Beta-ketoacyl-ACP synthase II variants

Abstract

Provided are non-natural or variant beta-ketoacyl-acyl carrier protein (ACP) synthase (KAS) II enzymes (KASII), polynucleotides encoding such variant KASII, host cells expressing such variant KASII, oils and oil products produced by such cells, and methods of making and using such variant KASII.

Claims

1. A polynucleotide encoding a non-natural variant β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) II enzyme (KASII), wherein the non-natural KASII comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid residues 39-469 of SEQ ID NO:2 and comprises an X at the position corresponding to position 162; wherein X is an amino acid residue selected from the group consisting of cysteine (C), aspartate (D), glutamate (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y), wherein the positions are with reference to SEQ ID NO:2, and wherein the non-natural KASII catalyzes the elongation of C14 acyl-ACP to C16 acyl-ACP or C16 acyl-ACP to C18 acyl-ACP.

2. The polynucleotide of claim 1, wherein the non-natural KASII preferentially produces C18 acyl-ACP, or wherein the non-natural KASII facilitates the production of increased levels of C18, C20, C22, and/or C24 fatty acids in comparison to a wild-type KASII having SEQ ID NO: 1, or wherein the non-natural KASII facilitates the production of an increased level of C18:1 fatty acid in comparison to a wild-type KASII having SEQ ID NO: 1, and/or wherein the non-natural KASII facilitates a decreased level of C16:0 in comparison to a wild-type KASII having SEQ ID NO: 1.

3. The polynucleotide of claim 1, wherein the X at position 162 is an amino acid residue selected from cysteine (C), glutamate (E), lysine (K), methionine (M), serine (S), tryptophan (W), valine (V), asparagine (N), aspartate (D), glutamine (Q), histidine (H), leucine (L), phenylalanine (F), threonine (T), and tyrosine (Y).

4. A host cell comprising the polynucleotide of claim 3, further comprising a polynucleotide encoding a fatty acyl-ACP thioesterase.

5. A method of producing triglyceride lipids comprising predominantly C18, C20, C22, and/or C24 fatty acids, comprising cultivating a microalgal host cell comprising: (a) a polynucleotide encoding a fatty acyl-ACP thioesterase; and (b) the polynucleotide of claim 3, so as to produce triglyceride lipids comprising at least about 40% C18, C20, C22, and/or C24 fatty acids, or optionally at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or more, C18, C20, C22, and/or C24 fatty acids.

6. The polynucleotide of claim 1, wherein the X at position 162 is an amino acid selected from valine (V), asparagine (N), aspartate (D), glutamine (Q), histidine (H), leucine (L), phenylalanine (F), threonine (T), and tyrosine (Y).

7. A host cell comprising the polynucleotide of claim 6, further comprising a polynucleotide encoding a fatty acyl-ACP thioesterase.

8. A method of producing triglyceride lipids comprising predominantly C18, C20, C22, and/or C24 fatty acids, comprising cultivating a microalgal host cell comprising: (a) a polynucleotide encoding a fatty acyl-ACP thioesterase; and (b) the polynucleotide of claim 6, so as to produce triglyceride lipids comprising at least about 40% C18, C20, C22, and/or C24 fatty acids, or optionally at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or more, C18, C20, C22, and/or C24 fatty acids.

9. The polynucleotide of claim 1, wherein the non-natural or variant KASII comprises a plastid transit peptide.

10. The polynucleotide of claim 9, wherein the plastid transit peptide comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

11. The polynucleotide of claim 1, comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to: a) nucleic acid residues 115-1407 of SEQ ID NO:6; b) nucleic acid residues 1-1407 of SEQ ID NO:6; or, c) SEQ ID NO:6.

12. The polynucleotide of claim 11, wherein the polynucleotide comprises a thymine (T) at residue position 1080.

13. The polynucleotide of claim 1, comprising codon bias for improved expression in a microalgal host cell, optionally in a Prototheca or Chlorella microalgal host cell.

14. A host cell comprising the polynucleotide of claim 1, further comprising a polynucleotide encoding a fatty acyl-ACP thioesterase.

15. The host cell of claim 14, wherein the thioesterase preferentially hydrolyzes C18 acyl-ACP substrates.

16. The host cell of claim 14, wherein the thioesterase preferentially hydrolyzes C18:0 acyl-ACP, C18:1 acyl-ACP or C18:2 acyl-ACP substrates.

17. The host cell of claim 14, wherein the thioesterase is from a plant genus selected from Brassica, Carthamus, Camelina, Garcinia, Glycine, Mangifera, Helianthus, Madhura, Arachis, Monts, Ricinus, Herrania, Corchorus, Corchorus, Jatropha, Ziziphus, Trema orientalis, Hevea, Theobroma, Quercus, Cucurbita, Dorcoceras, Rosa, Asparagus, Cephalotus, Parasponia, Carica, Arabidopsis, Capsella and Eutrema.

18. The host cell of claim 14, wherein the polynucleotide encodes a Garcinia mangostana (GmFATA) thioesterase, optionally comprising one or more amino acid substitutions selected from the group consisting of L91F, L91K, L91S, G96A, G96T, G96V, G108A, G108V, S111A, S111V, T156F, T156A, T156K, T156V and V193A, wherein the amino acid positions are with reference to SEQ ID NO:11.

19. The host cell of claim 14, wherein the host cell is a microalga cell of the genus Prototheca or Chlorella.

20. The host cell of claim 19, wherein the microalga cell is selected from the group consisting of Prototheca moriformis, Prototheca krugani, Prototheca stagnora, Prototheca zopfii and Chlorella protothecoides.

21. A method of producing triglyceride lipids comprising predominantly C18, C20, C22, and/or C24 fatty acids, comprising cultivating a microalgal host cell comprising the polynucleotide of claim 1 so as to produce triglyceride lipids comprising at least about 40% C18, C20, C22, and/or C24 fatty acids, or optionally at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or more, C18, C20, C22, and/or C24 fatty acids.

22. A method of producing triglyceride lipids comprising predominantly C18, C20, C22, and/or C24 fatty acids, comprising cultivating a microalgal host cell comprising: (a) a polynucleotide encoding a fatty acyl-ACP thioesterase; and (b) the polynucleotide of claim 1, so as to produce triglyceride lipids comprising at least about 40% C18, C20, C22, and/or C24 fatty acids, or optionally at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or more, C18, C20, C22, and/or C24 fatty acids.

Description

DETAILED DESCRIPTION

(1) 1. Introduction

(2) Provided are heterologous KAS II enzymes that preferentially produce C18 acyl-ACP to facilitate the production of appreciable levels of C18 fatty acids, e.g., stearic (C18:0) acid, oleic (C18:1) acid and/or linoleic acid (C18:2), as well as increasing levels of C20 (e.g., eicosapentaenoic acid, arachidonic acid), C22 (e.g., docosahexaenoic acid, erucic acid) and/or C24 fatty acids, in microalgal (e.g., Prototheca) host cells. The KAS II enzyme variants can coordinate with a heterologous thioesterase that preferentially hydrolyzes C18-acyl ACP substrates into stearic (C18:0) acid, oleic (C18:1) acid and/or linoleic acid (C18:2) and acyl carrier proteins (ACPs), so that the resulting stearic, oleic and/or linoleic fatty acids can be incorporated into triglycerides (TAGs). Here, we report on the identification of a more active variant, PmKASII.sup.A162X, for strain engineering. By over-expressing one or more copies of the PmKASII.sup.A162X gene variant in a Prototheca host strain, the resulting transgenic strains can produce triglyceride oils with modified fatty acid profiles, for example, fatty acid profiles comprising greater than 80%, 85%, 90%, or more, C18, C20, C22, and/or C24 fatty acids. These triglyceride oils also comprise a high level of monosaturated fatty acids (e.g., C18:1 fatty acid) and provide health benefits compared with saturated, hydrogenated (trans fats) often found in conventional food products.

(3) 2. ρ-Ketoacyl-ACP Synthase (KAS) II Variants

(4) The variant β-ketoacyl-ACP synthase (KAS) II enzymes (KASII) can be used in genetic constructs and genetically engineered oleaginous cells (e.g., plants, algae, microalgae) with one or more exogenous genes to produce fatty acids, acyl glycerides, or derivatives thereof. For example, microalgae or oilseed crops that would naturally, or through genetic modification, produce high levels of triglycerides can be engineered (or further engineered) to express an exogenous variant KASII, which can catalyze the elongation of C14 acyl-ACP or C16 acyl-ACP to long-chain fatty acyl-ACP, e.g., from C14 to C16 or from C16 to C18, optionally producing C18 acyl-ACP, and/or facilitating the production of increased levels of C18, C20, C22, and/or C24 fatty acids, e.g., in comparison to a wild-type KASII. The fatty acids synthesized may be incorporated into acyl glycerides including triglycerides (TAGs, triacylglycerol). The triglycerides can be recovered or, through further enzymatic processing within the cell, or in vitro, yield other useful compounds.

(5) Generally, the variant KASII enzymes described herein have preferential substrate specificity for C14 ACP or C16 ACP-fatty acyl substrates (e.g., to promote the elongation of C16 fatty acids to C18 fatty acids, and therefore, the production of C18 fatty acids). The variant KASII enzymes described herein catalyze the elongation of C14 acyl-ACP or C16 acyl-ACP to long-chain fatty acyl-ACP, e.g., from C14 to C16 or from C16 to C18 carbon atoms in length, and are categorized as EC 2.3.1.179 (β-ketoacyl-acyl-carrier-protein (ACP) synthase II).

(6) In some embodiments, the non-natural KASII comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid residues 39-469 of SEQ ID NO:2, e.g., to amino acid residues 1-469 of SEQ ID NO:2, and comprises an X at the position corresponding to position 162; wherein X is an amino acid residue selected from the group consisting of cysteine (C), aspartate (D), glutamate (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y), wherein the positions are with reference to SEQ ID NO:2, and wherein the non-natural KASII catalyzes the elongation of C14 acyl-ACP, e.g., from C14 to C16. In some embodiments, the non-natural KASII comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid residues 39-469 of SEQ ID NO:2, e.g., to amino acid residues 1-469 of SEQ ID NO:2, and comprises an X at the position corresponding to position 162; wherein X is an amino acid residue selected from the group consisting of cysteine (C), aspartate (D), glutamate (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W) and tyrosine (Y), wherein the positions are with reference to SEQ ID NO:2, and wherein the non-natural KASII catalyzes the elongation of C16 acyl-ACP, e.g., from C16 to C18. In some embodiments, the non-natural KASII preferentially produces C18 acyl-ACP, e.g., C18:0 acyl-ACP, C18:1 acyl-ACP and/or C18:2 acyl-ACP. In some embodiments, the non-natural KASII facilitates the production of increased levels of C18, C20, C22, and/or C24 fatty acids in comparison to a wild-type KASII.

(7) In some embodiments, the amino acid X at position 162 is an amino acid residue selected from serine (S), threonine (T) and valine (V). In some embodiments, the amino acid X at position 162 is an amino acid residue selected from isoleucine (I), leucine (L) and valine (V). In some embodiments, the amino acid X at position 162 is a valine (V) residue.

(8) In some embodiments, the amino acid X at position 162 is an amino acid residue selected from cysteine (C), glutamate (E), lysine (K), methionine (M), serine (S), tryptophan (W), valine (V), asparagine (N), aspartate (D), glutamine (Q), histidine (H), leucine (L), phenylalanine (F), threonine (T), and tyrosine (Y).

(9) In another embodiment, the amino acid at position at position 162 is selected from valine (V), asparagine (N), aspartate (D), glutamine (Q), histidine (H), leucine (L), phenylalanine (F), threonine (T), and tyrosine (Y).

(10) In some embodiments, the non-natural or variant KASII comprises a plastid transit peptide. In some embodiments, the plastid transit peptide comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to amino acid residues 1-35 of SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the plastid transit peptide is encoded by a polynucleotide comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9 or SEQ ID NO:10.

(11) In some embodiments, the polynucleotide comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleic acid residues 115-1407 of SEQ ID NO:6, e.g., wherein the nucleic acid base at position 1080 is a thymine (T). In some embodiments, the polynucleotide comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleic acid residues 1-1407 of SEQ ID NO:6, e.g., wherein the nucleic acid base at position 1080 is a T. In some embodiments, the polynucleotide comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6. In some embodiments, the polynucleotide comprises codon bias for improved expression in a microalgal host cell, e.g., a Prototheca or Chlorella microalgal host cell.

(12) In certain embodiments, provided is a fragment of any of the above-described proteins or nucleic acids (including fragments of protein or nucleic acid variants), wherein the protein fragment has activity, e.g., to catalyze the elongation of C14 acyl-ACP or C16 acyl-ACP, e.g., from C14 to C16 or from C16 to C18, optionally producing C18:0 acyl-ACP, C18:1 acyl-ACP and/or C18:2 acyl-ACP, and/or facilitating the production of increased levels of C18, C20, C22, and/or C24 fatty acids, e.g., in comparison to a wild-type KASII. Also contemplated are nucleic acid fragments encoding such protein fragments. In other embodiments, the fragment includes a domain of the KASII enzyme that mediates a particular function, e.g., elongation of C14 to C16 or C16 to C18 fatty acids. Illustrative fragments can be produced by C-terminal and/or N-terminal truncations and include at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the full-length sequences disclosed herein.

(13) 3. Co-Expression with C18-Preferring Thioesterases

(14) In some embodiments, the variant KASII enzyme is co-expressed with a heterologous or exogenous fatty acyl-ACP thioesterase. In an embodiment, polynucleotide encoding a variant KASII enzyme, as described herein, is co-expressed in a host cell with an exogenous FATA or FATB acyl-ACP thioesterase gene. FATA genes encode enzymes that preferentially, but not exclusively, hydrolyze long-chain fatty acids with highest activity towards C18:1, and in some instances C18:0. FATB genes encode a group of enzymes with more heterogeneous substrate specificities but generally show higher activity toward saturated fatty acids. The substrate specificities of FATB enzymes are quite heterogenous; there are a number of FATB enzymes that show high activity towards C18:0 and C18:1. FATA and FATB enzymes terminate the synthesis of fatty acids by hydrolyzing the thioester bond between the acyl moiety and the acyl carrier protein (ACP).

(15) In some embodiments, the thioesterase preferentially hydrolyzes C18 acyl-ACP substrates, e.g., catalyzes the production of increased levels of C18 (e.g., C18:0, C18:1, C18:2), C20, C22, and/or C24 fatty acids and/or has increased specificity for C18 fatty acids in comparison to a wild-type thioesterase. Illustrative C18-preferring thioesterases of use for co-expression include without limitation a thioesterase from a plant genus selected from Brassica, Carthamus, Camelina, Garcinia, Glycine, Mangifera, Helianthus, Madhura, Arachis, Morus, Ricinus, Herrania, Corchorus, Corchorus, Jatropha, Ziziphus, Trema orientalis, Hevea, Theobroma, Quercus, Cucurbita, Dorcoceras, Rosa, Asparagus, Cephalotus, Parasponia, Carica, Arabidopsis, Capsella and Eutrema. In some embodiments, the variant KASII is co-expressed with a thioesterase from a plant species selected from Brassica napus, Brassica campestris, Brassica juncea, Carthamus tinctorius, Camelina saliva, Garcinia mangostana, Glycine max, Mangifera indica, Helianthus annuus, Madhura longifolia, Arachis hypogaea, Morus notabilis, Ricinus communis, Herrania umbratical, Corchorus olitorius, Corchorus capsularis, Jatropha curcas, Ziziphus jujube, Trema orientalis, Hevea brasiliensis, Theobroma cacao, Quercus suber, Cucurbita maxima, Cucurbita pepo, Cucurbita moschata, Dorcoceras hygrometricum, Rosa chinensis, Asparagus officinalis, Cephalotus follicularis, Parasponia andersonii, Carica papaya, Arabidopsis thaliana, Capsella rubella and Eutrema salsugineum. In some embodiments, the variant KASII is co-expressed with a Garcinia mangostana (GmFATA) thioesterase, optionally comprising one or more amino acid substitutions selected from the group consisting of L91F, L91K, L91S, G96A, G96T, G96V, G108A, G108V, S111A, S111V, T156F, T156A, T156K, T156V and V193A, wherein the amino acid positions are with reference to SEQ ID NO:11. In some embodiments, the variant KASII is co-expressed with Brassica napus BnOTE (Accession No. CAA52070) and its variants, e.g., as described in US2016/0083758, which is hereby incorporated herein by reference in its entirety for all purposes.

(16) 4. Co-Expression with Other Lipid Biosynthesis Enzymes

(17) In some embodiments, the variant KASII enzyme is co-expressed with one or more heterologous or exogenous lipid biosynthesis enzymes. In some embodiments, the variant KASII enzyme is co-expressed with one or more heterologous or exogenous lipid biosynthesis enzymes selected from the group consisting of a fatty acyl thioesterase A (FATA), a fatty acyl thioesterase B (FATB), a fatty acyl desaturase (FAD), including without limitation phosphatidylglycerol desaturase (FAD4), plastidial oleate desaturase (FADE), plastidial linoleate desaturase (FAD7/FAD8), endoplasmic reticulum oleate desaturase (FAD2), endoplasmic reticulum linolate desaturase (FAD3), delta 12 fatty acid desaturase (412 FAD), delta 15 fatty acid desaturase (Δ15 FAD), and stearoyl-ACP desaturase 2 (SAD2); a 1-acylglycerol-3-phosphate O-acyltransferase (LPAAT), a glycerol-3-phosphate acyltransferase (GPAT), an acyl CoA:diacylglycerol acyltransferase (DGAT), a fatty acid and a fatty acid elongase (FAE), and a long-chain acyl-CoA synthetase (LACS). In some embodiments, the variant KASII enzyme is co-expressed with one or more exogenous or heterologous enzymes, such as a sucrose invertase, an alpha galactosidase, and a 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THIC). Recombinant expression of heterologous or exogenous lipid biosynthesis enzymes is described, e.g., in U.S. Patent Publ. No. 2014/0178950, which is incorporated herein by reference in its entirety for all purposes. For example, one or more polynucleotides encoding one or more of the aforementioned lipid biosynthesis enzymes 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 genes can be included in an expression cassette that includes a promoter (e.g., a heterologous promoter) and downstream regulatory element. The vector can include flanking sequences for homologous recombination. For example, the vector can cause insertion into a chromosome of the host cell, where it can be stably expressed. The proteins produced by the genes can be used in vivo or in purified form. In an embodiment, an expression cassette comprises a homologous promoter, a CDS operable to express one or more lipid biosynthesis enzymes and a 3′UTR. The 3′UTR can comprise a polyadenylation site.

(18) In some embodiments, one or more lipid biosynthesis enzymes endogenous to the host cell selected from the group consisting of a fatty acyl thioesterase A (FATA), a fatty acyl thioesterase B (FATB), a fatty acyl desaturase (FAD), including without limitation phosphatidylglycerol desaturase (FAD4), plastidial oleate desaturase (FADE), plastidial linoleate desaturase (FAD7/FAD8), endoplasmic reticulum oleate desaturase (FAD2), endoplasmic reticulum linolate desaturase (FAD3), delta 12 fatty acid desaturase (Δ12 FAD), delta 15 fatty acid desaturase (Δ15 FAD), and stearoyl-ACP desaturase 2 (SAD2); a 1-acylglycerol-3-phosphate O-acyltransferase (LPAAT), a glycerol-3-phosphate acyltransferase (GPAT), an acyl CoA:diacylglycerol acyltransferase (DGAT), a fatty acid elongase (FAE) and a long-chain acyl-CoA synthetase (LACS) are deleted, knocked out or knocked down. For example, one or more polynucleotides encoding one or more of the aforementioned lipid biosynthesis enzymes can also be used to prepare antisense, or inhibitory RNA (e.g., RNAi or hairpin RNA) to inhibit complementary genes in the microalgal host cell. For example, armed with the knowledge of a gene sequence encoding one of the aforementioned proteins, one can engineer a microalgal host cells with the same or similar gene to express an RNAi construct, gene knockout, knockdown, point mutation, or the like, and thereby reduce the expression and/or activity of one or more of the enzymes in the microalgal host cell. As a result, the microalgae can produce an oil with an altered fatty acid profile in which the mean chain length is decreased or increased, depending on the presence of other fatty acid synthesis genes. In some embodiments, a mutation (including knockout) or inhibition (e.g., using antisense or RNAi) of one or more endogenous desaturase genes (e.g., a stearoyl-ACP desaturase or fatty acid desaturase including a delta 12 fatty acid desaturase) can reduce or eliminate desaturase activity to produce a more fully saturated oil profile.

(19) Depending on the desired properties of the lipid molecule to be produced, one or more genes encoding enzymes that utilize fatty acids or fatty acyl molecules as substrates to produce lipid molecules may be attenuated or over-expressed in the host cell (e.g., microalga), for example using RNAi, hairpin constructs, knockdowns, double or single knockouts or replacement (e.g., replacing an endogenous gene with a heterologous gene).

(20) 5. Codon-Bias for Improved Expression in Microalgal Host Cells

(21) DNA encoding a polypeptide to be expressed in a microorganism, e.g., a KASII variant, optionally with an exogenous lipid biosynthesis enzyme, e.g., a fatty acyl-ACP thioesterase, and selectable marker can be codon-optimized cDNA. Methods of recoding genes for expression in microalgae are described in U.S. Pat. No. 7,135,290. Additional information for codon optimization is available, e.g., at the Codon Usage Database at kazusa.or.jp/codon/. The table for Prototheca preferred codon usage is also provided in U.S. Patent Publ. No. 2012/0283460. Preferred codon usage in Prototheca and Chlorella protothecoides is provided in Tables A and B.

(22) TABLE-US-00001 TABLE A Preferred codon usage in Prototheca strains. Amino Usage Acid Codon Frequency Ala GCG  36% GCA   7% GCT  11% GCC  46% Arg AGG   6% AGA   2% CGG  18% CGA   8% CGT   9% CGC  57% Asn AAT   4% AAC  96% Asp GAT  12% GAC  88% Cys TGT  10% TGC  90% Gin CAG  82% CAA  18% Glu GAG  96% GAA   4% Gly GGG  12% GGA   7% GGT  10% GGC  71% His CAT  21% CAC  79% He ATA   1% ATT   8% ATC  91% Lys AAG  98% AAA   2% Leu TTG   4% TTA   0% CTG  61% CTA   3% CTT   6% CTC  26% Met ATG 100% Phe TTT  29% TTC  71% Pro CCG  29% CCA   9% CCT  13% CCC  49% Ser AGT   3% AGC  22% TCG  28% TCA   6% TCT  10% TCC  31% Thr ACG  38% ACA   5% ACT   5% ACC  52% Tyr TAT   5% TAC  95% Tip TGG 100% Val GTG  50% GTA   1% GTT   6% GTC  43% Stop TGA/TAG/TAA

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

(24) In various embodiments, the nucleic acids encoding the KASII variants, and optionally the exogenous co-expressed lipid biosynthesis enzyme, e.g., fatty acyl-ACP thioesterase, can be codon biased for improved expression in a target host cell. For expression in a Prototheca or a Chlorella host cell, the encoding polynucleotide can be recoded, using the preferred codons identified in Tables A or B, respectively. For example, in some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codons used in the encoding polynucleotide can be the most preferred codon according to Tables A or B. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codons used in the encoding polynucleotide can be the first or second most preferred codon according to Tables A and B. In some embodiments, the non-natural or variant β-ketoacyl-ACP synthase (KAS) II enzyme (KASII) is encoded by a polynucleotide comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to nucleic acid residues 115-1407 of SEQ ID NO:6, e.g., nucleic acid residues 1-1407 of SEQ ID NO:6. In some embodiments, the non-natural or variant β-ketoacyl-ACP synthase (KAS) II enzyme (KASII) is encoded by a polynucleotide comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6.

(25) 6. Expression and Targeting to Plastids

(26) Heterologous or exogenous proteins expressed in the nuclear genome of Prototheca can be targeted to the plastid using plastid targeting signals. Plastid targeting sequences endogenous to Chlorella are known, such as genes in the Chlorella nuclear genome that encode proteins that are targeted to the plastid; see for example GenBank Accession numbers AY646197 and AF499684, and in one embodiment, such control sequences are used in the vectors described herein, e.g., to target expression of a protein to a Prototheca plastid.

(27) The Examples below describe the use of algal plastid targeting sequences to target heterologous proteins to the correct compartment in the host cell. cDNA libraries were made using Prototheca moriformis and Chlorella protothecoides cells and are described in the Examples of U.S. Patent Publ. No. 2012/0283460 and in PCT Application No. PCT/US2009/066142. Amino acid sequences of the algal plastid targeting sequences identified from the cDNA libraries useful plastid targeting of recombinantly expressed variant KASII enzymes are provided in U.S. Patent Publ. No. 2012/0283460 and herein. In some embodiments, the plastid transit peptide comprises an amino acid sequence selected from the group consisting of MATASTFSAFNARCGDLRRSAGSGPRRPARPLPVRGRA (SEQ ID NO:15), SGPRRPARPLPVR (SEQ ID NO:16), SGPRRPARPLPVRAAIASEVPVATTSPR (SEQ ID NO:17), RPARPLPVRGRA (SEQ ID NO:18), RPARPLPVRAAIASEVPVATTSPR (SEQ ID NO:19), RCGDLRRSAGSGPRRPARPLPVRGRA (SEQ ID NO:20), RCGDLRRSAGSGPRRPARPLPVRAAIASEVPVATTSPR (SEQ ID NO:21), PARPLPVR (SEQ ID NO:22), PARPLPVRAAIASEVPVATTSPR (SEQ ID NO:23), RRPARPLPVR (SEQ ID NO:24), and RRPARPLPVRAAIASEVPVATTSPR (SEQ ID NO:25). In some embodiments, the plastid transit peptide comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to amino acid residues 1-33 of SEQ ID NO:1, amino acid residues 1-33 of SEQ ID NO:2, SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the plastid transit peptide is encoded by a polynucleotide comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9 or SEQ ID NO:10.

(28) Where novel KASII variants are disclosed here, it will be understood that a variety of heterologous plastid transit peptides can be used. In other words, the non-targeting peptide domain is more highly conserved. Accordingly, embodiments described herein feature the novel KASII enzymatic domain with or without a plastid targeting sequence. For example, where a percent identity to a novel KASII gene is given herein, the same identity can be applied (where specified) to the same sequence absent the targeting peptide. A substitute targeting peptide can optionally be used in connection with such a sequence.

(29) 7. Host Cells—Oil- or Lipid-Producing Microorganisms

(30) Any species of organism that produces suitable triglycerides can be used, although microorganisms that naturally produce high levels of suitable triglyceride are preferred. Considerations for the selection of microorganisms include, in addition to production of suitable triglycerides for production of oils and oleochemicals: (1) high lipid content as a percentage of dry cell weight; (2) ease of growth; (3) ease of genetic engineering; and (4) ease of biomass processing. In particular embodiments, the wild-type, classically improved or genetically engineered microorganism yields cells that are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% or more lipid as a percentage of their dry cell weight. Preferred organisms grow heterotrophically (on sugars in the absence of light) or can be engineered to do so using, for example, methods disclosed herein. The ease of transformation and availability of selectable markers and promoters, constitutive or inducible, that are functional in the microorganism affect the ease of genetic engineering. Examples of selectable markers useful in microalgae include sucrose invertase, alpha galactosidase (for selection on melibiose) and antibiotic resistance genes. Processing considerations can include, for example, the availability of effective means for lysing the cells.

(31) Microalgae

(32) In some embodiments, the microorganism is a microalga. Non-limiting examples of microalgae that can be used for expression of variant KASII enzymes include, e.g., Achnanthes orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphora coffeiformis tenuis, Amphora delicatissima, Amphora delicatissima capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus fakatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata, Chlorella Antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides (including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25), Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pvrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

(33) Illustrative host cells 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 lipid biosynthesis pathway, including plastidic oleaginous cells such as those of oleaginous algae. Specific examples of cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorpophya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae are provided in Published PCT Patent Applications WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/150411, 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 dry cell weight, ±5%. 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. In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous sucrose invertase gene so as to allow the cells to produce oil from a sucrose feedstock.

(34) The oleaginous cells produce a storage oil, which may be stored in storage vesicles of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. 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. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, as animal feed, for human nutrition, or for fertilizer.

(35) Where a fatty acid profile of a triglyceride cell oil is given, 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). Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the triglycerides in the cell.

(36) In some embodiments, the host cell is a plastidic cell, e.g., a heterotrophic microalga of the phylum Chlorpophya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. In some embodiments, the cell is oleaginous and capable of accumulating at least 40% oil by dry cell weight. The cell can be an obligate heterotroph, such as a species of Prototheca, including Prototheca moriformis or Prototheca zopfii. The nucleic acid encoding the variant KASII enzymes described herein can also be expressed in autotrophic algae or plants. Optionally, the cell is capable of using sucrose to produce oil and a recombinant invertase gene may be introduced to allow metabolism of sucrose, as described in PCT Publications WO2008/151149, WO2010/06032, WO2011/150410, WO2011/150411, and international patent application PCT/US12/23696. The invertase can be codon-biased and integrated into a chromosome of the cell, as well as any of the genes mentioned herein. Codon usage for different algal and plant species of interest is known in the art and can be found, e.g., on the internet at the Codon Usage Database at kazusa.or.jp/codon/.

(37) The polynucleotides encoding the variant KASII enzymes described herein further can be expressed in a wide variety of plant and microalgal host cells. Of particular interest are plant cells of plants involved in the production of vegetable oils for edible and industrial uses, including e.g., temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, Cuphea, soybean, peanut, coconut and oil palms, and corn. See, U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; and U.S. Pat. Nos. 5,344,771; 5,304,481.

(38) 8. Methods of Culturing Microorganisms

(39) Microorganisms are cultured both for purposes of conducting genetic manipulations and for subsequent production of oil or triglycerides (TGs, triacylglycerols, TAGs, or triacylglycerides). The former type of culture is conducted on a small scale and initially, at least, under conditions in which the starting microorganism can grow. For example, if the starting microorganism is a photoautotroph the initial culture is conducted in the presence of light. The culture conditions can be changed if the microorganism is evolved or engineered to grow independently of light. Culture for purposes of oil or triglyceride production is usually conducted on a large scale. Preferably a fixed carbon source is present. The culture can also be exposed to light some or all of the time.

(40) Microalgae can be cultured in liquid media. The culture can be contained within a bioreactor. Optionally, the bioreactor does not allow light to enter. Alternatively, microalgae can also be cultured in photobioreactors that contain a fixed carbon source and allow light to strike the cells. Exposure of microalgal cells to light, even in the presence of a fixed carbon source that the cells transport and utilize (i.e., mixotrophic growth), nonetheless accelerates growth compared to culturing cells in the dark. Culture condition parameters can be manipulated to increase or improve total triglyceride production, the combination of triglyceride species produced, and/or production of a triglyceride species. In some instances it is preferable to culture cells in the dark, such as, for example, when using extremely large (e.g., 10,000 L, 40,000 L, 100,000 L, 500,000 L, or larger, bioreactors) fermentors that do not allow light to strike the culture.

(41) Microalgal culture media typically contain components such as a fixed nitrogen source, trace elements, vitamins (e.g., thiamine), optionally a buffer for pH maintenance, and phosphate. Other components can include a fixed carbon source such as acetate or glucose, and salts such as sodium chloride, particularly for seawater microalgae. Examples of trace elements include zinc, boron, cobalt, copper, manganese, and molybdenum in, for example, the respective forms of ZnCl.sub.2, H.sub.3BO.sub.3, CoCl.sub.2.6H.sub.2O, CuCl.sub.2.2H.sub.2O, MnCl.sub.2.4H.sub.2O and (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O.

(42) For organisms able to grow on a fixed carbon source, the fixed carbon source can be, for example, glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside, and/or glucuronic acid. The one or more carbon source(s) can be supplied at a concentration of at least about 50 μM, at least about 100 μM, at least about 500 μM, at least about 5 mM, at least about 50 mM, and at least about 500 mM, of one or more exogenously provided fixed carbon source(s). Some microalgal species can grow by utilizing a fixed carbon source such as glucose or acetate in the absence of light. Such growth is known as heterotrophic growth. For Chlorella and/or Prototheca, for example, heterotrophic growth results in high production of biomass and accumulation of high lipid content in cells.

(43) Some microorganisms naturally grow on or can be engineered to grow on a fixed carbon source that is a heterogeneous source of compounds such as municipal waste, secondarily treated sewage, wastewater, and other sources of fixed carbon and other nutrients such as sulfates, phosphates, and nitrates. The sewage component serves as a nutrient source in the production of triglycerides, and the culture provides an inexpensive source of triglycerides.

(44) Other culture parameters can also be manipulated, such as the pH of the culture media, the identity and concentration of trace elements and other media constituents.

(45) Heterotrophic Growth

(46) As an alternative to photosynthetic growth of microorganisms, some microorganisms can be cultured under heterotrophic growth conditions in which a fixed carbon source provides energy for growth and lipid accumulation.

(47) In one heterotrophic culture method, crude, partially purified, or purified glycerol produced as a byproduct of lipid transesterification can be employed as a feedstock for fermenting, for example, lipid-producing microbial cultures. Thus, the methods can involve culturing a microbe (e.g., a microalga) in a first microbial culture; recovering microbial lipid from the culture; subjecting the microbial lipid to transesterification to produce fatty acid ester(s) and glycerol, as described above; and adding the glycerol to a second microbial culture as a feedstock. The first and second microbial cultures can, but need not, be cultures of the same microbe. If desired, a continuous system can be devised whereby glycerol produced from the lipid recovered from a culture can be fed back into the same culture.

(48) Provided are significantly improved culture parameters incorporating the use of glycerol for fermentation of multiple genera of both eukaryotic and prokaryotic microbes, including microbes of the genera Prototheca, Chlorella, Navicula, Scenedesmus, and Spirulina. Standard methods for the growth and propagation of Chlorella and/or Prototheca are known (see for example Miao and Wu, J. Biotechnology, 2004, 11:85-93 and Miao and Wu, Biosource Technology (2006) 97:841-846). In addition, multiple species of Chlorella and/or Prototheca and multiple strains within a species can be grown, e.g., in the presence of a sugar (e.g., glucose, sucrose, xylose) and/or glycerol, including glycerol byproduct from biodiesel transesterification.

(49) For oil production, cells, including recombinant cells described herein, are preferably cultured or fermented in large quantities. The culturing may be in large liquid volumes, such as in suspension cultures as an example. Other examples include starting with a small culture of cells which expand into a large biomass in combination with cell growth and propagation as well as oil production. Bioreactors or steel fermentors can be used to accommodate large culture volumes. A fermentor similar to those used in the production of beer and/or wine is suitable, as are extremely large fermentors used in the production of ethanol.

(50) Appropriate nutrient sources for culture in a fermentor are provided. These include raw materials such as one or more of the following: a fixed carbon source such as glucose, corn starch, depolymerized cellulosic material, sucrose, sugar cane, sugar beet, lactose, milk whey, or molasses; a fat source, such as fats or vegetable oils; a nitrogen source, such as protein, soybean meal, cornsteep liquor, ammonia (pure or in salt form), nitrate or nitrate salt, or molecular nitrogen; and a phosphorus source, such as phosphate salts. Additionally, a fermentor allows for the control of culture conditions such as temperature, pH, oxygen tension, and carbon dioxide levels. Optionally, gaseous components, like oxygen or nitrogen, can be bubbled through a liquid culture. Other starch (polymerized glucose) sources such as wheat, potato, rice, and sorghum. Other carbon sources include process streams such as technical grade glycerol, black liquor, organic acids such as acetate, and molasses. Carbon sources can also be provided as a mixture, such as a mixture of sucrose and depolymerized sugar beet pulp.

(51) A fermentor can be used to allow cells to undergo the various phases of their growth cycle. As an example, an inoculum of oil-producing cells can be introduced into a medium followed by a lag period (lag phase) before the cells begin growth. Following the lag period, the growth rate increases steadily and enters the log, or exponential, phase. The exponential phase is in turn followed by a slowing of growth due to decreases in nutrients and/or increases in toxic substances. After this slowing, growth stops, and the cells enter a stationary phase or steady state, depending on the particular environment provided to the cells.

(52) Oil production by cells disclosed herein can occur during the log phase or thereafter, including the stationary phase wherein nutrients are supplied, or still available, to allow the continuation of oil production in the absence of cell division.

(53) In some embodiments, microorganisms grown using conditions described herein and comprise at least about 20% by weight of lipids, preferably at least about 40% by weight, at least about 50% by weight, and more preferably at least about 60% by weight, even more preferably at least about 70%, 75%, 80% or 85% by weight.

(54) In one heterotrophic growth method, sucrose, produced by example from sugar cane or sugar beet, is used as a feedstock. Oil production can be facilitated or made more efficient through the engineering of microbes such as Chlorella and/or Prototheca, to utilize sucrose as a carbon source. For example, expression of a sucrose transporter and a sucrose invertase allows Chlorella and/or Prototheca to transport sucrose into the cell from the culture media and hydrolyze sucrose to yield glucose and fructose. Optionally, a fructokinase can be expressed as well in instances where endogenous hexokinase activity is insufficient for maximum phosphorylation of fructose. Examples of suitable sucrose transporters are Genbank accession numbers CAD91334, CAB92307, and CAA53390. Examples of suitable sucrose invertases are Genbank accession numbers CAB95010, NP012104 and CAA06839. Examples of suitable fructokinases are Genbank accession numbers P26984, P26420 and CAA43322. Vectors for transformation of microalgae, including Chlorella and/or Prototheca, encoding one or more of such genes can be designed as described herein.

(55) Secretion of a sucrose invertase can obviate the need for expression of a transporter that can transport sucrose into the cell. This is because a secreted invertase catalyzes the conversion of a molecule of sucrose into a molecule of glucose and a molecule of fructose, both of which can be transported and utilized by microbes disclosed herein. For example, expression of a sucrose invertase with a secretion signal generates invertase activity outside the cell. See Hawkins et al., Current Microbiology Vol. 38 (1999), pp. 335-341 for examples of secretion signals active in Chlorella and/or Prototheca. Expression of such a protein, as enabled by the genetic engineering methodology disclosed herein, allows cells already capable of utilizing extracellular glucose as an energy source to utilize sucrose as an extracellular energy source. Chlorella and/or Prototheca cells can use both extracellular fructose and extracellular glucose as an energy source, secretion of an invertase can provide the sole catalytic activity necessary for use of sucrose as an efficient, inexpensive energy source.

(56) For example, Chlorella and/or Prototheca cells can be engineered with a sucrose invertase gene under the regulatory control of one of three promoters (Cauliflower mosaic virus 35S promoter (CMV), Chlorella virus promoter (CV), or Chlorella HUP1 promoter (HUP 1)). The sucrose invertase gene used in this example comprises codon-bias to the S. cerevisiae SUC2 gene to improve expression in a C. protothecoides host cell. Expression of a secretable sucrose invertase, such as that described herein, permits the use of molasses, sugar cane juice, and other sucrose-containing feedstocks for cell fermentation.

(57) Alternatively, a sucrose invertase can also be expressed intracellularly in cells that express a sucrose transporter, as well as in cells that express any carbohydrate transporter that allows sucrose to enter the cell.

(58) Bioreactors can be employed for use in heterotrophic growth methods. As will be appreciated, provisions made to make light available to the cells in photosynthetic growth methods are unnecessary when using a fixed-carbon source in the heterotrophic growth methods described herein.

(59) The specific examples of process conditions and heterotrophic growth methods described herein can be combined in any suitable manner to improve efficiencies of microbial growth and/or lipid production. Additionally, conditions and heterotrophic growth methods are useful in the selection and/or genetic engineering of microbes, such as microalgae, to produce microbes that are even more suitable for use in the above-described methods. For example, the microbes having a greater ability to utilize any of the above-described feedstocks for increased proliferation and/or lipid (e.g., fatty acid) production are within the scope of the compositions and methods described herein.

(60) Growth Media

(61) Microorganisms useful in accordance with the methods described herein are found in various locations and environments throughout the world. As a consequence of their isolation from other species and their resulting evolutionary divergence, the particular growth medium for optimal growth and generation of triglyceride constituents can be difficult to predict. In some cases, certain strains of microorganisms may be unable to grow on a particular growth medium because of the presence of some inhibitory component or the absence of some essential nutritional requirement required by the particular strain of microorganism.

(62) Solid and liquid growth media are generally available from a wide variety of sources, and instructions for the preparation of particular media that is suitable for a wide variety of strains of microorganisms can be found, for example, online at utex.org/, a site maintained by the University of Texas at Austin for its culture collection of algae (UTEX). For example, various fresh water and salt water media are provided in U.S. Patent Publ. No. 2012/0288930, hereby incorporated herein by reference in its entirety for all purposes.

(63) In a particular example, a medium suitable for culturing Chlorella and/or Prototheca cells comprises Proteose Medium. This medium is suitable for axenic cultures, and a 1 L volume of the medium (pH .about.6.8) can be prepared by addition of 1 g of proteose peptone to 1 liter of Bristol Medium. Bristol medium comprises 2.94 mM NaNO.sub.3, 0.17 mM CaCl.sub.22H.sub.2O, 0.3 mM MgSO.sub.47H.sub.2O, 0.43 mM, 1.29 mM KH.sub.2PO.sub.4, and 1.43 mM NaCl in an aqueous solution. For 1.5% agar medium, 15 g of agar can be added to 1 L of the solution. The solution is covered and autoclaved, and then stored at a refrigerated temperature prior to use.

(64) Other suitable media for use with the methods described herein can be readily identified by consulting the URL identified above, or by consulting other organizations that maintain cultures of microorganisms, such as SAG, CCAP, or CCALA. SAG refers to the Culture Collection of Algae at the University of Gottingen (Gottingen, Germany), CCAP refers to the culture collection of algae and protozoa managed by the Scottish Association for Marine Science (Scotland, United Kingdom), and CCALA refers to the culture collection of algal laboratory at the Institute of Botany (ccala.butbn.cas.cz/, Czech Republic).

(65) Increasing Production of Lipids

(66) Process conditions can be adjusted to increase the production of lipids suitable for a particular use and/or to reduce production cost. For example, in certain embodiments, an oleaginous cell (e.g., a plant, an algae, a microalga) is cultured in the presence of a limiting concentration of one or more nutrients, such as, for example, carbon and/or nitrogen, phosphorous, or sulfur, while providing an excess of fixed carbon energy such as glucose. Nitrogen limitation tends to increase microbial lipid production over microbial lipid production in a culture in which nitrogen is provided in excess. In particular embodiments, the increase in lipid production is at least about: 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500%. The oleaginous cells (e.g., plant cells, algae cells, microalgal cells) can be cultured in the presence of a limiting amount of a nutrient for a portion of the total culture period or for the entire period. In particular embodiments, the nutrient concentration is cycled between a limiting concentration and a non-limiting concentration at least twice during the total culture period.

(67) In another embodiment, lipid production is increased by culturing oleaginous cells or an oleaginous organism (e.g., plants, algae, microalgae) in the presence of one or more cofactor(s) for a lipid biosynthesis enzyme (e.g., a fatty acid synthetic enzyme). Generally, the concentration of the cofactor(s) is sufficient to increase microbial lipid (e.g., fatty acid) production over microbial lipid production in the absence of the cofactor(s). In a particular embodiment, the cofactor(s) are provided to the culture by including in the culture oleaginous cells (e.g., plant cells, algae cells, microalgal cells) containing an exogenous gene encoding the cofactor(s). Alternatively, cofactor(s) may be provided to a culture by including an oleaginous cell (e.g., a plant, an algae, a microalga) containing an exogenous gene that encodes a protein that participates in the synthesis of the cofactor. In certain embodiments, suitable cofactors include any vitamin required by a lipid biosynthesis enzyme, such as, for example: biotin, pantothenate. Genes encoding cofactors suitable for use in the present compositions and methods or that participate in the synthesis of such cofactors are well known and can be introduced into oleaginous cells (e.g., plant cells, algae cells, microalgal cells), using constructs and techniques such as those described above and herein.

(68) In some embodiments, the cells can be fully auxotrophic or partially auxotrophic (i.e., synthetic sickness or lethality) with respect to one or more types of fatty acid. The cells are cultured with supplementation of the fatty acid(s) so as to increase the cell number, then allowing the cells to accumulate oil (e.g., to at least 40% by dry cell weight). Alternatively, the cells comprise a regulatable fatty acid synthesis gene that can be switched in activity based on environmental conditions and the environmental conditions during a first, cell division, phase favor production of the fatty acid and the environmental conditions during a second, oil accumulation phase disfavor production of the fatty acid.

(69) As a result of applying either of these supplementation or regulation methods, a cell oil may be obtained from the cell that has low amounts of one or more fatty acids essential for optimal cell propagation. Specific examples of oils that can be obtained include those low in stearic, linoleic and/or linolenic acids. Optionally, the cells are oleaginous plastidic microbes such as those of the division Chlorophyta.

(70) Accordingly, in some embodiments, provided are methods for producing an oil or fat. The method comprises cultivating a recombinant oleaginous cell in a growth phase under a first set of conditions that is permissive to cell division so as to increase the number of cells due to the presence of a fatty acid, cultivating the cell in an oil production phase under a second set of conditions that is restrictive to cell division but permissive to production of an oil that is enriched in C18, C20, C22 and/or C24 fatty acids. The cell can be cultivated heterotrophically. In some embodiments, the cell can be a microalgal cell and may produce at least 40%, 50%, 60%, 70%, 80%, or 90% oil by dry cell weight.

(71) 9. Oils with Non-Naturally Occurring Fatty Acid Profiles

(72) Oils disclosed herein are distinct from other naturally occurring oils that are high in C18 long-chain fatty acids, including corn, peanut, cotton seed, apricot seed, mustard seed, neem, olive, palm, safflower, sesame, soybean and sunflower oils. For example, levels of contaminants such as carotenoids are far higher in corn, peanut, cotton seed, apricot seed, mustard seed, neem, olive, palm, safflower, sesame, soybean and sunflower oils than in the oils described herein. Plant oils in particular contain alpha and beta carotenes and lycopene in much higher amounts than are in the oils described herein. The oils described herein contain very few carotenoids species and very low levels. In addition, the levels of vitamin E compounds such as tocotrienols are far higher in plant oils than in the oils described herein.

(73) In some cases, the oleaginous cells (e.g., Prototheca strains) containing a transgene encoding a variant KASII has a fatty acid profile characterized by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or more, C18, C20, C22, and/or C24 fatty acids. In other cases, the Prototheca strains containing a transgene encoding a variant KASII, has activity towards fatty acyl-ACP substrates of chain length C16 and produces fatty acids of the chain length C18 with a ratio of C18 to the sum of other fatty acids (e.g., C8+C10+C12+C14+C16+C20+C22+C24) of at least about 6.0, e.g., at least about 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or higher. The oleaginous cells can optionally co-express an exogenous lipid biosynthesis enzyme, e.g., a C18-preferring fatty acyl-ACP thioesterase,

(74) In some instances, keeping the transgenic Prototheca strains under constant and high selective pressure to retain exogenous genes is advantageous due to the increase in the desired fatty acid of a specific chain length. High levels of exogenous gene retention can also be achieved by inserting exogenous genes into the nuclear chromosomes of the cells using homologous recombination vectors and methods disclosed herein. Recombinant cells containing exogenous genes integrated into nuclear chromosomes are also contemplated.

(75) Microalgal oil can also include other constituents produced by the microalgae, or incorporated into the microalgal oil from the culture medium. These other constituents can be present in varying amounts depending on the culture conditions used to culture the microalgae, the species of microalgae, the extraction method used to recover microalgal oil from the biomass and other factors that may affect microalgal oil composition. Non-limiting examples of such constituents include carotenoids, present from 0.1-0.4 micrograms/ml, chlorophyll present from 0-0.02 milligrams/kilogram of oil, gamma tocopherol present from 0.4-0.6 milligrams/100 grams of oil, and total tocotrienols present from 0.2-0.5 milligrams/gram of oil.

(76) The other constituents can include, without limitation, phospholipids, tocopherols, tocotrienols, carotenoids (e.g., alpha-carotene, beta-carotene, lycopene, etc.), xanthophylls (e.g., lutein, zeaxanthin, alpha-cryptoxanthin and beta-cryptoxanthin), and various organic or inorganic compounds. Additionally, microalgal oils contain long-chain polyunsaturated fatty acids, particularly eicosapentaenoic (EPA).

(77) Generally, higher carotenoid levels accumulate in algae grown photosynthetically than in algae grown heterotrophically. In some cases, the oil extracted from Prototheca species comprises no more than 0.02 mg/kg chlorophyll. In some cases, the oil extracted from Prototheca species comprises no more than 0.4 mcg/ml total carotenoids. In some cases the Prototheca oil comprises between 0.40-0.60 milligrams of gamma tocopherol per 100 grams of oil. In other cases, the Prototheca oil comprises between 0.2-0.5 milligrams of total tocotrienols per gram of oil.

(78) Oils produced from host cells expressing a variant KASII, optionally co-expressing an exogenous lipid biosynthesis enzyme, e.g., a C18-preferring fatty acyl-ACP thioesterase, will have an isotopic profile that distinguishes it, e.g., from blended oils from other sources. The stable carbon isotope value δ13C is an expression of the ratio of 13C/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 δ13C (0/00) of the oils can be related to the δ13C 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 δ13C (0/00) of the oil is from 10 to −17 0/00 or from 13 to −16 0/00.

(79) The oils produced according to the above methods in some cases are made using a microalgal host cell. As described above, the microalga can be, without limitation, fall in the classification of Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. Microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides has been found to contain ergosterol and brassicasterols as major sterols. Both of these sterols feature C24β stereochemistry, in contrast to the C24α stereochemistry found in the majority of common plant sterols. Additional minor sterols present in Chlorella are also believed to primarily have C24β stereochemistry. Thus, the oils produced by the microalgae described above can be distinguished from plant oils by the preponderance of sterols with C24β stereochemistry in the sterols present. For example, the oils produced may contain 22, 23-dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in β-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of Δ.sup.7-poriferasterol. Accordingly, in some embodiments, the oils produced according to the methods described herein lack C24-α sterols.

(80) In one embodiment, 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.

(81) Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, β-sitosterol, and stigamsterol are common plant sterols, with β-sitosterol being a principle plant sterol. For example, β-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).

(82) 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, August 1983. Results of the analysis are shown below (units in mg/100 g) in Table C:

(83) TABLE-US-00003 TABLE C Refined, Refined & bleached, & Sterol Crude Clarified bleached deodorized 1 Ergosterol 384 398 293 302 (56%) (55%) (50%) (50%) 2 5,22-cholestadien- 15 19 14 15 24-methyl-3-ol (2.1%) (2.6%) (2.4%) (2.5%) (Brassicasterol) 3 Other sterols 287 302 283 284 Total sterols 686 719 590 601

(84) 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. Ergosterol is a sterol 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. In summary, Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of β-sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol:β-sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.

(85) 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% β-sitosterol. In other embodiments the oil is free from β-sitosterol.

(86) In some embodiments, the oil is free from one or more of β-sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from β-sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.

(87) 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.

(88) 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.

(89) In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.

(90) 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 β-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).

(91) In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than β-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.

(92) 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.

(93) In some embodiments, oleaginous cells expressing one or more of the polynucleotides described herein can produce an oil with at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or more, C18, C20, C22, and/or C24 fatty acids.

(94) In some embodiments, a recombinant cell comprises nucleic acids operable to express an exogenous gene encoding a variant KASII that catalyzes the elongation of C14 acyl-ACP or C16 acyl-ACP to long-chain fatty acids, optionally co-expressing an exogenous lipid biosynthesis enzyme, e.g., C18-preferring fatty acyl-ACP thioesterase. In some embodiments, the oil produced has a fatty acid profile that is elevated in C18, C20, C22, and/or C24 fatty acids and reduced in C8, C10, C12, C14 and/or C16 fatty acids as a result of the expression of the recombinant nucleic acids. In some embodiments, the increase in C18 (e.g., C18:0, C18:1, C18:2), C20, C22 and/or C24 fatty acids is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, from 75-85%, from 70-90%, from 90-200%, from 200-300%, from 300-400%, from 400-500%, or greater than 500%. In some embodiments, the increase in C18, C20, C22, and/or C24 fatty acids is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or more, in comparison to an untransformed microalga or a microalga transformed with a wild-type KASII enzyme.

(95) The high long-chain oils or fatty acids derived from hydrolysis of these oils may be particularly useful in food, fuel and oleochemical applications including the production of lubricants and surfactants. For example, fatty acids derived from the cells can be esterified, cracked, reduced to an aldehyde or alcohol, aminated, sulfated, sulfonated, or subjected to other chemical process known in the art.

(96) The following examples, which are offered to illustrate, but not to limit, the compositions and methods described herein.

EXAMPLES

(97) The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Improved Variants of Prototheca moriformis KAS II Enzyme

(98) Random mutations were introduced into the wild-type PmKASII gene and the nucleotide sequence encoding the heterologous Chlorella protothecoides SAD1 transit peptide (CpSAD1tp) (SEQ ID NO: 4) via error-prone PCR (epPCR) using the commercially-available GeneMorph II Random Mutagenesis Kit (Agilent Technologies). Construct pSZ6014 (SEQ ID NO: 4) was used as the PCR template, while the oligonucleotides 5′-CGCGCGCAGGATAGACT-3′ and 5′-AAACACCCACCTCCAACG-3′ were used as the forward and reverse primers, respectively, for the mutagenesis reaction. The template concentration and the number of PCR cycles were adjusted to produce a library of variant genes containing an average of 4-5 mutations. This library was then subjected to gel purification using techniques familiar to those skilled in the art. The resulting isolated insert (˜1.5-kb) was cloned into a pUC19-based vector (˜8-kb) via SLIC, a sequence and ligase independent cloning technique familiar to those skilled in the art, at an insert to vector ratio of 20:1. This vector was obtained from the digestion of construct pSZ6014 with restriction enzymes, SpeI and PacI, followed by gel purification. Both the insert and vector share homologous sequences of approximately 40 bp at their 5′ and 3′ ends. The SLIC reaction was transformed into electrocompetent Escherichia coli cells, and the resulting transformants were grown on LB agar with 100 mg/L ampicillin for selection.

(99) Quality control was performed on 20 randomly selected E. coli transformants via colony PCR and Sanger sequencing, techniques that are familiar to those skilled in the art, to confirm the mutagenized inserts they harbor exhibit high mutational diversity but with minimal frame-shift mutations. Plasmid DNA for the epPCR library was then prepared from an E. coli culture that was inoculated with approximately 16,000 transformants and grown in LB medium with 200 mg/L ampicillin for selection. This DNA was digested with the PmeI restriction enzyme to yield a ˜6.6-kb fragment of interest for transformation into the P. moriformis high oleic base strain, S8758, via a suitable technique (as described in U.S. Pat. Nos. 8,846,352; 9,328,351 and 9,649,368). This fragment includes: 1) the 5′ and 3′ homology arms for targeted integration into the DAO1b locus of P. moriformis, 2) the PmSAD2-Iv3 promoter and the PmATP 3′-UTR for expression of the PmKASII variant genes, and 3) an expression cassette for the S. cerevisiae SUC2 gene to enable algal transformants to grow on medium containing sucrose as the sole carbon source. S8758 is a classically-improved derivative of the wild-type strain UTEX 1435, which was obtained from the University of Texas collection and was classically mutagenized to increase the oleic acid content of its oil.

(100) Approximately 1,100 P. moriformis transformants were selected from agar plates containing growth medium (as detailed in U.S. Pat. Nos. 8,846,352; 9,328,351 and 9,649,368) with 20 g/L sucrose as the sole carbon source. These transformants were cultured individually in nitrogen-replete growth medium at pH 5 and 28° C. in microtiter plates until they reached mid-log phase. They were then pooled and used to inoculate 10-mL cultures containing nitrogen-replete growth medium that was supplemented with 80-120 μM cerulenin, a known inhibitor of KASII, to enrich for clones harboring PmKASII variants with increased activity. After approximately 40 h of growth at pH 5 and 28° C., the enrichment culture was plated for clonal isolation.

(101) The resulting clones were grown at pH 5 and 28° C. for 3 or 5 days and screened under low-nitrogen conditions suitable for lipid production (as detailed in the Examples of PCT Patent Application WO 2018/067849). Those that consistently produced oils with a combined C14+C16 fatty acid content that was at least 3 standard deviations below the mean of the control strain S8786, which is also derived from strain S8758 but over-expresses one copy of the wild-type PmKASII gene, were considered as potential hits. Their PmKASII variant genes were PCR-amplified from their genomic DNA and subsequently sequenced to identify the causative mutations that led to their shift in lipid composition compared to strain S8786 using techniques familiar to those skilled in the art. Clones that harbored more than one PmKASII variant gene according to the sequencing data were eliminated from further evaluation.

(102) The PmKASII variant gene (PmKASII.sup.A162V) that conferred the greatest decrease in C14+C16 fatty acid content and increase in long-chain and very long-chain fatty acid content (C18+C20+C22+C24) contains two nucleotide mutations (SEQ ID NO: 5). One of them consists of a cytosine to thymine change at nucleotide position 485, which results in the replacement of an alanine to a valine at amino-acid position 162 of the encoded enzyme. The other, which consists of the cytosine to thymine change at nucleotide position 1080, is a silent mutation. To confirm the improved activity of the PmKASII.sup.A162V enzyme (SEQ ID NO: 2) in P. moriformis, the variant gene was re-cloned into the same expression vector that can be obtained through excision of construct pSZ6014 (as described above). The resulting construct pSZ6256 (SEQ ID NO: 3) was then re-transformed into strain S8758, and 12 clonally-purified transformants were selected for evaluation in 5-day lipid production cultures at pH 5 and 28° C. under low-nitrogen conditions. The transformants over-expressing one copy of the PmKASII.sup.A162V variant gene produced oils that on average showed a decrease in C14+C16 content from 3.9% to 2.8% and an increase in C18+C20+C22+C24 content from 95.3% to 96.9% compared to the control strain S8786, which over-expresses one copy of the wild-type PmKASII gene (Table 1). Despite the fact that a very low C14+C16 content can already be reached with the wild-type PmKASII (SEQ ID NO: 1), a further drop in C14+C16 content that is of statistical significance (t-test, p<0.01) was achieved with the PmKASII.sup.A162V variant. These results highlight the enhanced elongation activity of the PmKASII.sup.A162V variant and its utility for producing oils with reduced levels of C14 or C16 fatty acids and elevated levels of long-chain or very long-chain fatty acids.

(103) TABLE-US-00004 TABLE 1 Comparison of Overexpression of PmKASII.sup.A162V and Wild-type PmKASII in P. moriformis strain S8758 on Oil Composition.* Strain/ Fatty Acid Profile (%) Transfor- C16:1 C16:1 C14 + mant PmKASII n C14:0 C16:0 cis-7 cis-9 C16 C18:0 C18:1 C18:2 S8758- A162V 9 Avg 0.3 2.4 0.0 0.1 2.8 6.5 81.5 5.5 pSZ6256 Std — 0.07 — — 0.08 0.15 0.25 0.01 Dev S8786 Wild- 3 Avg 0.5 3.2 0.0 0.1 3.9 5.6 82.3 5.9 (Control) type Std — 0.01 — — 0.01 0.04 0.04 0.05 Dev S8758 None 3 Avg 0.4 10.9 0.0 0.3 11.7 3.6 77.5 5.8 (High Oleic Std — 0.08 — — 0.09 0.03 0.07 0.00 Base Strain) Dev Strain/ Fatty Acid Profile (%) Transfor- C18:3 C18 + C20 + mant PmKASII n α C20:0 C20:1 C20:2 C22:0 C24:0 C22 + C24 S8758- A162V 9 Avg 0.6 0.7 1.9 0.0 0.1 0.1 96.9 pSZ6256 Std — — 0.06 — — — 0.09 Dev S8786 Wild- 3 Avg 0.6 0.4 0.3 0.0 0.1 0.1 95.3 (Control) type Std — — — — — — 0.01 Dev S8758 None 3 Avg 0.5 0.2 0.3 0.0 0.0 0.0 88.0 (High Oleic Std — — — — — — 0.08 Base Strain) Dev *All strains and transformants were tested in 5-day lipid production cultures at pH 5 and 28° C. under low-nitrogen conditions. Data for outlier transformants are not included.

Example 2

Saturation Mutagenesis at Amino Acid Residue 162 of the Prototheca moriformis KAS II Enzyme

(104) Example 1 demonstrated that overexpression of the wild-type PmKASII enzyme (SEQ NO ID: 1) can decrease the C14+C16 fatty acid content of the oil produced by P. moriformis, while increasing the long-chain and very long-chain fatty acid content (C18+C20+C22+C24). It also showed that an additional shift in oil composition toward long-chain and very long-chain fatty acids can be achieved by overexpressing a PmKASII enzyme variant that contains an Ala to Val mutation at amino acid residue 162 (SEQ NO ID: 2). In this example, results from saturation mutagenesis at position 162 of the PmKASII enzyme confirm the importance of this amino acid position in producing oils with elevated levels of long-chain (C18) and/or very long-chain (C20+C22+C24) fatty acids.

(105) An expression construct that targets integration of the wild-type PmKASII gene (pSZ6014, SEQ NO ID: 12), the PmKASII.sup.A162V gene variant (pSZ7122, SEQ NO ID: 13), or the remaining eighteen PmKASII.sup.A162X gene variants (pSZ7104-pSZ7121, SEQ NO ID: 13) to the DAO1 locus in P. moriformis was transformed into the base strain S7485. These constructs also contain an expression cassette for the S. cerevisiae SUC2 (ScSUC2) gene, which enables growth of the resulting transformants on medium containing sucrose as the sole carbon source. Expression of the PmKASII.sup.A162X variants in the transgenic strains is driven by the constitutive PmSAD2-1 promoter. S7485 is a classically-improved derivative of the wild-type strain UTEX 1435, which was classically mutagenized to increase oil yield and productivity. The classical mutagenesis did not substantively alter the oleic acid content of the oil produced by S7485 when compared to UTEX 1435.

(106) Clonally-purified transformants derived from strain S7485 and expressing a single copy of one of the PmKASII.sup.A162X variants (pSZ6014, pSZ7122, or pSZ7104-pSZ7121) were evaluated in 5-day lipid production cultures at pH 5 (as detailed in the Examples of PCT Patent Application WO 2018/067849) to determine their lipid profiles. The parental strain S7485 was also assessed as a control under the same conditions. The fatty acid profile of the oils produced by these transformants and strain are presented in Table 2. Compared to the parental strain S7485, the transformants expressing one additional copy of the wild-type PmKASII (pSZ6014) showed a prominent increase in C18+C20+C22+C24 fatty acid content from 65% to 84% with a concomitant decrease in C14+C16 fatty acid content from 35% to 16%. Overexpressing the A162V variant (pSZ7122) instead of the wild-type gene further increased the C18+C20+C22+C24 fatty acid content to 90%, while decreasing the C14+C16 fatty acid content to 10%. Likewise, overexpression of the A162N (pSZ7105), A162D (pSZ7106), A162Q (pSZ7108), A162H (pSZ7111), A162L (pSZ7113), A162F (pSZ7116), A162T (pSZ7119), or A162Y (pSZ7121) variant resulted in a similar shift in the fatty acid profile. The overexpression of many of the remaining variants—including A162C (pSZ7107), A162E (pSZ7109), A162K (pSZ7114), A162M (pSZ7115), A162S (pSZ7118), and A162W (pSZ7120)—also led to marked increases in the C18+C20+C22+C24 fatty acid content compared to the wild-type PmKASII with concomitant decreases in the C14+C16 fatty acid content, though the observed impact was less pronounced than that achieved with the A162V variant. Taken together, these results demonstrate the importance of position 162 of the PmKASII enzyme on the biosynthesis of triglycerides with elevated levels of long-chain and very long chain fatty acids (C18+C20+C22+C24).

(107) TABLE-US-00005 Comparison of Overexpression of PmKASII.sup.A162X and Wild-type PmKASII in P. moriformis strain S7485 on Oil Composition.* Fatty Acid Profile (%) Strain/ C16:1 C16:1 C14 + Transformant PmKASII n C14:0 C16:0 cis-7 cis-9 C16 C18:0 C18:1 C18:2 S7485 None 4 Avg 1.9 32.2 0.0 0.8 34.8 3.8 55.2 4.8 Std 0.00 0.08 0.01 0.02 0.09 0.01 0.01 0.03 Dev S7485- Wild- 4 Avg 2.0 13.4 0.1 0.3 15.7 6.0 71.6 5.1 pSZ6014 type Std 0.01 0.08 0.01 0.01 0.08 0.02 0.12 0.05 Dev S7485- A162V 5 Avg 1.1 8.3 0.1 0.2 9.6 7.1 76.4 5.1 pSZ7122 Std 0.01 0.05 0.01 0.01 0.07 0.07 0.14 0.08 Dev S7485- A162R 4 Avg 1.5 13.6 0.1 0.3 15.5 6.1 71.9 5.1 pSZ7104 Std 0.02 0.21 0.02 0.01 0.22 0.09 0.25 0.07 Dev S7485- A162N 4 Avg 1.3 9.0 0.1 0.2 10.5 7.2 75.4 5.1 pSZ7105 Std 0.02 0.14 0.01 0.01 0.16 0.07 0.23 0.09 Dev S7485- A162D 6 Avg 1.2 9.3 0.1 0.2 10.8 7.0 75.3 5.1 pSZ7106 Std 0.03 0.21 0.00 0.00 0.24 0.12 0.39 0.06 Dev S7845- A612C 6 Avg 1.6 12.1 0.1 0.2 13.9 6.2 73.2 5.1 pSZ7107 Std 0.06 0.10 0.03 0.01 0.13 0.18 0.11 0.08 Dev S7845- A612Q 6 Avg 1.2 8.1 0.1 0.2 9.6 7.0 76.7 5.0 pSZ7108 Std 0.01 0.07 0.01 0.01 0.07 0.03 0.01 0.08 Dev S7845- A612E 6 Avg 1.4 11.3 0.1 0.2 13.0 6.4 73.8 5.1 pSZ7109 Std 0.02 0.12 0.01 0.01 0.15 0.10 0.16 0.05 Dev S7845- A612G 6 Avg 2.1 14.8 0.1 0.3 17.3 5.7 70.4 5.1 pSZ7110 Std 0.02 0.12 0.01 0.01 0.15 0.09 0.13 0.05 Dev S7845- A612H 5 Avg 1.2 8.6 0.1 0.2 10.1 6.8 76.3 5.1 pSZ7111 Std 0.02 0.13 0.01 0.01 0.16 0.13 0.35 0.10 Dev S7845- A612I 8 Avg 1.9 13.2 0.1 0.3 15.5 5.9 72.0 5.1 pSZ7112 Std 0.05 0.23 0.01 0.01 0.28 0.08 0.36 0.08 Dev S7845- A612L 7 Avg 1.1 8.7 0.1 0.2 10.0 7.1 76.0 5.2 pSZ7113 Std 0.01 0.12 0.01 0.01 0.12 0.12 0.20 0.13 Dev S7845- A612K 6 Avg 1.3 10.0 0.1 0.2 11.6 6.7 74.9 5.1 pSZ7114 Std 0.02 0.13 0.01 0.01 0.16 0.03 0.17 0.03 Dev S7845- A612M 8 Avg 1.3 11.1 0.1 0.2 12.7 6.4 74.2 5.1 pSZ7115 Std 0.04 0.51 0.01 0.03 0.52 0.24 0.53 0.09 Dev S7845- A612F 7 Avg 1.1 7.9 0.1 0.2 9.3 7.0 76.9 5.1 pSZ7116 Std 0.02 0.08 0.01 0.00 0.09 0.05 0.14 0.04 Dev S7845- A612P 8 Avg 1.9 13.1 0.1 0.3 15.4 6.0 72.0 5.1 pSZ7117 Std 0.03 0.10 0.01 0.01 0.11 0.10 0.26 0.03 Dev S7845- A612S 6 Avg 1.5 10.3 0.1 0.2 12.2 6.5 74.5 5.2 pSZ7118 Std 0.03 0.16 0.01 0.01 0.20 0.12 0.08 0.04 Dev S7845- A612T 7 Avg 1.1 7.3 0.1 0.1 8.7 7.3 77.0 5.2 pSZ7119 Std 0.04 0.36 0.01 0.01 0.40 0.13 0.33 0.07 Dev S7845- A612W 6 Avg 1.3 10.8 0.1 0.2 12.3 6.4 74.5 5.1 pSZ7120 Std 0.03 0.13 0.01 0.01 0.15 0.04 0.19 0.06 Dev S7845- A612Y 8 Avg 1.1 7.7 0.1 0.2 9.1 6.9 77.2 5.1 pSZ7121 Std 0.03 0.14 0.01 0.01 0.19 0.22 0.30 0.11 Dev Fatty Acid Profile (%) Strain/ C18:3 C18 + C20 + Transformant PmKASII n α C20:0 C20:1 C20:2 C22:0 C24:0 C22 + C24 S7485 None 4 Avg 0.4 0.3 0.0 0.0 0.1 0.0 64.6 Std 0.01 0.01 0.02 0.00 0.01 0.00 0.09 Dev S7485- Wild- 4 Avg 0.4 0.4 0.1 0.0 0.1 0.0 83.7 pSZ6014 type Std 0.01 0.01 0.10 0.00 0.01 0.00 0.12 Dev S7485- A162V 5 Avg 0.4 0.5 0.1 0.0 0.1 0.0 89.6 pSZ7122 Std 0.01 0.01 0.17 0.00 0.01 0.00 0.13 Dev S7485- A162R 4 Avg 0.4 0.4 0.1 0.0 0.1 0.0 84.0 pSZ7104 Std 0.01 0.01 0.10 0.00 0.01 0.00 0.28 Dev S7485- A162N 4 Avg 0.4 0.5 0.2 0.0 0.1 0.0 88.9 pSZ7105 Std 0.01 0.01 0.14 0.00 0.01 0.00 0.22 Dev S7485- A162D 6 Avg 0.4 0.5 0.1 0.0 0.1 0.0 88.5 pSZ7106 Std 0.01 0.01 0.18 0.00 0.01 0.00 0.21 Dev S7845- A612C 6 Avg 0.4 0.4 0.0 0.0 0.1 0.0 85.4 pSZ7107 Std 0.02 0.03 0.07 0.00 0.01 0.00 0.17 Dev S7845- A612Q 6 Avg 0.4 0.5 0.3 0.0 0.1 0.0 89.9 pSZ7108 Std 0.02 0.01 0.13 0.00 0.01 0.00 0.17 Dev S7845- A612E 6 Avg 0.4 0.5 0.1 0.0 0.1 0.0 86.4 pSZ7109 Std 0.01 0.01 0.17 0.00 0.01 0.00 0.12 Dev S7845- A612G 6 Avg 0.4 0.4 0.0 0.0 0.1 0.0 82.0 pSZ7110 Std 0.01 0.02 0.07 0.00 0.01 0.00 0.22 Dev S7845- A612H 5 Avg 0.4 0.5 0.1 0.0 0.1 0.0 89.2 pSZ7111 Std 0.01 0.01 0.15 0.00 0.01 0.00 0.24 Dev S7845- A612I 8 Avg 0.4 0.4 0.0 0.0 0.1 0.0 83.9 pSZ7112 Std 0.01 0.01 0.04 0.00 0.01 0.00 0.29 Dev S7845- A612L 7 Avg 0.4 0.5 0.1 0.0 0.1 0.0 89.3 pSZ7113 Std 0.01 0.02 0.17 0.00 0.01 0.00 0.21 Dev S7845- A612K 6 Avg 0.4 0.5 0.0 0.0 0.1 0.0 87.7 pSZ7114 Std 0.01 0.01 0.10 0.00 0.01 0.00 0.17 Dev S7845- A612M 8 Avg 0.4 0.4 0.1 0.0 0.1 0.0 86.6 pSZ7115 Std 0.01 0.01 0.10 0.00 0.01 0.00 0.47 Dev S7845- A612F 7 Avg 0.4 0.5 0.2 0.0 0.1 0.0 90.2 pSZ7116 Std 0.01 0.01 0.14 0.00 0.01 0.00 0.16 Dev S7845- A612P 8 Avg 0.4 0.4 0.1 0.0 0.1 0.0 84.0 pSZ7117 Std 0.01 0.02 0.07 0.00 0.01 0.00 0.16 Dev S7845- A612S 6 Avg 0.4 0.5 0.1 0.0 0.1 0.0 87.1 pSZ7118 Std 0.01 0.02 0.12 0.00 0.01 0.00 0.28 Dev S7845- A612T 7 Avg 0.4 0.5 0.0 0.0 0.1 0.0 90.5 pSZ7119 Std 0.01 0.03 0.00 0.00 0.01 0.00 0.37 Dev S7845- A612W 6 Avg 0.4 0.4 0.0 0.0 0.1 0.0 87.0 pSZ7120 Std 0.01 0.02 0.09 0.00 0.01 0.00 0.14 Dev S7845- A612Y 8 Avg 0.4 0.5 0.0 0.0 0.1 0.0 90.2 pSZ7121 Std 0.01 0.02 0.11 0.00 0.01 0.00 0.16 Dev *The parental strain S7485 was transformed with construct pSZ6014, pSZ7122, or pSZ7104-pSZ7121 (Table 3). Each of these constructs expresses either the wild-type PmKASII or one of the enzyme variants containing an amino acid substitution at position 162. All clonally-purified transformants were tested in five-day lipid production cultures at pH 5 under low nitrogen conditions. Data for outlier transformants are not included. Fatty acid profiles of oils produced by single integrants of the PmKASII gene are shown. The highlighted variants represent the amino acid substitutions that confer the greatest increase in long-chain and very long-chain fatty acid content (C18 + C20 + C22 + C24). n = total of replicates or transformants tested.

(108) TABLE-US-00006 TABLE 3 Codons used to encode specific amino acids in various expression constructs. Construct Amino Acid Codon pSZ7104 Arg CGC pSZ7105 Asn AAC pSZ7106 Asp GAC pSZ7107 Cys TGC pSZ7108 Gin CAG pSZ7109 Glu GAG pSZ7110 Gly GGC pSZ7111 His CAC pSZ7112 Ile ATC pSZ7113 Leu CTG pSZ7114 Lys AAG pSZ7115 Met ATG pSZ7116 Phe TTC pSZ7117 Pro CCG pSZ7118 Ser AGC pSZ7119 Thr ACG pSZ7120 Trp TGG pSZ7121 Tyr TAC pSZ7122 Val GTC

(109) It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.