlorella vulgaris Nitrate reductase (NR) gene 3 UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScarMEL1 are indicated in bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3 UTR is indicated by lowercase underlined text. The second cassette containing the codon optimized CpauLPAAT1 gene from Cuphea paucipetala is driven by the P. moriformis AMT3 promoter and has the Chlorella vulgaris Nitrate reductase (NR) gene 3 UTR. In this cassette, the AMT3 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CpauLPAAT1 gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics. The 3 UTR is indicated by lowercase underlined text. The final construct was sequenced to ensure correct reading frame and targeting sequences.

TABLE-US-00014 SEQIDNO:19pSZ3840/D2554transformingconstruct(CpauLPAAT1) gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatga aatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgt cttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaa ttacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggc ttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagcca ggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggtatgggaaccacgggg [00001] [00002] [00003] [00004] [00005] [00006] [00007] [00008] [00009] [00010] [00011] gcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggacacggccga ccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctccggccgcgactccga cggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgtt cggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacgcccagttct tcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcccgagatctcctacca ccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccctgtgcaactggggccaggacctga ccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcggagttcacgcgccccgactcccgct gcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctgaacaaggccgccccc atgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaacctgacggacgacga ggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaacaacctgaaggcctcct cctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtctggcgctacta cgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtggc gctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctccaagaa gctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgcaaca agaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttcgg ccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttctaccgcctgcgcccc tcctccTGAtacgtactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgcc acacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagtt gctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacg ctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtact [00012] [00013] [00014] [00015] [00016] [00017] [00018] [00019] [00020] [00021] [00022] [00023] [00024] [00025] atcaacctgttccaggccagtgatcgtgaggtgtggcccagtccaagaacgcctaccgccgcatcaaccgcgtgttcgccg agctgctgctgtccgagctgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttcc gcctgatgggcaaggagcacgccctggtgatcatcaaccacatgaccgagctggactggatgctgggctgggtgatgggcca gcacctgggctgcctgggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttct ccgagtacctgtacatcgagcgctcctgggccaaggaccgcaccaccctgaagtcccacatcgagcgcctgaccgactacccc ctgcccttctggatggtgatcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcct ccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgccc gccgtgtacgacgtgaccgtggccttccccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcgtgc tgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagtt cgtggagaaggacgccctgctggacaagcacaacgccgaggacaccttctccggccaggaggtgcaccgcaccggctcccg ccccatcaagtccctgctggtggtgatctcctgggtggtggtgatcaccttcggcgccctgaagttcctgcagtggtcctcctgga agggcaaggccttctccgtgatcggcctgggcatcgtgaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctc ctccaaccccgccaaggtggcccaggccaagctgaagaccgagctgtccatctccaagaaggccaccgacaaggagaacT GActcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgcctt gacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtg ctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatcc ctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaac cagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttgagctcagcggcgacggtcctgctacc gtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcct ggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgc cagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactct cgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaag ggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttctt agctcttc

[0126] The sequence for all of the other LPAAT constructs are identical to that of pSZ3840 with the exception of the encoded LPAAT. The LPAAT sequence alone with flanking SpeI and XhoI restriction sites is provided for the remaining LPAAT constructs are shown below. The amino acid sequence of the LPAAT proteins is provided below.

TABLE-US-00015 pSZ3841/D2555(CpaiLPAAT1) SEQIDNO:20 actagt gccatcccctccgccgccgtggtgttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccag gccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgcccctgga gttcctgtggctgttccactggtgcgccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaagga gcacgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctg ggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccggctacctgttcctg gagcgctcctgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgcccttctggctgatc atcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgcccc gcaacgtgctgatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgacc gtggccttccccaagacctcccccccccccaccatgctgaagctgttcgagggccagtccgtggagctgcacgtgcacatcaag cgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgcc ctgctggacaagcacaactccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggt ggtgatctcctgggtggtggtgatcatcttcggcgccctgaagttcctgctgtggtcctccctgctgtcctcctggaagggcaagg ccttctccgtgatcggcctgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgaggg ctccaaccccgtgaaggccgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaac ctcgag pSZ3842/D2556(CigneaLPAAT1) SEQIDNO:21 actagt gccatcgccgccgccgccgtgatcttcctgttcggcctgctgttcttcgcctccggcatcatcatcaacctgttccag gccctgtgcttcgtgctgatctggcccctgtccaagaacgtgtaccgccgcatcaaccgcgtgttcgccgagctgctgctgatgg acctgctgtgcctgttccactggtgggccggcgccaagatcaagctgttcaccgaccccgagaccttccgcctgatgggcatgg agcacgccctggtgatcatgaaccacaagaccgacctggactggatggtgggctggatcctgggccagcacctgggctgcct gggctccatcctgtccatcgccaagaagtccaccaagttcatccccgtgctgggctggtccgtgtggttctccgagtacctgttcc tggagcgctcctgggccaaggacaagtccaccctgaagtcccacatggagaagctgaaggactaccccctgcccttctggctg gtgatcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgc cccgcaacgtgctgatcccccacaccaagggcttcgtgtcctgcgtgtccaacatgcgctccttcgtgcccgccgtgtacgacgt gaccgtggccttccccaagtcctcccccccccccaccatgctgaagctgttcgagggccagtccatcgtgctgcacgtgcacatc aagcgccacgccctgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggac gccctgctggacaagcacaacgccgaggacaccttctccggccaggaggtgcaccacatcggccgccccatcaagtccctgct ggtggtgatcgcctgggtggtggtgatcatcttcggcgccctgaagttcctgcagtggtcctccctgctgtccacctggaagggc aaggccttctccgtgatcggcctgggcatcgccaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctccaacc ccgccaaggtggccaag ctcgag pSZ3844/D2557(ChookLPAAT1) SEQIDNO:22 actagt gccatcccctccgccgccgtggtgttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccag gccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgcccctgga gttcctgtggctgttccactggtgcgccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaagga gcacgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctg ggctccatcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccgagtacctgttcctg gagcgctcctgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgcccttctggctgatc atcttcgtggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgcccc gcaacgtgctgatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgacc gtggccttccccaagacctcccccccccccaccatgctgaagctgttcgagggccagtccgtggagctgcacgtgcacatcaag cgccacgccatgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgcc ctgctggacaagcacaactccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggt ggtgatctcctgggtggtggtgatcatcttcggcgccctgaagttcctgctgtggtcctccctgctgtcctcctggaagggcaagg ccttctccgtgatcggcctgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgaggg ctccaaccccgtgaaggccgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaac ctcgag

[0127] To determine the impact of the CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1 genes on mid-chain fatty acid accumulation, the above constructs containing the codon optimized CpauLPAAT1, CigneaLPAAT1, ChookLPAAT1, and CpaiLPAAT1 genes were transformed into strain S6511. Primary transformants were clonally purified and grown under standard lipid production conditions at pH7.0 (all the strains require growth at pH 7.0 to allow for maximal expression of the LPAAT gene driven by the pH-regulated AMT3 promoter). The resulting profiles from a set of representative clones arising from these transformations are shown in Table 6.

TABLE-US-00016 TABLE 6 Transformants of pSZ3840 (CpauLPAAT1), pSZ3841 (CpaiLPAAT1), pSZ3842 (CigneaLPAAT1), and pSZ3844 (ChookLPAAT1). The fatty acid profiles for transgenic strains expressing LPAATs derived from C. paucipetala, C. painteri, C. ignea, and C. hookeriana. Sample ID C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3a Parent S6511a 14.4 27.7 0.6 1.3 8.8 1.6 38.2 5.4 0.4 S6511b 14.5 27.7 0.6 1.3 8.6 1.6 38.4 5.3 0.4 pSZ3840 CpauLPAAT1 S6511; T792; D2554-20 16.6 29.9 0.7 1.3 8.0 1.0 35.2 5.2 0.5 S6511; T792; D2554-17 14.6 28.7 0.6 1.3 8.4 1.7 37.1 5.7 0.5 S6511; T792; D2554-41 15.2 28.5 0.7 1.3 8.3 1.4 37.5 5.2 0.4 S6511; T792; D2554-35 14.7 28.4 0.6 1.3 8.6 1.6 37.3 5.6 0.5 S6511; T792; D2554-27 15.2 27.6 0.7 1.3 9.5 1.5 37.1 5.1 0.4 pSZ3841 CpaiLPAAT1 S6511; T792; D2555-34 17.3 29.5 0.7 1.3 7.8 1.2 35.1 5.1 0.4 S6511; T792; D2555-43 17.5 29.1 0.7 1.3 8.0 0.9 35.4 5.0 0.5 S6511; T792; D2555-10 15.7 28.3 0.7 1.3 8.6 1.6 36.2 5.7 0.5 S6511; T792; D2555-22 16.0 27.9 0.7 1.3 8.4 0.9 37.8 5.0 0.4 S6511; T792; D2555-44 15.3 27.5 0.6 1.3 8.1 1.8 38.2 5.4 0.4 pSZ3842 CigneaLPAAT1 S6511; T792; D2556-38 16.2 29.2 0.7 1.3 8.1 1.3 36.1 5.2 0.5 S6511; T792; D2556-22 14.3 28.5 0.7 1.3 8.5 1.6 37.6 5.7 0.5 S6511; T792; D2556-44 13.6 28.4 0.7 1.4 9.0 1.5 36.3 6.7 0.7 S6511; T792; D2556-14 14.1 28.0 0.6 1.3 8.6 1.7 38.0 5.6 0.5 S6511; T792; D2556-36 14.3 28.0 0.6 1.3 8.6 1.7 37.9 5.7 0.5 pSZ3844 ChookLPAAT1 S6511; T792; D2557-47 15.8 29.3 0.7 1.3 8.2 1.2 36.5 5.0 0.5 S6511; T792; D2557-24 16.8 28.8 0.7 1.3 8.1 1.2 35.8 5.4 0.5 S6511; T792; D2557-30 15.2 28.3 0.7 1.3 8.5 1.6 36.8 5.7 0.5 S6511; T792; D2557-39 14.7 28.2 0.7 1.3 8.7 1.5 37.3 5.7 0.5 S6511; T792; D2557-26 15.3 27.7 0.7 1.4 8.7 0.9 37.7 5.4 0.5

[0128] The transformants in Table 6 display a marked increase in the production of C8:0 and C10:0 fatty acids upon expression of the heterologous LPAATs. To determine if expression of the heterologous LPAAT genes affected the regiospecificity of fatty acids at the sn-2 position, we analyzed TAGs from representative D2554 (CpauLPAAT1), D2555 (CpaiLPAAT1), D2556 (CigneaLPAAT1), and D2557 (ChookLPAAT1) strains utilizing the porcine pancreatic lipase method. Cells were grown under conditions to maximize midchain fatty acid levels and to generate sufficient biomass for TAG analysis. TAG and sn-2 profiles are shown in Table 7.

[0129] Table 7: Inclusion of C8:0 and C10:0 fatty acids at the sn-2 position of TAGs. Selected transformants were subjected to porcine pancreatic lipase determination of fatty acid inclusion at the sn-2 position. The general fatty acid distribution in triacylglycerols (TAG) is shown to indicate fatty acid abundance for each transformant. In addition, the sn-2-specific distribution is shown. Numbers highlighted in bold and italic reflect significantly increased inclusion of the noted fatty acid compared to the parent S6511.

TABLE-US-00017 TABLE 7 Strain: S6511; T792; S6511; T792; S6511; T792; S6511; T792; D2554-20 D2555-34 D2556-38 D2557-24 S6511 (CpauLPAAT1) (CpaiLPAAT1) (CigneaLPAAT1) (ChookLPAAT1) Analysis TAG sn-2 TAG sn-2 TAG sn-2 TAG sn-2 TAG sn-2 Fatty Acid C8:0 14.4 8.5 16.6 12.8 17.3 16.2 10.0 16.8 (area %) C10:0 27.7 26.4 29.9 29.5 22.2 29.2 28.8 19.4 C12:0 0.6 0.4 0.7 0.3 0.7 0.4 0.7 0.4 0.7 0.3 C14:0 1.3 1.0 1.3 1.0 1.3 0.9 1.3 1.2 1.3 0.9 C16:0 8.8 0.9 8.0 1.1 7.8 1.1 8.1 1.2 8.1 0.9 C18:0 1.6 0.2 1.0 0.4 1.2 0.5 1.3 0.5 1.2 0.3 C18:1 38.2 52.5 35.2 37.8 35.1 43.6 36.1 42.2 35.8 40.7 C18:2 5.4 8.9 5.2 6.2 5.1 7.9 5.2 7.0 5.4 7.1 C18:3 0.4 0.8 0.5 0.7 0.4 0.9 0.5 0.8 0.5 0.7 C8 + C10 42.2 34.9 46.4 51.8 46.8 44.5 45.5 46.1 45.6 48.5 sum

[0130] As disclosed in Table 7, the CpauLPAAT1 and CigneaLPAAT1 genes show remarkable specificity towards C10:0 fatty acids. D2554-20 exhibits 39.0% of C10:0 in the sn-2 position versus just 26.4% in the S6511 base strain without the heterologous LPAAT, demonstrating a 1.5 fold increase in C10:0 inclusion at the sn-2 position. D2556-38 exhibits 36.2% of C10:0 in the sn-2 position versus 26.4% in the S6511 base strain, demonstrating a 1.4 fold increase in C10:0 inclusion at the sn-2 position. Although there is a small increase in C8:0 levels in the D2554-20 and D2555-34 strains, the vast majority of sn-2 targeting is C10:0-specific. Similarly, CpaiLPAAT1 and ChookLPAAT1 show remarkable specificity towards C8:0 fatty acids. D2555-34 exhibits 22.3% C8:0 in the sn-2 position versus just 8.5% in the S6511 base strain without the heterologous LPAAT, demonstrating a 2.6 fold increase in C8:0 inclusion at the sn-2 position. D2557-24 exhibits 29.1% C8:0 in the sn-2 position versus 8.5%, demonstrating a 3.4 fold increase in C8:0 inclusion at the sn-2 position. We teach that CpauLPAAT1 and CigneaLPAAT1 are C10:0-specific LPAATs and that CpaiLPAAT1 and ChookLPAAT1 are C8:0-specific LPAATs. Knutzon D S, Lardizabal K D, Nelsen J S, Bleibaum J L, Davies H M, Metz J G (1995) Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates. Plant Physiol 109:999-1006

Amino Acid Sequences for Novel LPAAT Genes

[0131]

TABLE-US-00018 SEQIDNO:23CpauLPAAT1 MAIPAAAVIFLFGLLFFTSGLIINLFQALCFVLVWPLSKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWML GWVMGQHLGCLGSILSVAKKSTKFLPVLGWSMWFSEYLYIERSWAKDRTT LKSHIERLTDYPLPFWMVIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSCVSHMRSFVPAVYDVTVAFPKTSPPPTLLNLFEGQSIVLHV HIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHRTG SRPIKSLLVVISWVVVITFGALKFLQWSSWKGKAFSVIGLGIVTLLMHML ILSSQAERSSNPAKVAQAKLKTELSISKKATDKEN SEQIDNO:24CprocLPAAT1 MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTQTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSCVSHMRSFVPAVYDLTVAFPKTSPPPTLLNLFEGQSVVLHV HIKRHAMKDLPESDDEVAQWCRDKFVEKDALLDKHNAEDTFSGQELQHTG RRPIKSLLVVISWVVVIAFGALKFLQWSSWKGKAFSVIGLGIVTLLMHML ILSSQAERSKPAKVAQAKLKTELSISKTVTDKEN SEQIDNO:25CprocLPAAT1b MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPISKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWNKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTQTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSCVSHMRSFVPAVYDLTVAFPKTSPPPTLLNLFEGQSVVLHV HIKRHAMKDLPESDDEVAQWCRDKFVEK SEQIDNO:26CprocLPAAT2a IVNLVQAVCFVLVRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKV FTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKS SKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPFWLALFV EGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAI YDVTVAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDAVAQWC RDIFVEKDALLDKHNADDTFSGQELQDTGRPIKSLLVVISWAVLEVFGAV KFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAK AKIEGESSKTEMEKEK SEQIDNO:27CprocLPAAT2b IVNLVQAVCFVLVRPLSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKV FTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKS SKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDYPLPFWLALFV EGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAI YDVTVAIPKTSPPPTLIRMFKGQSSVLHVHLKRHVMKDLPESDDAVAQWC RDIFVEKDALLDKHNADDTFSGQELQDTGRPIKSLLV SEQIDNO:28CpaiLPAAT1 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVFA ELLPLEFLWLFHWCAGAKLKLFTDPETFRLMGKEHALVIINHKIELDWMV GWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSGYLFLERSWAKDKIT LKSHIESLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSVSHMRSFVPAIYDVTVAFPKTSPPPTMLKLFEGQSVELHV HIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNSEDTFSGQEVHHVG RPIKALLVVISWVVVIIFGALKFLLWSSLLSSWKGKAFSVIGLGIVAGIV TLLMHILILSSQAEGSNPVKAAPAKLKTELSSSKKVTNKEN SEQIDNO:29ChookLPAAT1 MAIPSAAVVFLFGLLFFTSGLIINLFQAFCFVLISPLSKNAYRRINRVFA ELLPLEFLWLFHWCAGAKLKLFTDPETFRLMGKEHALVIINHKIELDWMV GWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSEYLFLERSWAKDKIT LKSHIESLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSVSHMRSFVPAIYDVTVAFPKTSPPPTMLKLFEGQSVELHV HIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNSEDTFSGQEVHHVG RPIKALLVVISWVVVIIFGALKFLLWSSLLSSWKGKAFSVIGLGIVAGIV TLLMHILILSSQAEGSNPVKAAPAKLKTELSSSKKVTNKEN SEQIDNO:30ChookLPAAT2a LSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLI DWWAGVKIKVFTDHETFNLMGKEHALVVCNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDY PLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SHMRSFVPAIYDVTVAIPKTSVPPTMLRIFKGQSSVLHVHLKRHLMKDLP ESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLLVVIS WAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSER STPAKVAPAKPKNEGESSKTEMEKEH SEQIDNO:31ChookLPAAT2b QIKVFTDHETFNLMGKEHALVVCNHKSDIDWLVGWVLAQWSGCLGSTLAV MKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPFWL ALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSF VPAIYDVTVAIPKTSVPPTMLRIFKGQSSVLHVHLKRHLMKDLPESDDAV AQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLLVVISWAVLVI FGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKV APAKLKKEGESSKPETDKQN SEQIDNO:32ChookLPAAT3a LSLLFFVSGLIVNLVQAVCFVLIRPLLKNTYRRINRVVAELLWLELVWLI DWWAGIKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SQMRSFVPAIYDVTVAIPKTSPPPTLLRMFKGQSSVLHVHLKRHLMNDLP ESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVVIS WATLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSER STPAKVAPAKPKNEGESSKTEMEKEH SEQIDNO:33ChookLPAAT3b LSLLFFVSGLIVNLVQAVCFVLIRPLLKNTYRRINRVVAELLWLELVWLI DWWAGIKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SQMRSFVPAIYDVTVAIPKTSPPPTLLRMFKGQSSVLHVHLKRHLMNDLP ESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLLVVIS WAVLEIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSER STPAKVAPAKPKKEGESSKPETDKEN SEQIDNO:34CigneaLPAAT1 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVFA ELLLMDLLCLFHWWAGAKIKLFTDPETFRLMGMEHALVIMNHKTDLDWMV GWILGQHLGCLGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKDKST LKSHMEKLKDYPLPFWLVIFVEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPHTKGFVSCVSNMRSFVPAVYDVTVAFPKSSPPPTMLKLFEGQSIVLHV HIKRHALKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHHIG RPIKSLLVVIAWVVVIIFGALKFLQWSSLLSTWKGKAFSVIGLGIATLLM HMLILSSQAERSNPAKVAK SEQIDNO:35CigneaLPAAT2 MAIAAAAVIFLFGLLFFASGIIINLFQALCFVLIWPLSKNVYRRINRVFA ELLLMDLLCLFHWWAGAKIKLFTDPETFRLMGMEHALVIMNHKTDLDWMV GWILGQHLGCLGSILSIAKKSTKFIPVLGWSVWFSEYLFLERSWAKDEST LKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPKNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSAPPTLLRMFKGQSSVLHV HLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELHDIG RPVKSLLVVISWAMLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLM HILILFSQSERSTPAKVAPAKQKNNEGESSKTEMEKEH SEQIDNO:36DcLPAAT1 SGLVVNLIQAFFFVLVRPFSKNAYRKINRVVAELLWLELIWLIDWWAGVK IQLYTDPETFKLMGKEHALVICNHKSDIDWLVGWILAQRSGCLGSALAVM KKSSKFLPVIGWSMWFSEYLFLERSWAKDENTLKSGFQRLRDFPHAFWLA LFVEGTRFTQAKLLAAQEYASSMGLPAPRNVLIPRTKGFVTAVTHMRPFV PAVYDVTLAIPKTSPPPTMLRLFKGQSSVVHIHLKRHLMSDLPKSDDSVA QWCKDAFVVKDNLLDKHKENDSFGDGVLQDTGRPLNSLVVVISWACLLIF GALKFFQWSSILSSWKGLAFSAVGLGIVTVLMQILIQFSQSERSNRPMPS KHAK SEQIDNO:37DcLPAAT2 MAIPTAAYVVPLGAIFFFSGLLVNLIQAFFFITVWPLSKKTYIRINKVIV ELLWLEFVWLADWWAGLKIEVYADAETFQLMGKEHALVICNHKSDIDWLV GWILAQRAGCLGSSFAVTKKSARYLPVVGWSIWFSGAIFLERSWEKDENT LKAGFQRLREFPCAFWLGLFVEGTRFTQAKLLAAQEYASTMGLPFPRNVL IPRTKGFIAAVNHMREFVPAIYDLTFAFPKDSPPPTMLRLLKGQPSVVHV HIKRHLMKDLPEKNEAVAQWCKDVFLVKDKLLDKHKDDGSFGDGELHEIG RPLKSLVVVTTWACLLILGTLKFLLWSSLLSSWKGLIFSATGLAVLTVLM QFLIQSTQSERSNPASLSK SEQIDNO:38CcrLPAAT1a LGLLFFISGLAVNLIQAVCFVFLRPLSKNTYRKINRVLAELLWLQLVWLV DWWAGVKIKVFADRESFNLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSSLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKEGLRRLKDF PRPFWLALFVEGTRFTQAKLLAAQEYATSQGLPVPRNVLIPRTKVHVHVK RHLMKELPETDEAVAQWCKDLFVEKDKLLDKHVAEDTFSDQPLQDIGRPV KPLLVVSSWACLVAYGALKFLQWSSLLSSWKGIAVSAVALAIVTILMQIM ILFSQSERSIPAKVA SEQIDNO:39CcrLPAAT1b LGLLFFISGLAVNLIQAVCFVFLRPLSKNTYRKINRVLAELLWLQLVWLV DWWAGVKIKVFADRESFNLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSSLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKEGLRRLKDF PRPFWLALFVEGTRFTQAKLLAAQEYATSQGLPVPRNVLIPRTKGFVSAV SHMRSFVPAVYDMTVAIPKSSPSPTMLRLFKGQSSVVHVHVKRHLMKELP ETDEAVAQWCKDLFVEKDKLLDKHVAEDTFSDQPLQDIGRPVKPLLVVSS WACLVAYGALKFLQWSSLLSSWKGIAVSAVALAIVTILMQIMILFSQSER SIPTKVA SEQIDNO:40CcrLPAAT2a MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVFA ELLLLELLWLFHWRAGAKLKLFADPETFRLFGKEHALVICNHRTDLDWMV GWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKDKST LKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKLHVHIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFS GQEVHHIGRPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIG LGIVTLLVNILILSSQAERSNPAKVAPAKLKTELSPSKKVTNKEN SEQIDNO:41CcrLPAAT2b MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVFA ELLLLELLWLFHWRAGAKLKLFADPETFRLFGKEHALVICNHRTDLDWMV GWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKDKST LKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSMSHMRSFVPAVYDLTVAFPKTSPPPTLLKLFEGQSVVLHV HIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFSGQEVHHIG RPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIGLGIVTLLV NILILSSQAERSNPAKVAPAKLKTELSPSKKVTNKEN SEQIDNO:42BrLPAAT1a AAAVIVPLGILFFISGLVVNLLQAICYVLIRPLSKNTYRKINRVVAETLW LELVWIVDWWAGVKIQVFADNETFNRMGKEHALVVCNHRSDIDWLVGWIL AQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSG LQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLIPRT KGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKC HSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQNIGRPIK SLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIITLCMQILI RSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE SEQIDNO:43BrLPAAT1b AAAVIVPLGILFFISGLVVNLLQAVCYVLVRPMSKNTYRKINRVVAETLW LELVWIVDWWAGVKIQVFADDETFNRMGKEHALVVCNHRSDIDWLVGWIL AQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTLKSG LQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLIPRT KGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVHIKC HSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQNIGRPIK SLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIITLCMQILI RSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE SEQIDNO:44BrLPAAT1c MAIAAAVIVPLGLLFFISGLLMNLLQAICYVLVRPLSKNTYRKINRVVAE TLWLELVWIVDWWAGVKIKVFADNETFSRMGKEHALVVCNHRSDIDWLVG WILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDESTL KSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSELPVPRNVLI PRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFKGQPSVVHVH IKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFPGQQEQNIGR PIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALGLGIITLCMQ ILIRSSQSERSTPAKVVPAKPKDNHNDSGSSSQTE SEQIDNO:45BjLPAAT1a INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRIVIGKEHALVVCNH RSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSE LPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFK GQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFP GQKEQNIGRPIKSLAVSLIKTFPWLHPHQLTNIFVLFQVVVSWACLLTLG AMKFLHWSNLFSSWKGIALSAFGLGIITLCMQILIRSSQSERSTPAKVAP AKPK SEQIDNO:46BjLPAAT1b INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRIVIGKEHALVVCNH RSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSE LPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFK GQPSVVHVHIKCHSMKDLPEPEDEIAQWCRDQFVAKDALLDKHIAADTFP GQKEQNIGRPIKSLAVVVSWACLLTLGAMKFLHWSNLFSSWKGIALSAFG LGIITLCMQILIRSSQSERSTPAKVAPAKPK SEQIDNO:47BjLPAAT1c INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRIVIGKEHALVVCNH RSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSE LPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFK GQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFP GQQEQNIGRPIKSLAVVLSWSCLLILGAMKFLHWSNLFSSWKGIAFSALG LGIITLCMQILIRSSQSERSTPAKVVPAKPKDNHNDSGS5SQTE SEQIDNO:48BjLPAAT1d INLVVAETLWLELVWIVDWWAGVKIQVFADDETFNRIVIGKEHALVVCNH RSDIDWLVGWILAQRSGCLGSALAVMKKSSKFLPVIGWSMWFSEYLFLER NWAKDESTLKSGLQRLNDFPRPFWLALFVEGTRFTEAKLKAAQEYAASSE LPVPRNVLIPRTKGFVSAVSNMRSFVPAIYDMTVAIPKTSPPPTMLRLFK GQPSVVHVHIKCHSMKDLPESDDAIAQWCRDQFVAKDALLDKHIAADTFP GQQEQNIGRPIKSLAVSLS SEQIDNO:49CcLPAAT1a MAIGVAAIVVPLGLLFILSGLMVNLIQAICFILVRPLSKNMYRRVNRVVV ELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDWLV GWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLRRLKDFPRPFWLALFVEGTRFTQAKLLAAREYAASTGLPIPRNVL IPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQPSVVHV HIKRHSMNQLPQTDEGVGQWCKDIFVAKDALLDRHLAE SEQIDNO:50CcLPAAT1b MAIGVAAIVVPLGLLFILSGLMVNLIQAICFILVRPLSKNMYRRVNRVVV ELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDWLV GWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLRRLKDFPRPFWLALFVEGTRFTQAKLLAAREYAASTGLPIPRNVL IPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQPSVVHV HIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEKEFKRIR RPIKSLLVISSWSFLLMFGVFKFLKWSALLSTWKGVAVSTTVLLLVTVVM YMFILFSQSERSSPRKVAPSGPENG SEQIDNO:51UcLPAAT1a MAIGVAAIVVPLGLLFILSGLIINLIQAICFILVRPLSKNMYRKVNRVVV ELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDWLV GWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIPRNVL IPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQPSVVHV HIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEKEFKLIR RPIKSLLVISSWSFLLMFGVFKFLKWSALLSTWKGVAVSTAVLLLVTVVM YMFILFSQSERSSPRKVAPIGPENG SEQIDNO:52UcLPAAT1b MAIGVAAIVVPLGLLFILSGLIINLIQAICFILVRPLSKNMYRKVNRVVV ELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHRSDIDWLV GWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIPRNVL IPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQPSVVHV HIKRHSMNQLPQTDEGVAQWCKDIFVAKDALLDRHLAE SEQIDNO:53LdLPAAT1 SLLFFMSGLVVNFIQAVFYVLVRPISKNTYRRINTLVAELLWLELVWVID WWAGVKVQLYTDTESFRLMGKEHALLICNHRSDIDWLIGWVLAQRCGCLS SSIAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDENTLKSGLQRLNDFP KPFWLALFVEGTRFTKAKLLAAQEYAASAGLPVPRNVLIPRTKGFVSAVS NMRSFVPAIYDLTVAIPKTTEQPTMLRLFRGKSSVVHVHLKRHLMKDLPK TDDGVAQWCKDQFISKDALLDKHVAEDTFSGLEVQDIGRPMKSLVVVVSW MCLLCLGLVKFLQWSALLSSWKGMMITTFVLGIVTVLMHILIRSSQSEHS TPAK SEQIDNO:54CaequLPAAT1a QRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGL KRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTK GFVSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRH LMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKS LLVVISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILIL FSQSERSTPAKVAPAKPKKEGESSKTETEKEN SEQIDNO:55CaequLPAAT1b DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDY PLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKDLP ESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLV SEQIDNO:56CaequLPAAT1c DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDY PLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKDLP ESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLVVIS WAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSER STPAKVAPAKPKKEGESSKTETEKEN SEQIDNO:57CaequLPAAT1d QRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGL KRLKDYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTK GFVSSVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRH LMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKS LLV SEQIDNO:58CglutLPAAT1a LSLLFFVSGLFVNLVQAVCFVLIRPFSKNTYRRINRVVAELLWLELVWLI DWWAGVKIKVFTDHETLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCL GSTLAVIVIKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLKRLK DYPLPFWLALFVEGTRFTQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVS SVSHMRSFVPAIYDVTVAIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKD LPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPVKSLLVV ISWAVLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQS ERSTPAKVAPAKPKKEGESSKTETEKEN SEQIDNO:59CglutLPAAT1b QAVCFVLIRPFSKNTYRRINRVVAELLWLELVWLIDWWAGVKIKVFTDHE TLSLMGKEHALVISNHKSDIDWLVGWVLAQRSGCLGSTLAVMKKSSKFLP VIGWSMWFSEYLFLERSWAKDESTLKSGLKRLKDYPLPFWLALFVEGTRF TQAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSVSHMRSFVPAIYDVTV AIPKMSTPPTMLRIFKGQSSVLHVHLKRHLMKDLPESDDAVAQWCRDIFV EKDALLDKHNAEDTFSGQELQDIGRPVKSLLVVISWAVLVIFGAVKFLQW SSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSERSTPAKVAPAKPKKEG ESSKTETEKEN SEQIDNO:60CprLPAAT1 MAIAAAAVVFLFGLLFFTSGLIINLAQAVCFVLIWPLSKNAYRRINRVFA ELLLLELLWLFHWRAGAKLKLFADPETFRLFGKEHALVICNHRTDLDWMV GWVLGQHFGCLGSILSVAKKSTKFLPVLGWSMWFSEYLFLERSWAKDKST LKSHTERLKDYPLPFWLGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSMSHMRSFVPAVYDLTVAFPKTSPPPTLLKLFEGQSVVLHV HIKRYAMKDLPESDDAVAQWCRDIYVEKDAFLDKHNAEDTFSGQEVHHIG RPIKSLLVVISWVVVIIFGALKFLRWSSLLSSWKGKAFSVIGLGIVTLLV NILILSSQAERSNPAKVVPAKLKTELSPSKKVTNKEN SEQIDNO:61ChsLPAAT1 MAIPSAAVVFLFGLLFFASGLIINLVQAVCFVLIWPLSKNTCRRINIVFQ DMLLSELLWLFHWRAGAKLKFFTDPETYRHMGKEHALVITNHRTDLDWMI GWVLGEHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWFGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSVSHMRSFVPAVYETTMTFPKTSPPPTLLKLFEGQPLVLHI HMKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDTFGGLEVHIGR SIKSLMVVICWVVVIIFGALKFLQWSSLLSSWKGIAFIGIGLGIVNLLVH VLILSSQAERSAPTKVAPAKLKTKLLSSKKITNKEN SEQIDNO:62ChsLPAAT2 MAIPSAAVVFLFGLLFFASGLIINLVQAVCFVLIWPLSKNTCRRINIVFQ DMLLSELLWLFHWRAGAKLKFFTDPETYRHMGKEHALVITNHRTDLDWMI GWVLGEHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWFGIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHV HLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIG RPIKSLVVVISWAALVVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLM HILILFSQSERSTPAKVAPAKPKREGESSKTEMDKEN SEQIDNO:63CcalcLPAAT1a MAIPAAAVVFLFGLLFFPSGLIINLFQAVCFVLIWPFSRNTCRRINIVFQ EMLLSELLWLFHWRAGAKLKLFADPETYRHMGKEHALLITNHRTDLDWMI GWALGQHLGCLGSILSVVKKSTKFLPSHIERLEDFPQPFWMAIFVEGTRF TRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSCVSHMRSFVPAVYETTM TFPKTSPPPTLLKLFEGQPIVLHVHMKRHAMKDIPESDEAVAQWCRDKFV EKDSLLDKHNAGDTFSCQEIHIGRPIKSLMVVISWVVVIIFGALKFLQWS SLLSSWKGIAFSGIGLGIVTLLVHILILSSQAERSTPAKVAPAKLKTELS SSTKVTNKEN SEQIDNO:64CcalcLPAAT1b MAIPAAAVVFLFGLLFFPSGLIINLFQAVCFVLIWPFSRNTCRRINIVFQ EMLLSELLWLFHWRAGAKLKLFADPETYRHMGKEHALLITNHRTDLDWMI GWALGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPRTKGFVSCVSHMRSFVPAVYETTMTFPKTSPPPTLLKLFEGQPIVLHV HMKRHAMKDIPESDEAVAQWCRDKFVEKDSLLDKHNAGDTFSCQEIHIGR PIKSLMVVISWVVVIIFGALKFLQWSSLLSSWKGIAFSGIGLGIVTLLVH ILILSSQAERSTPAKVAPAKLKTELSSSTKVTNKEN SEQIDNO:65CcalcLPAAT2 LSLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLI DWWAGVKIKVFTDHETFRLMGTEHALVISNHKSDIDWLVGWVLAQRSGCL GSTLAVIVIKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLK DYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVS SVSHMRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHVHLKRHLMKD LPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDIGRPIKSLVVV ISWAALVVFGAVKFLQWSSLLSSWKGLAFSGIALGIITLLMHILILFSQS ERSTPAKVAPAKPKKEGESSKTETDKEN SEQIDNO:66ChtLPAAT1a MAIPAAAVIFLFSILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVFQ EMLLSELLGLFHWRAGAKLKLYTDPETYPLLGKEHALLMINHRTDLDWMI GWVLGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKGFVSTVSHMRSFVPAVYDTTLTFPKTSPPPTLLNLFAGQPIVLHI HIKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDAFSDQEFPISR SIKSLMVVISWVMVIIFGALKFLQWSSLLSSWKGKAFSVIAVGIVTLLMH MSILSSQAERSNPAKVALPKLKTELPSSKKVLNKEN SEQIDNO:67ChtLPAAT1b MAIPAAAVIFLFSILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVFQ EMLLSELLGLFHWRAGAKLKLYTDPETYPLLGKEHALLMINHRTDLDWMI GWVLGQHLGCLGSILSVVKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKGFVSTVSHMRSFVPAVYDTTLTFPKTSPPPTLLNLFAGQPIVLHI HIKRHAMKDIPESDDAVAQWCRDKFVEKDALLDKHNAEDAFSDQEFPISR SIKSLMVVISWVMVIIFGALKFLQWSSLLSSWKGIAFSGIGLGIVTLLMH ILILSSQAERSTPAKVAQAKVKTELPSSTKVTNKGN SEQIDNO:68CwLPAAT1 MAIPAAAVIFLFGILFFASGLIINLVQAVCFVLIWPLSKNTCRRINLVFQ EMLLSELLWLFHWRAGAELKLFTDPETYRLLGKEHALVMTNHRTDLDWMI GWVTGQHLGCLGSILSIAKKSTKFLPVLGWSMWFSEYLFLERNWAKDKST FKSHIERLEDFPQPFWMAIFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSVCHMRSFVPAVYDTTLTFPKNSPPPTLLNLFAGQPIVLHI HIKRHAMKDMPKSDDAVAQWCRDKFVKKDALLDKHNTEDTFSDQEFPIGR PIKSLMVVISWVVVIIFGTLKFLQWSSLLSSWKGIAFSGIGLGIVTLLVH ILILSSQAERSTPPKVAPAKLKTELSSTTKVINKGN SEQIDNO:69CwLPAAT2b LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRLNRVVAELLWLELVWLI DWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SHMIRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVDALLDKHNADDTF SGQELHDIGRPIKSLLVVISWAVLVVFGAVKFLQWSSLLSSWKGIAFSGI GLGIVTLLVHILILSSQAERSTSAKVAQAKVKTELSSSKKVKNKGN SEQIDNO:70CwLPAAT2a LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRLNRVVAELLWLELVWLI DWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDESTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVLIPRTKGFVSSV SHMIRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHVHLKRHLMKDL PESDDAVAQWCRDIFVEKDVLLDKHNAEDTFSGQELQDIGRPVKSLLVVI SWTLLVIFGAVKFLQWSSLLSSWKGLAFSGIGLGIVTLLMHILILFSQSE RSTPAKVAPAKPKKEGESSKMETDKEN SEQIDNO:71CgLPAAT1a LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDES TLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASLGLPVPRNV LIPRTKGFVSSVSHMIRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSSVL HVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQD TGRPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITL LMHILILFSQSERSTPAKVAPAKPKNEGESSKAEMEKEK SEQIDNO:72CgLPAAT1b LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDES TLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASLGLPVPRNV LIPRTKGFVSSVSHMIRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSSVL HVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQD TGRPIKSLLVRCFLVLSLIYLNGIMLKLRGPCLQVVISWAVLEVFGAVKF LQWSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKPK NEGESSKAEMEKEK SEQIDNO:73CgLPAAT1c LAGWMGSSSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDES TLKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASLGLPVPRNV LIPRTKGFVSSVSHMIRSFVPAIYDVTVAIPKTSPPPTMIRMFKGQSSVL HVHLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQD TGRPIKSLLVVTSWAVLVISGAVKFLQWSSLLSSWKGLAFSGIGLGIVTL LMHILILFSQSERSTPAKVAPAKPKKEGESSKTEKDKEN SEQIDNO:74CpalLPAAT1 LGLLFFVSGLIVNLVQAVCFVLIRPLSKNTYRRINRVVAELLWLELVWLI DWWAGVKIKVFTDHETLSLMGKEHALVICNHKSDIDWLVGWVLAQRSGCL GSTLAVMKKSSKFLPVIGWSMWFSEYLFLERSWAKDENTLKSGLNRLKDY PLPFWLALFVEGTRFTRAKLLAAQQYATSSGLPVPRNVLIPRTKGFVSSV SHMIRSFVPAIYDVTVAIPKTSPPPTMLRMFKGQSSVLHVHLKRHLMKDL PESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTGRPIKSLLVVI SWAVLVIFGAVKFLQWSSLLSSWKGLAFSGVGLGIITLLMHILILFSQSE RSTPAKVAPAKPKKDGESSKTEIEKEN SEQIDNO:75CaLPAAT1 MAIAAAAVIVPVSLLFFVSGLIVNLVQAVCFVLIRPLFKNTYRRINRVVA ELLWLELVWLIDWWAGVKIKVFTDHETFHLMGKEHALVICNHKSDIDWLV GWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYLFLERNWAKDEST LKSGLNRLKDYPLPFWLALFVEGTRFTRAKLLAAQQYAASSGLPVPRNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLLRMFKGQSSVLHV HLKRHQMNDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLIVISWAVLVVFGAVKFLQWSSLLSSWKGLAFSGIGLGVITLLM HILILFSQSERSTPAKVAPAKPKIEGESSKTEMEKEH SEQIDNO:76CaLPAAT3 MTIASAAVVFLFGILLFTSGLIINLFQAFCSVLVWPLSKNAYRRINRVFA EFLPLEFLWLFHWWAGAKLKLFTDPETFRLMGKEHALVIINHKIELDWMV GWVLGQHLGCLGSILSVAKKSTKFLPVFGWSLWFSEYLFLERNWAKDKKT LKSHIERLKDYPLPFWLIIFVEGTRFTRTKLLAAQQYAASAGLPVPRNVL IPHTKGFVSSVSHMRSFVPAIYDVTVAFPKTSPPPTMLKLFEGHFVELHV HIKRHAMKDLPESEDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHHVG RPIKSLLVVISWVVVIIFGALKFLQWSSLLSSWKGIAFSVIGLGTVALLM QILILSSQAERSIPAKETPANLKTELSSSKKVTNKEN SEQIDNO:77SalLPAAT1 MAIGAAAIVVPLGLLFMLSGLMVNLIQAICFILVRPLSKNMYRRVNRVVV ELLWLELIWLIDWWGGVKVDVYADSETFQSLGKEHALVVSNHKSDIDWLV GWVLAQRSGCLGSTLAVMKKSSKFLPVIGWSMWFSEYVFLERSWAKDEST LKSGLQRLKDFPRPFWLALFVEGTRFTQAKLLAAQEYAASTGLPIPRNVL IPRTKGFVSAVSNMRSFVPAIYDVTVAIPKTQPSPTMLRIFNRQPSVVHV RIKRHSMNQLPPTDEGVAQWCKDIFVAKDALLDRHLAEGKFDEKEFKRIR RPIKSLLVISSWSFLLLFGVFKFLKWSALLSTWKGVAVSTAVLLLVTVVM YMFILFSQSERSSPRKVAPSGPENG SEQIDNO:78CleptLPAAT1 MAIPAAVVIFLFGLLFFSSGLIINLFQALCFVLIWPLSKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSCVNHMRSFVPAVYDLTVAFPKTSPPPTLLNLFEGQSVVLHV HIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSSQEVHHTG SRPIKSLLVVISWVVVITFGALKFLQWSSWKGKAFSVIGLGIVTLLMHML ILSSQAERSKPAKVTQAKLKTELSISKKVTDKEN SEQIDNO:79ClopLPAAT1 MAIAAAAVIFLFGLLFFASGLIINLFQALCFVLIRPLSKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETLRLMGKEHALIIINHMTELDWMV GWVMGQHFGCLGSIISVAKKSTKFLPVLGWSMWFSEYLYLERSWAKDKST LKSHIERLKDYPLPFWLVIFVEGTRFTRTKLLAAQEYAASSGLPVPRNVL IPRTKGFVSCVNHMRSFVPAVYDVTVAFPKTSPQPTLLNLFEGRSIVLHV HIKRHAMKDLPESDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHHTG RRPIKSLLVVMSWVVVTTFGALKFLQWSSWKGKAFSVIGLGIVTLLMHVL ILSSQAERSNPAKVVQAELNTELSISKKVTNKGN SEQIDNO:80CcrasLPAAT1a MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHV HLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLM HILILFSQSERSTPAKVAPAKAK SEQIDNO:81CcrasLPAAT1b MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHV HLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVRCFLVLSLIYLNGIILKLCGLCLQVVISWAVLEVFGAVKFLQ WSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKAK SEQIDNO:82CcrasLPAAT1c MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHV HLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVVISWAVLEVFGAVKFLQWSSLLSSWKGLAFSGIGLGIITLLM HILILFSQSERSTPAKVAPAKAKMEGESSKTEMEMEK SEQIDNO:83CcrasLPAAT1d MAIPAAAVIFLFGLIFFASGLIINLFQALCFVLIWPLWKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVIINHMTELDWMV GWVMGQHFGCLGSILSVAKKSTKFLPVLGWSMWFTEYLYIERSWDKDKST LKSHIERLKDYPLPFWLVIFAEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPRTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHV HLKRHVMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQDTG RPIKSLLVRCFLVLSLIYLNGIILKLCGLCLQVVISWAVLEVFGAVKFLQ WSSLLSSWKGLAFSGIGLGIITLLMHILILFSQSERSTPAKVAPAKAKME GESSKTEMEMEK SEQIDNO:84CkoeLPAAT1 MAIAAAPVIFLFGLLFFASGLIINLFQAICFVLIWPLSKNAYRRINRVFA ELLLSELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVITNHKIDLDWMI GWILGQHFGCLGSVISIAKKSTKFLPIFGWSLWFSEYLFLERNWAKDKRT LKSHIERMKDYPLPLWLILFVEGTRFTRTKLLAAQQYAASSGLPVPRNVL IPHTKGFVSSVSHMRSFVPAIYDVTVAIPKTSPPPTLIRMFKGQSSVLHV HLKRHLMKDLPESDDAVAQWCRDIFVEKDALLDKHNAEDTFSGQELQETG RPIKSLLVVISWAVLEVYGAVKFLQWSSLLSSWKGLAFSGIGLGLITLLM HILILFSQSERSTPAKVAPAKPKKEGESSKTEMEKEK SEQIDNO:85CkoeLPAAT2 MHVLLEMVTFRFSSFFVFDNVQALCFVLIWPLSKSAYRKINRVFAELLLS ELLCLFDWWAGAKLKLFTDPETFRLMGKEHALVITNHKIDLDWMIGWILG QHFGCLGSVISIAKKSTKFLPIFGWSLWFSEYLFLERNWAKDKRTLKSHI ERMKDYPLPLWLILFVEGTRFTRTKLLAAQQYAASSGLPVPRNVLIPHTK GFVSSVSHMRSFVPAVYDVTVAFPKTSPPPTMLSLFEGQSVVLHVHIKRH AMKDLPDSDDAVAQWCRDKFVEKDALLDKHNAEDTFSGQEVHHVGRPIKS LLVVISWMVVIIFGALKFLQWSSLLSSWKGKAFSAIGLGIATLLMHVLVV FSQADRSNPAKVPPAKLNTELSSSKKVTNKEN

Example 5: Expression of LPAATs to Improve Sn-2 Selectivity in Prototheca Moriformis

[0132] In the example we disclose genetically engineered Prototheca moriformis strains in which we have modified fatty acid and triacylglycerol biosynthesis to maximize the accumulation of Stearoyl-Oleoyl-Stearoyl (SOS) TAGs, and minimize the production of trisaturated TAGs. Oils from these strains resemble plant seed oils known as structuring fats, which have high proportions of Saturated-Oleate-Saturated TAGs and low levels of trisaturates. These structuring fats (often called butters) are generally solid at room temperature but melt sharply between 35-40 C.

[0133] Strains with high SOS and low trisaturates were obtained by three successive transformations, beginning with S5100, a classically improved derivative of S376 (improved to increase lipid titer), a wild type isolate of Prototheca moriformis. S5100 was transformed with a construct to which increased expression of PmKASII-1 and ablated the SAD2-1 allele. The resultant strain, S5780, produced oil with increased C18:0 and lower C16:0 content relative to S5100. S5780 was prepared according to the methods disclosed in co-owned application WO2013/158938 and as described below. C18:0 levels were increased further by transformation of S5780 with a construct overexpressing the C18:0-specific FATA1 thioesterase gene from Garcinia mangostana (GarmFATA1), generating strain S6573. S6573 was disclosed in co-owned application WO2015/051319. Finally, accumulation of trisaturated TAGs was reduced by expression of genes encoding LPAATs from Brassica napus, Theobroma cacao, Garcinia hombororiana or Garcinia indica in S6573 as described below.

Construct Used for SAD2 Knockout and PmKASII-1 Overexpression in S5100 to Produce S5780

[0134] The sequence of the transforming DNA from the SAD2-1 ablation, PmKASII over-expression construct, pSZ2624, is shown below. The construct is written as: pSZ2624:SAD2-1vD::PmKASII-1tp_PmKASII-1_FLAG-CvNR:CpACT-AtTHIC-CpEF1a::SAD2-1vE Relevant restriction sites are indicated in lowercase, bold, and are from 5-3 PmeI, SpeI, AscI, ClaI, SacI, AvrII, EcoRV, AflII, KpnI, XbaI, MfeI, BamHI, BspQI and PmeI. Underlined sequences at the 5 and 3 flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the SAD2-1 locus. The SAD2-1 5 integration flank contained the endogeneous SAD2-1 promoter, enabling the in situ activation of the PmKASII gene. Proceeding in the 5 to 3 direction, the region encoding the PmKASII plastid targeting sequence is indicated by lowercase, underlined italics. The sequence that encodes the mature PmKASII polypeptide is indicated with lowercase italics, while a 3FLAG epitope encoding sequence is in bold italics. The initiator ATG and terminator TGA for PmKASII-FLAG are indicated by uppercase italics. The 3 UTR of the Chlorella vulgaris nitrate reductase (CvNR) gene is indicated by small capitals. Two spacer regions are represented by lowercase text. The CpACT promoter driving the expression of the AtTHIC gene (encoding 4-amino-5-hydroxymethyl-2-methylpyrimidine synthase activity, thereby permitting the strain to grow in the absence of exogeneous thiamine) is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3 UTR of the Chlorella protothecoides EF1a (CpEF1a) gene is indicated by small capitals. The use of THIC as a selection marker was described in co-owned applications WO2011/150410 and WO2013/150411.

TABLE-US-00019 pSZ2624NucleotidesequenceofthetransformingDNA SEQIDNO:86 gtttaaacGCCGGTCACCACCCGCATGCTCGTACTACAGCGCACGCACCGCTTCGTGA TCCACCGGGTGAACGTAGTCCTCGACGGAAACATCTGGTTCGGGCCTCCTGCTTG CACTCCCGCCCATGCCGACAACCTTTCTGCTGTTACCACGACCCACAATGCAACG CGACACGACCGTGTGGGACTGATCGGTTCACTGCACCTGCATGCAATTGTCACAA GCGCTTACTCCAATTGTATTCGTTTGTTTTCTGGGAGCAGTTGCTCGACCGCCCGC GTCCCGCAGGCAGCGATGACGTGTGCGTGGCCTGGGTGTTTCGTCGAAAGGCCA GCAACCCTAAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGTTTGGACC AGATCCGCCCCGATGCGGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCT TTCGTAAATGCCAGATTGGTGTCCGATACCTGGATTTGCCATCAGCGAAACAAGA CTTCAGCAGCGAGCGTATTTGGCGGGCGTGCTACCAGGGTTGCATACATTGCCCA TTTCTGTCTGGACCGCTTTACTGGCGCAGAGGGTGAGTTGATGGGGTTGGCAGGC ATCGAAACGCGCGTGCATGGTGTGCGTGTCTGTTTTCGGCTGCACGAATTCAATA GTCGGATGGGCGACGGTAGAATTGGGTGTGGCGCTCGCGTGCATGCCTCGCCCC GTCGGGTGTCATGACCGGGACTGGAATCCCCCCTCGCGACCATCTTGCTAACGCT CCCGACTCTCCCGACCGCGCGCAGGATAGACTCTTGTTCAACCAATCGACAactagt ATGcagaccgcccaccagcgcccccccaccgagggccactgcttcggcgcccgcctgcccaccgcctcccgccgcgccgtgc gccgcgcctggtcccgcatcgcccgcgggcgcgccgccgccgccgccgacgccaaccccgcccgccccgagcgccgcgtggt gatcaccggccagggcgtggtgacctccctgggccagaccatcgagcagttctactcctccctgctggagggcgtgtccggcatct cccagatccagaagttcgacaccaccggctacaccaccaccatcgccggcgagatcaagtccctgcagctggacccctacgtgc ccaagcgctgggccaagcgcgtggacgacgtgatcaagtacgtgtacatcgccggcaagcaggccctggagtccgccggcctg cccatcgaggccgccggcctggccggcgccggcctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggcat gacctccttcgccgccggcgtggaggccctgacccgcggcggcgtgcgcaagatgaaccccttctgcatccccttctccatctcca acatgggcggcgccatgctggccatggacatcggcttcatgggccccaactactccatctccaccgcctgcgccaccggcaacta ctgcatcctgggcgccgccgaccacatccgccgcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcc cctccggcatcggcggcttcatcgcctgcaaggccctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggac gccgaccgcgacggcttcgtgatgggcgagggcgccggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcg ccaccatcctggccgagctggtgggcggcgccgccacctccgacgcccaccacatgaccgagcccgacccccagggccgcgg cgtgcgcctgtgcctggagcgcgccctggagcgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacct ccacccccgccggcgacgtggccgagtaccgcgccatccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagt ccatgatcggccacctgctgggcggcgccggcgccgtggaggccgtggccgccatccaggccctgcgcaccggctggctgcac cccaacctgaacctggagaaccccgcccccggcgtggaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacc tggacgtggtgctgtccaactccttcggcttcggcggccacaactcctgcgtgatatccgcaagtacgacgag TGAatcgatAGATCTCTT AAGGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGAT GGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTAT CAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCT GCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGC TTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCT GCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCT GGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGG ATGGGAACACAAATGGAAAGCTTAATTAAgagctccgcgtctcgaacagagcgcgcagaggaacgct gaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagc gtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctaggtg atatccatcttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgta agtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagatac agcgcgagccagacacggagtgccgagctatgcgcacgctccaactaggtaccagtttaggtccagcgtccgtggggggggacg ggctgggagcttgggccgggaagggcaagacgatgcagtccctctggggagtcacagccgactgtgtgtgttgcactgtgcggccc gcagcactcacacgcaaaatgcctggccgacaggcaggccctgtccagtgcaacatccacggtccctctcatcaggctcaccttgct cattgacataacggaatgcgtaccgctattcagatctgtccatccagagaggggagcaggctccccaccgacgctgtcaaacttgctt cctgcccaaccgaaaacattattgtttgagggggggggggggggggcagattgcatggcgggatatctcgtgaggaacatcactgg gacactgtggaacacagtgagtgcagtatgcagagcatgtatgctaggggtcagcgcaggaagggggcctttcccagtctcccatgc cactgcaccgtatccacgactcaccaggaccagcttcttgatcggcttccgctcccgtggacaccagtgtgtagcctctggactccagg tatgcgtgcaccgcaaaggccagccgatcgtgccgattcctgggtggaggatatgagtcagccaacttggggctcagagtgcacact ggggcacgatacgaaacaacatctacaccgtgtcctccatgctgacacaccacagcttcgctccacctgaatgtgggcgcatgggcc cgaatcacagccaatgtcgctgctgccataatgtgatccagaccctctccgcccagatgccgagcggatcgtgggcgctgaatagatt cctgtttcgatcactgtttgggtcctttccttttcgtctcggatgcgcgtctcgaaacaggctgcgtcgggctttcggatcccttttgctccct ccgtcaccatcctgcgcgcgggcaagttgcttgaccctgggctgataccagggttggagggtattaccgcgtcaggccattcccagcc cggattcaattcaaagtctgggccaccaccctccgccgctctgtctgatcactccacattcgtgcatacactacgttcaagtcctgatcca ggcgtgtctegggacaaggtgtgatgagtttgaatctcaaggacccactccagcacagctgctggttgaccccgccctcgcaatcta gaATGgccgcgtccgtccactgcaccctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaac tcctccctgctgcccggcttcgacgtggtggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacg ctgacgttcgacccccccacgaccaactccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttcca gcccatcccctccttcgaggagtgcttccccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcct gaaggtgcccttccgccgcgtgcacctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaa cgcccacatcggcctggcgaagctgcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatg tactacgcgaagcagggcatcatcacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctc cgaggtcgcgcggggccgcgccatcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcc tggtgaaggtgaacgcgaacatcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccacc atgtggggcgccgacaccatcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgc ggtccccgtgggcaccgtccccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttcc gcgagacgctgatcgagcaggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctga ccgccaagcgcctgacgggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcg cctacgagcactgggacgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggct ccatctacgacgccaacgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaagga cgtgcaggtgatgaacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgc aacgaggcgcccttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcg gccaacatcggcgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgt gaaggcgggcgtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtggga cgacgcgctgtccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttcc acgacgagacgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatca cggaggacatccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgt ccgaggagttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtc ctacgtcaaggccgcgcagaagTGAcaattgACGGAGCGTCGTGCGGGAGGGAGTGTGCCGAG CGGGGAGTCCCGGTCTGTGCGAGGCCCGGCAGCTGACGCTGGCGAGCCGTACGC CCCGAGGGTCCCCCTCCCCTGCACCCTCTTCCCCTTCCCTCTGACGGCCGCGCCTG TTCTTGCATGTTCAGCGACggatccTAGGGAGCGACGAGTGTGCGTGCGGGGCTGGC GGGAGTGGGACGCCCTCCTCGCTCCTCTCTGTTCTGAACGGAACAATCGGCCACC CCGCGCTACGCGCCACGCATCGAGCAACGAAGAAAACCCCCCGATGATAGGTTG CGGTGGCTGCCGGGATATAGATCCGGCCGCACATCAAAGGGCCCCTCCGCCAGA GAAGAAGCTCCTTTCCCAGCAGACTCCTTCTGCTGCCAAAACACTTCTCTGTCCA CAGCAACACCAAAGGATGAACAGATCAACTTGCGTCTCCGCGTAGCTTCCTCGG CTAGCGTGCTTGCAACAGGTCCCTGCACTATTATCTTCCTGCTTTCCTCTGAATTA TGCGGCAGGCGAGCGCTCGCTCTGGCGAGCGCTCCTTCGCGCCGCCCTCGCTGAT CGAGTGTACAGTCAATGAATGGTCCTGGGCGAAGAACGAGGGAATTTGTGGGTA AAACAAGCATCGTCTCTCAGGCCCCGGCGCAGTGGCCGTTAAAGTCCAAGACCG TGACCAGGCAGCGCAGCGCGTCCGTGTGCGGGCCCTGCCTGGCGGCTCGGCGTG CCAGGCTCGAGAGCAGCTCCCTCAGGTCGCCTTGGACGGCCTCTGCGAGGCCGG TGAGGGCCTGCAGGAGCGCCTCGAGCGTGGCAGTGGCGGTCGTATCCGGGTCGC CGGTCACCGCCTGCGACTCGCCATCCgaagagcgtttaaac

[0135] Construct D1683 (pSZ2624), was transformed into S5100. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. Integration of pSZ2624 at the SAD2-1 locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 8). Simultaneous ablation of SAD2-1 and over-expression of PmKASII (driven in situ by the SAD2-1 promoter) resulted in C18:0 levels up to 26.1%. C16:0 accumulation was reduced from 15.3% in S5100 to 6% the strains derived from D1683, demonstrating that PmKASII-1 over-expression promoted the elongation of C16:0 to C18:0. S5780 was chosen for further development as it had the highest lipid titer relative to the S5100 parent.

TABLE-US-00020 TABLE 8 Fatty acid profiles of SAD2-1 ablation, PmKASII-1 overexpression strains derived from D1683-1, compared to the S5100 parent. Primary S5100; T531; D1683.1 Strain S5100 S5780 S5781 S5782 S5783 S5784 Fatty Acid C14:0 0.7 0.7 0.8 0.7 0.7 0.7 Area % C16:0 15.3 5.9 6.0 6.0 5.8 5.8 C16:1 0.5 0.1 0.0 0.1 0.0 0.0 C18:0 4.0 25.6 26.1 26.0 25.0 25.3 C18:1 55.7 54.5 54.6 56.3 55.6 C18:2 7.3 8.0 8.5 8.5 8.1 8.4 C18:3 0.5 0.7 0.8 0.8 0.7 0.7 C20:0 0.3 1.8 1.9 1.8 1.8 1.8 C20:1 0.2 0.6 0.6 0.6 0.7 0.7 C22:0 0.1 0.2 0.3 0.3 0.3 0.2 C24:0 0.1 0.4 0.4 0.4 0.4 0.4 saturates 20.6 34.7 35.6 35.4 34.1 34.5

[0136] We disclose additional methods of elevating C18:0 levels that can be used in conjunction with SAD2 knockout and KASII over-expression. Previously we described acyl-ACP thioesterases from Brassica napus (BnFATA) (Co-owned application WO2012/106560), Garcinia mangostana (GarmFATA1) (Co-owned application WO2015/051319) and Theobroma cacao (TcFATA) (Co-owned application WO2013/158938) with specificity towards cleavage of C18:0-ACP, and we observed that average C18:0 levels were higher in strains in which we replaced the native BnFATA transit peptide with the Chlorella protothecoides SAD1 transit peptide (CpSAD1tp). A DNA construct was made for expression of a chimeric gene encoding CpSAD1tp fused to the predicted GarmFATA1 mature polypeptide and a FLAG tag sequence.

[0137] The sequence of the transforming DNA from the GarmFATA1 expression construct pSZ3204 is shown below. The construct is written as pSZ3204:6SA::CrTUB2-ScSUC2-CvNR:PmSAD2-2-CpSAD1_tp_GarmFATA1_FLAG-CvNR::6SB. Relevant restriction sites are indicated in lowercase, bold, and are from 5-3 BspQI, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, SpeI, AscI, ClaI, AflII, SacI and BspQI. Underlined sequences at the 5 and 3 flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the 6S locus. Proceeding in the 5 to 3 direction, the CrTUB2 promoter driving the expression of Saccharomyces cerevisiae SUC2 (ScSUC2) gene, enabling strains to utilize exogeneous sucrose, is indicated by lowercase, boxed text. The initiator ATG and terminator TGA of ScSUC2 are indicated by uppercase italics, while the coding region is represented by lowercase italics. The 3 UTR of the CvNR gene is indicated by small capitals. A spacer region is represented by lowercase text. The P. moriformis SAD2-2 (PmSAD2-2) promoter driving the expression of the chimeric CpSAD1tp_GarmFATA1_FLAG gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding CpSAD1tp is represented by lowercase, underlined italics; the sequence encoding the GarmFATA1 mature polypeptide is indicated by lowercase italics; and the 3 FLAG epitope tag is represented by uppercase, bold italics. A second CvNR 3 UTR is indicated by small capitals.

TABLE-US-00021 pSZ3204 SEQIDNO:87 gctcttcGCCGCCGCCACTCCTGCTCGAGCGCGCCCGCGCGTGCGCCGCCAGCGCCTT GGCCTTTTCGCCGCGCTCGTGCGCGTCGCTGATGTCCATCACCAGGTCCATGAGG TCTGCCTTGCGCCGGCTGAGCCACTGCTTCGTCCGGGCGGCCAAGAGGAGCATG AGGGAGGACTCCTGGTCCAGGGTCCTGACGTGGTCGCGGCTCTGGGAGCGGGCC AGCATCATCTGGCTCTGCCGCACCGAGGCCGCCTCCAACTGGTCCTCCAGCAGCC GCAGTCGCCGCCGACCCTGGCAGAGGAAGACAGGTGAGGGGGGTATGAATTGTA CAGAACAACCACGAGCCTTGTCTAGGCAGAATCCCTACCAGTCATGGCTTTACCT GGATGACGGCCTGCGAACAGCTGTCCAGCGACCCTCGCTGCCGCCGCTTCTCCCG CACGCTTCTTTCCAGCACCGTGATGGCGCGAGCCAGCGCCGCACGCTGGCGCTGC GCTTCGCCGATCTGAGGACAGTCGGGGAACTCTGATCAGTCTAAACCCCCTTGCG CGTTAGTGTTGCCATCCTTTGCAGACCGGTGAGAGCCGACTTGTTGTGCGCCACC CCCCACACCACCTCCTCCCAGACCAATTCTGTCACCTTTTTGGCGAAGGCATCGG CCTCGGCCTGCAGAGAGGACAGCAGTGCCCAGCCGCTGGGGGTTGGCGGATGCA CGCTCAggtaccctttcttgcgctatgacacttccagcaaaaggtagggegggctgcgagacggcttcceggcgctgcatgcaa caccgatgatgcttcgaccccccgaagctccttcggggctgcatgggcgctccgatgccgctccagggcgagcgctgtttaaatagc caggcccccgattgcaaagacattatagcgagctaccaaagccatattcaaacacctagatcactaccacttctacacaggccactcga gettgtgatcgcactccgctaagggggcgcctatcctcttcgtttcagtcacaacccgcaaactctagaatatcaATGctgctgcag gccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcactt cacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagt acaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccag cccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttctt caacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcct acagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccg aaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccg acgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcga ggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttc aaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggact actacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactc cgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccgg agacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacac cacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaa caccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgc atgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcac caaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccaga acatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgt gaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCAGCA GCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGC CGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCT CAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTA TTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCA ACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTC ACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAA CCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACAC AAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcata caccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtgg caggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctagggatatcctgaagaatgggaggcaggtgttgttgattat gagtgtgtaaaagaaaggggtagagagccgtcctcagatccgactactatgcaggtagccgctcgcccatgcccgcctggctgaata ttgatgcatgcccatcaaggcaggcaggcatttctgtgcacgcaccaagcccacaatcttccacaacacacagcatgtaccaacgcac gcgtaaaagttggggtgctgccagtgcgtcatgccaggcatgatgtgctcctgcacatccgccatgatctcctccatcgtctcgggtgtt tccggcgcctggtccgggagccgttccgccagatacccagacgccacctccgacctcacggggtacttttcgagcgtctgccggtag tcgacgatcgcgtccaccatggagtagccgaggcgccggaactggcgtgacggagggaggagagggaggagagagagggggg ggggggggggggatgattacacgccagtctcacaacgcatgcaagacccgtttgattatgagtacaatcatgcactactagatggatg agcgccaggcataaggcacaccgacgttgatggcatgagcaactcccgcatcatatttcctattgtcctcacgccaagccggtcaccat ccgcatgctcatattacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacggaaacatctggctcgggcctcgt gctggcactccctcccatgccgacaacctttctgctgtcaccacgacccacgatgcaacgcgacacgacccggtgggactgatcggtt cactgcacctgcatgcaattgtcacaagcgcatactccaatcgtatccgtttgatttctgtgaaaactcgctcgaccgcccgcgtcccgc aggcagcgatgacgtgtgcgtgacctgggtgtttcgtcgaaaggccagcaaccccaaatcgcaggcgatccggagattgggatctg atccgagcttggaccagatcccccacgatgcggcacgggaactgcatcgactcggcgcggaacccagctttcgtaaatgccagattg gtgtccgataccttgatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgctaccagggttgcatacattgc ccatttctgtctggaccgctttaccggcgcagagggtgagttgatggggttggcaggcatcgaaacgcgcgtgcatggtgtgtgtgtct gttttcggctgcacaatttcaatagtcggatgggcgacggtagaattgggtgttgcgctcgcgtgcatgcctcgccccgtcgggtgtcat gaccgggactggaatcccccctcgcgaccctcctgctaacgctcccgactctcccgcccgcgcgcaggatagactctagttcaacca atcgacaactagtATGgccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccggg ccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaagg tgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgt cctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgc aggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctccaccacccccaccatgcgcaagctgcgcc tgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggcca gggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctcc aagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgccc ccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactc caagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctgg agtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacga cgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgcca acgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggcc gcaccgagtggcgcaagaagcccacccgc TGAatcgatagatctcttaagGCAGCAG CAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCC GCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTC AGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTAT TTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAA CCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCA CTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAAC CTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACA AATGGAaagcttaattaagagctcTTGTTTTCCAGAAGGAGTTGCTCCTTGAGCCTTTCATTC TCAGCCTCGATAACCTCCAAAGCCGCTCTAATTGTGGAGGGGGTTCGAATTTAAA AGCTTGGAATGTTGGTTCGTGCGTCTGGAACAAGCCCAGACTTGTTGCTCACTGG GAAAAGGACCATCAGCTCCAAAAAACTTGCCGCTCAAACCGCGTACCTCTGCTTT CGCGCAATCTGCCCTGTTGAAATCGCCACCACATTCATATTGTGACGCTTGAGCA GTCTGTAATTGCCTCAGAATGTGGAATCATCTGCCCCCTGTGCGAGCCCATGCCA GGCATGTCGCGGGCGAGGACACCCGCCACTCGTACAGCAGACCATTATGCTACC TCACAATAGTTCATAACAGTGACCATATTTCTCGAAGCTCCCCAACGAGCACCTC CATGCTCTGAGTGGCCACCCCCCGGCCCTGGTGCTTGCGGAGGGCAGGTCAACC GGCATGGGGCTACCGAAATCCCCGACCGGATCCCACCACCCCCGCGATGGGAAG AATCTCTCCCCGGGATGTGGGCCCACCACCAGCACAACCTGCTGGCCCAGGCGA GCGTCAAACCATACCACACAAATATCCTTGGCATCGGCCCTGAATTCCTTCTGCC GCTCTGCTACCCGGTGCTTCTGTCCGAAGCAGGGGTTGCTAGGGATCGCTCCGAG TCCGCAAACCCTTGTCGCGTGGCGGGGCTTGTTCGAGCTTgaagagc

[0138] Construct D1940 (pSZ3204), was transformed into the S5780 parent strain. Primary transformants were clonally purified and grown under standard lipid production conditions at pH 5. Integration of pSZ3204 at the 6S locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 9). Over-expression of GarmFATA1 (driven by the SAD2-2 promoter) resulted in C18:0 levels up to 54.3%. C16:0 levels were comparable in strains derived from D1940 and the S5780 parent. S6573 was chosen for further development as it had the highest lipid titer of the strains with >50% C18:0.

TABLE-US-00022 TABLE 9 Fatty acid profiles of GarmFATA1 overexpressing stable strains derived from D1940 primary transformants. Primary D1683.1 D1940.19 D1940.20 D1940.23 D1940.46 D1940.5 Strain S5100 S5780 S6571 S6572 S6573 S6574 S6575 S6578 S6580 Fatty Acid C14:0 0.7 0.0 0.8 0.0 0.8 0.7 0.7 0.0 0.0 Area % C16:0 18.0 5.9 6.3 6.6 6.3 5.0 5.1 5.0 5.3 C16:1 0.5 0.0 0.1 0.1 0.1 0.0 0.1 0.1 0.1 C18:0 3.9 29.0 52.7 54.3 53.7 43.1 46.0 45.4 47.9 C18:1 69.8 54.3 31.4 30.1 30.5 41.5 38.5 40.0 37.2 C18:2 5.9 6.4 5.7 5.8 5.6 6.3 6.2 6.1 6.2 C18:3 0.5 0.7 0.6 0.6 0.6 0.6 0.5 0.6 0.5 C20:0 0.3 2.4 1.8 1.6 1.7 2.1 2.0 2.0 2.0 C20:1 0.1 0.6 0.1 0.1 0.1 0.2 0.1 0.1 0.1 C22:0 0.1 0.3 0.2 0.2 0.2 0.3 0.3 0.2 0.2 C24:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 saturates 23.1 37.7 61.9 62.8 62.8 51.2 54.2 52.7 55.5

[0139] Lysophosphatidic acid acetyltransferase (LPAAT) enzymes are responsible for the transfer of acyl groups to the sn-2 position on the glycerol backbone. We disclose here that we can reduce the accumulation of excessive amounts of trisaturates in our high SOS strains by expressing heterologous LPAAT genes which were better than the endogenous acyltransferases at discriminating against saturated fatty acids. Expression of LPAT2 homologs from B. napus, T cacao, Garcinia hombroriana and Garcinia indica and their effect on the formation of trisaturated TAGs in the high-C18:0 S6573 strain is disclosed below.

[0140] The sequence of the transforming DNA from the BnLPAT2(Bn1.13) expression construct pSZ4198 is shown below The construct is written as pSZ4198:PLOOP::PmHXT1-ScarMEL1-CvNR:PmSAD2-2v2-BnLPAT2(Bn1.13)-CvNR::PLOOP. Relevant restriction sites are indicated in lowercase, bold, and are from 5-3 BspQI, KpnI, SpeI, SnaBI, EcoRI, SpeI, ClaI, BglII, AflII, HindIII, SacI and BspQI. Underlined sequences at the 5 and 3 flanks of the construct represent genomic DNA from P. moriformis that enable targeted integration of the transforming DNA via homologous recombination at the PLOOP locus. Proceeding in the 5 to 3 direction, the PmHXT1 promoter driving the expression of S. carlbergensis MEL1 (ScarMEL1) gene, enabling strains to utilize exogeneous melibiose, is indicated by lowercase, boxed text. The initiator ATG and terminator TGA of ScarMEL1 are indicated by uppercase italics, while the coding region is represented by lowercase italics. The 3 UTR of the CvNR gene is indicated by small capitals. The P. moriformis SAD2-2v2 promoter driving the expression of the BnLPAT2(Bn1.13) gene is indicated by lowercase, boxed text. The initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding BnLPAT2(Bn1.13) is represented by lowercase, underlined italics. A second CvNR 3 UTR is indicated by small capitals. The Brassica napus LPAAT2(BN1.13) sequence is from Genbank accession GU045434.

TABLE-US-00023 SEQIDNO:88:NucleotidesequenceofthetransformingDNAfrompSZ4198 gctcttccgctAACGGAGGTCTGTCACCAAATGGACCCCGTCTATTGCGGGAAACCACG GCGATGGCACGTTTCAAAACTTGATGAAATACAATATTCAGTATGTCGCGGGCGG CGACGGCGGGGAGCTGATGTCGCGCTGGGTATTGCTTAATCGCCAGCTTCGCCCC CGTCTTGGCGCGAGGCGTGAACAAGCCGACCGATGTGCACGAGCAAATCCTGAC ACTAGAAGGGCTGACTCGCCCGGCACGGCTGAATTACACAGGCTTGCAAAAATA CCAGAATTTGCACGCACCGTATTCGCGGTATTTTGTTGGACAGTGAATAGCGATG CGGCAATGGCTTGTGGCGTTAGAAGGTGCGACGAAGGTGGTGCCACCACTGTGC CAGCCAGTCCTGGCGGCTCCCAGGGCCCCGATCAAGAGCCAGGACATCCAAACT ACCCACAGCATCAACGCCCCGGCCTATACTCGAACCCCACTTGCACTCTGCAATG GTATGGGAACCACGGGGCAGTCTTGTGTGGGTCGCGCCTATCGCGGTCGGCGAA GACCGGGAAggtaccgcggtgagaatcgaaaatgcatcgtttctaggttcggagacggtcaattccctgctccggcgaatctg tcggtcaagctggccagtggacaatgttgctatggcagcccgcgcacatgggcctcccgacgcggccatcaggagcccaaacagc gtgtcagggtatgtgaaactcaagaggtccctgctgggcactccggccccactccgggggcgggacgccaggcattcgcggtcggt cccgcgcgacgagcgaaatgatgattcggttacgagaccaggacgtcgtcgaggtcgagaggcagcctcggacacgtctcgctag ggcaacgccccgagtccccgcgagggccgtaaacattgtttctgggtgtcggagtgggcattttgggcccgatccaatcgcctcatgc cgctctcgtctggtcctcacgttcgcgtacggcctggatcccggaaagggcggatgcacgtggtgttgccccgccattggcgcccac gtttcaaagtccccggccagaaatgcacaggaccggcccggctcgcacaggccatgctgaacgcccagatttcgacagcaacacca tctagaataatcgcaaccatccgcgttttgaacgaaacgaaacggcgctgtttagcatgtttccgacatcgtgggggccgaagcatgct ccggggggaggaaagcgtggcacagcggtagcccattctgtgccacacgccgacgaggaccaatccccggcatcagccttcatcg acggctgcgccgcacatataaagccggacgcctaaccggtttcgtggttatgactagtATGttcgcgttctacttcctgacggcctgc atctccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaaca cgttcgcctgcgacgtctccgagcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacatgggctaca agtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaacgg catgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtacacgtgcgccggct accccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgc tacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccg ccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccgg cgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttcc actgctccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaa cctggaggtcggcgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgat catcggcgcgaacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactcc aacggcatccccgccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtc cggccccctggacaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggagg agatcttcttcgactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaa ctccacggcgtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacg gcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccg cccacggcatcgcgttctaccgcctgcgcccctcctccTGAtacgtactcgagGCAGCAGCAGCTCGGATAGT ATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTG CCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATC TTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCAC CCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCT ACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCAC AGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGC ACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAagctgtag aattcctggctcgggcctcgtgctggcactccctcccatgccgacaacattctgctgtcaccacgacccacgatgcaacgcgacacg acccggtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcatactccaatcgtatccgtttgatttctgtgaaaactcg ctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtgacctgggtgtttcgtcgaaaggccagcaaccccaaatcgcaggc gatccggagattgggatctgatccgagcttggaccagatcccccacgatgcggcacgggaactgcatcgactcggcgcggaaccca gctttcgtaaatgccagattggtgtccgataccttgatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgct accagggttgcatacattgcccatttctgtctggaccgctttaccggcgcagagggtgagttgatggggttggcaggcatcgaaacgc gcgtgcatggtgtgtgtgtctgttttcggctgcacaatttcaatagtcggatgggcgacggtagaattgggtgttgcgctcgcgtgcatgc ctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccctcctgctaacgctcccgactctcccgcccgcgcgcag gatagactctagttcaaccaatcgacaactagtATGgccatggccgccgccgtgatcgtgcccctgggcatcctgttcttcatctcc ggcctggtggtgaacctgctgcaggccatctgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcg tggtggccgagaccctgtggctggagctggtgtggatcgtggactggtgggccggcgtgaagatccaggtgttcgccgacaacg agaccttcaaccgcatgggcaaggagcacgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcc tggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgt ggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgactt cccccgccccttctggctggccctgttcgtggagggcacccgcttcaccgaggccaagctgaaggccgcccaggagtacgccgc ctcctccgagctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgctccttcgt gcccgccatctacgacatgaccgtggccatccccaagacctcccccccccccaccatgctgcgcctgttcaagggccagccctcc gtggtgcacgtgcacatcaagtgccactccatgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcgacca gttcgtggccaaggacgccctgctggacaagcacatcgccgccgacaccttccccggccagcaggagcagaacatcggccgc cccatcaagtccctggccgtggtgctgtcctggtcctgcctgctgatcctgggcgccatgaagttcctgcactggtccaacctgttctc ctcctggaagggcatcgccttctccgccctgggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcctcccagtccga gcgctccacccccgccaaggtggtgcccgccaagcccaaggacaaccacaacgactccggctcctcctcccagaccgaggtg gagaagcagaagTGAatcgatagatctcttaagGCAGCAGCAGCTCGGATAGTATCGACACACT CTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGT GAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACG CGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCC CCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTG CTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTT GGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCT GATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagcttaattaagagctcAGCGG CGACGGTCCTGCTACCGTACGACGTTGGGCACGCCCATGAAAGTTTGTATACCGA GCTTGTTGAGCGAACTGCAAGCGCGGCTCAAGGATACTTGAACTCCTGGATTGAT ATCGGTCCAATAATGGATGGAAAATCCGAACCTCGTGCAAGAACTGAGCAAACC TCGTTACATGGATGCACAGTCGCCAGTCCAATGAACATTGAAGTGAGCGAACTGT TCGCTTCGGTGGCAGTACTACTCAAAGAATGAGCTGCTGTTAAAAATGCACTCTC GTTCTCTCAAGTGAGTGGCAGATGAGTGCTCACGCCTTGCACTTCGCTGCCCGTG TCATGCCCTGCGCCCCAAAATTTGAAAAAAGGGATGAGATTATTGGGCAATGGA CGACGTCGTCGCTCCGGGAGTCAGGACCGGCGGAAAATAAGAGGCAACACACTC CGCTTCTTAgctcttc

[0141] Additional transforming constructs to test the activity of LPAATs from B. napus, T. cacao, G. hombroriana and G. indica contained the same selectable marker, restriction sites, promoters and 3 UTR elements as pSZ4198. The coding sequences of BnLPAT2(Bn1.5), TcLPAT2, GhomLPAT2A, GhomLPAT2B, GhomLPAT2C, GindLPAT2A, GindLPAT2B and GindLPAT2C are shown in below. In each case the initiator ATG and terminator TGA are indicated by uppercase italics; the sequence encoding the LPAT2 homolog is represented by lowercase italics. The Brassica napus LPAAT2(BN1.13) sequence is from Genbank accession GU045435. The Theobroma cacao LPAAT2 sequence is from the cocoaGenDB database.

TABLE-US-00024 NucleotidesequenceoftheBnLPAT2(1.5)codingsequence, usedinthetransformingDNAfrompSZ4202 SEQIDNO:89 ATGgccatggccgccgccgccgtgatcgtgcccctgggcatcctgttcttcatctccggcctggtggtgaacctgctgcaggccgt gtgctacgtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagaccctgtggctggagctg gtgtggatcgtggactggtgggccggcgtgaagatccaggtgttcgccgacgacgagaccttcaaccgcatgggcaaggagca cgccctggtggtgtgcaaccaccgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctcc gccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgca actgggccaaggacgagtccaccctgaagtccggcctgcagcgcctgaacgacttcccccgccccttctggctggccctgttcgtg gagggcacccgcttcaccgaggccaagctgaaggccgcccaggagtacgccgcctcctcccagctgcccgtgccccgcaacgt gctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgctccttcgtgcccgccatctacgacatgaccgtggccat ccccaagacctcccccccccccaccatgctgcgcctgttcaagggccagccctccgtggtgcacgtgcacatcaagtgccactcc atgaaggacctgcccgagtccgacgacgccatcgcccagtggtgccgcgaccagttcgtggccaaggacgccctgctggacaa gcacatcgccgccgacaccttccccggccagaaggagcacaacatcggccgccccatcaagtccctggccgtggtggtgtcctg ggcctgcctgctgaccctgggcgccatgaagttcctgcactggtccaacctgttctcctccctgaagggcatcgccctgtccgccctg ggcctgggcatcatcaccctgtgcatgcagatcctgatccgctcctcccagtccgagcgctccacccccgccaaggtggcccccg ccaagcccaaggacaagcaccagtccggctcctcctcccagaccgaggtggaggagaagcagaagTGA NucleotidesequenceoftheTcLPAT2codingsequence,used inthetransformingDNAfrompSZ4206 SEQIDNO:90 ATGgccatcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttc gtgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggctggtgg actggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccctggtggtggccaacc accgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctcc aagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagaacaccctgaaggc cggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtggagggcacccgcttcacccaggccaagttcctggccgc ccaggagtacgccgcctcccagggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgc gctccttcgtgcccgccatctacgacatgaccgtggccatccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctc cgtggtgcacgtgcacatcaagcgctgcctgatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgttcg tggagaaggacaagctgctggacaagcacatcgccgaggacaccttctccgaccagcccatgcaggacctgggccgccccatcaagtcc ctgctggtggtggcctcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcg ccttcttcctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggc ccccggcaagcccaagaacgacggcgagacctccgaggcccgccgcgacaagcagcagTGA NucleotidesequenceoftheGhomLPAT2Acodingsequence, usedinthetransformingDNAfrompSZ4412. SEQIDNO:91 ATGgccatccccgccgccatcgtgatcgtgcccgtgggcctgctgttcttcatctccggcctgatcgtgaacctgctgcaggccctgtgcttcg tgctgatccgccccctgtccaagtccgcctaccgcaccatcaaccgccagctggtggagctgctgtggctggagctggtgtgcatcgtggac tggtgggcccgcgtgaagatccagctgttcaccgacaaggagaccctgaactccatgggcaaggagcacgccctggtgatgtgcaacca ccgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccaccgtggccgtgatgaagaagtcctcca aggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagtcc ggcctgcagcgcctgcgcgacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgcc caggagtacgccgcctccaccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccatcacccgc tccttcgtgcccgtgatctacgacatcaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctccg tggtgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgacgtggcccagtggtgccgcgaccagttcgtgg tgaaggactccctgctggacaagcacatcgccgaggacaccttctccgaccaggagctgcaggacatcggccgccccatcaagtccctgg tggtgttcacctcctgggtgtgcatcatcaccttcggcgccctgaagttcctgcagtggtcctccctgctgcactcctggaagggcatcgccat ctccgcctccggcctggccatcgtgaccgtgctgatgcacatcctgatccgcttctcccagtccgagcactccacctccgccaagatcgccgcc gagaagcacaagaacggcggcgtgtcccaggagatgggccgcgagaagcagcacTGA NucleotidesequenceoftheGhomLPAT2Bcodingsequence, usedinthetransformingDNAfrompSZ4413. SEQIDNO:92 ATGgagatccccgccgtggccgtgatcgtgcccatcggcatcctgttcttcatctccggcctgatcgtgaacctgatgcaggccatctgcttc ttcctgatccgccccctgtccaagaacacccaccgcatcgtgaaccgccagctggccgagctgctgtggctggagctgatctggatcgtgga ctggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcacctgatgggcaaggagcacgccctggtgatctgcaacc actcctccgacatcgactggctggtgggctggctgctgtgccagcgctccggctgcctgggctccgccctggccgtgatgaagtcctcctcca aggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagtccaccctgaagtcc ggcctgcagcgcctgaaggacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccaggccaagctgctggccgc ccaggagtacgccatgtccgccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgc gctccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccgtgcagcccaccatgctgcgcctgttcaagggccagtcctc cgtggtgcaggtgcacctgaagcgccactccatgaaggacctgcccgagtccgaggacgacgtggcccagtggtgccgcgaccgcttcgt ggtgaaggactccctgctggacaagcacaaggtggaggacaccttcaccgaccaggagctgcaggacctgggccgccccatcaagtccc tggtggtggtgacctgctgggcctgcatcatcatcttcggcatcctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatggc catctccgcctccggcctggccgtggtgaccttcctgatgcagatcctgatccgcttctcccagtccgagcgctccacccccgccaagatcgcc cccgccaagcccaacaaggccggcaactcctccgagaccgtgcgcgacaagcaccagTGA NucleotidesequenceoftheGhomLPAT2Ccodingsequence, usedinthetransformingDNAfrompSZ4414. SEQIDNO:93 ATGgccatccccgccgccatcatcatcgtgcccctgggcctgatcttcttcacctccggcctgatcatcaacctgatccaggccgtgtgctacg tgctgatccgccccctgtccaagtccaccttccgccgcatcaaccgcgagctggccgagctgctgtggctggagctggtgtgggtggtggac tggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcactccatgggcaaggagcacgccctggtgatctgcaaccac cgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacttcttcctggagcgcaactgggccatggacgagtccaccctgaagtccg gcctgcagcgcctgaaggacttcccccagcccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgccc aggagtacgccgcctccgccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgaacatcatgcgc tccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctccg tggtgcacgtgcacctgaagcgccacctgatggaggacctgcccgagaccgacgacgacgtggcccagtggtgccgcgaccgcttcgtgg tgaaggactccctgctggacaagtacgtggccgaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccctgg tggtggtgacctcctgggtgtgcatcatcgccttcggctccctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgtgat ctccgccgcctccctggccgtggtgaccgtgctgatgcagatcctgatccgcttctcccagtccgagcgctccacctccgccaagatcgccgcc gccaagcgcaagaacgtgggcgagcacTGA NucleotidesequenceoftheGindPAT2Acodingsequence, usedinthetransformingDNAfrompSZ4415. SEQIDNO:94 ATGgccatccccgtggtggtggtgatcgtgcccgtgggcctgctgttcttcatctccggcctgatcgtgaacctgctgcaggccctgtgcttc gtgctgatccgccccctgtccaagtccgcctaccgcaccatcaaccgccagctggtggagctgctgtggctggagctggtgtgcatcgtgga ctggtgggcccgcgtgaagatccagctgttcatcgacaaggagaccctgaactccatgggcaaggagcacgccctggtgatgtgcaacc accgctcctacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccaccgtggccgtgatgaagaagtcctcc aaggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccaaggacgagtccaccctgaagt ccggcctgcagcgcctgcgcgacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccg cccaggagtacgccgcctccaccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccatcaccc gctccttcgtgcccgtgatctacgacatcaccgtggccatccccaagtcctcctcccagcccaccatgctgaagctgttcaagggccagtcctc cgtggtgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgacgtggcccagtggtgccgcgcccagttcgt ggtgaaggactccctgctggacaagcacatcgccgaggacaccttctccgaccaggagctgcaggacatcggccgccccatcaagtccct ggtggtgttcacctcctgggtgtgcatcatcaccttcggcgccctgaagttcctgcagtggtcctccctgctgcactcctggaagggcatcgcc atctccgcctccggcctggccatcgtgaccgtgctgatgcacatcctgatccgcttctcccagtccgagcactccacctccgccaagatcgccg ccgagaagcacaagaacggcggcgtgtcccaggagatgggccgcgagaagcagcacTGA NucleotidesequenceoftheGindPAT2Bcodingsequence, usedinthetransformingDNAfrompSZ4416. SEQIDNO:95 ATGggcatccccgccgtggccgtgatcgtgcccatcggcatcctgttcttcatctccggcttcatcgtgaacctgatgcaggccatctgcttcg tgctgatccgccccctgtccaagaacacctaccgcatcgtgaaccgccagctggccgagttcctgtggctggagctgatctgggtggtggac tggtgggccggcgtgaagatccagctgttcaccgacaaggagaccctgcacctgatgggcaaggagcacgccctggtgatctgcaacca ccgctccgacatcgactggctggtgggctggctgctgtgccagcgctccggctgcctgggctccgccctggccgtgatgaagtcctcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagtccaccctgaagctgg gcctgcagcgcctgaaggacttcccccgccccttctggctggccctgttcgtggagggcacccgcttcacccaggccaagctgctggccgccc aggagtacgccatgtccgccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgtccaacatgcgc tccttcgtgcccgccatctacgacgtgaccgtggccatccccaagtcctccgtgcagcccaccatgctgggcctgttcaagggccagtcctgc gtggtgcaggtgcacctgaagcgccacctgatgaaggacctgcccgagtccgaggacgacgtggcccagtggtgccgcgagcgcttcgt ggtgaaggactccctgctggacaagcacaaggtggaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccct ggtggtggtgatctcctgggcctgcatcctgatcttctggatcctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgcc atctccgcctgcgccatggccgtgatcgccttcctgatgcagatcctgctgcgcttctcccagtccgagcgctccacccccgccaagatcgccc ccgccaagcccaacaacgcccgcaactcctccgagaccgtgcgcgacaagcaccagTGA SEQIDNO:96NucleotidesequenceoftheGindPAT2Ccodingsequence, usedinthetransformingDNAfrompSZ4417. ATGgccatccccgccgccatcatcatcgtgcccctgggcctgatcttcttcacctccggcttcatcatcaacctgatccaggccgtgtgctacg tgctgatccgccccctgtccaagtccaccttccgccgcatcaaccgccagctggccgagctgctgtggctggagctggtgtgggtggtggac tggtgggccggcgtgaagatccagctgttcaccaacaaggagaccctgcactccatcggcaaggagcacgccctggtgatctgcaaccag cgctccgacatcgactggctggtgggctggatcctggcccagcgctccggctgcctgggctccgccctggccgtgatgaagaagtcctccaa ggtgctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgcaactgggccatggacgagtccaccctgaagtccg gcctgcagtggctgaaggacttcccccagcccttctggctggccctgttcgtggagggcacccgcttcacccagcccaagctgctggccgcc caggagtacgccgcctccgccggcctgcccatcccccgcaacgtgctgatcccccgcaccaagggcttcgtgtccgccgtgaacatcatgcg ctccttcgtgcccgccgtgtacgacgtgaccgtggccatccccaagtcctccccccagcccaccatgctgcgcctgttcaagggccagtcctcc gtggtgcacgtgcacctgaagcgccacctgatggaggacctgcccgagaccgacgacgacgtggcccagtggtgccgcgaccgcttcgtg gtgaaggactccctgctggacaagcacctggccgaggacaccttctccgaccaggagctgcaggacctgggccgccccatcaagtccctg gtggtggtgacctcctgggtgtgcatcatcgccttcggcgccctgaagttcctgcagtggtcctccctgctgtactcctggaagggcatcgtg atctccgccgcctccctggccgtggtgaccgtgctgatgcagatcctgatccgcttctcccagtccgagcgctccacctccgccaaggtggtg gccgagaagcgcaagaacgtgggcgagcacTGA

[0142] Constructs D2971, D2973, D2975, D3219, D3221, D3223, D3225, D3227 and D3229, derived from pSZ4198, pSZ4202, pSZ4206, pSZ4412, pSZ4413, pSZ4414, pSZ4415, pSZ4416 and pSZ4417, respectively, were transformed into the S6573 parent strain. The fatty acid profiles of primary transformants are shown in Table 10. Also shown are the SOS/SSS ratios determined by LC/MS multiple response measurements. Expression of LPAT2 genes had no discernable effect on C16:0 or C18:0 accumulation, but C18:2 levels increased by 1-2% compared to the S6573 parent in strains when expressing the D2971, D2973, D2975, D3221, D3223, and D3227 constructs. Expression of LPAT2 genes increased C18:2 and also elevated ratios of SOS/SSS, showing reduced accumulation of trisaturated TAGs.

TABLE-US-00025 TABLE 10 Fatty acid profiles and SOS/SSS ratios of D2971, D2973, D2975, D3219, D3221, D3223, D3225, D3227 and D3229 primary transformants. Strain LPAAT gene SOS/SSS C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 saturates S5100 0.7 17.7 4.1 68.5 6.8 0.6 0.4 23.3 S6573.1 15 0.8 6.2 50.7 33.7 5.6 0.7 1.5 59.8 D2971.1 BnLPAT2(1.13) 23 0.8 6.1 51.4 30.5 8.6 0.6 1.4 60.2 D2971.2 16 0.8 6.1 54.3 28.9 7.0 0.6 1.5 63.3 D2971.4 16 0.8 6.4 53.3 29.5 7.3 0.6 1.4 62.6 S6573.2 14 0.8 6.6 52.8 31.7 5.2 0.6 1.5 62.3 D2973.2 BnLPAT2(1.5) 22 0.8 6.2 53.4 28.3 6.4 0.6 1.7 62.7 D2973.38 23 0.9 7.5 51.2 29.1 6.5 0.5 1.4 61.7 D2973.24 24 0.9 6.8 51.7 29.2 6.3 0.5 1.6 61.5 S6573.3 14 0.8 6.6 52.8 31.7 5.2 0.6 1.5 62.3 D2975.33 TcLPAT2 27 0.8 6.6 52.7 29.7 7.1 0.6 1.5 62.3 D2975.13 32 0.8 6.5 52.4 30.2 7.3 0.6 1.4 61.7 D2975.35 27 0.8 6.5 52.8 29.6 7.3 0.6 1.5 62.2 S6573.4 12 0.9 6.4 54.9 28.9 5.7 0.6 1.7 64.5 D3219.19 GhomLPAT2A 12 0.9 7.1 52.4 31.2 4.8 0.5 2.0 63.1 D3219.20 14 0.9 6.6 53.2 30.6 5.5 0.6 1.7 63.0 D3219.32 15 0.8 6.4 53.1 29.8 6.5 0.6 1.5 62.6 S6573.5 12 0.9 6.4 53.7 30.3 5.5 0.6 1.6 63.3 D3220.1 GhomLPAT2B 27 0.9 6.6 52.2 30.0 7.0 0.7 1.4 61.9 D3221.39 20 0.9 6.7 53.9 28.7 6.7 0.6 1.5 63.7 D3221.40 22 0.8 6.5 53.7 29.1 6.8 0.6 1.4 63.2 S6573.6 14 0.8 6.3 54.0 30.2 5.5 0.6 1.6 63.4 D3223.2 GhomLPAT2C 20 0.8 6.5 53.0 29.3 7.3 0.6 1.5 62.4 D3223.6 21 0.8 6.5 53.5 29.3 7.0 0.6 1.4 62.7 D3223.7 21 0.8 6.4 52.5 30.7 6.6 0.5 1.5 61.8 D3225.5 GindLPAT2A 13 0.9 6.6 53.5 30.2 5.6 0.6 1.6 63.2 S6573.7 12 0.9 6.5 53.5 29.9 5.7 0.6 1.8 63.3 D3227.6 GindLPAT2B 23 0.8 6.4 54.1 28.8 6.8 0.6 1.6 63.5 D3227.3 21 0.8 6.5 53.9 29.0 6.7 0.6 1.5 63.4 D3227.17 22 0.8 6.6 53.8 28.8 7.0 0.6 1.4 63.3 S6573.8 11 0.8 6.4 54.3 30.1 5.4 0.6 1.7 63.8 D3229.41 GindLPAT2C 11 0.9 6.6 54.2 29.7 5.6 0.6 1.7 63.9 D3229.27 13 0.8 6.4 54.1 30.0 5.6 0.6 1.7 63.6 D3229.33 12 0.8 6.4 54.0 30.2 5.5 0.6 1.7 63.5

[0143] Table 11 presents the TAG composition of the lipids produced by D2971, D2973, D2975, D3221, D3223, and D3227 primary transformants relative to the S6573 parent. SOS levels in the LPAT2-expressing strains were equivalent or slightly higher than in the S6573 controls. Trisaturates declined by up to 53%, and total Sat-Unsat-Sat levels improved in all of the strains expressing heterologous LPAT2 genes. Among the LPAT2 genes, the strains expressing the T. cacao LPAT2 homolog showed the greatest improvements in their TAG profiles).

TABLE-US-00026 TABLE 11 TAG composition of D2971, D2973, D2975, D3221, D3223, and D3227 primary transformants relative to the S6573 parent. LPAAT gene BnLPAT2 BnLPAT2 Ghom Ghom Gind (1.13) (1.5) TcLPAT2 LPAT2B LPAT2C LPAT2B Strain D2971.1 D2973.38 D2975.33 D2975.13 D3221.39 D3221.40 D3223.6 D3227.3 D3227.6 % S6573 TAG SOS 100 100 110 104 107 107 108 103 105 Sat-Sat-Sat 57 63 48 47 74 62 68 62 70 Sat-U-Sat 109 107 113 110 112 112 109 108 107 Sat-O-Sat 97 100 105 102 106 105 102 104 104 Sat-L-Sat 174 147 155 155 139 143 141 130 125 U-U-U/Sat 85 86 72 83 64 69 78 82 79

[0144] We analyzed the fatty acid profiles, TAG profiles and lipid titers from 50 mL shake flask cultures of stable lines generated from D2975-33. C18:0 and C16:0 levels were comparable between the strains and the S6573 control, and lipid titers ranged from 75-105% of the parent strain titer (Table 12). C18:2 levels increased by more than 2% in the TcLPAT2-expressing strains.

TABLE-US-00027 TABLE 12 Fatty acid profiles of TcLPAT2-expressing stable lines made from D2975-33. Primary D1940.19 D2975.33 Strain S6573 S7813 S7815 S7816 S7817 S7819 Fatty C12:0 0.2 0.2 0.2 0.2 0.2 0.2 Acid C14.0 0.9 0.7 0.8 0.8 0.7 0.7 Area % C16:0 6.5 5.9 6.1 5.9 6.1 6.0 C16:1 cis-9 0.1 0.1 0.1 0.1 0.1 0.1 C17:0 0.2 0.2 0.2 0.2 0.2 0.2 C18:0 56.1 55.6 55.9 56.2 53.9 53.9 C18:1 28.1 26.8 26.6 26.5 28.8 28.4 C18:2 5.5 8.1 7.7 7.9 7.7 7.8 C18:3 0.6 0.5 0.6 0.5 0.6 0.7 C20:0 1.5 1.5 1.4 1.3 1.3 1.5 C22:0 0.2 0.2 0.1 0.1 0.1 0.2 C24:0 0.1 0.1 0.1 0.1 0.1 0.1 saturates 65.7 64.4 65.0 64.9 62.8 62.9

[0145] The TAG profiles of S6573 and S57815 are compared in FIG. 1. SOS levels in the LPAT2-expressing strains were higher than in the S6573 control. Trisaturates were reduced from 10.2% in S6573 to 5.6% in S7815. Much of the improvement in total sat-unsat-sat levels in S7815 came from a 4% increase in stearate-linoleate-stearate (SLS) and a 1.5% increase in palmitate-linoleate-stearate (PLS), consistent with the enhanced C18:2 content of that strain. These results indicate that the T. cacoa LPAT2 reduces the incorporation of saturated fatty acids at the sn-2 position.

[0146] The performance of S7815 versus the S6573 parent strain was compared in high-density fermentations. The fatty acid profile of each strain at the two time points of the fermentations are shown in Table 13. The strains had very similar composition, with 5.5-5.7% C16:0, 56.4-56.8% C18:0, and 27.2-28.6% C18:1 as the major fatty acids. As was observed in the shake flask assays, (see Table 12), C18:2 levels increased from 5.5% in S6573 to 7.7% in S7815 (Table 13). Normalized lipid titers and yields were comparable between the two strains, indicating that expression of the TcLPAT2 gene in S7815 did not have deleterious effects on growth or lipid accumulation.

TABLE-US-00028 TABLE 13 Fatty acid profiles of S7815 versus S6573 fermentations. Strain S6573 S7815 Fermentation 140207F25 140208F26 Fatty Acid C12:0 0.19 0.20 0.20 0.21 Area % C14:0 0.71 0.72 0.66 0.66 C16:0 5.69 5.73 5.57 5.54 C16:1 cis-7 0.05 0.05 0.05 0.06 C16:1 cis-9 0.07 0.06 0.05 0.05 C17:0 0.11 0.11 0.12 0.11 C18:0 56.01 56.78 55.50 56.37 C18:1 29.31 28.58 27.92 27.19 C18:2 5.56 5.51 7.75 7.70 C18:3 0.34 0.32 0.40 0.37 C20:0 1.51 1.50 1.35 1.34 C22:0 0.16 0.16 0.14 0.14 C24:0 0.10 0.09 0.09 0.08 sum C18 91.22 91.19 91.57 91.63 saturates 64.54 65.34 63.69 64.51 unsaturates 35.46 34.64 36.30 35.49

[0147] Table 13 compares the TAG profiles of the lipids produced during high-density fermentation of S7815 versus S6573. SOS and Sat-Oleate-Sat levels were almost identical between S7815 and the S6573 control. However, Sat-Linoleate-Sat levels increased by more than 7%, and di-unsaturated and tri-unsaturated TAGs (U-U-U/Sat) declined by more than 3% in S7815 compared to S6573. Trisaturates at the end points of the fermentations were reduced from 10.1% in S6573 to 6.1% in S7815. These results indicate that the activity of T. cacoa LPAT2 drives the transfer of unsaturated fatty acids towards the sn-2 position and discriminates against the incorporation of saturated fatty acids at sn-2.

Example 6: Identification and Expression of Novel LPAAT, GPAT, DGAT, LPCAT and PLA2 with Specificity for Mid-Chain Fatty Acids

[0148] In this example, we demonstrate the effect of expression of LPAAT, GPAT, DGAT, LPCAT and PLA2 enzymes involved in triacylglycerol biosynthesis (in previously described P. moriformis (UTEX 1435) transgenic strains, S7858 and S8174. S7858 and S8174 were prepared according to co-owned WO2015/051319, herein incorporated by reference. In addition co-owned WO2010/063031 and WO2010/063032 teach the expression Cuphea hookerianas FATB2. Briefly, strain S7858 is a strain that express sucrose invertase and a Cuphea. hookeriana FATB2. To make S7858, the construct pSZ4329 (SEQ ID NO: 197) was engineered into S3150, a strain classically mutagenized to increase lipid yield. The plasmid, pSZ4329 is written as THI4a::CrTUB2-ScSUC2-PmPGH:PmAcp-Plp-CpSAD1_tp_trimmed_ChFATB2_FLAG-CvNR::THI4a The annotation of the coding portions of pSZ4329 is shown in the Table A below.

TABLE-US-00029 TABLE A Nucleotide Nucleotide Nucleotide pSZ4329 Identity Number Number Length THI4a 3 flank 3 flanking 5,692 6,394 703 sequences of endogenous THI4 CvNR 3UTR 5,278 5,679 402 ChFATB2 CDS 4,105 5,271 1,167 CpSAD1tp-trimmed CDS 3,991 4,104 114 PmACP-P1 promoter promoter 3,411 3,981 571 Buffer DNA 3,199 3,404 206 UTR04424 = PmPGH UTR 3UTR 2,749 3,192 444 ScSUC2(o) CDS 1,144 2,742 1,599 CrTUB2 promoter promoter 820 1,131 312 THI4a 5 flank 5 flanking 27 813 787 sequences of endogenous THI4

[0149] Strain S7858, accumulates C8:0 fatty acids to about 12% and C10:0 fatty acids to about 22-24%. Briefly, strain S8174 is a strain that express sucrose invertase and a Cuphea. Avigera var. pulcherrima FATB2. To make S8174, the construct pSZ5078 (SEQ ID NO: 198) was engineered into S3150, a strain classically mutagenized to increase lipid yield. pSZ5078 is written as THI4a5::CrTUB2_ScSUC2_PmPGH:PmAMT3_CpSAD1_tp_trimmed-CaFATB1_Flag_CvNR::THI4a3. Strain S8174 accumulates C8:0 fatty acids to about 24% and C10:0 fatty acids to about 10%. The annotation of the coding portions of pSZ5078 is shown in the Table B below.

TABLE-US-00030 TABLE B Nucleotide Nucleotide Nucleotide pSZ5078 Identity Number Number Length THI4a 3 flank 3 flanking 6,200 6,902 703 sequences of endogenous THI4 CvNR 3UTR 5,786 6,187 402 CaFATB1 CDS 4,602 5,771 1,170 wild-type CpSAD1tp CDS 4,488 4,601 114 AMT3 promoter eukaryotic 3,411 4,481 1,071 Buffer DNA misc_feature 3,199 3,404 206 PmPGH 3UTR 2,749 3,192 444 ScSUC2(o) CDS 1,144 2,742 1,599 CrTUB2 promoter 820 1,131 312 promoter THI4a 5 flank 5 flanking 27 813 787 sequences of endogenous THI4

[0150] The pool of acyl-CoAs in the ER can be utilized for the synthesis of TAGs as well as phospholipids and long chain fatty acids. The enzymes involved in the synthesis of TAGS and phospholids actively compete against each other for the same substrates. Acyl-CoAs can associate with lysophosphatidate to form phosphatidate which is converted to phosphatidylcholine (PC) and other phospholipid species. PC can be desaturated by FAD2 and FAD3 enzymes to generate polyunsaturated fatty acids, which can be cleaved by phosphotransferases and reenter the acyl-CoA pool. Acyl-CoAs can also be generated from PC directly by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT). LPCAT can also catalyze the reverse reaction to consume acyl-CoA. Removal of fatty acids from PC to form acyl-CoAs can also be catalyzed by phospholipase A.sub.2 (PLA2). TAG formation in the ER from acyl-CoAs requires action of glycerol phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT) and diacyl glycerol acyltransferase (DGAT).

[0151] The endogenous P. moriformis TAG biosynthesis machinery has evolved to function with the longer chain fatty acids that the strain normally makes. We introduced heterologous acyltransferases and phospholipases from species that naturally accumulate high levels of short chain fatty acids into Prototheca to increase accumulation of C8:0 fatty acids. We identified the following plant enzymes in NCBI as shown in Table 14 below.

TABLE-US-00031 TABLE 14 Genes representing target enzymes identified from higher plants that produce high amounts of C8:0 and C10:0. All these genes were synthesized with codon usage optimized for expression in Prototheca. Species Gene Enzyme Cocos nucifera CnLPAAT1 LPAAT Cuphea paucipetala CpauLPAAT1 Cuphea procumbens CprocLPAAT1 Cuphea painteri CpaiLPAAT1 Cuphea hookeriana ChookLPAAT1 Cuphea ignea CigneaLPAAT1 Cuphea avigera var. pulcherrima CavigLPAAT1 Cuphea avigera var. pulcherrima CavigLPAAT2 Cuphea palustris CpalLPAAT1 Cuphea koehneana CkoeLPAAT1 Cuphea koehneana CkoeLPAAT2 Cuphea procumbens CprocLPAAT2 Cuphea PSR23 CuPSRLPAAT2 Cuphea avigera var. pulcherrima CavigGPAT9 GPAT Cuphea hookeriana ChookGPAT9-1 Cuphea ignea CignGPAT9-1 Cuphea ignea CignGPAT9-2 Cuphea palustris CpalGPAT9-1 Cuphea palustris CpalGPAT9-2 Cuphea avigera var. pulcherrima CavigDGAT1 DGAT Cuphea hookeriana ChookDGAT1-1 Cuphea avigera var. pulcherrima CavigLPCAT LPCAT Cuphea palustris CpalLPCAT Cuphca paucipetala CpauLPCAT Cuphea schumanii CschuLPCAT1 Cuphea avigera var. pulcherrima CavigPLA2-1 PLA2 Cuphea ignea CignPLA2-1 Cuphea procumbens CprocPLA2-2 Cuphea PSR23 CuPSR23PLA2-2

[0152] We made a set of constructs expressing heterologous short chain specific acyltransferases and PLA2s as shown in Table 15. The genes were codon optimized to reflect UTEX 1435 codon usage.

TABLE-US-00032 TABLE 15 List of constructs transformed into S7858 or S8174 D# Strain Construct D4289 S7858 SAD2-1vD::CpauLPAAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4290 S7858 SAD2-1vD::CpaiLPAAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4291 S7858 SAD2-1vD::CigneaLPAAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4292 S7858 SAD2-1vD::CprocLPAAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4293 S7858 SAD2-1vD::ChookLPAAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4404 S7858 SAD2-1vD::CnLPAAT-PmATP:PmHXT1-ScarMEL1-PmPGK::SAD2Bex D4517 S8174 SAD2-1vD::CavigLPAAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4518 S8174 SAD2-1vD::CavigLPAAT2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4519 S8174 SAD2-1vD::CpalLPAAT1-PmATP:PmHXT-ScarMEL-PmPGK::SAD2Bex D4690 S8174 SAD2-1vD::CuPSR23 LPAAT2-1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4728 S8174 SAD2-1vD::CkoeLPAAT-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4729 S8174 SAD2-1vD::CkoeLPAAT2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4730 S8174 SAD2-1vD::CprocLPAAT2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4551/D4683 S8174 SAD2-1vD::CavigGPAT9-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4552/D4684 S8174 SAD2-1vD::ChookGPAT9-1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4553/D4685 S8174 SAD2-1vD::CignGPAT9-1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4554/D4686 S8174 SAD2-1vD::CignGPAT9-2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4724 S8174 SAD2-1vD::CpalGPAT9-1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4725 S8174 SAD2-1vD::CpalGPAT9-2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4549 S8174 SAD2-1vD::CavigDGAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4681 S8174 SAD2-1vD::CavigDGAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4555/D4688 S8174 SAD2-1vD::CavigLPCAT-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4726 S8174 SAD2-1vD::CpalLPCAT-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4556/D4689 S8174 SAD2-1vD::CpauLPCAT-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4727 S8174 SAD2-1vD::CschuLPCAT1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4732 S8174 SAD2-1vD::CavigPLA2-1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4734 S8174 SAD2-1vD::CignPLA2-1-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4735 S8174 SAD2-1vD::CuPSR23PLA2-2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex D4736 S8174 SAD2-1vD::CprocPLA2-2-PmATP:PmHXT1-ScarMEL-PmPGK::SAD2Bex

[0153] All the constructs shown in Table 15 can be written as SAD2-1vD::gene of interest-PmATP-PmHXT1-ScarMEL-PmPGK::SAD2B, and were made to target the transforming DNA to the SAD2 locus on the genome, thereby disrupting the expression of at least one allele of the endogenous stearoyl ACP desaturase. Sequences of all the transforming DNAs are provided below. The relevant restriction sites in the construct from 5-3 are Pme I, BspQ I, Kpn I, Xho I, Avr II, Spe I, SnaB I, EcoR V, Sac I, BspQ I, Pme I respectively are indicated in lowercase, bold, and underlined. Pme I sites delimit the 5 and 3 ends of the transforming DNA. Bold, lowercase sequences at the 5 and 3 end of the construct represent genomic DNA from UTEX 1435 that target integration to the SAD2 locus via homologous recombination, wherein the SAD2 5 flank provides the promoter for the gene of interest downstream. The primary construct was made with the previously characterized CnLPAAT gene as shown below and all other constructs were made by replacing the CnLPAAT gene with other genes of interest using the restriction sites, Kpn I and Xho I that span the gene on either side. Proceeding in the 5 to 3 direction, the first cassette has the codon optimized Cocos nucifera LPAAT and the Prototheca moriformis ATP synthase (PmATP) gene 3 UTR. The initiator ATG and terminator TGA for cDNAs are indicated by uppercase italics, while the coding region is indicated with lowercase italics. The 3 UTR is indicated by lowercase underlined text. The second cassette containing the selection gene melibiose from Saccharomyces carlsbergensis (ScarMEL1) is driven by the endogenous HXT1 promoter, and has the endogenous phosphoglycerate kinase (PmPGK) gene 3 UTR. In this cassette, the PmHXT1 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the ScarMEL1 gene are indicated in uppercase italics, while the coding region is indicated by lowercase italics. The 3 UTR is indicated by lowercase underlined text. All the final constructs were sequenced to ensure correct reading frames and targeting sequences.

TABLE-US-00033 SEQIDNO:97pSZX61SequenceofthetransformingDNAexpressing CnLPAATdownstreamoftheSAD2promoterinthecassettefollowedbytheScarMEL1 geneforselectiondownstreamofthePmHXT1promoterinthesecondcassette. gtttaaacgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacgg aaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgaca cgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagc agttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcg caggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcgg aacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttgg cgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcagg catcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtg gcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactc tcccgaccgcgcgcaggatagactcttgttcaaccaatcgacaggtaccATGgacgcctccggcgcctcctccttcctgcgcggccgct gcctggagtcctgcttcaaggcctccttcggctacgtaatgtcccagcccaaggacgccgccggccagccctcccgccgccccgccgacgcc gacgacttcgtggacgacgaccgctggatcaccgtgatcctgtccgtggtgcgcatcgccgcctgcttcctgtccatgatggtgaccaccatc gtgtggaacatgatcatgctgatcctgctgccctggccctacgcccgcatccgccagggcaacctgtacggccacgtgaccggccgcatgct gatgtggattctgggcaaccccatcaccatcgagggctccgagttctccaacacccgcgccatctacatctgcaaccacgcctccctggtgg acatcttcctgatcatgtggctgatccccaagggcaccgtgaccatcgccaagaaggagatcatctggtatcccctgttcggccagctgtac gtgctggccaaccaccagcgcatcgaccgctccaacccctccgccgccatcgagtccatcaaggaggtggcccgcgccgtggtgaagaag aacctgtccctgatcatcttccccgagggcacccgctccaagaccggccgcctgctgcccttcaagaagggcttcatccacatcgccctccag acccgcctgcccatcgtgccgatggtgctgaccggcacccacctggcctggcgcaagaactccctgcgcgtgcgccccgcccccatcaccgt gaagtacttctcccccatcaagaccgacgactgggaggaggagaagatcaaccactacgtggagatgatccacgccctgtacgtggacc acctgcccgagtcccagaagcccctggtgtccaagggccgcgacgcctccggccgctccaactccTGAttaattaactcgagatgtggaga tgtagggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttccca acgccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcg tctggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggaga gcgtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcg [00026] [00027] [00028] [00029] [00030] [00031] [00032] [00033] [00034] [00035] [00036] cgtctccccctcctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccga gcagctgctgctggacacggccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgact gctggtcctccggccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgac cacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggcc gcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagt tcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccct gtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcacggcgg agttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatga acatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcg tcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaa cgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatcccc gccacgcgcgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctgg acaacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttc gactccaacctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggc gtccgccatcctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtcca agaacgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggc atcgcgttctaccgcctgcgcccctcctccTGAtacaacttattacgtattctgaccggcgctgatgtggcgcggacgccgtcgtac tctttcagactttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgcaattaattgtgtgatgaagaaaggg tggcacaagatggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctggctcaatcttgtcgcatgt ccggcgcaatgtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactgatcgcattgcc atcccgtcaactcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaagcgt caggaaatcgtctcggcagctggaagcgcatggaatgcggagcggagatcgaatcagatatcAAGCTCCATCgagctccagc cacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgaggg ccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctacgccaacatga tgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaacctcttcgccga cttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaag gtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgcc gagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagg gagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccg cgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcaca tcaaagggcccctccgccagagaagaagctcctttcccagcagactcctgaagagcgtttaaac.

[0154] The sequence for all of the other acyltransferase constructs are identical to that of pSZEX61 with the exception of the encoded acyltransferase. The acyltransferase sequence alone is provided below for the remaining acyltransferase constructs.

TABLE-US-00034 CpauLPAAT1 SEQIDNO:98 ggtaccATGgccatccccgccgccgccgtgatcttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccagg ccctgtgcttcgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgagc tgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagca cgccctggtgatcatcaaccacatgaccgagctggactggatgctgggctgggtgatgggccagcacctgggctgcctgggctcc atcctgtccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacatcgagcgct cctgggccaaggaccgcaccaccctgaagtcccacatcgagcgcctgaccgactaccccctgcccttctggatggtgatcttcgtg gagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtg ctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggccttcc ccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcgtgctgcacgtgcacatcaagcgccacgccat gaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaag cacaacgccgaggacaccttctccggccaggaggtgcaccgcaccggctcccgccccatcaagtccctgctggtggtgatctcct gggtggtggtgatcaccttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggccttctccgtgatcggcctgggc atcgtgaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctcctccaaccccgccaaggtggcccaggccaagc tgaagaccgagctgtccatctccaagaaggccaccgacaaggagaacTGActcgag CprocLPAAT1 SEQIDNO:99 ggtacc ctcgag CpaiLPAAT1 SEQIDNO:100 ggtaccATGgccatcccctccgccgccgtggtgttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccagg ccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgcccctggagtt cctgtggctgttccactggtgcgccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagcac gccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctcca tcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccggctacctgttcctggagcgctcc tgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgcccttctggctgatcatcttcgtgga gggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgct gatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccttcccc aagacctcccccccccccaccatgctgaagctgttcgagggccagtccgtggagctgcacgtgcacatcaagcgccacgccatg aaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaagc acaactccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtgatctcctgggt ggtggtgatcatcttcggcgccctgaagttcctgctgtggtcctccctgctgtcctcctggaagggcaaggccttctccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgagggctccaaccccgtgaaggc cgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag ChookLPAAT1 SEQIDNO:101 ggtaccATGgccatcccctccgccgccgtggtgttcctgttcggcctgctgttcttcacctccggcctgatcatcaacctgttccagg ccttctgcttcgtgctgatctcccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgcccctggagtt cctgtggctgttccactggtgcgccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagcac gccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctcca tcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccgagtacctgttcctggagcgctcc tgggccaaggacaagatcaccctgaagtcccacatcgagtccctgaaggactaccccctgcccttctggctgatcatcttcgtgga gggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgct gatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccttcccc aagacctcccccccccccaccatgctgaagctgttcgagggccagtccgtggagctgcacgtgcacatcaagcgccacgccatg aaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaagc acaactccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaaggccctgctggtggtgatctcctgggt ggtggtgatcatcttcggcgccctgaagttcctgctgtggtcctccctgctgtcctcctggaagggcaaggccttctccgtgatcggcc tgggcatcgtggccggcatcgtgaccctgctgatgcacatcctgatcctgtcctcccaggccgagggctccaaccccgtgaaggc cgcccccgccaagctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:102CignLPAAT1 ggtaccATGgccatcgccgccgccgccgtgatcttcctgttcggcctgctgttcttcgcctccggcatcatcatcaacctgttccag gccctgtgcttcgtgctgatctggcccctgtccaagaacgtgtaccgccgcatcaaccgcgtgttcgccgagctgctgctgatggac ctgctgtgcctgttccactggtgggccggcgccaagatcaagctgttcaccgaccccgagaccttccgcctgatgggcatggagca cgccctggtgatcatgaaccacaagaccgacctggactggatggtgggctggatcctgggccagcacctgggctgcctgggctc catcctgtccatcgccaagaagtccaccaagttcatccccgtgctgggctggtccgtgtggttctccgagtacctgttcctggagcgc tcctgggccaaggacaagtccaccctgaagtcccacatggagaagctgaaggactaccccctgcccttctggctggtgatcttcgt ggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgt gctgatcccccacaccaagggcttcgtgtcctgcgtgtccaacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggcctt ccccaagtcctcccccccccccaccatgctgaagctgttcgagggccagtccatcgtgctgcacgtgcacatcaagcgccacgcc ctgaaggacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaa gcacaacgccgaggacaccttctccggccaggaggtgcaccacatcggccgccccatcaagtccctgctggtggtgatcgcctg ggtggtggtgatcatcttcggcgccctgaagttcctgcagtggtcctccctgctgtccacctggaagggcaaggccttctccgtgatc ggcctgggcatcgccaccctgctgatgcacatgctgatcctgtcctcccaggccgagcgctccaaccccgccaaggtggccaag TGActcgag SEQIDNO:103CavigLPAAT1 ggtaccATGaccatcgcctccgccgccgtggtgttcctgttcggcatcctgctgttcacctccggcctgatcatcaacctgttccag gccttctgctccgtgctggtgtggcccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagttcctgcccctggag ttcctgtggctgttccactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagc acgccctggtgatcatcaaccacaagatcgagctggactggatggtgggctgggtgctgggccagcacctgggctgcctgggctc catcctgtccgtggccaagaagtccaccaagttcctgcccgtgttcggctggtccctgtggttctccgagtacctgttcctggagcgc aactgggccaaggacaagaagaccctgaagtcccacatcgagcgcctgaaggactaccccctgcccttctggctgatcatcttcg tggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgcctccgccggcctgcccgtgccccgcaac gtgctgatcccccacaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggcct tccccaagacctcccccccccccaccatgctgaagctgttcgagggccacttcgtggagctgcacgtgcacatcaagcgccacgc catgaaggacctgcccgagtccgaggacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggac aagcacaacgccgaggacaccttctccggccaggaggtgcaccacgtgggccgccccatcaagtccctgctggtggtgatctcc tgggtggtggtgatcatcttcggcgccctgaagttcctgcagtggtcctccctgctgtcctcctggaagggcatcgccttctccgtgat cggcctgggcaccgtggccctgctgatgcagatcctgatcctgtcctcccaggccgagcgctccatccccgccaaggagaccccc gccaacctgaagaccgagctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:104CavigLPAAT2 ggtaccATGgccatcgccgccgccgccgtgatcgtgcccgtgtccctgctgttcttcgtgtccggcctgatcgtgaacctggtgca ggccgtgtgcttcgtgctgatccgccccctgttcaagaacacctaccgccgcatcaaccgcgtggtggccgagctgctgtggctgg agctggtgtggctgatcgactggtgggccggcgtgaagatcaaggtgttcaccgaccacgagaccttccacctgatgggcaagg agcacgccctggtgatctgcaaccacaagtccgacatcgactggctggtgggctgggtgctggcccagcgctccggctgcctggg ctccaccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggag cgcaactgggccaaggacgagtccaccctgaagtccggcctgaaccgcctgaaggactaccccctgcccttctggctggccctgt tcgtggagggcacccgcttcacccgcgccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgca acgtgctgatcccccgcaccaagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccatctacgacgtgaccgtgg ccatccccaagacctcccccccccccaccctgctgcgcatgttcaagggccagtcctccgtgctgcacgtgcacctgaagcgcca ccagatgaacgacctgcccgagtccgacgacgccgtggcccagtggtgccgcgacatcttcgtggagaaggacgccctgctgg acaagcacaacgccgaggacaccttctccggccaggagctgcaggacaccggccgccccatcaagtccctgctgatcgtgatct cctgggccgtgctggtggtgttcggcgccgtgaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggccttctccgg catcggcctgggcgtgatcaccctgctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggccc ccgccaagcccaagatcgagggcgagtcctccaagaccgagatggagaaggagcacTGActcgag SEQIDNO:105CpalLPAAT1 ggtaccATGgccatcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcgtgtccggcctgatcgtgaacctggtgca ggccgtgtgcttcgtgctgatccgccccctgtccaagaacacctaccgccgcatcaaccgcgtggtggccgagctgctgtggctgg agctggtgtggctgatcgactggtgggccggcgtgaagatcaaggtgttcaccgaccacgagaccctgtccctgatgggcaagg agcacgccctggtgatctgcaaccacaagtccgacatcgactggctggtgggctgggtgctggcccagcgctccggctgcctggg ctccaccctggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgcccgagtcc gacgacgccgtggcccagtggtgccgcgacatcttcgtggagaaggacgccctgctggacaagcacaacgccgaggacacctt ctccggccaggagctgcaggacaccggccgccccatcaagtccctgctggtggtgatctcctgggccgtgctggtgatcttcggcg ccgtgaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggccttctccggcgtgggcctgggcatcatcaccctgct gatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggcccccgccaagcccaagaaggacggcga gtcctccaagaccgagatcgagaaggagaacgttcctggagcgctcctgggccaaggacgagaacaccctgaagtccggcct gaaccgcctgaaggactaccccctgcccttctggctggccctgttcgtggagggcacccgcttcacccgcgccaagctgctggcc gcccagcagtacgccacctcctccggcctgcccgtgccccgcaacgtgctgatcccccgcaccaagggcttcgtgtcctccgtgtc ccacatgcgctccttcgtgcccgccatctacgacgtgaccgtggccatccccaagacctcccccccccccaccatgctgcgcatgtt caagggccagtcctccgtgctgcacgtgcacctgaagcgccacctgatgaaggacctTGActcgag SEQIDNO:106CuPSR23LPAAT2 ggtaccATGgccatcgccgccgccgccgtgatcttcctgttcggcctgatcttcttcgcctccggcctgatcatcaacctgttccag gccctgtgcttcgtgctgatccgccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgccgagctgctgctgtccgag ctgctgtgcctgttcgactggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagc acgccctggtgatcatcaaccacatgaccgagctggactggatggtgggctgggtgatgggccagcacttcggctgcctgggctc catcatctccgtggccaagaagtccaccaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacctggagcg ctcctgggccaaggacaagtccaccctgaagtcccacatcgagcgcctgatcgactaccccctgcccttctggctggtgatcttcgt ggagggcacccgcttcacccgcaccaagctgctggccgcccagcagtacgccgtgtcctccggcctgcccgtgccccgcaacgt gctgatcccccgcaccaagggcttcgtgtcctgcgtgtcccacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggccttc cccaagacctcccccccccccaccctgctgaacctgttcgagggccagtccatcatgctgcacgtgcacatcaagcgccacgcca tgaaggacctgcccgagtccgacgacgccgtggccgagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaa gcacaacgccgaggacaccttctccggccaggaggtgtgccactccggctcccgccagctgaagtccctgctggtggtgatctcc tgggtggtggtgaccaccttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggccttctccgccatcggcctggg catcgtgaccctgctgatgcacgtgctgatcctgtcctcccaggccgagcgctccaaccccgccgaggtggcccaggccaagctg aagaccggcctgtccatctccaagaaggtgaccgacaaggagaacTGActcgag SEQIDNO:107CkoeLPAAT1 ggtaccATGgccatccccgccgccgtggccgtgatccccatcggcctgctgttcatcatctccggcctgatcgtgaacctgatcca ggccgtggtgtacgtgctgatccgccccctgtccaagaacctgcaccgcaagatcaacaagcccatcgccgagctgctgtggctg gagctgatctggctggtggactggtgggccggcatcaaggtggaggtgtacgccgactcccagaccctggagctgatgggcaag gagcacgccctgctgatctgcaaccaccgctccgacatcgactggctggtgggctgggtgctggcccagcgcgcccgctgcctgg gctccgccctggccatcatgaagaagtccgccaagttcctgcccgtgatcggctggtccatgtggttctccgactacatcttcctgga ccgcacctgggccaaggacgagaagaccctgaagtccggcttcgagcgcctggccgacttccccatgcccttctggctggccctg ttcgtggagggcacccgcttcaccaaggccaagctgctggccgcccaggagtacgccgcctcccgcggcctgcccgtgccccag aacgtgctgatcccccgcaccaagggcttcgtgaccgccgtgacccacatgcgctcctacgtgcccgccatctacgactgcaccg tggacatctccaaggcccaccccgccccctccatcctgcgcctgatccgcggccagtcctccgtggtgaaggtgcagatcacccg ccactccatgcaggagctgcccgagaccgccgacggcatctcccagtggtgcatggacctgttcgtgaccaaggacggcttcctg gagaagtaccactccaaggacatcttcggctccctgcccgtgcagaacatcggccgccccgtgaagtccctgatcgtggtgctgtg ctggtactgcctgatggccttcggcctgttcaagttcttcatgtggtcctccctgctgtcctcctgggagggcatcctgtccctgggcctg atcctgctggccgtggccatcgtgatgcagatcctgatccagtccaccgagtccgagcgctccacccccgtgaagtccatccaga aggacccctccaaggagaccctgctgcagaacTGActcgag SEQIDNO:108CkoeLPAAT2 ggtaccATGcacgtgctgctggagatggtgaccttccgcttctcctccttcttcgtgttcgacaacgtgcaggccctgtgcttcgtgct gatctggcccctgtccaagtccgcctaccgcaagatcaaccgcgtgttcgccgagctgctgctgtccgagctgctgtgcctgttcga ctggtgggccggcgccaagctgaagctgttcaccgaccccgagaccttccgcctgatgggcaaggagcacgccctggtgatcac caaccacaagatcgacctggactggatgatcggctggatcctgggccagcacttcggctgcctgggctccgtgatctccatcgcca agaagtccaccaagttcctgcccatcttcggctggtccctgtggttctccgagtacctgttcctggagcgcaactgggccaaggaca agcgcaccctgaagtcccacatcgagcgcatgaaggactaccccctgcccctgtggctgatcctgttcgtggagggcacccgctt cacccgcaccaagctgctggccgcccagcagtacgccgcctcctccggcctgcccgtgccccgcaacgtgctgatcccccacac caagggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgccgtgtacgacgtgaccgtggccttccccaagacctcccc cccccccaccatgctgtccctgttcgagggccagtccgtggtgctgcacgtgcacatcaagcgccacgccatgaaggacctgccc gactccgacgacgccgtggcccagtggtgccgcgacaagttcgtggagaaggacgccctgctggacaagcacaacgccgagg acaccttctccggccaggaggtgcaccacgtgggccgccccatcaagtccctgctggtggtgatctcctggatggtggtgatcatct tcggcgccctgaagttcctgcagtggtcctccctgctgtcctcctggaagggcaaggccttctccgccatcggcctgggcatcgcca ccctgctgatgcacgtgctggtggtgttctcccaggccgaccgctccaaccccgccaaggtgccccccgccaagctgaacaccga gctgtcctcctccaagaaggtgaccaacaaggagaacTGActcgag SEQIDNO:109CprocLPAAT2 ggtaccATGgccatccccgccgccgtggccgtgatccccatcggcctgctgttcatcatctccggcctgatcgtgaacctgatcca ggccgtggtgtacgtgctgatccgccccctgtccaagaacctgtaccgcaagatcaacaagcccatcgccgagctgctgtggctg gagctgatctggctggtggactggtgggccggcatcaaggtggaggtgtacgccgactccgagaccctggagtccatgggcaag gagcacgccctgctgatctgcaaccaccgctccgacatcgactggctggtgggctgggtgctggcccagcgcgcccgctgcctgg gctccgccctggccatcatgaagaagtccgccaagttcctgcccgtgatcggctggtccatgtggttctccgactacatcttcctgga ccgcacctgggagaaggacgagaagaccctgaagtccggcttcgagcgcctggccgacttccccatgcccttctggctggccct gttcgtggagggcacccgcttcaccaaggccaagctgctggccgcccaggagttcgccgcctcccgcggcctgcccgtgcccca gaacgtgctgatcccccgcaccaagggcttcgtgaccgccgtgacccacatgcgctcctacgtgcccgccatctacgactgcacc gtggacatctccaaggcccaccccgccccctccatcctgcgcctgatccgcggccagtcctccgtggtgaaggtgcagatcaccc gccactccatgcaggagctgcccgagacccccgacggcatctcccagtggtgcatggacctgttcgtgaccaaggacgccttcct ggagaagtaccactccaaggacatcttcggctccctgcccgtgcacgacatcggccgccccgtgaagtccctgatcgtggtgctgt gctggtactccctgatggccttcggcttctacaagttcttcatgtggtcctccctgctgtcctcctgggagggcatcctgtccctgggcct ggtgctgatcgtgatcgccatcgtgatgcagatcctgatccagtcctccgagtccgagcgctccacccccgtgaagtccgtgcaga aggacccctccaaggagaccctgctgcagaacTGActcgag SEQIDNO:110CavigGPAT9 ggtaccATGgccaccggcggctccctgaagccctcctcctccgacctggacctggaccaccccaacatcgaggactacctgcc ctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccgc cggcgccatcgtggacgactccttcacccgctgcttcaagtccatcccccgcgagccctggaactggaacctgtacctgttccccct gtggtgcatcggcgtgctgatccgctacttcatcctgttccccggccgcgtgatcgtgctgaccatgggctggatcaccgtgatctcct ccttcatcgccgtgcgcgtgctgctgaagggccacgacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tccttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttcttcatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctgc tgcagtccaccctgctggagtccgtgggctgcatctggttcgaccgcgccgaggccaaggaccgcggcatcgtggccaagaagc tgtgggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaacaactactccgtga tgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgccttctgg aactccaagaagcagtccttcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagcc ccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgcccgcgccggcctgaaga aggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaccttcgccgagtcc gtgctgcagcgcctggaggagTGActcgag SEQIDNO:111ChookGPAT9-1 ggtaccATGgccaccgccggctccctgaagccctcccgctccgagctggacttcgaccgccccaacatcgaggactacctgcc ctccggctcctccatcatcgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccgcc ggcgccatcgtggacgactccttcacccgctgcttcaagtccaacccccccgagccctggaactggaacatctacctgttccccct gtggtgcttcggcgtgctgatccgctacctgatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcatcttcctgtcctc cttcatccccgtgcacctgctgctgaagggccacgacgccctgcgcatcaagctggagcgcctgctggtggagctgatctgctcctt cttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaaccac acctccatgatcgacttcttcatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctgctg cagtccaccctgctggagtccgtgggctgcatctggttcgaccgcgccgaggccaaggaccgcggcatcgtggccaagaagctg tgggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaacaactactccgtgatg ttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgccttctggaa ctccaagaagcagtccttcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagcccc agaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctgaagaag gtgccctgggacggctacctgaagtactcccgcccctcccccaagcacaccgagcgcaagcagcagaacttcgccgagtccgt gctgcagcgcctggagaagaagTGActcgag SEQIDNO:112CignGPAT9-1 ggtaccATGgccaccggcggccgcctgaagccctcctcctccgagctggacctggaccgcgccaacaccgaggactacctgc cctccggctcctccatcaacgagcccgtgggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccg ccggcgccatcgtggacgactccttcacccgctgcttcaagtccatcccccccgagccctggaactggaacatctacctgttccccc tgtggtgcttcggcgtgctgatccgctacttcatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcaccgtgatctcct ccttcaccgccgtgcgcttcctgctgaagggccacaacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tccttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttcctgatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctg ctgcagtccaccctgctggagtccgtgggctgcatctggttcaaccgcgccgaggccaaggaccgcgagatcgtggccaagaag ctgtgggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaaccactactccgtg atgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgccttctg gaactcccgcaagcagtccttcaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagc cccagaccctgaagcccggcgagaccgccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctgaag aaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagtccaagcagcagtccttcgccgagtcc gtgctgcgccgcctggaggagaagTGActcgag SEQIDNO:113CignGPAT9-2 ggtaccATGgccaccggcggccgcctgaagccctcctcctccgagctggacctggaccgcgccaacaccgaggactacctgc cctccggctcctccatcaacgagcccgtgggcaagctgcgcctgcgcgacctgctggacatctcccccaccctgaccgaggccg ccggcgccatcgtggacgactccttcacccgctgcttcaagtccatcccccccgagccctggaactggaacatctacctgttccccc tgtggtgcttcggcgtgctgatccgctacttcatcctgttccccgcccgcgtgatcgtgctgaccatcggctggatcaccgtgatctcct ccttcaccgccgtgcgcttcctgctgaagggccacaacgccctgcagatcaagctggagcgcctgatcgtgcagctgctgtgctcc tccttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgccccaagcaggtgtacgtggccaacc acacctccatgatcgacttcctgatcctggaccagatgaccgtgttctccgtgatcatgcagaagcaccccggctgggtgggcctg ctgcagtccaccctgctggagtccgtgggctgcatctggttcaaccgcgccgaggccaaggaccgcgagatcgtggccaagaag ctgtgggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaaccactactccgtg atgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgccttctg gaactccaagaagcactccttcacccgccacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttggagc cccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccgacctgaag aaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaagttcgccgagtc cgtgctgcgccgcctggaggagaagTGActcgag SEQIDNO:114CpalGPAT9-1 ggtaccATGgccaccgccggccgcctgaagccctcctcctccgagctggagctggacctggaccgccccaacatcgaggact acctgccctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccatgctgaccga ggccgccggcgccatcgtggacgactccttcacccgctgcttcaagtccatcccccccgagccctggaactggaacatctacctgt tccccctgtggtgcttcggcgtgctgatccgctacctgatcctgttccccgcccgcgtgatcgtgctgaccgtgggctggatcaccgtg atctcctccttcatcaccgtgcgcttcctgctgaagggccacgactccctgcgcatcaagctggagcgcctgatcgtgcagctgttct gctcctccttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgcccccagcaggtgtacgtggcc aaccacacctccatgatcgacttcatcatcctgaaccagatgaccgtgttctccgccatcatgcagaagcaccccggctgggtggg cctgatccagtccaccatcctggagtccgtgggctgcatctggttcaaccgcgccgaggccaaggaccgcgagatcgtggccaa gaagctgctggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaaccactactc cgtgatgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgcct tctggaactccaagaagcagtccttcaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttgg agccccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctg aagaaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagtccttcgccga gtccgtgctgcgccgcctggagaagcgcTGActcgag SEQIDNO:115CpalGPATt9-2 ggtaccATGgccaccgccggccgcctgaagccctcctcctccgagctggagctggacctggaccgccccaacatcgaggact acctgccctccggctcctccatcaacgagcccgccggcaagctgcgcctgcgcgacctgctggacatctcccccatgctgaccga ggccgccggcgccatcgtggacgactccttcacccgctgcttcaagtccatcccccccgagccctggaactggaacatctacctgt tccccctgtggtgcttcggcgtgctgatccgctacctgatcctgttccccgcccgcgtgatcgtgctgaccgtgggctggatcaccgtg atctcctccttcatcaccgtgcgcttcctgctgaagggccacgactccctgcgcatcaagctggagcgcctgatcgtgcagctgttct gctcctccttcgtggcctcctggaccggcgtggtgaagtaccacggcccccgcccctccatccgcccccagcaggtgtacgtggcc aaccacacctccatgatcgacttcatcatcctgaaccagatgaccgtgttctccgccatcatgcagaagcaccccggctgggtggg cctgatccagtccaccatcctggagtccgtgggctgcatctggttcaaccgcgccgaggccaaggaccgcgagatcgtggccaa gaagctgctggaccacgtgcacggcgagggcaacaaccccctgctgatcttccccgagggcacctgcgtgaacaaccactactc cgtgatgttcaagaagggcgccttcgagctgggctgcaccgtgtgccccgtggccatcaagtacaacaagatcttcgtggacgcct tctggaactccaagaagctgtccttcaccatgcacctgctgcagctgatgacctcctgggccgtggtgtgcgacgtgtggtacttgg agccccagaccctgaagcccggcgagacccccatcgagttcgccgagcgcgtgcgcgacatcatctccgtgcgcgccggcctg aagaaggtgccctgggacggctacctgaagtactcccgcccctcccccaagcaccgcgagcgcaagcagcagaccttcgccg agtccgtgctgcgccgcctggaggagaagggcaacgtggtgcccaccgtgaacTGActcgag SEQIDNO:116CavigDGAT1 ggtaccATGgccatcgccgacggcggcatcatcggcgccgccggctccatctccgccctgaccgccgacaccgaccccccct ccctgcgccgccgcaacgtgcccgccggccaggcctccgccgtgtccgccttctccaccgagtccatggccaagcacctgtgcga cccctcccgcgagccctccccctcccccaagtcctccgacgacggcaaggaccccgacatcggctccgtggactccctgaacga gaagccctcctcccccgccgccggcaagggccgcctgcagcacgacctgcgcttcacctaccgcgcctcctcccccgcccaccg caaggtgaaggagtcccccctgtcctcctccaacatcttcaagcagtcccacgccggcctgttcaacctgtgcgtggtggtgctggt ggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggcctgctgatcaagaccggcttctggttctcctcccgctccct gcgcgactggcccctgttcatgtgctgcctgtccctgcccatcttccccctggccgccttcctggtggagaagctggcccagaagaa ccgcctgcaggagcccaccgtggtgtgctgccacgtgctgatcacctccgtgtccatcctgtaccccgtgctggtgatcctgcgctg cgactccgccgtgctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtcctacgcccactccaactac gacatgcgctacgtggccaagtccctggacaagggcgagcccgtggtggactccgtgatcgccgaccacccctaccgcgtgga ctacaaggacctggtgtacttcatggtggcccccaccctgtgctaccagctgtcctaccccctgaccccctgcgtgcgcaagtcctg gatcgcccgccaggtgatgaagctggtgctgttcaccggcgtgatgggcttcatcgtggagcagtacatcaaccccatcgtgcag aactccaagcaccccctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaacctgtacgtgtggc tgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgatctgcttcggcgaccgcgagttctacaaggactgg tggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgccacatctacttcccct gcctgcgcaacggcatcccccgcggcgtggccgtgctgatcgccttcctggtgtccgccgtgttccacgagctgtgcatcgccgtgc cctgccacgtgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctggtgtccaactgcctgcagaagaagtt ccagtcctccatggccggcaacatgttcttctggttcatcttctgcatcttcggccagcccatgtgcgtgctgctgtactaccacgacct gatgaaccgcaagggctcccgcatcgacTGActcgag SEQIDNO:117ChookDGAT1-1 ggtaccATGgccatcgccgacggcggctccgccggcgccgccggctccatctccggctccgacccctccccctccaccgcccc ctccctgcgccgccgcaacgcctccgccggccaggccttctccaccgagtccatggcccgcgacctgtgcgacccctcccgcga gccctccctgtcccccaagtcctccgacgacggcaaggaccccgccgacgacatcggcgccgccgactccgtggactccggcg gcgtgaaggacgagaagccctcctcccaggccgccgccaaggcccgcctggagcacgacctgcgcttcacctaccgcgcctcc tcccccgcccaccgcaaggtgaaggagtcccccctgtcctcctccaacatcttcaagcagtcccacgccggcctgttcaacctgtg cgtggtggtgctggtggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggcctgctgatcaagaccggcttctggtt ctcctcccgctccctgcgcgactggcccctgttcatgtgctgcctgtccctgcccatcttccccctggccgccttcctggtggagaagc tggcccagaagaaccgcctgcaggagcccaccgtggtgtgctgccacgtgatcatcacctccgtgtccatcctgtaccccgtgctg gtgatcctgcgctgcgactccgccgtgctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtcctacg cccacgccaactacgacatgcgctccgtggccaagtccctggacaagggcgagaccgtggccgactccgtgatcgtggaccac ccctaccgcgtggactacaaggacctggtgtacttcatggtggcccccaccctgtgctaccagctgtcctaccccctgaccccctac gtgcgcaagtcctgggtggcccgccaggtgatgaagctggtgctgttcaccggcgtgatgggcttcatcgtggagcagtacatcaa ccccatcgtgcagaactccaagcaccccctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaa cctgtacgtgtggctgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgacctgcttcggcgaccgcgagt tctacaaggactggtggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgc cacatctacttcccctgcctgcgcaacggcatcccccgcggcgtggccgtgctgatcgccttcctggtgtccgccgtgttccacgag ctgtgcatcgccgtgccctgccacgtgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctggtgtccaactg cctgcagaagaagttccagtcctccatggccggcaacatgttcttctggttcatcttctgcatcttcggccagcccatgtgcgtgctgct gtactaccacgacctgatgaaccgcaagggctcccgcatcgacTGActcgag SEQIDNO:118CavigLPCAT ggtaccATGggcctggtgtccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctggccaccat ccccgtgtccttcctgtggcgcctggtgcccggccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgtcctacct gtccttcggcgcctcctccaacctgcacttcatcgtgcccatgaccctgggctacctgtccatgctgttcttccgccccttctccggcct gctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcggcatcg acgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatgaactacaacgacggcctgctgaaggaggagg gcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacttcggctactgcctgtgctgcggctc ccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcatctggtcccgctcccagaagg agcccaagccctcccccttcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgtacctgtacctggtgccc caccaccccctgacccgcttcaccgagcccgtgtactacgagtggggcttcttccgccgcctgtcctaccagtacatggccgccctg accgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggaccgagt cctccccccccaagccccgctgggaccgcgccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgcagctgc ccctggtgtggaacatccaggtgtccatctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccccggctt cttccagctgctggccacccagaccgtgtccgccgtgtggcacggcctgtaccccggctacatcatcttcttcgtgcagtccgccctg atgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccccaagatgggcctggtgaagaacatcttcgtgttctt caacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcctacgg ctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccgcccgctccaa ggcccacaaggagcagTGActcgag SEQIDNO:119CpalLPCAT ggtaccATGgagctgggctccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctggccaccat ccccgtgtccttcctgtggcgcctggtgcccggccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgtcctacct gtccttcggcccctcctccaacctgcacttcatcgtgcccatgaccctgggctacctgtccatgctgttcttccgccccttctccggcct gctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcggcatcg acgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggaggagg gcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacatcggctactgcctgtgctgcggctc ccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcgtgtggtcccactccgagaagg agcccaagccctcccccttcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgtacatgtacctggtgccc caccaccccctgtcccgcttcaccgagcccgtgtactacgagtggggcttcttccgccgcctgtcctaccagtacatggccggcctg accgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggaccgagt cctccccccccaagccccgctgggaccgcgccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgcagctgc ccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccccggctt cttccagctgctggccacccagaccgtgtccgccatctggcacggcctgtaccccggctacatcatcttcttcgtgcagtccgccctg atgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccccaagatgggcctggtgaagaacatcttcgtgttctt caacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcctacgg ctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccgcccgctccaa ggcccacaaggagcagTGActcgag SEQIDNO:120CpauLPCAT ggtaccATGgagctggagatcggctccgtggccgccgccatcggcgtgtccgtgcccgtggcccgcttcctgctgtgcttcctgg ccaccatccccgtgtccttcctgtgccgcctgctgcccgcccgcctgcccaagcacctgtactccgccgcctccggcgccatcctgt cctacctgtccttcggcccctcctccaacctgcacttcatcgtgcccatgtccctgggctacctgtccatgctgttcttccgccccttctcc ggcctgctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcgg catcgacgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggag gagggcctgcgcgagtcccagaagaagaaccgcctgaccaagatgccctccctgatcgagtacttcggctactgcctgtgctgcg gctcccacttcgccggccccgtgtacgagatgaaggactacctggagtggaccgagggcaagggcatctggtcccgctccgaga aggaccccaagccctcccccttcggcggcgccctgcgcgccatcatccaggccgccgtgtgcatggccatgcacatgtacctggt gccccaccaccccctgacccgcttcaccgagcccgtgtactacgagtggggcttcttccgccgcctgtcctaccagtacatggccg cccagaccgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggac cgagtcctccccccccaagccccgctgggacaaggccaagaacgtggacatcatcggcgtggagttcgccaagtcctccgtgca gctgcccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccc cggcttcttccagctgctggccacccagaccgtgtccgccgtgtggcacggcctgtaccccggctacatcatcttcttcgtgcagtcc gccctgatgatcgccggctcccgcgtgatctaccgctggcagcaggccgtgccccagaagatgggcctggtgaagaacatcttcg tgttcttcaacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctcc tacggctccgtgtactacatcggcaccatcctgcccatcaccctgatcctgctgtcctacgtgatcaagcccggcaagcccacccg ctccaaggtgcacaaggagcagTGActcgag SEQIDNO:121CschuLPCAT ggtaccATGgagctggagatggagcccctggccgccgccatcggcgtgtccgtggccgtgttccgcttcctggtgtgcttcatcg ccaccatccccgtgtccttcatctgccgcctggtgcccggcggcctgccccgccacctgttctccgccgcctccggcgccgtgctgtc ctacctgtccttcggcttctcctccaacctgcacttcctggtgcccatgaccctgggctacctgtccatgatcctgttccgccgcttctgc ggcatcctgaccttcttcctgggcttcggctacctgatcggctgccacgtgtactacatgtccggcgacgcctggaaggagggcgg catcgacgccaccggcgccctgatggtgctgaccctgaaggtgatctcctgctccatcaactacaacgacggcctgctgaaggag gagggcctgcgcgagtcccagaagaagaaccgcctgatccgcctgccctccctgatcgagtacttcggctactgcctgtgctgcg gctcccacttcgccggccccgtgtacgagatgaaggactacctggactggaccgagggcaagggcatctggtcccactccgaga agggccccaagccctcccccctgcgcgccgccctgcgcgccatcatccaggccggcttctgcatggccatgtacctgtacctggtg ccccactaccccctgacccgcttcaccgaccccgtgtactacgagtggggcatcctgcgccgcctgtcctaccagtacatggcctc cttcaccgcccgctggaagtactacttcatctggtccatctccgaggcctccctgatcatctccggcctgggcttctccggctggacc gagtcctccccccccaagccccgctgggaccgcgccaagaacgtggacatcctgggcgtggagctggccaagtcctccgtgca gatccccctggtgtggaacatccaggtgtccacctggctgcgccactacgtgtacgaccgcctggtgcagaacggcaagcgccc cggcttcctgcagctgctggccacccagaccgtgtccgccatctggcacggcgtgtaccccggctacctgatcttcttcgtgcagtcc gccctgatgatcgccggctcccgcgccatctaccgctggcagcaggccgtgccccccaagatgtccctggtgaagaacaccctg gtgttcttcaacttcgcctacaccctgctggtgctgaactactccgccgtgggcttcatggtgctgtccatgcacgagaccctggcctc ctacggctccgtgtactacgtgggcaccatcctgcccgtgaccctgatcctgctgggctacgtgatcaagcccggcaagtcccccc gctccaaggcctccaaggagcagTGActcgag SEQIDNO:122CavigPLA2-1 ggtaccATGaacttcgacttcctgtccaacatcccctggttcggcgccaaggcctccgacaacgccggctcctccttcggctccg ccaccatcgtgatccagcagcccccccccgtgtcccgcggcttcgacatccgccactggggctggccctggtccgtgctgtccgtg ctgccctggggcaagcccggctgcgacgagctgcgcgccccccccaccaccatcaaccgccgcctgaagcgcaacgccacct ccatgcactcctccgccgtgcgcggcaacgccgaggccgcccgcgtgcgcttccgcccctacgtgtccaaggtgccctggcaca ccggcttccgcggcctgctgtcccagctgttcccccgctacggccactactgcggccccaactggtcctccggcaagaacggcgg ctcccccgtgtgggaccagcgccccatcgactggctggactactgctgctactgccacgacatcggctacgacacccacgacca ggccaagctgctggaggccgacctggccttcctggagtgcctggagcgcccctcctaccccaccaagggcgacgcccacgtgg cccacatgtacaagaccatgtgcgtgaccggcctgcgcaacgtgctgatcccctaccgcacccagctgctgcgcctgaactcccg ccagcccctgatcgacttcggctggctgtccaacgccgcctggaagggctggaacgcccagaagtccTGActcgag SEQIDNO:123CiPLA2-1 ggtaccATGaacctggacttcctgtccaagatcccctggttcgaggccaaggcctccgagaaccccggcctgaacctgggctcc accaccatcgtgatcaagcagccccgccagggcttcgacatccgccactggggctggccctggtccgtgctgacctggggcaac cgcgtgaccgacgaggtgcacgccccccccaccaccatcaaccgccgcctgaagcgcaacgccaccggccccgccgtgcag ggcgacaccgaggccgcccgcctgcgcttccgcccctacgtgtccaaggtgccctggcacaccggcttccgcggcctgctgtccc agctgttcccccgctacggccactactgcggccccaactggtcctccggcaagaacggcggctcccccgtgtgggaccagcgcc ccatcgactggctggactactgctgctactgccacgacatcggctacgacacccacgaccaggccaagctgctggaggccgacc tggccttcctggagtgcctggagcgcccctcctaccccaccaccggcgacgcccacgtggcccacatgtacaagaccatgtgcgt gaccggcctgcgcaacgtgctgatcccctaccgcacccagctgctgcgcctgaacttccgccagcccctgatcgacttcggctggc tgtccaacgccgcctggaagggctggtccgcccagaagaccTGActcgag SEQIDNO:124CuPSR23PLA2-2 ggtaccATGgtgcacctgccccacaccctgaagctgggcctggtgatcgccatctccatctccggcctgtgcttctcctccacccc cgcccgcgccctgaacgtgggcatccaggccgccggcgtgaccgtgtccgtgggcaagggctgctcccgcaagtgcgagtccg acttctgcaaggtgccccccttcctgcgctacggcaagtactgcggcctgatgtactccggctgccccggcgagaagccctgcgac ggcctggacgcctgctgcatgaagcacgacgcctgcgtgcaggccaagaacaacgactacctgtcccaggagtgctcccagaa cctgctgaactgcatggcctccttccgcatgtccggcggcaagcagttcaagggctccacctgccaggtggacgaggtggtggac gtgctgaccgtggtgatggaggccgccctgctggccggccgctacctgcacaagcccTGActcgag SEQIDNO:125CprocPLA2-2 ggtaccATGgtgcacctgccccacaccctgaagctgggcctggtgatcgccatctccatctccggcctgtgcctgtcctccacccc cgcccgcgccctgaacgtgggcatccaggccgccggcgtgaccgtgtccgtgggcaagggctgctcccgcaagtgcgagtccg acttctgcaaggtgccccccttcctgcgctacggcaagtactgcggcctgatgtactccggctgccccggcgagaagccctgcgac ggcctggacgcctgctgcatgaagcacgacgcctgcgtgcaggccaagaacgacgactacctgtcccaggagtgctcccagaa cctgctgaactgcatggcctccttccgcatgtccggcggcaagcagttcaagggctccacctgccaggtggacgaggtggtggac gtgctgaccgtggtgatggaggccgccctgctggccggccgctacctgcacaagcccTGActcgag

[0155] The constructs containing the codon optimized genes described above driven by the UTEX 1453 SAD2 promoter, were transformed into strain S57858 or S8714. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as described herein. The transgenic strains were selected for their ability to grow on melibiose. Stable transformants were grown under standard lipid production conditions at pH5 (for transgenic strains generated in the strain S7858) or at pH7 (for the transgenic strains generated in the strain S8174) for fatty acid analysis.

Expression of LPAATs

[0156] In WO2013/158938 we disclosed that Cocos nucifera LPAAT enzymes exhibit chain length specificity for the fatty acid acyl-CoA that it attach to the glycerol backbone. We disclosed the impact of expressing CnLPAAT in a transgenic strain also expressing a laurate specific thioesterase. In this example we transformed 5 LPAAT enzymes derived from C8-C10 rich Cuphea species and the CnLPAAT into S7858, and the remaining 8 LPAAT enzymes were transformed into S8174. The resulting fatty acid profiles from a set of representative transgenic lines arising from these transformations are shown in Tables 16 and 17. Expression of these genes as shown in Table 16 resulted in increases in C8:0 and/or -C10:0 fatty acid accumulation.

TABLE-US-00035 TABLE 16 Fatty acid profiles of representative transgenic strains of S7858 expressing optimized versions of the CpauLPAAT1, CpalLPAAT1, CignLPAAT1, CprocLPAAT1, ChookLPAAT1 and CnLPAAT1. Sample ID C8:0 C10:0 C12:0 C8-C10 S6165 0.00 0.00 0.05 0.00 S7858 11.70 23.36 0.48 35.06 CpauLPAAT1 @ SAD2-1vD locus S7858; D4289-7 12.69 25.06 0.51 37.75 S7858; D4289-12 11.98 24.54 0.48 36.52 S7858; D4289-2 11.68 24.14 0.49 35.82 S7858; D4289-13 11.53 24.18 0.49 35.71 S7858; D4289-11 11.47 23.85 0.46 35.32 CpaiLPAAT1 @ SAD2-1vD locus S7858; D4290-3 13.43 25.04 0.52 38.47 S7858; D4290-25 12.98 24.75 0.51 37.73 S7858; D4290-5 12.27 25.00 0.52 37.27 S7858; D4290-12 11.98 24.21 0.48 36.19 S7858; D4290-22 11.91 23.86 0.49 35.77 CignLPAAT1 @ SAD2-1vD locu S7858; D4291-13 12.95 24.78 0.52 37.73 S7858; D4291-20 12.13 24.63 0.49 36.76 S7858; D4291-15 12.12 24.35 0.47 36.47 S7858; D4291-22 11.94 24.50 0.47 36.44 S7858; D4291-7 12.11 23.14 0.50 35.25 CprocLPAAT1 @ SAD2-1vD locus S7858; D4292-15 11.86 24.05 0.46 35.91 S7858; D4292-11 11.49 24.01 0.48 35.50 S7858; D4292-22 11.49 23.81 0.47 35.30 S7858; D4292-3 11.46 23.76 0.46 35.22 S7858; D4292-24 11.38 23.64 0.46 35.02 ChookLPAAT1 @ SAD2-1vD locus S7858; D4293-4 11.09 24.48 0.51 35.57 S7858; D4293-16 12.03 24.24 0.48 36.27 S7858; D4293-6 11.83 23.79 0.48 35.62 S7858; D4293-2 11.81 23.69 0.47 35.50 S7858; D4293-12 11.65 23.11 0.49 34.76 CnLPAAT1 @ SAD2-1vD locus S7858; D4404-11 12.30 24.31 0.47 36.61 S7858; D4404-6 12.03 24.02 0.46 36.05 S7858; D4404-13 11.48 23.98 0.46 35.46 S7858; D4404-2 11.54 23.71 0.46 35.25 S7858; D4404-1 11.76 23.36 0.48 35.12

TABLE-US-00036 TABLE 17 Fatty acid profiles of representative transgenic strains of S8174 expressing CavigLPAAT1, CavigLPAAT2, CpalLPAAT1, CuPSR23LPAAT1, CkoeLPAAT1, CkoeLPAAT2, CprocLPAAT1 and CprocLPAAT2 before lipase treatment. Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.32 9.24 0.37 33.56 CavigLPAAT1 @ SAD2-1vD locus S8174: D4517-23 25.42 9.63 0.39 35.05 S8174: D4517-9 25.44 9.61 0.39 35.05 S8174: D4517-8 25.09 9.84 0.39 34.93 S8174: D4517-18 25.20 9.65 0.39 34.85 S8174: D4517-2 25.20 9.57 0.37 34.77 CavigLPAAT2 @ SAD2-1vD locus S8174: D4518-2 24.25 9.97 0.42 34.22 S8174: D4518-45 24.09 9.65 0.39 33.74 S8174: D4518-34 23.94 9.71 0.38 33.65 S8174: D4518-10 24.11 9.50 0.37 33.61 S8174: D4518-4 23.93 9.59 0.39 33.52 CpalLPAAT1 @ SAD2-1vD locus S8174: D4519-27 25.06 9.75 0.37 34.81 S8174: D4519-4 23.05 10.74 0.47 33.79 S8174: D4519-28 24.11 9.54 0.37 33.65 S8174: D4519-10 23.57 9.51 0.38 33.08 S8174: D4519-12 23.55 9.49 0.38 33.04 CuPSR23LPAAT2-1 @ SAD2-1vD locus S8174; D4690-2 25.88 10.62 0.43 36.50 S8174; D4690-1 24.60 9.82 0.44 34.42 S8174; D4690-3 24.13 9.62 0.47 33.75 S8174; D4690-4 23.38 9.97 0.41 33.35 CkoeLPAAT1 @ SAD2-1vD locus S8174; D4728-8 25.44 10.31 0.46 35.75 S8174; D4728-10 24.15 9.51 0.43 33.66 S8174; D4728-5 23.88 9.56 0.45 33.44 S8174; D4728-6 23.58 9.28 0.40 32.86 S8174; D4728-9 23.47 9.25 0.40 32.72 CkoeLPAAT2-1 @ SAD2-1vD locus S8174; D4729-2 25.20 9.81 0.43 35.01 S8174; D4729-1 23.49 10.60 0.46 34.09 S8174; D4729-4 22.25 9.45 0.40 31.70 S8174; D4729-5 18.24 8.22 0.35 26.46 CprocLPAAT2 @ SAD2-1vD locus S8174: D4730-14 24.97 9.92 0.41 34.89 S8174: D4730-13 23.26 10.72 0.49 33.98 S8174: D4730-1 23.79 10.15 0.49 33.94 S8174: D4730-7 23.42 10.13 0.36 33.55 S8174: D4730-5 23.69 9.49 0.42 33.18 CuPSR23LPAAT4 @ SAD2-1vD locus S8174; D4731-1 25.94 10.87 0.56 36.81 S8174; D4731-3 22.79 11.52 0.59 34.31 S8174; D4731-5 22.89 11.22 0.53 34.11 S8174; D4731-2 22.99 11.07 0.45 34.06 S8174; D4731-4 21.15 9.63 0.43 30.78

[0157] To assess the regiospecific activity of novel LPAAT enzymes, oil extracted from some of these transformants were treated with porcine pancreatic lipase, which selectively hydrolyzes the fatty acids at the sn-1 and sn-3 positions from the glycerol unit of the triacylglycerol, leaving monoacylglycerols (MAGs) with fatty acids located only at the sn-2 position. The resulting mixture of monoacylglycrols (2-MAGs), were isolated by solid phase extraction on an amino propyl cartridge followed by transesterifcation to generate fatty acid methyl esters (FAMEs). The fatty acid profiles of these FAMEs, which represent the profile of fatty acids at the sn-2 position of the various TAGs, were determined by GC-FID. When compared to the fatty acid profiles from transesterification of the oil without lipase treatment, the sn-2 fatty acid profiles show that the expressed LPAAT are selective for the sn-2 position.

[0158] The sn-2 analyses after lipase treatment disclosed in Table 18 show that CavigLPAAT1, CpaiLPAAT exhibit selectivity for either C8:0 fatty acids and CpauLPAAT, CignLPAAT are selective for C10:0 fatty acids, demonstrating that the heterologous LPAATs expressed in these transgenic strains have activities that acylate at the sn-2 position with preference for C8:0 or C10:0.

TABLE-US-00037 TABLE 18 Fatty acid profiles & sn-2 analysis of representative transgenic strains of S7858 & S8174 expressing codon optimized versions of the CnLPAAT1, CpauLPAAT1, CpaiLPAAT1, CignLPAAT1, ChookLPAAT1 and CavigLPAAT1, CavigLPAAT2, CpalLPAAT1 pH 5; S7858; pH 5; S7858; pH 5; S7858; pH 5; S7858; pH 5; S7858 D4404-2; D4289-2 D4290-5 D4291-7 Fatty Acid FA profile sn-2 FA profile sn-2 FA profile sn-2 FA profile sn-2 FA profile sn-2 C8:0 11.08 8.6 13.54 6.8 11.68 8.1 12.27 10.5 12.11 7.4 C10:0 23.58 20.3 25.04 20.5 24.14 28.2 25.00 13.7 23.14 31.9 C12:0 0.47 0.2 0.49 0.2 0.49 0.2 0.52 0.2 0.50 0.2 C14:0 1.19 0.7 1.19 0.7 1.29 0.7 1.39 0.8 1.38 0.6 C16:0 11.63 1.2 10.28 1.0 12.57 1.2 12.72 1.5 12.63 1.2 C18:0 1.56 0.3 1.52 0.2 3.61 0.7 5.41 0.7 4.15 0.6 C18:1 44.49 63.1 42.25 63.1 39.69 52.9 38.50 63.2 39.50 50.2 C18:2 4.78 6.4 4.54 8.4 5.01 6.5 4.85 7.9 5.23 6.4 C18:3 0.31 0.7 0.25 0.7 0.50 1.0 0.54 1.2 0.49 1.2 CnLPAAT CpauLPAAT CpaiLPAAT CignLPAAT pH 7; S8174; pH 7; S8174; pH 7; S8174; pH 7; S8174 D4517-23; D4518-45; D4519-28; Fatty Acid FA profile sn-2 FA profile sn-2 FA profile sn-2 FA profile sn-2 C8:0 25.24 15.9 26.04 25.1 25.04 17.8 24.75 16.0 C10:0 9.33 8.8 9.02 7.2 9.01 9.0 8.94 8.7 C12:0 0.44 0.2 0.41 0.2 0.40 0.2 0.39 0.2 C14:0 2.48 1.4 2.45 1.2 2.45 1.4 2.45 1.4 C16:0 13.88 1.1 13.91 1.1 14.19 1.2 14.38 1.1 C18:0 1.33 0.3 3.43 0.4 3.35 0.4 3.52 0.4 C18:1 37.50 62.0 35.36 55.1 38.86 59.7 38.94 81.2 C18:2 8.52 8.4 5.87 8.0 6.08 8.4 6.14 9.1 C18:3 0.65 1.3 0.53 1.3 0.58 1.3 0.58 1.5 CavigLPAAT1 CavigLPAAT2 CpalLPAAT1

Expression of GPATs, DGATs, LPCATs and PLA2s:

[0159] The constructs expressing the other acyltransferases (GPAT, DGAT, LPCAT, and PLA2) were transformed into S8174. Stable transformants were grown under standard lipid production conditions at pH7 and analyzed for fatty acid profiles. Similar to the transgenic lines expressing LPAATs, expression of these genes (GPAT, DGAT, LPCAT, and PLA2) also resulted in increases in C8:0-C10:0 fatty acid accumulation (Tables 19a, 19b, and 20). The data presented shows that we have identified novel GPATs, DGATs, LPCATs and PLA2s that show high specificity for C8-C10 fatty acids. To determine the regiospecificity of the novel GPAT, DGAT, LPCAT, and PLA2 enzymes, sn-2 analysis is performed as disclosed in this example and elsewhere herein.

TABLE-US-00038 TABLE 19a Fatty acid profiles of representative transgenic strains of S8174 expressing GPATs and DGATs Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.61 9.10 0.42 33.71 CavigGPAT9 @ SAD2-1vD locus S8174; D4551-8 24.52 9.05 0.36 33.57 S8174; D4551-7 24.24 9.04 0.36 33.28 S8174; D4551-2 23.93 8.92 0.37 32.85 S8174; D4551-6 23.63 8.92 0.41 32.55 S8174; D4551-3 23.35 8.90 0.43 32.25 ChookGPAT9-1 @ SAD2-1vD locus S8174; D4552-6 23.57 9.00 0.36 32.57 S8174; D4552-4 23.62 8.87 0.37 32.49 S8174; D4552-9 23.39 8.97 0.40 32.36 S8174; D4552-8 23.28 8.80 0.40 32.08 S8174; D4552-11 23.18 8.80 0.44 31.98 CignGPAT9-1 @ SAD2-1vD locus S8174; D4553-12 25.19 9.42 0.40 34.61 S8174; D4685-1 24.33 10.24 0.46 34.57 S8174; D4553-15 25.11 9.33 0.41 34.44 S8174; D4553-1 24.56 9.50 0.44 34.06 S8174; D4553-6 24.74 9.16 0.40 33.90 CignGPAT9-2 @ SAD2-1vD locus S8174; D4554-9 24.49 9.13 0.45 33.62 S8174; D4554-3 24.28 8.90 0.42 33.18 S8174; D4554-7 23.86 8.96 0.43 32.82 S8174; D4554-8 23.99 8.81 0.39 32.80 S8174; D4554-4 23.87 8.78 0.4 32.65 CpalGPAT9-1 @ SAD2-1vD locus S8174; D4724-6 25.61 9.52 0.39 35.13 S8174; D4724-7 24.91 9.36 0.41 34.27 S8174; D4724-2 24.43 9.46 0.39 33.89 S8174; D4724-5 24.01 9.25 0.39 33.26 S8174; D4724-4 24.30 8.93 0.39 33.23 CpalGPAT9-2 @ SAD2-1vD locus S8174; D4725-5 24.24 10.30 0.48 34.54 S8174; D4725-6 24.81 9.29 0.41 34.10 S8174; D4725-7 24.35 9.51 0.42 33.86 S8174; D4725-8 24.37 9.39 0.40 33.76 S8174; D4725-9 24.28 9.29 0.41 33.57

TABLE-US-00039 TABLE 19b Fatty acid profiles of representative transgenic strains of S8174 expressing DGATs Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.61 9.10 0.42 33.71 Cavig DGAT1 @ SAD2-1vD locus S8174; D4549-7 24.89 9.28 0.36 34.17 S8174; D4549-6 24.53 9.04 0.47 33.57 S8174; D4549-4 23.93 8.99 0.41 32.92 S8174; D4549-1 23.93 8.97 0.38 32.90 S8174; D4549-3 23.76 8.9 0.36 32.66 Chook DGAT1 @ SAD2-1vD locus S8174; D4550-1 24.67 9.12 0.41 33.79 S8174; D4550-3 24.64 9.06 0.42 33.70 S8174; D4682-1 23.72 9.68 0.5 33.40 S8174; D4682-2 23.49 9.66 0.41 33.15 S8174; D4550-2 22.42 8.81 0.41 31.23

TABLE-US-00040 TABLE 20 Fatty acid profiles of representative transgenic strains of S8174 expressing LPCATs and PLA2s Sample ID C8:0 C10:0 C12:0 C8-C10 S7485 0.00 0.00 0.07 0.00 S8174 24.61 9.10 0.42 33.71 Cavig LPCAT @ SAD2-1vD locus S8174; D4555-1 26.6 9.38 0.47 35.98 S8174; D4555-3 26.4 9.47 0.39 35.87 S8174; D4688-1 25.95 9.67 0.44 35.62 S8174; D4688-3 25.47 9.89 0.44 35.36 S8174; D4555-2 25.52 9.55 0.36 35.07 Cpau LPCAT @ SAD2-1vD locus S8174; D4556-3 25.55 9.21 0.43 34.76 S8174; D4556-4 25.24 9.46 0.41 34.70 S8174; D4689-7 24.63 9.86 0.43 34.49 S8174; D4556-1 25.18 9.13 0.42 34.31 S8174; D4689-6 24.05 9.89 0.48 33.94 Cpal LPCAT @ SAD2-1vD locus S8174; D4726-4 26.34 9.76 0.41 36.10 S8174; D4726-2 25.92 9.9 0.44 35.82 S8174; D4726-3 26.15 9.62 0.41 35.77 S8174; D4726-5 26.09 9.55 0.41 35.64 S8174; D4726-1 25.64 9.57 0.39 35.21 Cschu LPCAT @ SAD2-1vD locus S8174; D4727-1 26.24 9.95 0.45 36.19 S8174; D4727-7 26.26 9.84 0.42 36.10 S8174; D4727-9 26.13 9.87 0.42 36.00 S8174; D4727-11 25.99 9.97 0.44 35.96 S8174; D4727-16 26.28 9.68 0.44 35.96 Cavig PLA2-1 @ SAD2-1vD locus S8174; D4732-1 26.31 11.24 0.60 37.55 S8174; D4732-2 25.30 11.88 0.50 37.18 S8174; D4732-3 25.29 11.01 0.48 36.30 S8174; D4732-4 25.30 11.00 0.47 36.30 S8174; D4732-5 25.07 11.20 0.44 36.27 CignPLA2-1 @ SAD2-1vD locus S8174; D4734-6 26.39 11.34 0.47 37.73 S8174; D4734-1 26.17 10.90 0.46 37.07 S8174; D4734-5 25.58 11.12 0.57 36.70 S8174; D4734-4 25.48 11.17 0.57 36.65 S8174; D4734-2 24.75 11.32 0.46 36.07 CuPSR23PLA2-2 @ SAD2-1vD locus S8174; D4735-5 25.81 11.16 0.44 36.97 S8174; D4735-1 25.95 10.92 0.47 36.87 S8174; D4735-8 25.54 10.91 0.42 36.45 S8174; D4735-7 25.45 10.95 0.44 36.40 S8174; D4735-6 25.51 10.88 0.41 36.39 Cproc PLA2-2 @ SAD2-1vD locus S8174; D4736-2 25.60 10.87 0.42 36.47 S8174; D4736-4 25.55 10.76 0.40 36.31 S8174; D4736-3 25.40 10.87 0.36 36.27 S8174; D4736-5 25.45 10.46 0.39 35.91 S8174; D4736-1 24.34 11.06 0.48 35.40

Example 7: Expression of LPAAT and/or DGAT in Prototheca to Produce High SOS and Low Trisaturated Tags

[0160] In this example we describe genetically engineered Prototheca moriformis strains in which we have modified fatty acid and triacylglycerol biosynthesis to maximize the accumulation of Stearoyl-Oleoyl-Stearoyl (SOS) TAGs, and minimize the production of trisaturated TAGs. Tailored oils from these strains resemble plant seed oils known as structuring fats, which have high proportions of Saturated-Oleate-Saturated TAGs and low levels of trisaturates. These structuring fats (often called butters) are generally solid at room temperature but melt sharply between 35-40 C.

[0161] High-SOS strains were obtained by three successive transformations beginning with strain S5100, a classically improved derivative, of a wild type isolate of Prototheca moriformis, S376. Strain S5100 was transformed with plasmid pSZ5654 to generate strain S8754, which produces an oil with increased stearic acid (C18:0) content, lower palmitic acid (C16:0) and reduced linoleic acid (C18:2 cis9,12) content relative to S5100. In turn, strain S8754 was transformed with plasmid pSZ5868 to generate strain S8813, which produces oil with higher C18:0, lower C16:0 and improved sn-2 selectivity compared to S8754. Finally, strain S8813 was transformed with plasmids pSZ6383 or pSZ6384 to generate strains S9119, S9120 and S9121, producing oils rich in C18:0 with reduced levels of C18:2 cis9,12 and improved sn-3 selectivity.

[0162] Construct used for SAD2 knockout in S5100

[0163] The first intermediate strains were prepared by transformation of strain S5100 with integrative plasmid pSZ5654 (SAD2-1vD::PmKASII-1tp_PmKASII-1_FLAG-CvNR:CrTUB2-PmFAD2hpA-CvNR:PmHXT1-2v2-ScarMEL1-PmPGK::SAD2-1vE). The construct targeted ablation of allele 1 of the endogenous stearoyl-ACP desaturase 2 gene (SAD2), concomitant with expression of the PmKASII gene encoding P. moriformis -keto-acyl-ACP synthase, and a RNAi hairpin sequence to down-regulate fatty acid desaturase (FAD2) gene expression. Deletion of one allele of SAD2 reduced SAD activity, resulting in elevated levels of C18:0. Overexpression of PmKASII stimulated elongation of C16:0 to C18:0, further increasing C18:0. FAD2 is responsible for the conversion of C18:1 cis9 (oleic) to C18:2 cis9,12 (linoleic) fatty acids, and RNAi of FAD2 resulted in decreased C18:2. Thus, the first intermediate strains had higher levels of C18:0 and decreased C16:0 and C18:2 fatty acid levels relative to the S5100 parent. The Saccharomyces carlsbergensis MEL1 gene, encoding a secreted melibiase served as a selectable marker as part of plasmid pSZ5654, enabling the strain to grow on melibiose.

[0164] The sequence of the pSZ5654 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlining and are 5-3 PmeI, SpeI, AscI, ClaI, SacI, AvrII, EcoRV, EcoRI, SpeI, BsiWI, XhoI, SacI, KpnI, SnaBI, BspQI and PmeI, respectively. PmeI sites delimit the 5 and 3 ends of the transforming DNA. Bold, lowercase sequences represent SAD2-1 5 genomic DNA that permit targeted integration at the SAD2-1 locus via homologous recombination. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent SAD2-1 5 genomic DNA sequences that permit targeted integration at the FATA-1 locus via homologous recombination. The initiator ATG of the sequence encoding the P. moriformis KASII-1 transit peptide (PmKASII-1tp) is indicated by uppercase, bold italics, and the PmKASII-1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The PmKASII-1 coding region is indicated by lowercase italics. A sequence encoding a 3 FLAG tag fused to the C-terminus of PmKASII-1 is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The Chlorella vulgaris nitrate reductase (NR) gene 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The C. reinhardtii TUB2 promoter, driving expression of the PmFAD2hpA sequence is indicated by boxed text. Bold italics denote the PmFAD2hpA sequence followed by lowercase underlined text representing C. vulgaris nitrate reductase 3 UTR. A second spacer sequence is represented by lowercase text. The P. moriformis HXT1 promoter driving the expression of the S. carlbergensis MEL1 gene is indicated by boxed text. The initiator ATG and terminator TGA for MEL1 gene are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis PGK 3 UTR is indicated by lowercase underlined text. The SAD2-1 3 genomic region indicated by bold, lowercase text.

TABLE-US-00041 SEQIDNO:126NucleotidesequenceoftransformingDNAcontainedin pSZ5654 gtttaaacgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacgg aaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgaca cgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagc agttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcg caggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcgg aacccagctttcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttgg cgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcagg catcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtg gcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgctaacgctcccgactc tcccgaccgcgcgcaggatagactcttgttcaaccaatcgacaactagtATGcagaccgcccaccagcgcccccccaccgagg gccactgcttcggcgcccgcctgcccaccgcctcccgccgcgccgtgcgccgcgcctggtcccgcatcgcccgcgggcgcgccgc cgccgccgccgacgccaaccccgcccgccccgagcgccgcgtggtgatcaccggccagggcgtggtgacctccctgggccag accatcgagcagttctactcctccctgctggagggcgtgtccggcatctcccagatccagaagttcgacaccaccggctacacc accaccatcgccggcgagatcaagtccctgcagctggacccctacgtgcccaagcgctgggccaagcgcgtggacgacgtga tcaagtacgtgtacatcgccggcaagcaggccctggagtccgccggcctgcccatcgaggccgccggcctggccggcgccgg cctggaccccgccctgtgcggcgtgctgatcggcaccgccatggccggcatgacctccttcgccgccggcgtggaggccctgac ccgcggcggcgtgcgcaagatgaaccccttctgcatccccttctccatctccaacatgggcggcgccatgctggccatggacatc ggcttcatgggccccaactactccatctccaccgcctgcgccaccggcaactactgcatcctgggcgccgccgaccacatccgcc gcggcgacgccaacgtgatgctggccggcggcgccgacgccgccatcatcccctccggcatcggcggcttcatcgcctgcaag gccctgtccaagcgcaacgacgagcccgagcgcgcctcccgcccctgggacgccgaccgcgacggcttcgtgatgggcgagg gcgccggcgtgctggtgctggaggagctggagcacgccaagcgccgcggcgccaccatcctggccgagctggtgggcggcg ccgccacctccgacgcccaccacatgaccgagcccgacccccagggccgcggcgtgcgcctgtgcctggagcgcgccctggag cgcgcccgcctggcccccgagcgcgtgggctacgtgaacgcccacggcacctccacccccgccggcgacgtggccgagtaccg cgccatccgcgccgtgatcccccaggactccctgcgcatcaactccaccaagtccatgatcggccacctgctgggcggcgccgg cgccgtggaggccgtggccgccatccaggccctgcgcaccggctggctgcaccccaacctgaacctggagaaccccgcccccg gcgtggaccccgtggtgctggtgggcccccgcaaggagcgcgccgaggacctggacgtggtgctgtccaactccttcggcttc ggcggccacaactcctgcgtgatcttccgcaagtacgacgagATGGACTACAAGGACCACGACGGCGACTACAA GGACCACGACATCGACTACAAGGACGACGACGACAAGTGAatcgatgcagcagcagctcggatagtatcgaca cactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcc tcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctc gtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgca cagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtggga tgggaacacaaatggagagctccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcg gcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcg [00037] [00038] [00039] [00040] [00041] [00042] [00043] [00044] [00045] [00046] [00047] [00048] cacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacag cctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccc tcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcg cacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgg gatgggaacacaaatggaaagctgtagagctcgatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccat gtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggca [00049] [00050] [00051] [00052] [00053] [00054] [00055] ctacaacggcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgc tggacacggccgaccgcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctcc ggccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcaca acaactccttcctgttcggcatgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggagg aggacgcccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgctacaacaagggccagttcggcacgcc cgagatacctaccaccgctacaaggccatgtccgacgccctgaacaagacgggccgccccatcttctactccagtgcaactg gggccaggacctgaccttctactggggctccggcatcgcgaactcaggcgcatgtccggcgacgtcacggcggagttcacgc gccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgctccatcatgaacatcctga acaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgtcggcaac ctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtgaaca acctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcg cgtctggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggc gaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaac ctgggctccaagaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccat cctgggccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgac acccgcctgttcggccagaagatcggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacggcatcgcgttct accgcctgcgcccctcctccTGAtacaacttattacgtattctgaccggcgctgatgtggcgcggacgccgtcgtactctttcagact ttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgcaattaattgtgtgatgaagaaagggtggcacaaga tggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctggctcaatcttgtcgcatgtccggcgcaat gtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactgatcgcattgccatcccgtcaa ctcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaagcgtcaggaaatcg tctcggcagctggaagcgcatggaatgcggagcggagatcgaatcaggatccttagggagcgacgagtgtgcgtgcggggctggc gggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacga agaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaa gctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctc cgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgct cgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtcctgggcgaagaacgagggaatttg tgggtaaaacaagcatcgtctctcaggccccggcgcagtggccgttaaagtccaagaccgtgaccaggcagcgcagcgcgtccgt gtgcgggccctgcctggcggctcggcgtgccaggctcgagagcagctccctcaggtcgccttggacggcctctgcgaggccggtga gggcctgcaggagcgcctcgagcgtggcagtggcggtcgtatccgggtcgccggtcaccgcctgcgactcgccatccgaagagcg tttaaac

[0165] Construct pSZ5654 was transformed into S5100. Primary transformants were clonally purified and screened under standard lipid production conditions at pH 5. Integration of pSZ5654 at the SAD2-1 locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 21). 58754 was selected as the lead strain for additional rounds of genetic engineering. As shown in Table 21, C16:0 decreased from 17.6% to less than 6%, C18:0 increased from 4.3% to about 28%, C18:2 decreased from 5.8% to 1.3%.

TABLE-US-00042 TABLE 21 Fatty acid profiles of SAD2-1 ablation strains. Sample ID S5100 S8741 S8742 S8743 S8744 S8745 S8746 S8752 S8753 S8754 C14:0 0.7 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 C16:0 17.6 5.9 5.9 5.8 5.9 5.9 5.9 5.9 5.8 5.9 C16:1 cis-9 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 C18:0 4.3 28.2 28.1 27.7 27.8 27.4 28.2 28.3 28.3 28.1 C18:1 69.8 60.1 60.2 60.6 60.5 60.9 60.0 60.0 60.0 60.0 C18:2 5.8 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.2 1.3 C18:3 0.5 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 C20:0 0.3 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2 2.2 saturates 23.2 37.5 37.5 37.1 37.2 36.8 37.7 37.7 37.7 37.6 lipid (g/L) 13.5 12.8 12.5 12.5 12.5 12.3 12.3 12.3 12.4 12.3

Construct Used for FATA-1 Knockout in S8754

[0166] The second intermediate strains were prepared by transformation of strain S8754 with integrative plasmid pSZ5868 (FATA-1vB::CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1:PmG3PDH-1-TcLPAT2-PmATP:CrTUB2-ScSUC2-PmPGH::FATA-1vC). This construct targeted ablation of allele 1 of the endogenous fatty acyl-ACP thioesterase gene (FATA-1), and contained expression modules for GarmFATA1 (G108A), encoding a variant of the Garcinia mangostana FATA1 thioesterase with improved activity, and TcLPAT2 encoding the Theobroma cacao lysophosphatidic acid acyltransferase (LPAAT). Deletion of one copy of FATA-1 reduced endogenous thioesterase activity, further reducing C16:0 accumulation. Expression of GarmFATA1(G108A) stimulated C18:0-ACP hydrolysis, further increasing C18:0. TcLPAT2 had superior specificity for transfer of C18:1 to the sn-2 position of triacylglycerides than the endogeneous LPAAT, leading to reduced accumulation of trisaturates. The second intermediate strains had increased C18:0 and lower C16:0 compared their parent, S8754. The S. cerevisiae SUC2 gene encoding a secreted sucrose invertase, served as a selectable marker as part of plasmid pSZ5868 and enabled the strain to grow on sucrose.

[0167] The sequence of the pSZ5868 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlining and are 5-3 BspQI, PmeI, SpeI, AscI, ClaI, SacI, AvrII, NdeI, NsiI, AflII, KpnI, XbaI, MfeI, BamHI, BspQI and PmeI, respectively. BspQI and PmeI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FATA-1 5 genomic DNA that permit targeted integration at the FATA-1 locus via homologous recombination. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1 (G108A) coding region is indicated by lowercase italics. A sequence encoding a 3 FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis G3PDH-1 promoter, driving expression of the TcLPAT2 sequence is indicated by boxed text. The initiator ATG and terminator TGA codons of the TcLPAT2 gene are indicated by uppercase, bold italics, while the remainder of the coding region is represented with italics. Lowercase underlined text represents the P. moriformis ATP 3 UTR. A second spacer sequence is represented by lowercase text. The C. reinhardtii TUB2 promoter driving the expression of the S. cerevisiae SUC2 gene is indicated by boxed text. The initiator ATG and terminator TGA for SUC2 are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis PGH 3 UTR is indicated by lowercase underlined text. The FATA-1 3 genomic region indicated by bold, lowercase text.

TABLE-US-00043 SEQIDNO:127NucleotidesequenceoftransformingDNAcontainedin pSZ5868 gaagagcgcccaatgtttaaacctcttttgctgcgtctcctcaggcttgggggcctccttgggcttgggtgccgccatgatctgcgcg catcagagaaacgttgctggtaaaaaggagcgcccggctgcgcaatatatatataggcatgccaacacagcccaacctcactcg ggagcccgtcccaccacccccaagtcgcgtgccttgacggcatactgctgcagaagcttcatgagaatgatgccgaacaagaggg gcacgaggacccaatcccggacatccttgtcgataatgatctcgtgagtccccatcgtccgcccgacgctccggggagcccgccga tgctcaagacgagagggccctcgaccaggaggggctggcccgggcgggcactggcgtcgaaggtgcgcccgtcgttcgcctgca gtcctatgccacaaaacaagtcttctgacggggtgcgtttgctcccgtgcgggcaggcaacagaggtattcaccctggtcatgggg agatcggcgatcgagctgggataagagatacggtcccgcgcaaggatcgctcatcctggtctgagccggacagtcattctggcaa gcaatgacaacttgtcaggaccggaccgtgccatatatttctcacctagcgccgcaaaacctaacaatttgggagtcactgtgcca ctgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtcagcaccgccagccggccgaggacccgagtcata [00056] ggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctc ctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgacc gaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacc atcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggcttctccaccacccccacc atgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtgga gatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggt gatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcga cgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctgaagaagatctccaagct ggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtg acctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccctggactaccg ccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccaca acggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAAGGACCACGACGGCG [00057] gcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatc gagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgcca gagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaact tgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggc gagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaatgaatggtgagctccgcgtctcgaaca gagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttg gttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggt [00058] [00059] [00060] [00061] [00062] [00063] tcgccgccgccgccgtgatcgtgcccctgggcctgctgttcttcatctccggcctggtggtgaacctgatccaggccctgtgcttcg tgctgatccgccccctgtccaagaacacctaccgcaagatcaaccgcgtggtggccgagctgctgtggctggagctgatctggc tggtggactggtgggccggcgtgaagatcaaggtgttcatggaccccgagtccttcaacctgatgggcaaggagcacgccct ggtggtggccaaccaccgctccgacatcgactggctggtgggctggctgctggcccagcgctccggctgcctgggctccgccct ggccgtgatgaagaagtcctccaagttcctgcccgtgatcggctggtccatgtggttctccgagtacctgttcctggagcgctcct gggccaaggacgagaacaccctgaaggccggcctgcagcgcctgaaggacttcccccgccccttctggctggccttcttcgtg gagggcacccgcttcacccaggccaagttcctggccgcccaggagtacgccgcctcccagggcctgcccatcccccgcaacgt gctgatcccccgcaccaagggcttcgtgtccgccgtgtcccacatgcgctccttcgtgcccgccatctacgacatgaccgtggcc atccccaagtcctccccctcccccaccatgctgcgcctgttcaagggccagccctccgtggtgcacgtgcacatcaagcgctgcct gatgaaggagctgcccgagaccgacgaggccgtggcccagtggtgcaaggacatgttcgtggagaaggacaagctgctgg acaagcacatcgccgaggacaccttctccgaccagcccatgcaggacctgggccgccccatcaagtccctgctggtggtggcc tcctgggcctgcctgatggcctacggcgccctgaagttcctgcagtgctcctccctgctgtcctcctggaagggcatcgccttcttc ctggtgggcctggccatcgtgaccatcctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtgg [00064] agggtggtcgactcgttggaggtgggtgtttttttttatcgagtgcgcggcgcggcaaacgggtccctttttatcgaggtgttcccaac gccgcaccgccctcttaaaacaacccccaccaccacttgtcgaccttctcgtttgttatccgccacggcgccccggaggggcgtcgtc tggccgcgcgggcagctgtatcgccgcgctcgctccaatggtgtgtaatcttggaaagataataatcgatggatgaggaggagagc gtgggagatcagagcaaggaatatacagttggcacgaagcagcagcgtactaagctgtagcgtgttaagaaagaaaaactcgctg ttaggctgtattaatcaaggagcgtatcaataattaccgaccctatacctttatctccaacccaatcgcggcttaaggatctaagtaa gattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgtagtgaccgccaatgtaagtgggctggcgtttccctgtacg tgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagatacagcgcgagccagacacggagtg [00065] [00066] [00067] [00068] [00069] ccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgc caagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacg acctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtg gactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccg gagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccg ccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccag gactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcgg ctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccat caaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaa ccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccct gggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaag ttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagca acgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcac cggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctgg ttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaac agcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaac gacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccacc aacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgaca [00070] cgaaacaagcccctggagcatgcgtgcatgatcgtctctggcgccccgccgcgcggtttgtcgccctcgcgggcgccgcggccgcg ggggcgcattgaaattgttgcaaaccccacctgacagattgagggcccaggcaggaaggcgttgagatggaggtacaggagtcaa gtaactgaaagtttttatgataactaacaacaaagggtcgtttctggccagcgaatgacaagaacaagattccacatttccgtgtag aggcttgccatcgaatgtgagcgggcgggccgcggacccgacaaaacccttacgacgtggtaagaaaaacgtggcgggcactgtc cctgtagcctgaagaccagcaggagacgatcggaagcatcacagcacaggatcctgaggacagggtggttggctggatggggaa acgctggtcgcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtgcacacaca caacccaaccggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatcctcctccctgcc gcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacaggccccttgtgcgcagtg ctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtgtggccaagcgggctgctgggcgc gccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttctgtaagccacgcgcttgctgctttgggaagagaagg gggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttggggaggcagggagagttggaaaatgt aagtggcacgacgggcaaggagaatggtgagcatgtgcatggtgatgtcgttggtcgaggacgatcctgcacgcgtgtatctgat gtagaatacggcaatcaccctagtctacatctataccttctccgtataacgccctttccaaatgccctcccgtttctctcctattcttg atccacatgatgaccctggcactatttcaagggctggagaagagcgtttaaac

[0168] Construct pSZ5868 was transformed into 58754. Primary transformants were clonally purified and screened under standard lipid production conditions at pH 5. Integration of pSZ5868 at the FATA-1 locus was verified by DNA blot analysis. The fatty acid profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 22). 58813 was selected as the lead strain for the final round of genetic engineering. As shown in Table 22 as compared to strain S8754, C16:0 decreased from 5.9% to 3.4%, and C18:0 increased from 27.3% to about 45%. C18:2 increased slightly from 1.3% to about 1.6% due to the activity of the T. cacao LPAAT.

TABLE-US-00044 TABLE 22 Fatty acid profiles of FATA-1 ablation strains. Strain S5100 S8754 S8813 S8814 C14:0 0.7 0.6 0.5 0.5 C16:0 18.8 5.9 3.4 3.4 C16:1 cis-9 0.5 0.0 0.0 0.0 C18:0 4.0 27.3 45.3 44.8 C18:1 68.3 60.9 45.9 46.3 C18:2 6.3 1.3 1.5 1.6 C18:3 0.6 0.3 0.3 0.3 C20:0 0.3 2.4 2.0 2.1 saturates 24.2 37.0 52.0 51.5 lipid (g/L) 12.7 11.9 11.9 11.9

Constructs Used for FAD2 Knockout in S8813

[0169] The high-SOS strains were generated by transformation of strain S8813 with integrative plasmid pSZ6383 (FAD2-1vA::PmLDH1-AtTHIC-PmHSP90:PmSAD2-2v2-TcDGAT1-CvNR:PmSAD2-1v3-CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1::FAD2-1vB), plasmid pSZ6384 (FAD2-1vA::PmLDH1-AtTHIC-PmHSP90:PmSAD2-2v2-TcDGAT2-CvNR:PmSAD2-1v3-CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1::FAD2-1vB), or plasmid pSZ6377 (FAD2-1vA::PmLDH1-AtTHIC-PmHSP90: PmSAD2-1v3-CpSAD1tp_GarmFATA1(G108A)_FLAG-PmSAD2-1::FAD2-1vB). These constructs targeted ablation of allele 1 of the endogenous fatty acid desaturase 2 gene (FAD2-1), and contained expression modules for a second copy of GarmFATA1(G108A), and either TcDGAT1 encoding the Theobroma cacao diacylglycerol O-acyltransferase 1 (pSZ6383) or TcDGAT2 encoding the Theobroma cacao diacylglycerol O-acyltransferase 2 (pSZ6384). Deletion of one allele of FAD2 further reduced C18:2 accumulation. Expression of GarmFATA1(G108A) stimulated C18:0-ACP hydrolysis, further increasing C18:0. TcDGAT1 and TcDGAT2 had superior specificity for transfer of C18:0 to the sn-3 position of triacylglycerides than the endogeneous DGAT, leading to an increase in C18:0 and lipid titer, and a reduction in trisaturated TAGs. The final strains had higher C18:0, lower C16:0 and lower C18:2 than their parent, S8813. The Arabidopsis thaliana THIC gene (AtTHIC) catalyzes the conversion of 5-aminoimidazole ribotide (AIR) to 4-amino-5-hydroxymethylpyrimidine (HMP), providing the pyrimidine ring structure for the biosynthesis of thiamine. AtTHIC served as a selectable marker as part of plasmids pSZ6383 and pSZ6384, allowing the strains to grow in the absence of exogenous thiamine.

[0170] The sequence of the pSZ6383 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlined text, and are 5-3 BspQI, KpnI, XbaI, SnaBI, BamHI, AvrII, SpeI, ClaI, AflII, EcoRI, SpeI, AscI, ClaI, SacI and BspQ I, respectively. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FAD2-1 5 genomic DNA that permits targeted integration at the FAD2-1 locus via homologous recombination. The P. moriformis LDH1 promoter driving the expression of the Arabidopsis thaliana THIC gene is indicated by boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis HSP90 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis SAD2-2 promoter, driving expression of the TcDGAT1 sequence is indicated by boxed text. The initiator ATG and terminator TGA codons of the TcDGAT1 gene are indicated by uppercase, bold italics, while the remainder of the coding region is represented with italics. Lowercase underlined text represents the C. vulgaris NR 3 UTR. A second spacer sequence is represented by lowercase text. The P. moriformis SAD2-1 promoter, indicated by boxed italicized text, is utilized to drive the expression of the G. mangostana FATA1 gene. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3 FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. The FAD2-1 3 genomic region is indicated by bold, lowercase text.

TABLE-US-00045 SEQIDNO:128NucleotidesequenceoftransformingDNAcontainedin pSZ6383 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00071] [00072] [00073] [00074] [00075] [00076] [00077] [00078] [00079] [00080] [00081] [00082] ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00083] cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00084] [00085] [00086] [00087] [00088] [00089] [00090] [00091] [00092] cgagatcctgggctccaccgccaccgtgacctcctcctcccactccgactccgacctgaacctgctgtccatccgccgccgcacct ccaccaccgccgccgcccgcgcccccgaccgcgacgactccggcaacggcgaggccgtggacgaccgcgaccgcgtggagt ccgccaacctgatgtccaacgtggccgagaacgccaacgagatgcccaactcctccgacacccgcttcacctaccgcccccgcg tgcccgcccaccgccgcatcaaggagtcccccctgtcctccggcgccatcttcaagcagtcccacgccggcctgttcaacctgtgc atcgtggtgctggtggccgtgaactcccgcctgatcatcgagaacctgatgaagtacggctggctgatccgctccggcttctggt tctcctcccgctccctgtccgactggcccctgttcatgtgctgcctgaccctgcccatcttccccctggccgccttcgtggtggagaa gctggtgcagcgcaactacatctccgagcccgtggtggtgttcctgcacgccatcatctccaccaccgccgtgctgtaccccgtg atcgtgaacctgcgctgcgactccgccttcctgtccggcgtggccctgatgctgttcgcctgcatcgtgtggctgaagctggtgtc ctacgcccacaccaacaacgacatgcgcgccctggccaagtccgccgagaagggcgacgtggacccctcctacgacgtgtcct tcaagtccctggcctacttcatggtggcccccaccctgtgctaccagcagtcctacccccgcacccccgccgtgcgcaagtcctgg gtggtgcgccagttcatcaagctgatcgtgttcaccggcctgatgggcttcatcatcgagcagtacatcaaccccatcgtgcag aactcccagcaccccctgaagggcaacctgctgtacgccatcgagcgcgtgctgaagctgtccgtgcccaacctgtacgtgtgg ctgtgcatgttctactgcttcttccacctgtggctgaacatcctggccgagctgctgcgcttcggcgaccgcgagttctacaagga ctggtggaacgccaagaccgtggaggagtactggcgcatgtggaacatgcccgtgcacaagtggatggtgcgccacatctac ttcccctgcctgcgcaacggcatccccaagggcgtggccatcgtgatcgccttcctggtgtccgccgtgttccacgagctgtgcat cgccgtgccctgccacatgttcaagctgtgggccttcatcggcatcatgttccaggtgcccctggtgctgatcaccaactacctgc aggacaagttccgctcctccatggtgggcaacatgatcttctggttcatcttctccatcctgggccagcccatgtgcgtgctgctgt [00093] gacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaac agcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttc cctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccct cgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagt gggatgggaacacaaatggacttaaggatctaagtaagattcgaagcgctcgaccgtgccggacggactgcagccccatgtcgta gtgaccgccaatgtaagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggacca ggcatcgcgagatacagcgcgagccagacacggagtgccgagctatgcgcacgctccaactagatatcatgtggatgatgagcat [00094] [00095] [00096] [00097] [00098] [00099] [00100] aatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgc catccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctgg ccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggc atcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctact ccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctaca agtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactgga tcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctg cagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaaca actcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctgg acatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacga gctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccg aggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaactt cctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcAT GGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAA [00101] tcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagat ccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaacacttctctgtcca cagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcactattat cttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagt caatgaatggtgagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgccttgtcttttgcacgcgcgactccgt cgcttcgcgggtggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtacccccaaccacccacctgcacct ctattattggtattattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttcagctggctcccaccattgtaaatt cttgctaaaatagtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggttttcgtgctgatctcgggcacaag gcgtcgtcgacgtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcctttactccgcactccaaacgact gtcgctcgtatttttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaaaggcctcgcggccgtgctcgtgg tgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcatggtgagtgcgcatcacaagatg catgtcttgttgtctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgctttggtcattacagaaattgcacaag ggcgtcctccgggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcaggagcgcggcgcatgacgacctacc cacatgcgaagagc

[0171] The sequence of the pSZ6384 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlined text, and are 5-3 BspQI, KpnI, XbaI, SnaBI, BamHI, AvrII, SpeI, ClaI, AflII, EcoRI, SpeI, AscI, ClaI, SacI and BspQ I, respectively. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FAD2-1 5 genomic DNA that permits targeted integration at the FAD2-1 locus via homologous recombination. The P. moriformis LDH1 promoter driving the expression of the Arabidopsis thaliana THIC gene is indicated by boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis HSP90 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis SAD2-2 promoter, driving expression of the TcDGAT2 sequence is indicated by boxed text. The initiator ATG and terminator TGA codons of the TcDGAT2 gene are indicated by uppercase, bold italics, while the remainder of the coding region is represented with italics. Lowercase underlined text represents the C. vulgaris NR 3 UTR. A second spacer sequence is represented by lowercase text. The P. moriformis SAD2-1 promoter, indicated by boxed italicized text, is utilized to drive the expression of the G. mangostana FATA1 gene. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3 FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. The FAD2-1 3 genomic region is indicated by bold, lowercase text.

TABLE-US-00046 NucleotidesequenceoftransformingDNAcontainedinpSZ6384 SEQIDNO:129 [00102] cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00103] ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00104] tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00105] gaggagcgcaaggccaccggctaccgcgagttctccggccgccacgagttcccctccaacaccatgcacgccctgctggccat gggcatctggctgggcgccatccacttcaacgccctgctgctgctgttctccttcctgttcctgcccttctccaagttcctggtggtgt tcggcctgctgctgctgttcatgatcctgcccatcgacccctactccaagttcggccgccgcctgtcccgctacatctccaagcacg cctgctcctacttccccatcaccctgcacgtggaggacatccacgccttccaccccgaccgcgcctacgtgttcggcttcgagccc cactccgtgctgcccatcggcgtggtggccctggccgacctgaccggcttcatgcccctgcccaagatcaaggtgctggcctcct ccgccgtgttctacacccccttcctgcgccacatctggacctggctgggcctgacccccgccaccaagaagaacttctcctccctg ctggacgccggctactcctgcatcctggtgcccggcggcgtgcaggagaccttccacatggagcccggctccgagatcgccttc ctgcgcgcccgccgcggcttcgtgcgcatcgccatggagatgggctcccccctggtgcccgtgttctgcttcggccagtcccacgt gtacaagtggtggaagcccggcggcaagttctacctgcagttctcccgcgccatcaagttcacccccatcttcttctggggcatct tcggctcccccctgccctaccagcaccccatgcacgtggtggtgggcaagcccatcgacgtgaagaagaacccccagcccatc gtggaggaggtgatcgaggtgcacgaccgcttcgtggaggccctgcaggacctgttcgagcgccacaaggcccaggtgggc [00106] aagtgggctggcgtttccctgtacgtgagtcaacgtcactgcacgcgcaccaccctctcgaccggcaggaccaggcatcgcgagat [00107] tcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcctggccgaccgcctgcg cctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagacc gccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgccggctt ctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatctacaagtaccccgcctg gtccgacgtggtggagatcgagtcctggggccagggcgagggcaagatcggcacccgccgcgactggatcctgcgcgacta cgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtgga cgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaacaactcctccctga agaagatctccaagctggaggacccctcccagtactccaagctgggcctggtgccccgccgcgccgacctggacatgaacca gcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagacc atcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccctccgaggacgccga ggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaacgaccacggctgccgcaacttcctgcacctgct gcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcATGGACTACAA [00108] tggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtacccccaaccacccacctgcacctctattattggta ttattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttcagctggctcccaccattgtaaattcttgctaaaat agtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggttttcgtgctgatctcgggcacaaggcgtcgtcgac gtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcctttactccgcactccaaacgactgtcgctcgtatt tttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaaaggcctcgcggccgtgctcgtggtgggggccgcg agcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcatggtgagtgcgcatcacaagatgcatgtcttgttg tctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgctttggtcattacagaaattgcacaagggcgtcctccg [00109]

[0172] The sequence of the pSZ6377 transforming DNA is provided below. Relevant restriction sites in the construct are indicated in lowercase, bold and underlined text, and are 5-3 BspQI, KpnI, XbaI, SnaBI, BamHI, AvrII, SpeI, AscI, ClaI, SacI and BspQ I, respectively. BspQI sites delimit the 5 and 3 ends of the transforming DNA. Proceeding in the 5 to 3 direction, bold, lowercase sequences represent FAD2-1 5 genomic DNA that permits targeted integration at the FAD2-1 locus via homologous recombination. The P. moriformis LDH1 promoter driving the expression of the Arabidopsis thaliana THIC gene is indicated by boxed text. The initiator ATG and terminator TGA for AtTHIC are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis HSP90 3 UTR is indicated by lowercase underlined text. A spacer sequence is represented by lowercase text. The P. moriformis SAD2-1 promoter, indicated by boxed italicized text, is utilized to drive the expression of the G. mangostana FATA1 gene. The initiator ATG of the sequence encoding the C. protothecoides SAD1 transit peptide (CpSAD1tp) is indicated by uppercase, bold italics, and the remainder of the CpSAD1tp sequence located between the ATG and the AscI site is indicated with lowercase, underlined italics. The GarmFATA1(G108A) coding region is indicated by lowercase italics. A sequence encoding a 3 FLAG tag fused to the C-terminus of GarmFATA1(G108A) is represented by uppercase italics, and the TGA terminator codon is indicated with uppercase, bold italics. The P. moriformis SAD2-1 3 UTR is indicated by lowercase underlined text. The FAD2-1 3 genomic region is indicated by bold, lowercase text.

TABLE-US-00047 NucleotidesequenceoftransformingDNAcontainedinpSZ6377 SEQIDNO:130 gctcttcgcgaaggtcattttccagaacaacgaccatggcttgtcttagcgatcgctcgaatgactgctagtgagtcgtacgctcga cccagtcgctcgcaggagaacgcggcaactgccgagcttcggcttgccagtcgtgactcgtatgtgatcaggaatcattggcattg gtagcattataattcggcttccgcgctgtttatgggcatggcaatgtctcatgcagtcgaccttagtcaaccaattctgggtggccag ctccgggcgaccgggctccgtgtcgccgggcaccacctcctgccatgagtaacagggccgccctctcctcccgacgttggccaact gaataccgtgtcttggggccctacatgatgggctgcctagtcgggcgggacgcgcaactgcccgcgcaatctgggacgtggtctga atcctccaggcgggtttccccgagaaagaaagggtgccgatttcaaagcagagccatgtgccgggccctgtggcctgtgttggcgc ctatgtagtcaccccccctcacccaattgtcgccagtttgcgcaatccataaactcaaaactgcagcttctgagctgcgctgttcaa [00110] [00111] [00112] [00113] [00114] [00115] [00116] [00117] [00118] [00119] [00120] [00121] ctgatgtccgtggtctgcaacaacaagaaccactccgcccgccccaagctgcccaactcctccctgctgcccggcttcgacgtgg tggtccaggccgcggccacccgcttcaagaaggagacgacgaccacccgcgccacgctgacgttcgacccccccacgaccaa ctccgagcgcgccaagcagcgcaagcacaccatcgacccctcctcccccgacttccagcccatcccctccttcgaggagtgcttc cccaagtccacgaaggagcacaaggaggtggtgcacgaggagtccggccacgtcctgaaggtgcccttccgccgcgtgcac ctgtccggcggcgagcccgccttcgacaactacgacacgtccggcccccagaacgtcaacgcccacatcggcctggcgaagct gcgcaaggagtggatcgaccgccgcgagaagctgggcacgccccgctacacgcagatgtactacgcgaagcagggcatcat cacggaggagatgctgtactgcgcgacgcgcgagaagctggaccccgagttcgtccgctccgaggtcgcgcggggccgcgc catcatcccctccaacaagaagcacctggagctggagcccatgatcgtgggccgcaagttcctggtgaaggtgaacgcgaac atcggcaactccgccgtggcctcctccatcgaggaggaggtctacaaggtgcagtgggccaccatgtggggcgccgacacca tcatggacctgtccacgggccgccacatccacgagacgcgcgagtggatcctgcgcaactccgcggtccccgtgggcaccgtc cccatctaccaggcgctggagaaggtggacggcatcgcggagaacctgaactgggaggtgttccgcgagacgctgatcgag caggccgagcagggcgtggactacttcacgatccacgcgggcgtgctgctgcgctacatccccctgaccgccaagcgcctgac gggcatcgtgtcccgcggcggctccatccacgcgaagtggtgcctggcctaccacaaggagaacttcgcctacgagcactggg acgacatcctggacatctgcaaccagtacgacgtcgccctgtccatcggcgacggcctgcgccccggctccatctacgacgcca acgacacggcccagttcgccgagctgctgacccagggcgagctgacgcgccgcgcgtgggagaaggacgtgcaggtgatg aacgagggccccggccacgtgcccatgcacaagatccccgagaacatgcagaagcagctggagtggtgcaacgaggcgcc cttctacaccctgggccccctgacgaccgacatcgcgcccggctacgaccacatcacctccgccatcggcgcggccaacatcgg cgccctgggcaccgccctgctgtgctacgtgacgcccaaggagcacctgggcctgcccaaccgcgacgacgtgaaggcgggc gtcatcgcctacaagatcgccgcccacgcggccgacctggccaagcagcacccccacgcccaggcgtgggacgacgcgctgt ccaaggcgcgcttcgagttccgctggatggaccagttcgcgctgtccctggaccccatgacggcgatgtccttccacgacgaga cgctgcccgcggacggcgcgaaggtcgcccacttctgctccatgtgcggccccaagttctgctccatgaagatcacggaggac atccgcaagtacgccgaggagaacggctacggctccgccgaggaggccatccgccagggcatggacgccatgtccgagga gttcaacatcgccaagaagacgatctccggcgagcagcacggcgaggtcggcggcgagatctacctgcccgagtcctacgtc [00122] cgcacgcatccaacgaccgtatacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgagatgtctcaggcttggtgc atcctcgggtggccagccacgttgcgcgtcgtgctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggcccgttttt ttgctcccatttcctttccgctacatcttgaaagcaaacgacaaacgaagcagcaagcaaagagcacgaggacggtgaacaagtct gtcacctgtatacatctatttccccgcgggtgcacctactctctctcctgccccggcagagtcagctgccttacgtgacggatcccgcg tctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacga atgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtgga [00123] [00124] [00125] [00126] [00127] [00128] [00129] ccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccgggccccggcgcccagcgaggc ccctccccgtgcgcgggcgcgccatccccccccgcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgag gccgtggtgtcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttcatc gtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacctgctgcaggaggtgggctgcaac cacgcccagtccgtgggctactccaccgccggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgccc gcatgcacatcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaaga tcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcgccacctccaagtgggtgatgatg aaccaggacacccgccgcctgcagaaggtggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcc tggccttccccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcctg gtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggctgggtgctggagtccatgcccca ggagatcatcgacacccacgagctgcagaccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactcc ctgacctcccccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgccaa cgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagatcaaccgcggccgcaccgagtggc gcaagaagcccacccgcATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACA [00130] ctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgc ggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgct gccaaaacacttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttg caacaggtccctgcactattatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgc cctcgctgatcgagtgtacagtcaatgaatggtgagctcctcactcagcgcgcctgcgcggggatgcggaacgccgccgccgcctt gtcttttgcacgcgcgactccgtcgcttcgcgggtggcacccccattgaaaaaaacctcaattctgtttgtggaagacacggtgtac ccccaaccacccacctgcacctctattattggtattattgacgcgggagcgggcgttgtactctacaacgtagcgtctctggttttca gctggctcccaccattgtaaattcttgctaaaatagtgcgtggttatgtgagaggtatggtgtaacagggcgtcagtcatgttggtt ttcgtgctgatctcgggcacaaggcgtcgtcgacgtgacgtgcccgtgatgagagcaataccgcgctcaaagccgacgcatggcc tttactccgcactccaaacgactgtcgctcgtatttttcggatatctattttttaagagcgagcacagcgccgggcatgggcctgaa aggcctcgcggccgtgctcgtggtgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggaggaacgcat ggtgagtgcgcatcacaagatgcatgtcttgttgtctgtactataatgctagagcatcaccaggggcttagtcatcgcacctgcttt ggtcattacagaaattgcacaagggcgtcctccgggatgaggagatgtaccagctcaagctggagcggcttcgagccaagcagg agcgcggcgcatgacgacctacccacatgcgaagagc

[0173] Constructs pSZ6383, pSZ6384 and pSZ6377 were transformed into S8813. Primary transformants were clonally purified and screened under standard lipid production conditions at pH 5. Integration of pSZ6383 or pSZ6384 at the FAD2-1 locus was verified by DNA blot analysis. The fatty acid profiles, sn-2 profiles and lipid titers of lead strains were assayed in 50-mL shake flasks (Table 23). FAD2-1 ablation reduced C18:2 to <1% in most strains. Expression of a second copy of GarmFATA1(G108A) and TcDGAT1 (S8990, 58992, 58998 & S8999), or TcDGAT2 (S8994, 59000 & S9047) elevated C18:0 to >56%. The D5393-28 strain, expressing a second copy of GarmFATA1(G108A) without either of the cocoa DGAT genes (pSZ6377) had a similar fatty acid profile, but lower lipid titer. As shown in Table 23, as compared to strain S8813, for strains expressing either TcDGAT1 or TcDGAT2, C16:0 increased from 3.2% to 3.7%-4.0%, C18:0 increased from 45.8% to about 56%, C18:2 decreased from 1.4% to about 1.0%.

TABLE-US-00048 TABLE 23 Fatty acid profiles of FAD2-1 ablation strains. Strain S8813 D5393-28 S8990 S8992 S8998 S8999 S8994 S9000 S9047 C12:0 0.1 0.2 0.2 0.2 0.1 0.2 0.1 0.1 0.2 C14:0 0.4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 C16:0 3.2 3.8 3.7 3.8 3.9 4.0 3.7 3.8 3.5 C16:1 cis-7 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C16:1 cis-9 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C17:0 0.1 0.2 0.2 0.1 0.2 0.1 0.2 0.2 0.2 C18:0 45.8 56.0 56.6 56.0 56.2 56.0 56.3 56.4 56.5 C18:1 45.9 35.8 35.4 35.9 35.7 35.5 35.9 35.7 35.9 C18:2 1.4 1.0 0.9 1.0 0.9 1.1 0.9 0.9 0.8 C18:3 0.3 0.3 0.3 0.2 0.3 0.2 0.2 0.3 0.3 C20:0 2.0 1.6 1.6 1.5 1.6 1.5 1.5 1.5 1.5 C22:0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 C24:0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 saturates 52.1 62.6 63.1 62.6 62.9 62.8 62.8 62.9 62.7

[0174] Liquid chromatography and mass spectrometry were used to analyze the TAG composition of final strains. The strains accumulated 68-71% SOS, with trisaturates ranging from 2.5-2.8%. The D5393-28 strain, expressing a second copy of GarmFATA1(G108A) without either of the cocoa DGAT genes had similar SOS content but slightly higher trisaturates. The TAG composition of a typical Shea stearin and a sample of Kokum butter are shown for comparison

TABLE-US-00049 TABLE 24 LC/MS TAG profiles of FAD2-1 ablation strains. Strain Shea Kokum D5393-28 S8990 S8992 S8998 S8999 S8994 S9000 S9047 stearin butter OOL 0.4 LLS 0.2 POL 0.3 OOO 1.3 1.7 SOL 1.0 0.4 LaOS + MOP 0.2 0.3 0.3 0.2 0.3 0.3 0.4 0.2 OOP 0.5 0.2 0.3 0.2 0.2 0.4 0.3 0.2 0.8 0.7 PLS (+SLnS) 0.6 0.7 0.7 0.7 0.7 0.6 0.6 0.4 0.6 0.3 POP (+MOS) 1.1 1.0 1.0 1.1 1.1 1.0 1.2 0.8 0.7 0.4 OOS 10.5 10.3 11.3 11.0 11.0 10.9 10.1 10.6 6.4 11.8 SLS (+PLA) 1.9 1.7 2.0 1.6 2.1 1.8 1.9 1.5 5.5 1.4 POS 8.4 8.5 8.4 8.7 8.9 8.4 10.0 7.7 6.3 4.8 MaOS 0.3 SOG 0.4 0.5 0.5 0.6 0.3 0.5 0.4 0.5 OOA 0.5 0.3 0.4 0.4 0.4 0.4 0.4 0.4 0.2 0.2 SOS (+POA) 68.4 69.7 68.7 69.1 68.3 69.4 68.0 71.4 69.7 76.6 SSP (+MSA) 0.5 0.5 0.5 0.4 0.5 0.5 0.5 0.4 0.2 SOA + POB 3.9 3.8 3.5 3.6 3.4 3.5 3.5 3.4 4.0 1.0 SSS (+PSA) 2.6 2.3 2.2 2.1 2.3 2.2 2.3 2.1 2.0 0.5 SOB + LgOP + AOA 0.4 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.4 SSA (+PBS) 0.2 SOLg (+POHx) 0.3 SUM (area %) 99.8 99.9 99.8 99.9 99.8 99.9 100.0 99.9 100.0 100.0 Sat-Sat-Sat 3.1 2.8 2.7 2.5 2.7 2.7 2.8 2.5 2.4 0.5 Sat-U-Sat 84.9 85.9 84.7 85.3 85.1 85.0 86.0 85.8 87.5 84.7 Sat-O-Sat 82.4 83.5 82.0 82.9 82.3 82.6 83.4 83.9 81.4 83.1 Sat-L-Sat 2.5 2.4 2.6 2.3 2.8 2.4 2.6 1.9 6.1 1.6 U-U-U/Sat 11.8 11.3 12.4 12.2 12.0 12.2 11.3 11.7 10.6 14.8 La = laurate (C12:0), M = myristate (C14:0), P = palmitate (C16:0), Ma = margarate (C17:0), S = stearate (C18:0), O = oleate (C18:1), L = linoleate (C18:2), Ln = -linolenate (C18:3 ), A = arachidate (C20:0), G = (C20:1), B = behenate (C22:0), Lg = lignocerate (C24:0), Hx = hexacosanoate (C26:0). Sat = saturated, U = unsaturated

Example 8 Variant Brassica Napus Thioeserase

[0175] In this example, we demonstrate the modification of the enzyme specificity of a FATA thioesterase originally isolated from Brassica napus (BnOTE, accession CAA52070), by site directed mutagenesis targeting two amino acids positions D124 and D209).

[0176] To determine the impact of each amino acid substitution on the enzyme specificity of the BnOTE, the wild-type and the mutant BnOTE genes were cloned into a vector enabling expression and expressed in P. moriformis strain S8588. Strain S8588 is a strain in which the endogenous FATA1 allele has been disrupted and expresses a Prototheca moriformis KASII gene and sucrose invertase. Recombinant strains with FATA1 disruption and co-expression of P. moriformis KASII and invertase were previously disclosed in co-owned applications WO2012/106560 and WO2013/15898, herein incorporated by reference.

[0177] Strains that express wild type or mutant BnOTE enzymes, contructs pSZ6315, pSZ6316, pSZ6317, or pSZ6318 were expressed in S8588. In these constructs, the Saccharomyces carlsbergensis MEL1 gene (Accession no: AAA34770) was utilized as the selectable marker to introduce the wild-type and mutant BnOTE genes into the FAD2-2 locus of P. moriformis strain S8588 by homologous recombination using previously described transformation methods (biolistics). The constructs that have been expressed in S8588 are listed in Table 25.

TABLE-US-00050 TABLE 25 DNA lot# and plasmid ID of DNA constructs that expressing wild-type and mutant BnOTE genes DNA Solazyme Lot# Plasmid Construct D5309 pSZ6315 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE-PmSAD2-1 utr::FAD2-2 D5310 pSZ6316 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE(D124A)-PmSAD2-1 utr::FAD2-2 D5311 PSZ6317 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE(D209A)-PmSAD2-1 utr::FAD2-2 D5312 pSZ6318 FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE(D124A, D209A)-PmSAD2-1 utr::FAD2-2

[0178] pSZ6315

[0179] The consruct psZ6315 can be written as FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE-PmSAD2-1 utr::FAD2-2. The sequence of the pSZ6315 transforming DNA is provided below. Relevant restriction sites in pSZ6315 are indicated in lowercase, bold and underlining and are 5-3 SgrAI, Kpn I, SnaBI, AvrII, SpeI, AscI, ClaI, Sac I, SK respectively. SgrAI and Sbff sites delimit the 5 and 3 ends of the transforming DNA. Bold, lowercase sequences represent FAD2-2 genomic DNA that permit targeted integration at FAD2-2 locus via homologous recombination. Proceeding in the 5 to 3 direction, the P. moriformis HXT1 promoter driving the expression of the Saccharomyces carlsbergensis MEL1 gene is indicated by boxed text. The initiator ATG and terminator TGA for MEL1 gene are indicated by uppercase, bold italics while the coding region is indicated in lowercase italics. The P. moriformis PGK 3 UTR is indicated by lowercase underlined text followed by the P. moriformis SAD2-2 V3 promoter, indicated by boxed italics text. The Initiator ATG and terminator TGA codons of the wild-type BnOTE are indicated by uppercase, bold italics, while the remainder of the coding region is indicated by bold italics in lower case. The three-nucleotide codon corresponding to the target amino acids, D124 and D209, are in lower case, italicized, bolded and wave underlined. The P. moriformis SAD2-1 3UTR is again indicated by lowercase underlined text followed by the FAD2-2 genomic region indicated by bold, lowercase text.

TABLE-US-00051 NucleotidesequenceoftransformingDNAcontainedinpSZ6315 SEQIDNO:131 caccggcgcgctgcttcgcgtgccgggtgcagcaatcagatccaagtctgacgacttgcgcgcacgcgccggatccttcaattccaaagtgtcg tccgcgtgcgcttcttcgccttcgtcctcttgaacatccagcgacgcaagcgcagggcgctgggcggctggcgtcccgaaccggcctcggcgcac gcggctgaaattgccgatgtcggcaatgtagtgccgctccgcccacctctcaattaagtttttcagcgcgtggttgggaatgatctgcgctcatg gggcgaaagaaggggttcagaggtgctttattgttactcgactgggcgtaccagcattcgtgcatgactgattatacatacaaaagtacagctc gcttcaatgccctgcgattcctactcccgagcgagcactcctctcaccgtcgggttgcttcccacgaccacgccggtaagagggtctgtggcctc gcgcccctcgcgagcgcatctttccagccacgtctgtatgattttgcgctcatacgtctggcccgtcgaccccaaaatgacgggatcctgcataa tatcgcccgaaatgggatccaggcattcgtcaggaggcgtcagccccgcgggagatgccggtcccgccgcattggaaaggtgtagagggggt [00131] [00132] [00133] [00134] [00135] [00136] gcctgggcctgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggacacggccgacc gcatctccgacctgggcctgaaggacatgggctacaagtacatcatcctggacgactgctggtcctccggccgcgactccgacggcttcctg gtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcggcatgtactcctccgc gggcgagtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggactacct gaagtacgacaactgctacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaaggccatgtccgacgccctgaacaa gacgggccgccccatcttctactccctgtgcaactggggccaggacctgaccttctactggggctccggcatcgcgaactcctggcgcatgtc cggcgacgtcacggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggcttccactgc tccatcatgaacatcctgaacaaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacctggacaacctggaggtcgg cgtcggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcgaacgtga acaacctgaaggcctcctcctactccatctactcccaggcgtccgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtct ggcgctactacgtgtccgacacggacgagtacggccagggcgagatccagatgtggtccggccccctggacaacggcgaccaggtcgtg gcgctgctgaacggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttcttcgactccaacctgggctccaagaagctga cctccacctgggacatctacgacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccg gcatcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaagaacgacacccgcctgttcggccagaagatcggctccctgtc [00137] accggcgctgatgtggcgcggacgccgtcgtactctttcagactttactcttgaggaattgaacctttctcgcttgctggcatgtaaacattggcgc aattaattgtgtgatgaagaaagggtggcacaagatggatcgcgaatgtacgagatcgacaacgatggtgattgttatgaggggccaaacctg gctcaatcttgtcgcatgtccggcgcaatgtgatccagcggcgtgactctcgcaacctggtagtgtgtgcgcaccgggtcgctttgattaaaactg atcgcattgccatcccgtcaactcacaagcctactctagctcccattgcgcactcgggcgcccggctcgatcaatgttctgagcggagggcgaag cgtcaggaaatcgtctcggcagctggaagcgcatggaatgcggagcggagatcgaatcaggatcccgcgtctcgaacagagcgcgcagagga acgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgt [00138] [00139] [00140] [00141] [00142] [00143] [00144] [00145] [00146] [00147] [00148] [00149] [00150] [00151] [00152] [00153] [00154] [00155] [00156] [00157] [00158] tcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcgg tggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactccttctgctgccaaaaca cttctctgtccacagcaacaccaaaggatgaacagatcaacttgcgtctccgcgtagcttcctcggctagcgtgcttgcaacaggtccctgcacta ttatcttcctgctttcctctgaattatgcggcaggcgagcgctcgctctggcgagcgctccttcgcgccgccctcgctgatcgagtgtacagtcaat gaatggtgagctccgcgcctgcgcgaggacgcagaacaacgctgccgccgtgtatttgcacgcgcgactccggcgcttcgctggtggcacccc cataaagaaaccctcaattctgtttgtggaagacacggtgtacccccacccacccacctgcacctctattattggtattattgacgcgggagtgg gcgttgtaccctacaacgtagcttctctagttttcagctggctcccaccattgtaaattcatgctagaatagtgcgtggttatgtgagaggtatag tgtgtctgagcagacggggcgggatgcatgtcgtggtggtgatctttggctcaaggcgtcgtcgacgtgacgtgcccgatcatgagagcaatac cgcgctcaaagccgacgcatagcctttactccgcaatccaaacgactgtcgctcgtattttttggatatctattttaaagagcgagcacagcgcc gggcatgggcctgaaaggcctcgcggccgtgctcgtggtgggggccgcgagcgcgtggggcatcgcggcagtgcaccaggcgcagacggag gaacgcatggtgcgtgcgcaatataagatacatgtattgttgtcctgcagg NucleotidesequenceofBnOTE(D124A)inpSZ6316 SEQIDNO:132 [00159] [00160] [00161] [00162] [00163] [00164] [00165] [00166] [00167] [00168] [00169] [00170] [00171] [00172]

[0180] The sequence of the pSZ6317 transforming DNA is same as pSZ6315 except the D209A point mutation, the BnOTE D209A DNA sequence is provided below. The three-nucleotide codon corresponding to the target two amino acids, D124 and D209, are in lower case, italicized, bolded and wave underlined. pSZ6317 is written as FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE (D209A)-PmSAD2-1 utr::FAD2-2

TABLE-US-00052 NucleotidesequenceofBnOTE(D209A)inpSZ6317: SEQIDNO:133 atggactacaaggaccac gacggcgactacaaggaccacgacatcgactacaaggacgacgacgaca ag

[0181] The sequence of the pSZ6318 transforming DNA is same as pSZ6315 except two point mutations, D124A and D209A, the BnOTE (D124A, D209A) DNA sequence is provided below. The three-nucleotide codon corresponding to the target two amino acids, D124 and D209, are in lower case, italicized, bolded and wave underlined. pSZ6318 is written as FAD2-2::PmHXT1-ScarMEL1-PmPGK:PmSAD2-2 V3-CpSADtp-BnOTE (D124A, D209A)-PmSAD2-1 utr::FAD2-2

[0182] SEQ ID NO: 134 Nucleotide Sequence of BnOTE (D124A, D209A) in pSZ6318

TABLE-US-00053 atggactacaagga ccacgacggcgactacaaggaccacgacatcgactacaaggacgacgac gacaag

[0183] The DNA constructs containing the wild-type and mutant BnOTE genes were transformed into the parental strain S8588. Primary transformants were clonally purified and grown under standard lipid production conditions at pH5.0. The resulting profiles from representative clones arising from transformations with pSZ6315, pSZ6316, pSZ6317, and pSZ6318 into S8588 are shown in Table 26. The parental strain S8588 produces 5.4% C18:0, when transformed with the DNA cassette expressing wild-type BnOTE, the transgenic lines produce 11% C18:0. The BnOTE mutant (D124A) increased the amount of C18:0 by at least 2 fold compared to the wild-type protein. In contrast, the BnOTE D209A mutation appears to have no impact on the enzyme activity/specificity of the BnOTE thioesterase. Finally, expression of the BnOTE (D124A, D209A) resulted in very similar fatty acid profile to what we observed in the transformants from S8588 expressing BnOTE (D124A), again indicating that D209A has no significant impact on the enzyme activity.

TABLE-US-00054 TABLE 26 Fatty acid profiles in S8588 and derivative transgenic lines transformed with wild-type and mutant BnOTE genes Fatty Acid Area % Transforming DNA Sample ID C16:0 C18:0 C18:1 C18:2 pH5; S8588 (parental strain) 3.00 5.43 81.75 6.47 D5309, pSZ6315; pH5; S8588, D5309-6; 3.86 11.68 76.51 5.06 wild-type BnOTE pH5; S8588, D5309-2; 3.50 11.00 77.80 4.95 pH5; S8588, D5309-9; 3.51 10.72 78.03 5.00 pH5; S8588, D5309-10; 3.55 10.69 78.06 4.96 pH5; S8588, D5309-11; 3.61 10.69 78.05 4.95 D5310, pSZ6316, pH5; S8588, D5310-6; 4.27 31.55 55.31 5.30 BnOTE (D124A) pH5; S8588, D5310-1; 4.53 30.85 54.71 6.03 pH5; S8588, D5310-5; 5.21 20.75 65.43 5.02 pH5; S8588, D5310-10; 4.99 19.18 67.75 5.00 pH5; S8588, D5310-2; 4.90 18.92 68.17 4.98 D5311, pSZ6317, pH5; S8588, D5311-3; 3.50 11.90 76.95 4.98 BnOTE (D209A) pH5; S8588, D5311-4; 3.63 11.35 77.44 4.94 pH5; S8588, D5311-14; 3.47 11.23 77.68 4.98 pH5; S8588, D5311-10; 3.60 11.20 77.53 5.00 pH5; S8588, D5311-12; 3.53 11.12 77.59 5.09 D5312, pSZ6318, pH5; S8588, D5312-20 4.79 37.97 47.74 6.01 BnOTE (D124A, pH5; S8588, D5312-40; 5.97 22.94 62.20 5.11 D209A) pH5; S8588, D5312-39; 6.07 22.75 62.24 5.17 pH5; S8588, D5312-16; 5.25 18.81 67.36 5.09 pH5; S8588, D5312-26; 4.93 18.70 68.37 4.96

Example 9 Variant Garcinia Mangostana Thioeserase

[0184] In this example, we demonstrate the ability to modify the activity and specificity of a FATA thioesterase originally isolated from Garcinia mangostana (GmFATA, accession 004792), using site directed mutagenesis targeting six amino acid positions within the enzyme and various combinations thereof. Facciotti et al (NatBiotech 1999) had previously altered three of the amino acids (G108, S111, V193). The remaining three amino acids targeted are L91, G96, and T156.

[0185] To test the impact of each mutation on the activity of the GmFATA, the wild-type and mutant genes were cloned into a vector enabling expression within the P. moriformis strain S3150. Table 27 summarizes the results from a three day lipid profile screen comparing the wild-type GmFATA with the 14 mutants. Three GmFATA mutants (DNA lot numbers D3998, D4000, D4003) increased the amount of C18:0 by at least 1.5 fold compared to the wild-type protein (DNA lot number D3997). D3998 and D4003 were mutations that had been described by Facciotti et al (NatBiotech 1999) as substitutions that increased the activity of the GmFATA. Strain S3150 expressing the mutations contained in DNA lot number D4000 was based on research at Solazyme which demonstrated this position influenced the activity of the FATB thioesterases. All of the constructs were codon optimized to reflect UTEX 1435 codon usage. Non-mutated GmFATA increases the fatty acid content of C18:0 and decreases the fatty acid content of C18:1 and C18:2. As can be seen in Table 27 the G90A mutant GmFATA increases the fatty acid content of C18:0 and decreases the fatty acid content of C18:1 and C18:2 when compared to the wild-type GmFATA.

TABLE-US-00055 TABLE 27 Algal Strain DNA # GmFATA C14:0 C16:0 C18:0 C18:1 C18:2 P. moriformis S3150 1.63 29.82 3.08 55.95 7.22 S3150 D3997 Wild-Type 1.79 29.28 7.32 52.88 6.21 pSZ5083 GmFATA D3998 S111A, 1.84 28.88 11.19 49.08 6.21 pSZ5084 V193A D3999 S111V, 1.73 29.92 3.23 56.48 6.46 pSZ5085 V193A D4000 G96A 1.76 30.19 12.66 45.99 6.01 pSZ5086 D4001 G96T 1.82 30.60 3.58 55.50 6.28 pSZ5087 D4002 G96V 1.78 29.35 3.45 56.77 6.43 pSZ5088 D4003 G108A 1.77 29.06 12.31 47.86 6.08 pSZ5089 D4007 G108V 1.81 28.78 5.71 55.05 6.26 pSZ5093 D4004 L91F 1.76 29.60 6.97 53.04 6.13 pSZ5090 D4005 L91K 1.87 28.89 4.38 56.24 6.35 pSZ5091 D4006 L91S 1.85 28.06 4.81 56.45 6.47 pSZ5092 D4008 T156F 1.81 28.71 3.65 57.35 6.31 pSZ5094 D4009 T156A 1.72 29.66 5.44 54.54 6.26 pSZ5095 D4010 T156K 1.73 29.95 3.17 56.86 6.21 pSZ5096 D4011 T156V 1.80 29.17 4.97 55.44 6.27 pSZ5097

[0186] Nucleotide sequence of the GmFATA wild-type parental gene expression vector is shown below (D3997, pSZ5083). The plasmid pSZ5083 can be written as THI4a::CrTUB2-NeoR-PmPGH:PmSAD2-2Ver3-CpSAD1tp_GarmFATA1_FLAG-CvNR::THI4a. The 5 and 3 homology arms enabling targeted integration into the Thi4 locus are noted with lowercase; the CrTUB2 promoter is noted in uppercase italic which drives expression of the neomycin selection marker noted with lowercase italic followed by the PmPGH 3UTR terminator highlighted in uppercase. The PmSAD2-1 promoter (noted in bold text) drives the expression of the GmFATA gene (noted with lowercase bold text) and is terminated with the CvNR 3UTR noted in underlined, lower case bold. Restriction cloning sites and spacer DNA fragments are noted as underlined, uppercase plain lettering. The nucleotide sequence for all of the GmFATA constructs disclosed in this example is identical to that of pSZ5083 with the exception of the encoded GmFATA. The promoter, 3UTR, selection marker and targeting arms are the same as described for pSZ5083. The individual GmFATA mutant sequences are shown below. The amino acid sequence of the unmutagenized GmFATA is showin in FIG. 1. The amino acid sequences of the altered GmFATA proteins are shown below.

TABLE-US-00056 pSZ5083 SEQIDNO:135 ccctcaactgcgacgctgggaaccttctccgggcaggcgatgtgcgtgggtttgcctccttg gcacggctctacaccgtcgagtacgccatgaggcggtgatggctgtgtcggttgccacttcg tccagagacggcaagtcgtccatcctctgcgtgtgtggcgcgacgctgcagcagtccctctg cagcagatgagcgtgactttggccatttcacgcactcgagtgtacacaatccatttttctta aagcaaatgactgctgattgaccagatactgtaacgctgatttcgctccagatcgcacagat agcgaccatgttgctgcgtctgaaaatctggattccgaattcgaccctggcgctccatccat gcaacagatggcgacacttgttacaattcctgtcacccatcggcatggagcaggtccactta gattcccgatcacccacgcacatctcgctaatagtcattcgttcgtgtcttcgatcaatctc aagtgagtgtgcatggatcttggttgacgatgcggtatgggtttgcgccgctggctgcaggg tctgcccaaggcaagctaacccagctcctctccccgacaatactctcgcaggcaaagccggt cacttgccttccagattgccaataaactcaattatggcctctgtcatgccatccatgggtct gatgaatggtcacgctcgtgtcctgaccgttccccagcctctggcgtcccctgccccgccca ccagcccacgccgcgcggcagtcgctgccaaggctgtctcggaGGTACCCTTTCTTGCGCTA TGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACAC CGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCA GGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAA GCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCA CTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACTCTAGAATATC Aatgatcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcg gctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcc cagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcagga cgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacg tggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctg tcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgca caccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgccc gcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctg gcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggt gacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttca tcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgac atcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgc ccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttctgaCAATTGACGC CCGCGCGGCGCACCTGACCTGTTCTCTCGAGGGCGCCTGTTCTGCCTTGCGAAACAAGCCCC TGGAGCATGCGTGCATGATCGTCTCTGGCGCCCCGCCGCGCGGTTTGTCGCCCTCGCGGGCG CCGCGGCCGCGGGGGCGCATTGAAATTGTTGCAAACCCCACCTGACAGATTGAGGGCCCAGG CAGGAAGGCGTTGAGATGGAGGTACAGGAGTCAAGTAACTGAAAGTTTTTATGATAACTAAC AACAAAGGGTCGTTTCTGGCCAGCGAATGACAAGAACAAGATTCCACATTTCCGTGTAGAGG CTTGCCATCGAATGTGAGCGGGCGGGCCGCGGACCCGACAAAACCCTTACGACGTGGTAAGA AAAACGTGGCGGGCACIGTCCCTGTAGCCTGAAGACCAGCAGGAGACGATCGGAAGCATCAC AGCACAGGATCCCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTC GCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCAT TAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGT GGAGCTGATGGICGAAACGTTCACAGCCTAGGGATATCGTGAAAACTCGCTCGACCGCCCGC GTCCCGCAGGCAGCGATGACGTGTGCGTGACCTGGGTGTTTCGTCGAAAGGCCAGCAACCCC AAATCGCAGGCGATCCGGAGATTGGGATCTGATCCGAGCTTGGACCAGATCCCCCACGATGC GGCACGGGAACTGCATCGACTCGGCGCGGAACCCAGCTTTCGTAAATGCCAGATTGGTGTCC GATACCTTGATTTGCCATCAGCGAAACAAGACTTCAGCAGCGAGCGTATTTGGCGGGCGTGC TACCAGGGTTGCATACATTGCCCATTTCTGTCTGGACCGCTTTACCGGCGCAGAGGGTGAGT TGATGGGGTTGGCAGGCATCGAAACGCGCGTGCATGGTGTGTGTGTCTGTTTTCGGCTGCAC AATTTCAATAGTCGGATGGGCGACGGTAGAATTGGGTGTTGCGCTCGCGTGCATGCCTCGCC CCGTCGGGTGTCATGACCGGGACTGGAATCCCCCCTCGCGACCCTCCTGCTAACGCTCCCGA CTCTCCCGCCCGCGCGCAGGATAGACTCTAGTTCAACCAATCGACAACTAGTatggccaccg catccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggcgggctccggg ccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatccccccccgcatcatcgt ggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtgtcctccggcc tggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaaggagaagttc atcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagaccatcgccaacct gctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggcggcttctcca ccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgcacatcgagatc tacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagggcgagggcaa gatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtgatcggccgcg ccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggtggacgtggac gtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttccccgaggagaa caactcctccctgaagaagatctccaagctggaggacccctcccagtactccaagctgggcc tggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgacctacatcggc tgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcagaccatcaccct ggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcccccgagccct ccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaacgtgtccgcc aacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacggcctggagat caaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaaggaccacgacg gcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtgaATCGATgcagca gcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccaca cttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgat cttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccaccccca gcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctg ctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctc cgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaag tagtgggatgggaacacaaatggaAAGCTTGAGCTCcagcgccatgccacgccctttgatgg cttcaagtacgattacggtgttggattgtgtgtttgttgcgtagtgtgcatggtttagaata atacacttgatttcttgctcacggcaatctcggcttgtccgcaggttcaaccccatttcgga gtctcaggtcagccgcgcaatgaccagccgctacttcaaggacttgcacgacaacgccgagg tgagctatgtttaggacttgattggaaattgtcgtcgacgcatattcgcgctccgcgacagc acccaagcaaaatgtcaagtgcgttccgatttgcgtccgcaggtcgatgttgtgatcgtcgg cgccggatccgccggtctgtcctgcgcttacgagctgaccaagcaccctgacgtccgggtac gcgagctgagattcgattagacataaattgaagattaaacccgtagaaaaatttgatggtcg cgaaactgtgctcgattgcaagaaattgatcgtcctccactccgcaggtcgccatcatcgag cagggcgttgctcccggcggcggcgcctggctggggggacagctgttctcggccatgtgtgt acgtagaaggatgaatttcagctggttttcgttgcacagctgtttgtgcatgatttgtttca gactattgttgaatgtttttagatttcttaggatgcatgatttgtctgcatgcgact AminoacidsequenceofGmFATAwild-typeparentalgene; D3997,pSZ5083.ThealgaltransitpeptideisunderlinedandtheFLAGepitopetagis uppercasebold SEQIDNO:136 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAS111A,V193Amutantgene; D3998,pSZ5084.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheS111A,V193Aresiduesarelower-casebold. SEQIDNO:137 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFaTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAS111V,V193Amutantgene; D3999,pSZ5085.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheS111V,V193Aresiduesarelower-casebold. SEQIDNO:138 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFvTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDaDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG96Amutantgene;D4000, pSZ5086.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheG96Aresidueislower-casebold. SEQIDNO:139 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVaCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG96Tmutantgene;D4001, pSZ5087.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheG96Tresidueislower-casebold. SEQIDNO:140 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVtCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG96Vmutantgene;D4002, pSZ5088.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheG96Vresidueislower-casebold. SEQIDNO:141 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVvCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG108Amutantgene; D4003,pSZ5089.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheG108Aresidueislower-casebold. SEQIDNO:142 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTaGESTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAL91Fmutantgene;D4004, pSZ5090.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheL91Fresidueislower-casebold. SEQIDNO:143 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANfLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAL91Kmutantgene;D4005, pSZ5091.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheL91Kresidueislower-casebold SEQIDNO:144 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANkLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK FIG.10.AminoacidsequenceofGmFATAL915mutant gene;D4006,pSZ5092.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheL915residueislower-casebold SEQIDNO:14 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANsLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAG108Vmutantgene; D4007,pSZ5093.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheG108Vresidueislower-casebold. SEQIDNO:146 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTvGESTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGTRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Fmutantgene; D4008,pSZ5094.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheT156Fresidueislower-casebold. SEQIDNO:147 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGfRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Amutantgene; D4009,pSZ5095.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheT156Aresidueislower-casebold. SEQIDNO:148 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGaRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Kmutantgene;D4010, pSZ5096.Thealgaltransitpeptideisunderlined,theFLAGepitopetagisuppercase boldandtheT156Kresidueislower-casebold. SEQIDNO:149 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGkRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK AminoacidsequenceofGmFATAT156Vmutantgene; D4011,pSZ5097.Thealgaltransitpeptideisunderlined,theFLAGepitopetagis uppercaseboldandtheT156Vresidueislower-casebold. SEQIDNO:150 MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRAIPPRIIVVSSSSSKVNPLKTEAVVSSGLADRLRLGSL TEDGLSYKEKFIVRCYEVGINKTATVETIANLLQEVGCNHAQSVGYSTGGFSTTPTMRKLRLIWVTARMHIEIYK YPAWSDVVEIESWGQGEGKIGvRRDWILRDYATGQVIGRATSKWVMMNQDTRRLQKVDVDVRDEYLVHCPRELRL AFPEENNSSLKKISKLEDPSQYSKLGLVPRRADLDMNQHVNNVTYIGWVLESMPQEIIDTHELQTITLDYRRECQ HDDVVDSLTSPEPSEDAEAVENHNGTNGSANVSANDHGCRNFLHLLRLSGNGLEINRGRTEWRKKPTRMDYKDHD GDYKDHDIDYKDDDDK NucleotidesequenceoftheGmFATAS111A,V193Amutantgene (D3998,pSZ5084).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesame aspSZ5083. SEQIDNO:151 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttcgccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAS111V,V193Amutantgene (D3999,pSZ5085).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesame aspSZ5083. SEQIDNO:152 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttcgtcaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgcggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG96Amutantgene(D4000, pSZ5086).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 SEQIDNO:153 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtggcgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG96Tmutantgene(D4001, pSZ5087).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 SEQIDNO:154 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgacgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG96Vmutantgene(D4002, pSZ5088).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. SEQIDNO:155 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtggtgtgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG108Amutantgene (D4003,pSZ5089).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesame aspSZ50836. SEQIDNO:156 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgcc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAL91Fmutantgene(D4004, pSZ5090).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 SEQIDNO:157 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacttcctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAL91Kmutantgene(D4005, pSZ5091).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. SEQIDNO:158 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacaagctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAL91Smutantgene(D4006, pSZ5092).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. SEQIDNO:159 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaactcgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAG108Vmutantgene (D4007,pSZ5093).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesame aspSZ5083. SEQIDNO:160 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccgtc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcacccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Fmutantgene(D4008, pSZ5094).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. SEQIDNO:161 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcttccgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Amutantgene(D4009, pSZ5095).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 SEQIDNO:162 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcgcgcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Kmutantgene(D4010, pSZ5096).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083. SEQIDNO:163 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcaagcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga NucleotidesequenceoftheGmFATAT156Vmutantgene(D4011, pSZ5097).Thepromoter,3UTR,selectionmarkerandtargetingarmsarethesameas pSZ5083 SEQIDNO:164 atggccaccgcatccactttctcggcgttcaatgcccgctgcggcgacctgcgtcgctcggc gggctccgggccccggcgcccagcgaggcccctccccgtgcgcgggcgcgccatcccccccc gcatcatcgtggtgtcctcctcctcctccaaggtgaaccccctgaagaccgaggccgtggtg tcctccggcctggccgaccgcctgcgcctgggctccctgaccgaggacggcctgtcctacaa ggagaagttcatcgtgcgctgctacgaggtgggcatcaacaagaccgccaccgtggagacca tcgccaacctgctgcaggaggtgggctgcaaccacgcccagtccgtgggctactccaccggc ggcttctccaccacccccaccatgcgcaagctgcgcctgatctgggtgaccgcccgcatgca catcgagatctacaagtaccccgcctggtccgacgtggtggagatcgagtcctggggccagg gcgagggcaagatcggcgtgcgccgcgactggatcctgcgcgactacgccaccggccaggtg atcggccgcgccacctccaagtgggtgatgatgaaccaggacacccgccgcctgcagaaggt ggacgtggacgtgcgcgacgagtacctggtgcactgcccccgcgagctgcgcctggccttcc ccgaggagaacaactcctccctgaagaagatctccaagctggaggacccctcccagtactcc aagctgggcctggtgccccgccgcgccgacctggacatgaaccagcacgtgaacaacgtgac ctacatcggctgggtgctggagtccatgccccaggagatcatcgacacccacgagctgcaga ccatcaccctggactaccgccgcgagtgccagcacgacgacgtggtggactccctgacctcc cccgagccctccgaggacgccgaggccgtgttcaaccacaacggcaccaacggctccgccaa cgtgtccgccaacgaccacggctgccgcaacttcctgcacctgctgcgcctgtccggcaacg gcctggagatcaaccgcggccgcaccgagtggcgcaagaagcccacccgcatggactacaag gaccacgacggcgactacaaggaccacgacatcgactacaaggacgacgacgacaagtga