CROSS REFERENCE TO RELATED APPLICATIONS This application is continuation of U.S. patent application Ser. No. 15/950,048, filed Apr. 10, 2018, entitled “Novel Ketoacyl ACP Synthase Genes and Uses Thereof”, which is a divisional of U.S. patent application Ser. No. 14/796,406, filed Jul. 10, 2015, entitled “Novel Ketoacyl ACP Synthase Genes and Uses Thereof”, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 62/023,112, filed Jul. 10, 2014, and U.S. Provisional Patent Application No. 62/081,143, filed Nov. 18, 2014, each of which is incorporated herein by reference in its entirety. This application includes subject matter related to that disclosed in U.S. provisional patent application No. 62/023,109 (attorney docket number 057518-448593), entitled “Tailored Oils,” filed Jul. 10, 2014, which is hereby incorporated by reference in its entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING This application includes include an electronic sequence listing in a file names “465964-Sequence.txt”, created on Sep. 28, 2015, and contains 235,869 bytes, which is hereby incorporated by reference in its entirety for all purposes.
TECHNICAL FIELD The present invention relates to novel β-ketoacyl ACP synthase genes and methods for using the genes including expressing the genes in oleaginous host cells to produce triglycerides with altered fatty acid profiles.
BACKGROUND Certain organisms including plants and some microalgae use a type II fatty acid biosynthetic pathway, characterized by the use of discrete enzymes in a multimeric complex for fatty acid synthesis. In contrast, mammals and fungi use a single, large, multifunctional protein.
In organisms that use a type II fatty acid biosynthetic pathway, β-ketoacyl-ACP synthase I (KAS I, EC 2.3.1.41) is one of the enzymes responsible for elongation of growing medium-chain fatty acyl-ACP from 4 to 16 carbon atoms in length. KAS I uses C2-C14 acyl-ACPs as substrates for condensation with a C2 unit derived from malonyl-ACP. KASIV is a related enzyme that serves a similar elongation function. Thus, KASI and KASIV can both be considered KASI-like enzymes.
Such genes have been introduced to plants using recombinant DNA technology. See for example U.S. Pat. Nos. 7,301,070, 6,348,642, 6,660,849, 6,770,465 and US2006/0094088 (of which ¶¶194-200 and the entirety of the document are hereby incorporated herein by reference). In plastidic cells such as those from plants, macroalgae and microalgae, KAS I-like enzymes are located in the chloroplasts or other plastids together with other enzyme of the fatty acid synthesis (FAS) pathway.
PCT publications WO2010/063032, WO2011/150411, WO2012/106560, and WO2013/158938 disclose genetic engineering of oleaginous microalgae including targeting of exogenous FAS gene products to the microalgal plastid.
SUMMARY In one aspect, embodiments of the invention include a non-natural, isolated polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to any one of SEQ ID NOs: 21-37, or 39-55, or encoding a KASI-like protein having at least 80, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% amino acid sequence identity to any one of SEQ ID NOs: 2-18, 62-72, or a mature protein produced therefrom, or the complement of the polynucleotide.
In another aspect, embodiments of the invention include a transformation vector comprising a cDNA molecule comprising a polynucleotide as discussed above. In some cases, the vector comprises promoter and 3′UTR sequences in operable linkage to the cDNA, and optionally a flanking sequence for homologous recombination. The promoter or the 3′UTR sequences are heterologous nucleotide sequences. The heterologous promoter or the heterologous 3′UTR sequences can be from a different organism than the organism from which the nucleotide sequences encoding KAS was first obtained.
In one aspect, the transfomration vector comprises a heterologous promoter or a heterologous 3′UTR sequence obtained from the same organism from which the KAS gene was first isolated. When the promoter sequence, the 3′UTR sequence and the KAS nucleotide sequences are from the same organism, the heterologous promoter does not naturally drive the expression of KAS, and the 3′UTR does not naturally occur downstream from the KAS nucleotide sequences in the source organism.
In yet another aspect, the transformation vector is used to express the KAS gene in the organism from which the KAS gene was first isolated. When the KAS gene is recombinantly expressed in the organism from which the KAS gene was first isolated, the gene is expressed in a different chromosomal locus than the natural chromosomal locus of the KAS gene. Alternatively, the KAS gene is expressed in the cytoplasm.
In another aspect, embodiments of the invention include a host cell comprising the polynucleotide and/or the vector discussed above, and expressing a functional KAS protein encoded by the cDNA. In some cases, the host cell further comprises an exogenous gene encoding a functional FATA acyl-ACP thioesterase or FATB acyl-ACP thioesterase. In one aspect, the FATB acyl-ACP thioesterase has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to any one of SEQ ID NOs: 1 or SEQ ID NO: 57. In some cases, the host cell produces a cell oil characterized by a fatty acid profile with (i) at least 30, 40, 50, or 55% C14:0, (ii) at least 7, 8, 9, 10, 11, 12, 13, or 14% C8:0, (iii) at least 10, 15, 20, 25, 30, or 35 area % for the sum of C8:0 and C10:0, or (iv) a C8/C10 ratio in the range of 2.2-2.5, 2.5-3.0, or 3.0-3.4. In some cases, the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway. In some cases, the host cell is a microalga. In some cases, the host cell is of Trebouxiophyceae, and optionally of the genus Chlorella or Prototheca. In some cases, the microalga is of the species Prototheca moriformis.
In another aspect, embodiments of the invention include a method for making a cell-oil, the method comprising cultivating a host cell as discussed above so as produce the cell-oil, wherein the oil comprises triglcyerides and microalgal sterols. In some cases, the cell oil comprises sterols characterized by a sterol profile and the sterol profile has an excess of ergosterol over β-sitosterol and/or the presence of 22, 23-dihydrobrassicasterol, poriferasterol or clionasterol.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a phylogenetic tree for KASI-like genes in connection with Example 3.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 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.
A “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 or cell fat 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 “cell oil” and “cell fat” encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching 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.
“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, for example, as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
“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.
“Microalgae” are microbial organisms that contain a chloroplast or other plastid, and optionally that are 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 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.
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.
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×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×drop-off 50; Expect: 10; Word Size: 3; Filter:
on.
Where multiple sequence identities are given for a strain having a pair of exogenous genes, this encompasses all combinations of sequence identities. For example, coexpression of a first gene encoding a first protein having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% with gene A and a second gene encoding a second protein having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% with gene A shall be understood to encompass (i) at least 85% identity with gene A and least 85% identity with gene B, (ii)) at least 85% identity with gene A and least 99% identity with gene B, (iii) at least 92% identity with gene A and least 95% identity with gene B, and all other combinations.
In connection with a cell oil, a “profile” is the distribution of particular species of 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). 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.
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).
“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 (host) 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 a gene product or suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) 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 nucleic by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Recombinant nucleic acids can also be produced in other ways; e.g., using chemical DNA synthesis. 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 “KAS I-like” gene or enzyme shall mean either a KAS I or KAS IV gene or enzyme.
Embodiments of the present invention relate to the use of KASI-like genes isolated from plants or other organisms, which can be expressed in a transgenic host cell in order to alter the fatty acid profile of a cell-oil produced by the host cell. Although the microalga Prototheca moriformis was used to screen the genes for ability to the alter fatty acid profile, the genes discovered are useful in a wide variety of host cells for which genetic transformation techniques are known. For example, the genes can be expressed in bacteria, cyanobacteria, other eukaryotic microalgae, or higher plants. The genes can be expressed in higher plants according to the methods disclosed in US patent Nos: U.S. Pat. Nos. 7,301,070, 6,348,642, 6,660,849, and 6,770,465. We have found that KASI-like transgenes can be used alone or in combination with a FatB transgene (encoding an active acyl-ACP thioesterase) can boost the levels of mid-chain fatty acids (e.g., capric, caprylic, lauric, myristic or palmitic acids) in the fatty acid profile of the cell oil. Combining an exogenous KASI-like gene with an exogenous FATA or FATB gene in a host cell can give levels of mid-chain fatty acids and/or long-chain fatty acids (e.g., stearic or oleic) greater than either exogenous gene alone. The fatty acids of the cell oil can be further converted to triglycerides, fatty aldehydes, fatty alcohols and other oleochemicals either synthetically or biosynthetically.
In specific embodiments, triglycerides are produced by a host cell expressing a novel KASI-like gene (from a novel cDNA and/or under control of a heterologous promoter). A cell oil can be recovered from the host cell. Typically, the cell oil comprises mainly triglycerides and sterols. The cell oil can be refined, degummed, bleached and/or deodorized. The oil, in its unprocesssed or processed form, can be used for foods, chemicals, fuels, cosmetics, plastics, and other uses. In other embodiments, the KASI-like gene may not be novel, but the expression of the gene in a microalga is novel.
The KAS 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 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 a KASI-like enzyme of Table 1 and a 3′UTR. The 3′UTR can comprise a polyadenylation site.
As described in the examples below, novel KAS genes are were discovered from cDNA produced from plant seed mRNA transcripts. Accordingly the gene sequences are non-natural because they lack introns that are present in the plant genes and mRNA transcripts of the genes prior to mRNA splicing. Accordingly, the invention comprises an isolated non-natural KASI-like gene of Table 1. Further departure from the natural gene is in the use of heterologous regulatory elements and expression in host cells for which such genes do not occur in nature.
For example, the gene can be prepared in an expression vector comprising an operably linked promoter and 5′UTR. Where a plastidic cell is used as the host, a suitably active plastid targeting peptide (also referred to below as a “transit peptide”) can be fused to the KASI-like gene, as in the examples below. The disclosed genes comprise a hydrophobic N-terminal plastid targeting sequence, which can be replaced with alternative targeting sequence and varied in length. Varying the plastid targeting peptide can improve cellular localization and enzyme activity for a given host-cell type. 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. Additional terminal or internal deletions may be made so-long as the enzymatic activity is retained. The targeting peptide can be cleaved by the host cell to produce a mature KASI-like protein that lacks the targeting peptide.
A selectable marker gene may be included in the vector to assist in isolating a transformed cell. Examples of selectable markers useful in microalgae include sucrose invertase, alpha galactosidase (for selection on melibiose) and antibiotic resistance genes.
The gene sequences disclosed can also be used to prepare antisense, or inhibitory RNA (e.g., RNAi or hairpin RNA) to inhibit complementary genes in a plant or other organism. For example, armed with the knowledge of a gene sequence of Table 1, one can engineer a plant with the same or similar KASI-like gene to express an RNAi construct, gene knockout, point mutation, or the like, and thereby reduce the KASI or KASIV activity of the plant's seed. As a result, the plant 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.
KASI-like genes/proteins found to be useful in producing desired fatty acid profiles in a cell are summarized below in Table 1, and related proteins discovered from transcript sequencing (as in Examples 1-2) are shown in Table 1a. Nucleic acids or proteins having the sequence of SEQ ID NOS: 2-18, 59, 62-72, 21-37 or 39-55 can be used to alter the fatty acid profile of a recombinant cell. Variant nucleic acids can also be used; e.g., variants having at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOS: 21-37 or 39-55. Codon optimization of the genes for a variety of host organisms is contemplated, as is the use of gene fragments. Preferred codons for Prototheca strains and for Chlorella protothecoides are shown below in Tables 2 and 3, respectively. Codon usage for Cuphea wrightii is shown in Table 4. Codon usage for Arabidopsis is shown in Table 5; for example, the most preferred codon for each amino acid can be selected. Codon tables for other organisms including microalgae and higher plants are known in the art. In some embodiments, the first and/or second most preferred Prototheca codons are employed for codon optimization. In specific embodiments, the novel amino acid sequences contained in the sequence listings below are converted into nucleic acid sequences according to the most preferred codon usage in Prototheca, Chlorella, Cuphea wrightii, or Arabidopsis as set forth in tables 2 through 3b or nucleic acid sequences having at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to these derived nucleic acid sequences. For example, the KASI-like gene can be codon optimized for Prototheca moriformis by substituting most preferred codons according to Table 2 for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of all codons. Likewise, the KASI-like gene can be codon optimized for Chlorella protothecoides by substituting most-preferred codons according to Table 3 for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of all codons. Alternately, the KASI-like gene can be codon optimized for Chlorella protothecoides or Prototheca moriformis by substituting first or second most-preferred codons according to Table 2 or 3 for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of all codons. Codon-optimized genes are non-naturally occurring because they are optimized for expression in a host organism.
In certain embodiments, percent sequence identity for variants of the nucleic acids or proteins discussed above can be calculated by using the full-length nucleic acid sequence (e.g., one of SEQ ID NOS: 21-37 or 39-55 or full-length amino acid sequence (e.g., one of SEQ ID NOS: 2-18) as the reference sequence and comparing the full-length test sequence to this reference sequence. For fragments, percent sequence identity for variants of nucleic acid or protein fragments can be calculated over the entire length of the fragment. In certain embodiments, there is a nucleic acid or protein fragment have at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to one of SEQ ID NOS: 21-37, 39-55 or 2-18.
Optionally, the plastidic targeting peptide can be swapped with another peptide that functions to traffic the KASI-like enzyme to a fatty acid synthesizing plastid of a plastidic host cell. Accordingly, in various embodiments of the invention, a transgene or transgenic host cell comprises a nucleotide or corresponding peptidic fusion of a plastic targeting sequence and an enzyme-domain sequence (the sequence remaining after deletion of the transit peptide), where the mature protein has at least 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to an mature protein sequence listed in Table 1 or Table 1a. Plastid transit/targeting peptides are underlined in the accompanying informal sequence listing. Examples of targeting peptides include those of Table 1 and others known in the art, especially in connection with the targeting of KAS I, KAS II, KAS III, FATA, FATB and SAD (stearoyl-ACP desaturase) gene products to chloroplasts or other plastids of plants and microalgae. See examples of Chorophyta given in PCT publications WO2010/063032, WO2011/150411, WO2012/106560, and WO2013/158938. Optionally, the KASI-like genes encode 450, 475 or 500 amino acids or more (with or without the transit peptide), or about 555 residues (with the transit peptide) in contrast to known truncated sequences.
TABLE 1
KASI-like genes: The expression cassette used to test the genes in
combination with a FATB transgene is given in SEQ ID NO: 38 (i.e.,
substituting the Cpal KASIV coding sequence of SEQ ID NO: 38 with
various other coding sequences of Table 1), except that the
Cuphea hookeriana KASIV was tested using the expression
cassette of SEQ ID NO: 61. See Examples 1-4.
nucleotide
coding
sequence
(from cDNA Prototheca
produced moriformis
from codon-
Amino seed mRNA, optimized
Acid not codon- nucleotide
Species Gene Name Sequence optimized) sequence
Cuphea KASIV 2 21 39
palustris
Cinnamonum KASIV 3 22 40
camphora
Cinnamonum KASI 4 23 41
camphora
Umbellularia KASI 5 24 42
californica
U. californica KASIV 6 25 43
Cuphea. KASAI 7 26 44
wrightii
Cuphea KASIVb 8 27 45
avigera
Cuphea KASIVb 9 28 46
paucipetala
C. ignea KASIVb 10 29 47
Cuphea KASIV 11 30 48
procumbens
C. paucipetala KASIVa 12 31 49
Cuphea KASIV 13 32 50
painteri
C. avigera KASIVa 14 33 51
C. ignea KASIVa 15 34 52
C. avigera KASIa 16 35 53
C. pulcherrima KASI 17 36 54
C. avigera mitochondrial 18 37 55
KAS
Cuphea KASIV 59 60, 61
hookeriana
TABLE 1a
Additional proteins encoded by cDNA discovered from transcript
profiling of seeds. Coding sequences can be derived from codon
tables for various host cells.
Amino
Acid
Species Gene Name Sequence
Various KASIV 69, 71
(Clade 1) consensus
sequence
Various KASIV 70, 72
(Clade 2) consensus
sequence
Cuphea KASIV 62
aequipetala
Cuphea KASIV 63
glassostoma
Cuphea KASIV 64
hookeriana
Cuphea KASIV 65
glassostoma
Cuphea KASIV 66, 67
carthagenesis
C. pulcherrima KASIV 68
TABLE 2
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) Pro CCG 161 (0.29)
GCC 442 (0.46) 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)
Phe TTT 89 (0.29) CGG 102 (0.18)
TTC 216 (0.71) CGA 49 (0.08)
Gly GGG 92 (0.12) CGT 51 (0.09)
GGA 56 (0.07) CGC 331 (0.57)
GGT 76 (0.10) Ser AGT 16 (0.03)
GGC 559 (0.71) AGC 123 (0.22)
His CAT 42 (0.21) TCG 152 (0.28)
CAC 154 (0.79) TCA 31 (0.06)
Ile ATA 4 (0.01) TCT 55 (0.10)
ATT 30 (0.08) TCC 173 (0.31)
ATC 338 (0.91) Thr ACG 184 (0.38)
Lys AAG 284 (0.98) ACA 24 (0.05)
AAA 7 (0.02) ACT 21 (0.05)
Leu TTG 26 (0.04) ACC 249 (0.52)
TTA 3 (0.00) Val GTG 308 (0.50)
CTG 447 (0.61) GTA 9 (0.01)
CTA 20 (0.03) GTT 35 (0.06)
CTT 45 (0.06) GTC 262 (0.43)
CTC 190 (0.26) Trp TGG 107 (1.00)
Met ATG 191 (1.00) Tyr TAT 10 (0.05)
TAC 180 (0.95)
Stop TGA/TAG/TAA
TABLE 3
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 4
Codon usage for Cuphea wrightii (codon, amino acid, frequency, per
thousand, number)
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 5
Codon usage for Arabidopsis (codon, amino acid, frequency, per thousand)
UUU F 0.51 21.8 UCU S 0.28 25.2 UAU Y 0.52 14.6 UGU C 0.60 10.5
UUC F 0.49 20.7 UCC S 0.13 11.2 UAC Y 0.48 13.7 UGC C 0.40 7.2
UUA L 0.14 12.7 UCA S 0.20 18.3 UAA * 0.36 0.9 UGA * 0.44 1.2
UUG L 0.22 20.9 UCG S 0.10 9.3 UAG * 0.20 0.5 UGG W 1.00 12.5
CUU L 0.26 24.1 CCU P 0.38 18.7 CAU H 0.61 13.8 CGU R 0.17 9.0
CUC L 0.17 16.1 CCC P 0.11 5.3 CAC H 0.39 8.7 CGC R 0.07 3.8
CUA L 0.11 9.9 CCA P 0.33 16.1 CAA Q 0.56 19.4 CGA R 0.12 6.3
CUG L 0.11 9.8 CCG P 0.18 8.6 CAG Q 0.44 15.2 CGG R 0.09 4.9
AUU I 0.41 21.5 ACU T 0.34 17.5 AAU N 0.52 22.3 AGU S 0.16 14.0
AUC I 0.35 18.5 ACC T 0.20 10.3 AAC N 0.48 20.9 AGC S 0.13 11.3
AUA I 0.24 12.6 ACA T 0.31 15.7 AAA K 0.49 30.8 AGA R 0.35 19.0
AUG M 1.00 24.5 ACG T 0.15 7.7 AAG K 0.51 32.7 AGG R 0.20 11.0
GUU V 0.40 27.2 GCU A 0.43 28.3 GAU D 0.68 36.6 GGU G 0.34 22.2
GUC V 0.19 12.8 GCC A 0.16 10.3 GAC D 0.32 17.2 GGC G 0.14 9.2
GUA V 0.15 9.9 GCA A 0.27 17.5 GAA E 0.52 34.3 GGA G 0.37 24.2
GUG V 0.26 17.4 GCG A 0.14 9.0 GAG E 0.48 32.2 GGG G 0.16 10.2
Gene Combinations In an embodiment, a gene/gene-product of Table 1 is co-expressed in a host cell with an exogenous FATA or FATB acyl-ACP thioesterase gene. In a specific embodiment, the FATB gene product has at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity to the Cuphea palustris FATB2 (“Cpal FATB2”, accession AAC49180, SEQ ID NO: 1) or C. hookeriana FATB2 (“Ch FATB2”, accession U39834, SEQ ID NO: 57) or fragment thereof. Optionally the FATB gene product has at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity to the non-transit-peptide domain of Cuphea palustris FATB2 (“Cpal FATB2”, accession AAC49180, SEQ ID NO: 1) or C. hookeriana FATB2 (“Ch FATB2”, accession U39834 SEQ ID NO: 57)).
FATA genes encode enzymes that preferentially, but not exclusively, hydrolyze long-chain fatty acids with highest activity towards C18:1. 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).
In an embodiment, a host cell is transformed to express both a FATA or FATB and KASI-like transgene. The host-cell produces a cell oil. Together, the FATA or FATB and KASI-like genes are expressed to produce their respective gene products and thereby alter the fatty acid profile of the cell oil. The two genes function either additively or synergistically with respect to control strains lacking one of the two genes. Optionally, the host cell is oleaginous and can be an oleaginous eukaryotic microalgae such as those described above or below. The fatty acid profile of the cell oil can be enriched (relative to an appropriate control) in C14:0 (myristic), C8:0, C10:0 or a combination of C8/C10.
In an embodiment, the fatty acid profile of the cell is enriched in C14:0 fatty acids. In this embodiment, the FATB gene expresses an acyl-ACP thioesterase enzyme having at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity percent amino acid identity to the enzyme of SEQ ID NO: 1. The co-expressed KASI-like gene encodes a beta-ketoacyl ACP synthase having at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity percent amino acid identity to the enzyme of SEQ ID NO: 2. Alternately The co-expressed KASI-like gene encodes a beta-ketoacyl ACP synthase having at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity percent amino acid identity to the enzyme of SEQ ID NO: 7. Optionally, the cell oil has a fatty acid profile characterized by at least 10%, 20%, 30%, 40%, 50% or at least 55% C14:0 (area % by FAME-GC-FID).
In another embodiment, the fatty acid profile of the cell is enriched in C8:0 and/or C10:0 fatty acids. In this embodiment, the FATB gene expresses an acyl-ACP thioesterase enzyme having at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity percent amino acid identity to the enzyme of SEQ ID NO: 57. The co-expressed KASI-like gene encodes a beta-ketoacyl ACP synthase having at least 85, 90, 91, 92, 93, 94, 9595.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity percent amino acid identity to an enzyme of one of SEQ ID NOs: 2, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 37. In a related embodiment, the co-expressed KASI-like gene encodes a beta-ketoacyl ACP synthase having at least 85, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5 97, 97.5, 98, 98.5 or 99% amino acid sequence identity percent amino acid identity to enzyme of one of SEQ ID NO: 2, 8, 11, 12, 13, 14, or 15. Optionally, the cell oil has a fatty acid profile characterized by at least 7, 8, 9, 10, 11, 12, 13, or 14 area % C8:0 (by FAME-GC-FID). Optionally, the cell oil has a fatty acid profile characterized by at least 10, 15, 20, 25, 30, or 35 area % for the sum of C8:0 and C10:0 fatty acids (by FAME-GC-FID). Optionally, the C8/C10 ratio of the cell oil is in the range of 2.2-2.5, 2.5-3.0, or 3.0-3.4.
Optionally, the oils produced by these methods can have a sterol profile in accord with those described below.
Host Cells The host cell can be a single cell (e.g., microalga, bacteria, yeast) or part of a multicellular organism such as a plant or fungus. Methods for expressing KASI-like genes in a plant are given in U.S. Pat. Nos. 7,301,070, 6,348,642, 6,660,849, and 6,770,465, or can be accomplished using other techniques generally known in plant biotechnology. Engineering of eukaryotic oleaginous microbes including eukaryotic microalgae (e.g., of Chlorophyta) is disclosed in WO2010/063032, WO2011/150411, and WO2012/106560 and in the examples below.
Examples of oleaginous host cells include plant cells and microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae. Specific examples of microalgal cells include heterotrophic or obligate heterotrophic eukaryotic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of eukaryotic oleaginous microalgae host cells 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 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. 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). When cultivated heterotrophically, the cells and cell oil can comprise less than 200 ppm, 20 ppm, or 2 ppm of color-generating impurities or of chlorophyll. 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. The cells can be cultivated on a depolymerized cellulosic feedstock such as acid or enzyme hydrolyzed bagasse, sugar beet pulp, corn stover, wood chips, sawdust or switchgrass. Optionally, the cells can be cultivated on a depolymerized cellulosic feedstock comprising glucose and at least 5, 10, 20, 30 or 40% xylose, while producing at least 20% lipid by dry weight. Optionally, the lipid comprises triglycerides having a fatty acid profile characterized by at least 10, 15 or 20% C12:0
Optionally, the host cell comprises 23S rRNA having at least 65, 70, 75, 80, 85, 90 or 95% nucleotide sequence identity to SEQ ID NO: 58.
Oils and Related Products 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.
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. WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/1504 disclose heterotrophic cultivation and oil isolation techniques. For example, oil may be obtained by cultivating, drying and pressing the cells. The cell oils produced may be refined, bleached and deodorized (RBD) as known in the seed-oil art or as described in WO2010/120939. The refining step may comprise degumming. The raw, refined, 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.
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.
The stable carbon isotope value 613C 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 613C (0/00) of the oils can be related to the 613C 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.
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, be a eukaryotic microalga falling in the classification of Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. It has been found that microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides (a close relative of Prototheca moriformis) was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and beta-sitosterol, when detected by GC-MS. However, it is believed that all sterols produced by Chlorella have C24β stereochemistry. Thus, it is believed that the molecules detected as campesterol, stigmasterol, and beta-sitosterol, are actually 22,23-dihydrobrassicasterol, proferasterol and clionasterol, respectively. Thus, the oils produced by the microalgae described above can be distinguished from plant oils by the presence of sterols with C24α stereochemistry and the absence of C24α stereochemistry in the sterols present. For example, the oils produced may contain 22, 23-dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in beta-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of Δ7-poriferasterol.
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.
Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, β-sitosterol, and stigmasterol 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).
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 6.
TABLE 6
Sterols in microalgal oil.
Refined,
Clari- Refined & bleached, &
Sterol Crude fied 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 (1.6%) (1.6%) (1.8%) (1.8%)
or 22,23-
dihydrobrassicasterol)
4 5,22-cholestadien-24- 57.7 59.2 46.8 49.9
ethyl-3-ol (8.4%) (8.2%) (7.9%) (8.3%)
(Stigmasterol
or poriferasterol)
5 24-ethylcholest-5-en- 9.64 9.92 9.26 10.2
3-ol (β-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
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, beta-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% beta-sitosterol was found to be present. Beta-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 beta-sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol:beta-sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.
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% beta-sitosterol. In other embodiments the oil is free from beta-sitosterol.
In some embodiments, the oil is free from one or more of beta-sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from beta-sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.
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.
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.
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.
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.
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.
In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.
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% beta-sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% beta-sitosterol. In some embodiments, the oil content further comprises brassicasterol. For any of the oils or cell-oils disclosed in this application, the oil can have the sterol profile of any column of Table 6, above, with a sterol-by-sterol variation of 30%, 20%, 10% or less.
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 beta-sitosterol and stigmasterol. In contrast, the sterol profile 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).
In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than beta-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.
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.
In embodiments of the present invention, oleaginous cells expressing one or more of the genes of Table 1 can produce an oil with at least 20, 40, 60 or 70% of C8, C10, C12, C14 or C16 fatty acids. In a specific embodiment, the level of myristate (C14:0) in the oil is greater than 30%.
Thus, in 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.
The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, detergents, soaps, etc). The oils, triglycerides, fatty acids from the oils may be subjected to C—H activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation, methylation, dimerization, thiolation, metathesis, hydro-alkylation, lactonization, or other chemical processes.
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: Screening KAS Genes in Combination with Cuphea palustris FATB2 Acyl-ACP Thioesterase A Prototheca moriformis strain expressing codon optimized Cuphea palustris (Cpal) FATB2 was constructed as described in WO2013/158938, example 53 (p. 231). The amino acid sequence of the Cpal FATB2 gene is given in SEQ ID NO: 1. This strain (S6336) produced a cell oil characterized by a fatty acid profile having about 38% myristic acid (C14:0).
Six KASI-like genes were cloned from seed oil genomes. Total RNA was extracted from dried mature seeds using a liquid-nitrogen-chilled mortar and pestle to break open the seed walls. RNA was then precipitated with an 8M urea, 3M LiCl solution followed by a phenol-chloroform extraction. A cDNA library was generated with oligo dT primers using the purified RNA and subjected to Next Generation sequencing. The novel KAS genes were identified from the assembled transcriptome using BLAST with known KAS genes as bait. The identified KAS gene sequences were codon optimized for expression in Prototheca and synthesized for incorporation into an expression cassette.
To test the impact on myristate accumulation, S6336 was transformed with a linearized plasmid designed for homologous recombination at the pLOOP locus and to express the KASI-like genes with coexpression of a selection marker (see WO2013/1589380). The vector is described in SEQ ID NO 38, the remaining codon optimized KAS genes were substituted into the KAS CDS segment of this vector prior to transformation. As shown in Table 7, increases in C14:0 levels in extracted cell oil were observed with the expression of the C. camphora KASIV (D3147), C. camphora KASI (D3148), U. cahfornica KASI (D3150) or U. cahfornica KASVI (D3152) genes in S6336. Even greater increases in C14:0 levels resulted from expression the KASI gene from C. palustris KASIV (D3145) or C. wrightii KASAI (D3153), with some individual lines producing >50% or >55% C14:0. The C14 production far exceeded the negligible amount found in the wild-type oil (see Table 7a).
TABLE 7
KAS genes that effect an increase in C14 fatty acids in eukaryotic
microalgal oil.
C14:0 (area %.
SEQ ID mean of 4 Highest C14:0
Gene (transformant ID) NOs: transformants) observed
C. camphora KASIV 3, 22, 40 38.0 40.3
C. camphora KASI 4, 23, 41 33.8 39.3
U. californica KASI 5, 24, 42 37.4 42.3
U. californica KASVI 6, 25, 43 38.4 41.6
C. palustris KASIV 2, 21, 39 45.4 58.4
C. wrightii KASAI 7, 26, 44 43.2 53.6
TABLE 7a
Fatty acid profile of wild-type Prototheca moriformis oil (area %).
C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3
0 0 0 2 38 4 48 5 1
Example 2: Screening KAS Genes in Combination with Cuphea hookeriana FATB Acyl-ACP Thioesterase P. moriformis strains were constructed that express ChFATB2 acyl-ACP thioesterase together with a KAS gene selected from ten KASI, one KASIII and one mitochondrial KAS were cloned from seed oil genomes, codon optimized and introduced into Prototheca as described in Example 1. The KAS genes were fused to an HA epitope TAG at the c-terminus of each KAS to allow confirmation of protein expression.
TABLE 8
Mean C8:0-C10:0 fatty acid profiles derived from transformation of
FATB2-expressing microalgal strain with KASI-like genes isolated
from seed oil genomes.
SEQ ID
NOS:
(amino acid,
CDS, codon C8:0 C10:0
optimized (mean (mean Sum C8:0 + C10/C8
KAS Gene CDS) area %) area %) C10:0 ratio
C. avigera 16, 35, 53 8.0 21.4 29.3 2.7
KASIa
C. pulcherrima 17, 36, 54 7.7 20.3 28.0 2.6
KASI
C. avigera NL, 37, 55 7.8 20.4 28.2 2.6
Mitochondrial
KAS
C. avigera 19, NL, 56 9.5 22.8 32.3 2.4
KAS III
C. paucipetala 9, 28, 46 7.9 22.5 30.3 2.9
KASIVb
C. ignea 10, 29, 47 6.6 18.7 25.4 2.8
KASIVb
C. painteri 13, 32, 50 9.0 22.4 31.4 2.5
KASIV
C. palustris 2, 21, 38 8.6 21.6 30.4 2.5
KASIVa
C. avigera 8, 27, 45 11.0 23.8 34.8 2.2
KASIVb
C. procumbens 11, 30, 48 8.2 25.8 34.0 3.2
KASIV
C. paucipetala 12, 31, 49 8.8 29.9 39.4 3.4
KASIVa
C. ignea 15, 34, 52 8.6 25.8 34.4 3.0
KASIVa
C avigera 14, 33, 51 10.0 23.0 32.9 2.3
KASIVa
C. hookeriana 59, NL, 61 14.5 27.81 42.6 3.0
KASIV
The parental strain is a stable microalgal strain expressing the C. hookeriana FATB2 under the control of the pH5-compatible PmUAPA1 promoter. The parental strain accumulates 27.8% C8:0-C10:0 with a C10/C8 ratio of 2.6. All transformants are derived from integrations of the KASI transgenes at the pLOOP locus of the parental strain. Means are calculated from at least 19 individual transformants for each KAS transgene (NL=not listed).
As can be seen from Table 8, expression of the following KAS genes significantly increased C8:0-C10:0 levels: C. avigera KASIVb (D3287), C. procumbens KASIV (D3290), C. paucipetala KASIVa (D3291), C. avigera KASIVa (D3293), and C. ignea KASIVa (D3294). Importantly, expression of the C. avigera KASIVb (D3287) augmented the accumulation of both C8:0 and C10:0 fatty acids, while only C10:0 levels were increased upon expression of D3290, D3291, D3293 and D3294. In some cases the sum of C8:0 and C10:0 fatty acids in the fatty acid profile was at least 30%, or at least 35% (area % by FAME-GC-FID). The midchain production far exceeded the negligible amount found in the wild-type oil (see Table 7a).
The mean C8/C10 ratios of Table 8 ranged from 2.2 to 3.4. The sum of mean C8 and C10 ranged from 25.4 to 39.4.
The highest C8:0 producing strain found was D3287, which combined C. avigera KASIV with C. hookeriana FATB2. The mean was 11.0% C8:0 with a range of 12.4 to 14.8. Thus, a cell oil with a fatty acid profile of greater than 14% C8 was produced. Furthermore, the C10/C8 ratio was less than 2.5.
Example 3: Identification of KAS Clades and Consensus Sequences The newly identified sequences of KASI-like genes were compared to those in the ThYme database of thioester-active enzymes maintained by Iowa State University (enzyme.cbirc.iastate.edu) using the blast algorithm and the top hits were extracted. The top 50 BLAST hits were downloaded and a multiple alignment was created using ClustalW alignment algorithm and a phylogenetic tree (FIG. 1) was created using that alignment with the Jukes-Cantor Neighbor-Joining method. The new KASIV genes grouped together with only 4 ThYme KAS genes internal to that group out of the 50 possible. The total ThYme KAS sequences were reduced to 12 because nearly all ThYme KAS grouped away from the new KAS sequences. The ThYme sequences are only 222 residues while the new KASIV are approximately 555 residues in length including the targeting peptide.
Two new clades were identified Clade 1 and Clade 2, characterized by consensus SEQ ID NO: 69 and SEQ ID NO:70, which include transit peptides. The clades can also be characterized by the sequences of the mature consensus proteins SEQ ID NO: 71 and SEQ ID NO: 72, respectively. The KAS genes of Clade 1 are associated with production of elevated C8 and C10 fatty acids based on based on transformations in P. moriformis in combination with a FATB acyl-ACP thioesterase as in Example 2. The KAS genes of Clade 2 are associated with production of elevated C10 fatty acids based on transformations in P. moriformis in combination with a FATB acyl-ACP thioesterase as in Example 2.
Although the above discussion discloses various exemplary embodiments of the invention, it should be apparent that those skilled in the art can make various modifications that will achieve some of the advantages of the invention without departing from the true scope of the invention.
SEQUENCE LISTING
SEQ ID NO: 1
Cuphea palustris FATB2 amino acid sequence
(Genbank Accession No. AAC49180.1)
MVAAAASAAFFSVATPRTNISPSSLSVPFKPKSNHNGGFQVKANASAHPKANGSAVS
LKSGSLETQEDKTSSSSPPPRTFINQLPVWSMLLSAVTTVFGVAEKQWPMLDRKSKR
PDMLVEPLGVDRIVYDGVSFRQSFSIRSYEIGADRTASIETLMNMFQETSLNHCKIIGL
LNDGFGRTPEMCKRDLIWVVTKMQIEVNRYPTWGDTIEVNTWVSASGKHGMGRD
WLISDCHTGEILIRATSVWAMMNQKTRRLSKIPYEVRQEIEPQFVDSAPVIVDDRKFH
KLDLKTGDSICNGLTPRWTDLDVNQHVNNVKYIGWILQSVPTEVFETQELCGLTLEY
RRECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIVKGRTEWRPKNAGAKG
AILTGKTSNGNSIS
SEQ ID NO: 2
Amino acid sequence of the C. palustris KASIV
(D3145 and D3295, pSZ4312). The algal transit
peptide is underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCLVTSYIDPCNQFSSSASL
SFLGDNGFASLFGSKPFRSNRGHRRLGRASHSGEAMAVALEPAQEVATKKKPLVKQ
RRVVVTGMGVVTPLGHEPDVYYNNLLDGVSGISEIEAFDCTQFPTRIAGEIKSFSTDG
WVAPKLSKRMDKFMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGLGGM
KLFSDSIEALRISYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFC
ILNSANHIVRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTKASRPWDSNR
DGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCI
EKALAQAGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHL
IGAAGGVEAVTVVQAIRTGWIHPNLNLEDPDKAVDAKVLVGPKKERLNVKVGLSNS
FGFGGHNSSILFAPYN
SEQ ID NO: 3
Amino acid sequence of the C. camphora KASIV
(D3147, pSZ4338).
MAMMAGSCSNLVIGNRELGGNGPSLLHYNGLRPLENIQTASAVKKPNGLFASSTAR
KSKAVRAMVLPTVTAPKREKDPKKRIVITGMGLVSVFGNDIDTFYSKLLEGESGIGPI
DRFDASSFSVRFAGQIHNFSSKGYIDGKNDRRLDDCWRYCLVAGRRALEDANLGPE
VLEKMDRSRIGVLIGTGMGGLSAFSNGVESLIQKGYKKITPFFIPYSITNMGSALLAID
TGVMGPNYSISTACATANYCFHAAANHIRRGEAEIMVTGGTEAAVSATGVGGFIACR
ALSHRNDEPQTASRPWDKDRDGFVMGEGAGVLVMESLHHARKRGANIIAEYLGGA
VTCDAHHMTDPRADGLGVSSCITKSLEDAGVSPEEVNYVNAHATSTLAGDLAEVNA
IKKVFKDTSEMKMNGTKSMIGHCLGAAGGLEAIATIKAINTGWLHPTINQFNIEPAVT
IDTVPNVKKKHDIHVGISNSFGFGGHNSVVVFAPFMP
Amino acid sequence of the C. camphora
KASI (D3148, pSZ4339).
SEQ ID NO: 4
MQILQTPSSSSSSLRMSSMESLSLTPKSLPLKTLLPLRPRPKNLSRRKSQNPRPISSSSSP
ERETDPKKRVVITGMGLVSVFGNDVDAYYDRLLSGESGIAPIDRFDASKFPTRFAGQI
RGFTSDGYIDGKNDRRLDDCLRYCIVSGKKALENAGLGPHLMDGKIDKERAGVLVG
TGMGGLTVFSNGVQTLHEKGYRKMTPFFIPYAITNMGSALLAIELGFMGPNYSISTAC
ATSNYCFYAAANHIRRGEADLMLAGGTEAAIIPIGLGGFVACRALSQRNDDPQTASR
PWDKDRDGFVMGEGAGVLVMESLEHAMKRDAPIIAEYLGGAVNCDAYHMTDPRA
DGLGVSTCIERSLEDAGVAPEEVNYINAHATSTLAGDLAEVNAIKKVFTNTSEIKINA
TKSMIGHCLGAAGGLEAIATIKAINTGWLHPSINQFNPEPSVEFDTVANKKQQHEVN
VAISNSFGFGGHNSVVVFSAFKP
SEQ ID NO: 5
Amino acid sequence of the U. californica KASI
(D3150, pSZ4341).
MESLSLTPKSLPLKTLLPFRPRPKNLSRRKSQNPKPISSSSSPERETDPKKRVVITGMGL
VSVFGNDVDAYYDRLLSGESGIAPIDRFDASKFPTRFAGQIRGFTSDGYIDGKNDRRL
DDCLRYCIVSGKKALENAGLGPDLMDGKIDKERAGVLVGTGMGGLTVFSNGVQTL
HEKGYRKMTPFFIPYAITNMGSALLAIDLGFMGPNYSISTACATSNYCFYAAANHIRR
GEADVMLAGGTEAAIIPIGLGGFVACRALSQRNDDPQTASRPWDKDRDGFVMGEGA
GVLVMESLEHAMKRDAPIIAEYLGGAVNCDAYHMTDPRADGLGVSTCIERSLEDAG
VAPEEVNYINAHATSTLAGDLAEVNAIKKVFTNTSEIKINATKSMIGHCLGAAGGLE
AIATIKAINTGWLHPSINQFNPEPSVEFDTVANKKQQHEVNVAISNSFGFGGHNSVVV
FSAFKP
SEQ ID NO: 6
Amino acid sequence of the U. californica KASIV
(D3152, pSZ4343).
MTQTLICPSSMETLSLTKQSHFRLRLPTPPHIRRGGGHRHPPPFISASAAPRRETDPKK
RVVITGMGLVSVFGTNVDVYYDRLLAGESGVGTIDRFDASMFPTRFGGQIRRFTSEG
YIDGKNDRRLDDYLRYCLVSGKKAIESAGFDLHNITNKIDKERAGILVGSGMGGLKV
FSDGVESLIEKGYRKISPFFIPYMIPNMGSALLGIDLGFMGPNYSISTACATSNYCIYAA
ANHIRQGDADLMVAGGTEAPIIPIGLGGFVACRALSTRNDDPQTASRPWDIDRDGFV
MGEGAGILVLESLEHAMKRDAPILAEYLGGAVNCDAHHMTDPRADGLGVSTCIESS
LEDAGVAAEEVNYINAHATSTPTGDLAEMKAIKNVFRNTSEIKINATKSMIGHCLGA
SGGLEAIATLKAITTGWLHPTINQFNPEPSVDFDTVAKKKKQHEVNVAISNSFGFGGH
NSVLVFSAFKP
SEQ ID NO: 7
Amino acid sequence of the C. wrightii KASAI
(D3153, pSZ4379). The algal transit peptide
is underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRYVFQCLVASCIDPCDQYRSSASL
SFLGDNGFASLFGSKPFMSNRGHRRLRRASHSGEAMAVALQPAQEAGTKKKPVIKQ
RRVVVTGMGVVTPLGHEPDVFYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDG
WVAPKLSKRMDKFMLYLLTAGKKALADGGITDEVMKELDKRKCGVLIGSGMGGM
KVFNDAIEALRVSYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSN
FCILNAANHIIRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNSDPTKASRPWDSN
RDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVIL
CIEKALAQAGVSKEDVNYINAHATSTSAGDIKEYQALARCFGQNSELRVNSTKSMIG
HLLGAAGGVEAVTVVQAIRTGWIHPNLNLEDPDKAVDAKLLVGPKKERLNVKVGL
SNSFGFGGHNSSILFAPCNV
SEQ ID NO: 8
Amino acid sequence of the C. avigera KASIVb
(D3287, pSZ4453).
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCYIGDNGFGSKPPRSNRG
HLRLGRTSHSGEVMAVAMQSAQEVSTKEKPATKQRRVVVTGMGVVTALGHDPDV
YYNNLLDGVSGISEIENFDCSQLPTRIAGEIKSFSADGWVAPKFSRRMDKFMLYILTA
GKKALVDGGITEDVMKELDKRKCGVLIGSGLGGMKVFSESIEALRTSYKKISPFCVPF
STTNMGSAILAMDLGWMGPNYSISTACATSNFCILNAANHITKGEADMMLCGGSDS
VILPIGMGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAKK
RGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIEKALAQSGVSREDVNYINAHATS
TPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGGAGGVEAVTVVQAIRTGWIHP
NINLDDPDEGVDAKLLVGPKKEKLKVKVGLSNSFGFGGHNSSILFAPCN
SEQ ID NO: 9
Amino acid sequence of the C. paucipetala KASIVb
(D3288, pSZ4454).
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCLGDIGFASLIGSKPPRSN
RNHRRLGRTSHSGEVMAVAMQPAHEASTKNKPVTKQRRVVVTGMGVATPLGHDP
DVYYNNLLDGVSGISQIENFDCTQFPTRIAGEIKSFSTEGYVIPKFAKRMDKFMLYLL
TAGKKALEDGGITEDVMKELDKRKCGVLIGSGMGGMKIINDSIAALNVSYKKMTPF
CVPFSTTNMGSAMLAIDLGWMGPNYSISTACATSNYCILNAANHIVRGEADMMLCG
GSDAVIIPVGLGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELE
HAKKRGATIYAEFLGGSFTCDAYHMTEPHPDGAGVILCIEKALAQSGVSREDVNYIN
AHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGAAGGVEAVTVVQAIR
TGWIHPNINLENPDEAVDAKLLVGPKKEKLKVKVGLSNSFGFGGHNSSILFAPYN
SEQ ID NO: 10
Amino acid sequence of the C. ignea KASIVb
(D3289, pSZ4455). The algal transit peptide
is underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTSQCLVTSYIDPCNKYCSSASL
SFLGDNGFASLFGSKPFRSNRGHRRLGRASHSGEAMAVALQPAQEVTTKKKPVIKQR
RVVVTGMGVVTPLGHEPDVYYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDGW
VAPKLSKRMDKFMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGMGGMK
LFNDSIEALRISYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCI
LNASNHIVRGEADMMLCGGSDSVTVPLGVGGFVACRALSQRNNDPTKASRPWDSN
RDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTSDAYHMTEPHPEGAGVILC
IEKALAQSGVSREDVNYINAHATSTPAGDIKEYQALARCFGQNSELRVNSTKSMIGH
LLGAAGGVEAVAVIQAIRTGWIHPNINLEDPDEAVDPKLLVGPKKEKLKVKVALSNS
FGFGGHNSSILFAPCN
SEQ ID NO: 11
Amino acid sequence of the C. procumbens KASIV
(D3290, pSZ4456). The algal transit peptide is
underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCLVTSHNDPCNQYCSSAS
LSFLGDNGFGSKPFRSNRGHRRLGRASHSGEAMAVALQPAQEVATKKKPAMKQRR
VVVTGMGVVTPLGHEPDVYYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDGWV
APKLSKRMDKFMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGMGGMKLF
NDSIEALRVSYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCIL
NAANHIVRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTKASRPWDSNRD
GFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIE
KALAQSGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLL
GAAGGVEAVTVIQAIRTGWIHPNLNLEDPDKAVDAKFLVGPKKERLNVKVGLSNSF
GFGGHNSSILFAPCN
SEQ ID NO: 12
Amino acid sequence of the C. paucipetala KASIVa
(D3291, pSZ4457). The algal transit peptide is
underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCLVNSHIDPCNQNVSSAS
LSFLGDNGFGSNPFRSNRGHRRLGRASHSGEAMAVALQPAQEVATKKKPAIKQRRV
VVTGMGVVTPLGHEPDVFYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDGWVA
PKLSKRMDKFMLYLLTAGKKALADAGITEDVMKELDKRKCGVLIGSGMGGMKLFN
DSIEALRVSYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCILN
AANHIIRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNSDPTKASRPWDSNRDGF
VMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPDGAGVILCIEKA
LAQSGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGA
AGGVEAVTVIQAIRTGWIHPNLNLEDPDEAVDAKFLVGPKKERLNVKVGLSNSFGFG
GHNSSILFAPYN
SEQ ID NO: 13
Amino acid sequence of the C. painteri KASIV
(D3292, pSZ4458). The algal transit peptide
is underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTPQCLDPCNQHCFLGDNGFAS
LIGSKPPRSNLGHLRLGRTSHSGEVMAVAQEVSTNKKHATKQRRVVVTGMGVVTPL
GHDPDVYYNNLLEGVSGISEIENFDCSQLPTRIAGEIKSFSTDGLVAPKLSKRMDKFM
LYILTAGKKALADGGITEDVMKELDKRKCGVLIGSGLGGMKVFSDSVEALRISYKKI
SPFCVPFSTTNMGSAMLAMDLGWMGPNYSISTACATSNFCILNAANHITKGEADMM
LCGGSDAAILPIGMGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLE
ELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPDGAGVILCIEKALAQSGVSREEVN
YINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGGAGGVEAVTVVQ
AIRTGWIHPNINLEDPDKGVDAKLLVGPKKEKLKVKVGLSNSFGFGGHNSSILFAPCN
SEQ ID NO: 14
Amino acid sequence of the C. avigera KASIVa
(D3293, pSZ4459). The algal transit peptide
is underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCLVTSYNDPCEQYRSSAS
LSFLGDNGFASLFGSKPFRSNRGHRRLGRASHSGEAMAVALQPAQEVGTKKKPVIKQ
RRVVVTGMGVVTPLGHEPDVYYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDG
WVAPKLSKRMDKFMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGLGGM
KVFSESIEALRTSYKKISPFCVPFSTTNMGSAILAMDLGWMGPNYSISTACATSNFCIL
NAANHITKGEADMMLCGGSDSVILPIGMGGFVACRALSQRNNDPTKASRPWDSNRD
GFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIE
KALAQSGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLL
GGAGGVEAVTVVQAIRTGWIHPNINLDDPDEGVDAKLLVGPKKEKLKVKVGLSNSF
GFGGHNSSILFAPCN
SEQ ID NO: 15
Amino acid sequence of the C. ignea KASIVa
(D3294, pSZ4460). The algal transit peptide
is underlined.
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTSQCLVTSYIDPCNKYCSSASL
SFLGDNGFASLFGSKPFRSNRGHRRLGRASHSGEAMAVALQPAQEVTTKKKPVIKQR
RVVVTGMGVVTPLGHEPDVYYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDGW
VAPKLSKRMDKFMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGMGGMK
LFNDSIEALRISYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCI
LNASNHIVRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTKASRPWDSNRD
GFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIE
KALAQAGVSKEDVNYINAHATSTPAGDIKEYQALAQCFGQNSELRVNSTKSMIGHL
LGAAGGVEAVTVVQAIRTGWIHPNLNLEDPDKAVDAKLLVGPKKERLNVKVGLSNS
FGFGGHNSSILFAPYN
SEQ ID NO: 16
Amino acid sequence of the C. avigera KASIa
(D3342, pSZ4511).
MQSLHSPALRASPLDPLRLKSSANGPSSTAAFRPLRRATLPNIRAASPTVSAPKRETDP
KKRVVITGMGLVSVFGSDVDAYYEKLLSGESGISLIDRFDASKFPTRFGGQIRGFNAT
GYIDGKNDRRLDDCLRYCIVAGKKALENSDLGGDSLSKIDKERAGVLVGTGMGGLT
VFSDGVQNLIEKGHRKISPFFIPYAITNMGSALLAIDLGLMGPNYSISTACATSNYCFY
AAANHIRRGEADLMIAGGTEAAIIPIGLGGFVACRALSQRNDDPQTASRPWDKDRDG
FVMGEGAGVLVMESLEHAMKRGAPIIAEYLGGAVNCDAYHMTDPRADGLGVSSCIE
SSLEDAGVSPEEVNYINAHATSTLAGDLAEINAIKKVFKNTKDIKINATKSMIGHCLG
ASGGLEAIATIKGITTGWLHPSINQFNPEPSVEFDTVANKKQQHEVNVAISNSFGFGG
HNSVVAFSAFKP
SEQ ID NO: 17
Amino acid sequence of the C. pulcherima KASI
(D3343, pSZ4512).
MHSLQSPSLRASPLDPFRPKSSTVRPLHRASIPNVRAASPTVSAPKRETDPKKRVVITG
MGLVSVFGSDVDAYYDKLLSGESGIGPIDRFDASKFPTRFGGQIRGFNSMGYIDGKN
DRRLDDCLRYCIVAGKKSLEDADLGADRLSKIDKERAGVLVGTGMGGLTVFSDGVQ
SLIEKGHRKITPFFIPYAITNMGSALLAIELGLMGPNYSISTACATSNYCFHAAANHIRR
GEADLMIAGGTEAAIIPIGLGGFVACRALSQRNDDPQTASRPWDKDRDGFVMGEGA
GVLVLESLEHAMKRGAPIIAEYLGGAINCDAYHMTDPRADGLGVSSCIESSLEDAGV
SPEEVNYINAHATSTLAGDLAEINAIKKVFKNTKDIKINATKSMIGHCLGASGGLEAI
ATIKGINTGWLHPSINQFNPEPSVEFDTVANKKQQHEVNVAISNSFGFGGHNSVVAFS
AFKP
SEQ ID NO: 18
Amino acid sequence of the C. avigera
mitochondrial KAS (D3344, pSZ4513).
MVFLPWRKMLCPSQYRFLRPLSSSTTFDPRRVVVTGLGMVTPLGCGVNTTWKQLIE
GKCGIRAISLEDLKMDAFDIDTQAYVFDQLTSKVAATVPTGVNPGEFNEDLWFNQKE
HRAIARFIAYALCAADEALKDANWEPTEPEEREMTGVSIGGGTGSISDVLDAGRMIC
EKKLRRLSPFFIPRILINMASGHVSMKYGFQGPNHAAVTACATGAHSIGDAARMIQF
GDADVMVAGGTESSIDALSIAGFCRSRALTTKYNSCPQEASRPFDTDRDGFVIGEGSG
VLVLEELDHARKRGAKMYAEFCGYGMSGDAHHITQPHSDGRGAILAMTRALKQSN
LHPDQVDYVNAHATSTSLGDAIEAKAIKTVFSDHAMSGSLALSSTKGAIGHLLGAAG
AVEAIFSILAIKNGLAPLTLNVARPDPVFTERFVPLTASKEMHVRAALSNSFGFGGTN
TTLLFTSPPQN
SEQ ID NO: 19
Amino acid sequence of the C. avigera KASIII
(D3345, pSZ4514).
MANAYGFVGSSVPTVGRAAQFQQMGSGFCSVDFISKRVFCCSAVQGADKPASGDSR
AEYRTPRLVSRGCKLIGSGSAIPTLQVSNDDLAKIVDTNDEWISVRTGIRNRRVLTGK
DSLTNLATEAARKALEMAQVDAEDVDMVLMCTSTPEDLFGSAPQIQKALGCKKNPL
SYDITAACSGFVLGLVSAACHIRGGGFNNVLVIGADSLSRYVDWTDRGTCILFGDAA
GAVLVQSCDAEEDGLFAFDLHSDGDGQRHLRAVITENETDHAVGTNGSVSDFPPRRS
SYSCIQMNGKEVFRFACRSVPQSIELALGKAGLNGSNIDWLLLHQANQRIIDAVATRL
EVPQERVISNLANYGNTSAASIPLALDEAVRGGKVKPGHLIATAGFGAGLTWGSAIV
RWG
SEQ ID NO: 20
HA Epitope TAG amino acid sequence
TMYPYDVPDYA
SEQ ID NO: 21
C. palustris KASIV CDS
ATGGCGGCCGCCGCTTCCATGGTTGCGTCCCCACTCTGTACGTGGCTCGTAGCCG
CTTGCATGTCCACTTCCTTCGACAACGACCCACGTTCCCCGTCCATCAAGCGTCTC
CCCCGCCGGAGGAGGACTCTCTCCCAATCCTCCCTCCGCGGCGGATCCACCTTCC
AATGCCTCGTCACCTCATACATCGACCCTTGCAATCAGTTCTCCTCCTCCGCCTCC
CTTAGCTTCCTCGGGGATAACGGATTCGCATCCCTTTTCGGATCCAAGCCTTTCCG
GTCCAATCGCGGCCACCGGAGGCTCGGCCGTGCTTCCCATTCCGGGGAGGCCATG
GCCGTGGCTTTGGAACCTGCACAGGAAGTCGCCACGAAGAAGAAACCTCTTGTC
AAGCAAAGGCGAGTAGTTGTTACAGGAATGGGCGTGGTGACTCCTCTAGGCCAT
GAACCTGATGTTTACTACAACAATCTCCTAGATGGAGTAAGCGGCATAAGTGAG
ATAGAGGCCTTCGACTGCACTCAGTTTCCCACGAGAATTGCCGGAGAGATCAAGT
CTTTTTCCACAGATGGATGGGTGGCCCCAAAGCTCTCCAAGAGGATGGACAAGTT
CATGCTTTACTTGTTGACTGCTGGCAAGAAAGCATTAGCGGATGGTGGAATCACC
GATGATGTGATGAAAGAGCTTGATAAAAGAAAGTGTGGAGTTCTCATTGGCTCC
GGATTGGGCGGCATGAAGCTGTTCAGTGATTCCATTGAAGCTCTGAGGATTTCAT
ATAAGAAGATGAATCCCTTTTGTGTACCTTTTGCTACTACAAATATGGGATCAGC
TATGCTTGCAATGGACTTGGGATGGATGGGTCCTAACTACTCGATATCAACTGCC
TGTGCTACAAGTAATTTCTGTATACTGAATTCTGCAAATCACATAGTCAGAGGCG
AAGCTGACATGATGCTTTGTGGTGGCTCGGATGCGGTCATTATACCTATTGGTTT
GGGAGGTTTTGTGGCGTGCCGAGCTTTGTCACAGAGGAATAATGACCCTACCAA
AGCTTCGAGACCATGGGACAGTAATCGTGATGGATTTGTAATGGGCGAAGGAGC
TGGAGTGTTACTTCTCGAGGAGTTAGAGCATGCAAAGAAAAGAGGTGCCACCAT
TTATGCGGAATTTTTAGGGGGCAGTTTCACTTGCGATGCCTACCATATGACCGAG
CCTCACCCTGAAGGTGCTGGAGTGATCCTCTGCATAGAGAAGGCCTTGGCTCAGG
CCGGAGTCTCTAGAGAAGACGTAAATTACATAAATGCGCATGCAACTTCCACTCC
TGCTGGAGATATCAAGGAATACCAAGCTCTCGCACACTGCTTCGGCCAAAACAG
TGAGCTGAGAGTGAATTCCACTAAATCGATGATCGGTCATCTTATTGGAGCAGCT
GGTGGTGTAGAAGCAGTTACCGTAGTTCAGGCGATAAGGACTGGGTGGATCCAT
CCAAATCTTAATTTGGAGGACCCGGACAAAGCCGTGGATGCAAAAGTGCTCGTA
GGACCTAAGAAGGAGAGACTAAATGTCAAGGTCGGTTTGTCCAATTCATTTGGGT
TCGGTGGTCATAACTCGTCCATACTCTTCGCCCCTTACAATTAG
SEQ ID NO: 22
C. camphora KASIV CDS
ATGGCAATGATGGCAGGTTCTTGTTCCAATTTGGTGATTGGAAACAGAGAATTGG
GTGGGAATGGGCCTTCTTTGCTTCACTACAATGGCCTCAGACCATTGGAAAATAT
TCAAACAGCCTCAGCTGTGAAAAAGCCAAATGGGTTATTTGCATCTTCTACAGCT
CGAAAATCCAAAGCTGTCAGAGCCATGGTATTGCCCACTGTAACAGCTCCAAAA
CGCGAAAAAGATCCCAAGAAGCGGATTGTAATAACAGGAATGGGCCTGGTTTCC
GTCTTTGGAAATGACATTGATACATTTTATAGTAAACTACTGGAAGGAGAGAGCG
GGATTGGCCCAATCGACAGATTTGATGCTTCTTCCTTCTCAGTGAGATTTGCTGGT
CAGATTCACAATTTCTCATCCAAAGGATACATTGATGGGAAGAATGATCGTCGGC
TAGATGACTGCTGGAGGTATTGCCTTGTGGCTGGAAGAAGAGCCCTTGAAGATG
CCAATCTTGGACCAGAGGTATTGGAAAAAATGGACCGATCTCGAATAGGGGTGC
TGATAGGGACAGGAATGGGTGGGTTGTCAGCCTTTAGCAATGGAGTTGAGTCTCT
GATCCAGAAGGGCTACAAGAAAATCACTCCATTTTTTATTCCTTACTCCATCACC
AATATGGGCTCTGCTCTTTTAGCAATCGACACGGGCGTAATGGGACCAAACTACT
CCATTTCAACAGCATGTGCAACCGCAAACTATTGCTTCCATGCTGCTGCAAATCA
TATAAGAAGGGGTGAAGCTGAAATCATGGTGACTGGAGGGACAGAGGCAGCAG
TCTCAGCTACTGGAGTTGGCGGATTCATAGCATGTAGAGCCTTATCGCACAGGAA
TGATGAGCCCCAGACGGCCTCGAGACCATGGGATAAAGATCGGGATGGTTTCGT
CATGGGCGAAGGCGCTGGTGTGCTGGTGATGGAGAGCTTGCATCATGCAAGAAA
GAGAGGAGCAAACATAATTGCAGAGTATTTAGGAGGAGCAGTAACATGTGATGC
ACATCACATGACAGATCCTCGAGCTGATGGTCTCGGGGTTTCTTCTTGCATAACC
AAGAGCTTAGAAGATGCAGGAGTCTCCCCAGAAGAGGTGAACTATGTGAATGCT
CATGCAACATCAACACTTGCAGGAGATTTAGCAGAGGTTAATGCCATAAAGAAG
GTCTTCAAGGACACATCTGAAATGAAAATGAATGGAACTAAGTCAATGATTGGA
CACTGTCTTGGAGCAGCTGGTGGATTAGAAGCCATTGCGACCATCAAAGCTATCA
ATACTGGCTGGCTACATCCAACCATCAATCAATTTAACATAGAACCAGCGGTAAC
TATCGACACGGTCCCAAATGTGAAGAAAAAGCATGATATCCATGTTGGCATCTCT
AACTCATTTGGCTTTGGTGGGCACAACTCGGTGGTCGTTTTTGCTCCCTTCATGCC
ATGA
SEQ ID NO: 23
C. camphora KASI CDS
ATGCAAATCCTCCAAACCCCATCATCATCATCGTCTTCTCTCCGCATGTCGTCCAT
GGAATCTCTCTCTCTCACCCCTAAATCTCTCCCTCTCAAAACCCTTCTTCCCCTTC
GTCCTCGCCCTAAAAACCTCTCCAGACGCAAATCCCAAAACCCTAGACCCATCTC
CTCCTCTTCCTCCCCCGAGAGAGAGACGGATCCCAAGAAGCGAGTCGTCATCACC
GGGATGGGCCTCGTCTCCGTCTTCGGCAACGATGTCGATGCCTACTACGACCGCC
TCCTCTCGGGAGAGAGCGGCATCGCCCCCATCGATCGCTTCGACGCCTCCAAGTT
CCCCACCAGATTCGCCGGTCAGATCCGAGGGTTCACCTCCGACGGCTACATTGAC
GGGAAGAACGACCGCCGGTTAGACGATTGTCTCAGATACTGTATTGTTAGTGGG
AAGAAGGCGCTCGAGAATGCCGGCCTCGGACCCCATCTCATGGACGGAAAGATT
GACAAGGAGAGAGCTGGTGTGCTTGTCGGGACAGGCATGGGTGGTCTTACAGTT
TTCTCTAATGGGGTCCAGACTCTACATGAGAAAGGTTACAGGAAAATGACTCCGT
TTTTCATCCCTTATGCCATAACAAACATGGGTTCTGCCTTGCTTGCAATTGAACTT
GGTTTTATGGGCCCAAACTATTCTATCTCAACTGCATGTGCTACCTCCAATTATTG
CTTTTATGCTGCTGCTAACCATATACGGAGAGGTGAGGCTGATCTGATGCTTGCT
GGTGGAACTGAAGCTGCAATTATTCCTATTGGATTAGGAGGCTTTGTTGCATGTA
GAGCTTTATCACAGAGAAATGATGACCCCCAGACAGCTTCAAGACCATGGGACA
AAGATCGAGACGGTTTTGTTATGGGTGAAGGTGCTGGAGTATTGGTAATGGAGA
GCTTGGAGCATGCTATGAAACGTGATGCACCAATTATTGCTGAGTATTTAGGAGG
TGCAGTGAACTGTGATGCGTATCATATGACGGATCCTAGAGCTGATGGGCTCGGG
GTTTCAACATGCATAGAAAGAAGTCTTGAAGATGCTGGTGTGGCACCTGAAGAG
GTTAACTACATAAATGCACATGCAACTTCCACTCTTGCAGGAGACCTGGCTGAGG
TGAATGCGATCAAAAAGGTTTTTACAAACACTTCAGAGATCAAAATCAATGCAA
CCAAGTCTATGATAGGGCACTGCCTTGGAGCGGCCGGGGGGTTAGAAGCCATTG
CCACAATCAAAGCAATAAATACTGGTTGGCTGCACCCTTCTATAAACCAATTTAA
TCCAGAGCCCTCTGTTGAGTTTGACACTGTAGCAAATAAAAAGCAGCAGCATGA
AGTGAATGTTGCCATTTCCAACTCTTTCGGGTTTGGCGGACACAACTCAGTCGTG
GTGTTTTCGGCATTCAAGCCTTGA
SEQ ID NO: 24
Umbellularia californica KASI CDS
ATGGAATCTCTCTCTCTCACCCCTAAATCTCTCCCTCTCAAAACCCTTCTTCCCTTT
CGTCCTCGCCCTAAAAACCTCTCCAGACGCAAATCCCAAAACCCTAAACCCATCT
CCTCCTCTTCCTCCCCGGAGAGAGAGACGGATCCCAAGAAGCGAGTCGTCATCAC
CGGGATGGGCCTCGTCTCCGTCTTCGGCAACGACGTCGATGCCTACTACGACCGC
CTCCTCTCCGGAGAGAGCGGCATCGCCCCCATCGATCGCTTCGACGCCTCCAAGT
TCCCCACCAGATTCGCCGGTCAGATCCGAGGGTTCACCTCCGACGGCTACATTGA
CGGGAAGAACGACCGCCGGTTAGACGATTGTCTCAGATACTGTATCGTTAGTGG
GAAGAAGGCGCTCGAGAATGCCGGCCTCGGACCCGATCTCATGGACGGAAAGAT
TGACAAGGAGCGAGCTGGTGTGCTTGTCGGGACAGGCATGGGTGGTCTTACAGT
TTTCTCTAATGGGGTTCAGACTCTCCATGAGAAAGGTTACAGGAAAATGACTCCG
TTTTTCATCCCTTATGCCATAACAAACATGGGTTCTGCCTTGCTTGCAATTGACCT
TGGTTTTATGGGCCCAAACTATTCTATCTCAACTGCATGTGCTACCTCCAATTATT
GCTTTTATGCTGCTGCTAACCATATACGGAGAGGTGAGGCTGATGTGATGCTTGC
TGGTGGAACTGAAGCTGCAATTATTCCTATTGGCTTAGGAGGCTTTGTTGCATGT
AGAGCTTTATCACAGCGAAATGATGACCCCCAGACAGCTTCAAGACCATGGGAC
AAAGATCGAGACGGTTTTGTTATGGGTGAAGGTGCTGGAGTATTGGTAATGGAG
AGCTTGGAGCATGCTATGAAACGTGATGCACCAATTATTGCTGAGTATTTAGGAG
GTGCAGTGAACTGTGATGCGTATCATATGACGGATCCTAGAGCTGATGGGCTCGG
GGTTTCAACATGCATAGAAAGAAGTCTTGAAGATGCTGGTGTGGCACCTGAAGA
GGTTAACTACATAAATGCACATGCAACTTCCACACTTGCAGGTGACCTGGCCGAG
GTGAATGCCATCAAAAAGGTTTTTACAAACACTTCAGAGATCAAAATCAATGCA
ACCAAGTCTATGATAGGGCACTGCCTTGGAGCGGCCGGGGGTTTAGAAGCCATT
GCCACAATCAAAGCAATAAATACTGGTTGGCTGCACCCTTCTATAAACCAATTTA
ATCCAGAGCCCTCTGTTGAGTTTGACACTGTAGCAAATAAAAAGCAGCAGCATG
AAGTGAATGTTGCCATTTCCAACTCTTTCGGGTTTGGTGGACACAACTCGGTCGT
GGTGTTTTCGGCATTCAAGCCTTGA
SEQ ID NO: 25
Umbellularia californica KASIV CDS
ATGACGCAAACCCTCATCTGCCCATCCTCCATGGAAACCCTCTCTCTTACCAAAC
AATCCCATTTCAGACTCAGGCTACCCACTCCTCCTCACATCAGACGCGGCGGCGG
CCATCGCCATCCTCCTCCCTTCATCTCCGCCTCCGCCGCCCCTAGGAGAGAGACC
GATCCGAAGAAGAGAGTCGTCATCACGGGAATGGGCCTCGTCTCCGTCTTCGGC
ACCAACGTCGATGTCTACTACGATCGCCTCCTCGCCGGCGAGAGCGGCGTTGGCA
CTATCGATCGCTTCGACGCGTCGATGTTCCCGACGAGATTCGGCGGCCAGATCCG
GAGGTTCACGTCGGAGGGGTACATCGACGGGAAGAACGACCGGCGGCTGGATGA
CTACCTCCGGTACTGCCTCGTCAGCGGGAAGAAGGCGATCGAGAGTGCTGGCTTC
GATCTCCATAACATCACCAACAAGATTGACAAGGAGCGAGCTGGGATACTTGTT
GGGTCAGGCATGGGCGGTCTTAAAGTTTTCTCTGATGGTGTTGAGTCTCTTATCG
AGAAAGGTTACAGGAAAATAAGTCCATTTTTCATCCCTTATATGATACCAAACAT
GGGTTCTGCTTTGCTTGGAATTGACCTTGGTTTCATGGGACCAAACTACTCAATTT
CAACTGCTTGTGCTACGTCAAATTATTGCATTTATGCTGCTGCAAATCATATCCGA
CAAGGTGATGCCGACCTAATGGTTGCTGGTGGAACTGAGGCTCCAATTATTCCAA
TTGGCTTAGGGGGCTTTGTAGCATGTAGAGCTTTGTCAACAAGAAATGATGATCC
CCAGACAGCTTCAAGGCCATGGGACATAGACCGAGATGGTTTTGTTATGGGCGA
AGGAGCTGGAATATTGGTATTGGAGAGCTTGGAACATGCAATGAAACGTGATGC
ACCAATTCTTGCTGAGTATTTAGGAGGTGCAGTTAACTGTGATGCTCATCATATG
ACAGATCCTCGAGCTGATGGGCTTGGGGTTTCAACATGCATTGAAAGCAGTCTTG
AAGATGCCGGCGTGGCAGCAGAAGAGGTTAACTATATAAATGCACACGCGACTT
CAACACCTACAGGTGACCTGGCTGAGATGAAGGCTATAAAAAATGTATTTAGGA
ACACTTCTGAGATCAAAATCAATGCAACCAAGTCTATGATTGGGCATTGCCTTGG
AGCGTCTGGGGGGCTAGAAGCCATTGCCACATTGAAAGCGATTACAACTGGTTG
GCTTCATCCAACTATAAACCAATTTAATCCAGAGCCTTCTGTTGACTTTGATACG
GTGGCAAAGAAAAAGAAGCAGCATGAAGTTAATGTTGCCATTTCAAACTCTTTTG
GATTCGGAGGACACAACTCAGTGTTGGTGTTTTCGGCATTCAAGCCTTGA
SEQ ID NO: 26
C. wrightii KASAI CDS (D3153, pSZ4379)
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcagggcccctgggg
cacgtcgctccggacggccagtcgccacccgcctgaggtacgtattccagtgcctggtggc
cagctgcatcgacccctgcgaccagtaccgcagcagcgccagcctgagcttcctgggcgac
aacggcttcgccagcctgttcggcagcaagcccttcatgagcaaccgcggccaccgccgcc
tgcgccgcgccagccacagcggcgaggccatggccgtggccctgcagcccgcccaggaggc
cggcaccaagaagaagcccgtgatcaagcagcgccgcgtggtggtgaccggcatgggcgtg
gtgacccccctgggccacgagcccgacgtgttctacaacaacctgctggacggcgtgagcg
gcatcagcgagatcgagaccttcgactgcacccagttccccacccgcatcgccggcgagat
caagagcttcagcaccgacggctgggtggcccccaagctgagcaagcgcatggacaagttc
atgctgtacctgctgaccgccggcaagaaggccctggccgacggcggcatcaccgacgagg
tgatgaaggagctggacaagcgcaagtgcggcgtgctgatcggcagcggcatgggcggcat
gaaggtgttcaacgacgccatcgaggccctgcgcgtgagctacaagaagatgaaccccttc
tgcgtgcccttcgccaccaccaacatgggcagcgccatgctggccatggacctgggctgga
tgggccccaactacagcatcagcaccgcctgcgccaccagcaacttctgcatcctgaacgc
cgccaaccacatcatccgcggcgaggccgacatgatgctgtgcggcggcagcgacgccgtg
atcatccccatcggcctgggcggcttcgtggcctgccgcgccctgagccagcgcaacagcg
accccaccaaggccagccgcccctgggacagcaaccgcgacggcttcgtgatgggcgaggg
cgccggcgtgctgctgctggaggagctggagcacgccaagaagcgcggcgccaccatctac
gccgagttcctgggcggcagcttcacctgcgacgcctaccacatgaccgagccccaccccg
agggcgccggcgtgatcctgtgcatcgagaaggccctggcccaggccggcgtgagcaagga
ggacgtgaactacatcaacgcccacgccaccagcaccagcgccggcgacatcaaggagtac
caggccctggcccgctgcttcggccagaacagcgagctgcgcgtgaacagcaccaagagca
tgatcggccacctgctgggcgccgccggcggcgtggaggccgtgaccgtggtgcaggccat
ccgcaccggctggattcaccccaacctgaacctggaggaccccgacaaggccgtggacgcc
aagctgctggtgggccccaagaaggagcgcctgaacgtgaaggtgggcctgagcaacagct
tcggcttcggcggccacaacagcagcatcctgttcgccccctgcaacgtgtga
SEQ ID NO: 27
C. avigera KASIVb CDS
ATGGCGGCCGCTTCTTGCATGGCTGCGTCCCCTTTCTGTACGTCGCTCGTGGCTGC
ATGCATGTCGACTTCATCCGACAACGACCCATGTCCCCTTTCCCGCCGCGGATCC
ACCTTCCAATGCTACATCGGGGATAACGGATTCGGATCGAAGCCTCCCCGTTCAA
ATCGTGGCCACCTGAGGCTCGGCCGCACTTCACATTCCGGAGAGGTGATGGCTGT
GGCTATGCAATCTGCACAAGAAGTCTCCACAAAGGAGAAACCTGCTACCAAGCA
AAGGCGAGTTGTTGTCACGGGTATGGGTGTGGTGACTGCTCTAGGCCATGACCCC
GATGTTTACTACAACAATCTCCTAGACGGAGTAAGCGGCATAAGCGAGATAGAA
AACTTTGACTGTTCTCAGCTTCCCACGAGAATTGCCGGAGAGATCAAGTCTTTTT
CTGCAGATGGGTGGGTGGCCCCGAAGTTCTCCAGGAGGATGGACAAGTTTATGC
TTTACATTCTGACTGCAGGCAAGAAAGCATTAGTAGATGGTGGAATCACTGAAG
ATGTGATGAAAGAGCTCGATAAAAGAAAGTGTGGAGTTCTCATTGGCTCCGGAT
TGGGCGGTATGAAGGTATTTAGCGAGTCCATTGAAGCTCTGAGGACTTCATATAA
GAAGATCAGTCCCTTTTGTGTACCTTTTTCTACCACGAATATGGGATCCGCTATTC
TTGCAATGGACTTGGGATGGATGGGCCCTAACTATTCGATATCGACTGCCTGTGC
AACAAGTAACTTCTGTATACTGAATGCTGCGAACCACATAACCAAAGGCGAAGC
AGACATGATGCTTTGTGGTGGCTCGGATTCGGTCATTTTACCTATTGGTATGGGA
GGTTTCGTAGCATGCCGAGCTTTGTCACAGAGGAATAATGACCCTACCAAAGCTT
CGAGACCATGGGACAGTAATCGTGATGGATTTGTGATGGGAGAAGGTGCTGGAG
TTTTACTTCTCGAGGAGTTAGAGCATGCAAAGAAAAGAGGCGCAACCATTTATGC
GGAATTTCTTGGTGGGAGTTTCACTTGCGATGCCTACCACATGACCGAGCCTCAC
CCTGAAGGAGCTGGAGTGATCCTCTGCATAGAGAAGGCCTTGGCTCAGTCCGGA
GTCTCGAGGGAAGACGTAAATTACATAAATGCGCATGCAACTTCCACTCCCGCTG
GAGATATCAAAGAATACCAAGCTCTCGCCCACTGTTTCGGCCAAAACAGTGAGTT
AAGAGTGAATTCCACCAAGTCGATGATCGGTCACCTTCTTGGAGGAGCCGGTGG
CGTAGAAGCAGTTACAGTCGTTCAGGCAATAAGGACTGGATGGATCCATCCAAA
TATTAATTTGGACGACCCGGACGAAGGCGTGGATGCAAAACTGCTCGTCGGCCCT
AAGAAGGAGAAACTGAAGGTCAAGGTCGGTTTGTCCAATTCATTCGGGTTCGGC
GGCCATAACTCATCCATACTCTTTGCCCCATGCAATTAG
SEQ ID NO: 28
C. paucipetala KASIVb CDS
ATGGCGGCCGCTTCATCAATGGTTGCCTCCCCATTCTCTACGTCCCTCGTAGCCGC
CTGCATGTCCACTTCATTCGACAACGACCCACGTTCCCTTTCCCACAACCGCATCC
GCCTCCGCGGATCCACCTTCCAATGCCTCGGGGATATCGGATTCGCTTCCCTCAT
CGGATCCAAGCCTCCGCGTTCAAATCGCAACCACCGGAGGCTCGGCCGCACTTCC
CATTCCGGGGAGGTCATGGCTGTGGCTATGCAACCTGCACATGAAGCTTCCACAA
AGAATAAACCTGTTACCAAGCAAAGGCGAGTAGTTGTGACAGGTATGGGCGTGG
CGACTCCTCTAGGCCATGACCCCGATGTTTACTACAACAATCTCCTAGACGGAGT
AAGTGGCATAAGTCAGATAGAGAACTTCGACTGCACTCAGTTTCCCACGAGAATT
GCCGGAGAGATCAAGTCTTTCTCCACAGAAGGGTATGTGATCCCGAAGTTCGCCA
AGAGGATGGACAAGTTCATGCTTTACTTGCTGACTGCAGGCAAGAAAGCATTAG
AAGATGGTGGAATCACTGAAGATGTGATGAAAGAGCTCGATAAAAGAAAGTGTG
GAGTTCTCATTGGCTCCGGAATGGGCGGTATGAAGATAATCAACGATTCCATTGC
AGCTCTGAATGTTTCATATAAGAAGATGACTCCCTTTTGTGTACCCTTTTCCACCA
CAAATATGGGATCCGCTATGCTTGCGATAGACTTGGGATGGATGGGCCCGAACT
ATTCGATATCAACTGCCTGTGCAACAAGTAACTACTGTATACTGAATGCTGCGAA
CCACATAGTCAGAGGCGAAGCAGATATGATGCTTTGTGGTGGCTCGGATGCGGT
CATTATACCTGTTGGTTTGGGAGGTTTCGTAGCATGCCGAGCTTTGTCACAGAGG
AACAATGACCCTACCAAAGCTTCGAGACCTTGGGACAGTAACCGTGATGGATTT
GTGATGGGAGAAGGAGCCGGAGTGTTACTTCTCGAGGAGTTAGAGCATGCAAAG
AAAAGAGGTGCAACCATTTATGCGGAATTTCTAGGTGGGAGTTTCACTTGCGATG
CCTACCACATGACCGAGCCTCACCCTGATGGAGCTGGAGTGATCCTCTGCATAGA
GAAGGCTTTGGCACAGTCCGGAGTCTCGAGGGAAGACGTCAATTACATAAATGC
GCATGCAACTTCTACTCCTGCTGGAGATATCAAGGAATACCAAGCTCTCGCCCAC
TGTTTCGGCCAAAACAGTGAGTTAAGAGTGAATTCCACCAAATCGATGATCGGTC
ACCTTCTTGGAGCTGCTGGTGGCGTAGAAGCAGTTACAGTAGTTCAGGCAATAAG
GACTGGGTGGATCCATCCAAATATTAATTTGGAAAACCCGGACGAAGCTGTGGA
TGCAAAATTGCTCGTCGGCCCTAAGAAGGAGAAACTGAAGGTCAAGGTCGGTTT
GTCCAATTCATTTGGGTTCGGTGGGCATAACTCATCCATACTCTTCGCCCCTTACA
ATTAG
SEQ ID NO: 29
C. ignea KASIVb CDS
ATGGCGGCGGCCGCTTCCATGTTTACGTCCCCACTCTGTACGTGGCTCGTAGCCT
CTTGCATGTCGACTTCCTTCGACAACGACCCACGTTCGCCGTCCGTCAAGCGTCT
CCCCCGCCGGAGGAGGATTCTCTCCCAATGCTCCCTCCGCGGATCCACCTCCCAA
TGCCTCGTCACCTCATACATCGACCCTTGCAATAAGTACTGCTCCTCCGCCTCCCT
TAGCTTCCTCGGGGATAACGGATTCGCATCCCTTTTCGGATCTAAGCCATTCCGG
TCCAATCGCGGCCACCGGAGGCTCGGCCGTGCTTCCCATTCCGGGGAGGCCATGG
CTGTGGCTCTGCAACCTGCACAGGAAGTCACCACGAAGAAGAAACCTGTGATCA
AGCAAAGGCGAGTAGTTGTTACAGGAATGGGCGTGGTGACTCCTCTAGGCCATG
AACCTGATGTTTACTACAACAATCTCCTAGATGGAGTAAGCGGCATAAGTGAGAT
AGAGACCTTCGACTGCACTCAGTTTCCCACGAGAATCGCCGGAGAGATCAAGTCT
TTTTCCACAGATGGGTGGGTGGCCCCAAAGCTCTCCAAGAGGATGGACAAGTTC
ATGCTTTACTTGTTGACTGCTGGCAAGAAAGCATTAGCAGATGGTGGAATCACCG
ATGATGTGATGAAAGAGCTTGATAAAAGAAAGTGTGGGGTTCTCATTGGCTCTG
GAATGGGCGGCATGAAGTTGTTCAACGATTCCATTGAAGCTCTGAGGATTTCATA
TAAAAAGATGAATCCCTTTTGTGTACCTTTTGCTACCACAAATATGGGATCAGCT
ATGCTTGCAATGGACTTGGGATGGATGGGTCCTAACTACTCGATATCAACTGCCT
GTGCAACAAGTAATTTCTGTATACTGAATGCTTCAAACCACATAGTCAGAGGCGA
AGCTGACATGATGCTTTGTGGTGGCTCGGATTCTGTCACTGTACCTTTAGGTGTG
GGAGGTTTCGTAGCATGCCGAGCTTTGTCACAGAGGAATAATGACCCTACCAAA
GCTTCGAGACCTTGGGACAGTAATCGGGATGGATTTGTGATGGGAGAAGGAGCT
GGAGTGTTACTTCTTGAGGAGTTAGAGCATGCAAAGAAAAGAGGTGCAACCATT
TATGCGGAATTTCTCGGTGGGAGCTTTACTTCTGATGCCTACCACATGACCGAGC
CTCACCCCGAAGGAGCTGGAGTGATTCTCTGCATTGAGAAGGCCTTGGCTCAGTC
CGGAGTCTCGAGGGAAGACGTGAATTATATAAATGCGCATGCAACTTCCACTCCT
GCTGGTGATATAAAGGAATACCAAGCTCTCGCCCGCTGTTTCGGCCAAAACAGTG
AGTTAAGAGTGAATTCCACCAAATCGATGATCGGTCACCTTCTTGGAGCAGCTGG
TGGCGTAGAAGCAGTTGCAGTAATTCAGGCAATAAGGACTGGATGGATCCATCC
AAATATTAATTTGGAAGACCCCGACGAAGCCGTGGATCCAAAATTGCTCGTCGG
CCCTAAGAAGGAGAAACTGAAGGTCAAGGTAGCTTTGTCCAATTCATTCGGGTTC
GGCGGGCATAACTCATCCATACTCTTTGCCCCTTGCAATTAG
SEQ ID NO: 30
C. procumbens KASIV CDS
ATGGCGGCGGCGCCCTCTTCCCCACTCTGTACGTGGCTCGTAGCCGCTTGCATGT
CCACTTCCTTCGACAACAACCCACGTTCGCCCTCCATCAAGCGTCTCCCCCGCCG
GAGGAGGGTTCTCTCCCAATGCTCCCTCCGTGGATCCACCTTCCAATGCCTCGTC
ACCTCACACAACGACCCTTGCAATCAGTACTGCTCCTCCGCCTCCCTTAGCTTCCT
CGGGGATAACGGATTCGGATCCAAGCCATTCCGGTCCAATCGCGGCCACCGGAG
GCTCGGCCGTGCTTCGCATTCCGGGGAGGCCATGGCTGTGGCCTTGCAACCTGCA
CAGGAAGTCGCCACGAAGAAGAAACCTGCTATGAAGCAAAGGCGAGTAGTTGTT
ACAGGAATGGGCGTGGTGACTCCTCTGGGCCATGAACCTGATGTTTACTACAACA
ATCTCCTAGATGGAGTAAGCGGCATAAGTGAGATAGAGACCTTCGACTGCACTC
AGTTTCCCACGAGAATCGCCGGAGAGATCAAGTCTTTTTCCACAGATGGATGGGT
GGCCCCAAAGCTCTCCAAGAGGATGGACAAGTTCATGCTTTACTTGTTGACTGCT
GGCAAGAAAGCATTAGCAGATGGTGGAATCACTGATGATGTGATGAAAGAGCTT
GATAAAAGAAAGTGTGGAGTTCTCATTGGCTCTGGAATGGGCGGCATGAAGTTG
TTCAACGATTCCATTGAAGCTCTGAGAGTTTCATATAAGAAGATGAATCCCTTTT
GTGTACCTTTTGCTACCACAAATATGGGATCAGCTATGCTTGCAATGGACTTGGG
ATGGATGGGTCCTAACTACTCGATATCAACTGCCTGTGCAACAAGTAATTTCTGT
ATACTGAATGCTGCAAACCACATAGTCAGAGGCGAAGCTGACATGATGCTTTGT
GGTGGCTCGGATGCGGTCATTATACCTATTGGTTTGGGAGGTTTTGTGGCGTGCC
GAGCTTTGTCACAGAGGAATAATGACCCTACCAAGGCTTCGAGACCATGGGATA
GTAATCGTGATGGATTTGTAATGGGCGAAGGAGCTGGAGTGTTACTTCTCGAGGA
GTTAGAGCATGCAAAGAAAAGAGGTGCAACCATTTATGCGGAATTTTTAGGGGG
CAGTTTCACTTGCGATGCCTACCATATGACCGAGCCTCACCCTGAAGGAGCTGGA
GTGATCCTCTGCATAGAGAAGGCCTTGGCTCAGTCCGGAGTCTCTAGAGAAGAC
GTAAATTACATAAATGCGCATGCAACTTCCACTCCTGCTGGAGATATCAAAGAAT
ACCAAGCTCTCGCCCACTGTTTCGGCCAAAACAGTGAGCTGAGAGTGAATTCCAC
TAAATCGATGATCGGTCATCTTCTTGGAGCAGCTGGTGGTGTAGAAGCAGTTACC
GTAATTCAGGCGATAAGGACTGGGTGGATCCATCCAAATCTTAATTTGGAAGACC
CGGACAAAGCCGTGGATGCAAAATTTCTCGTGGGACCTAAGAAGGAGAGACTGA
ATGTCAAGGTCGGTTTGTCCAATTCATTTGGGTTCGGGGGGCATAACTCATCCAT
ACTCTTTGCCCCTTGCAATTAG
SEQ ID NO: 31
C. paucipetala KASIVa CDS
ATGGCGGCGGCGGCCTCTTCCCCACTCTGCACATGGCTCGTAGCCGCTTGCATGT
CCACTTCATTCGACAACAACCCACGTTCGCCCTCCATCAAGCGTCTCCCCCGCCG
GAGGAGGGTTCTCTCCCAATGCTCCCTCCGCGGATCCACCTTCCAATGCCTCGTC
AACTCACACATCGACCCTTGCAATCAGAACGTCTCCTCCGCCTCCCTTAGCTTCCT
CGGGGATAACGGATTCGGATCCAATCCATTCCGGTCCAATCGCGGCCACCGGAG
GCTCGGCCGGGCTTCCCATTCCGGGGAGGCCATGGCTGTTGCTCTGCAACCTGCA
CAGGAAGTCGCCACGAAGAAGAAACCTGCTATCAAGCAAAGGCGAGTAGTTGTT
ACAGGAATGGGCGTGGTGACTCCTCTAGGCCATGAGCCTGATGTTTTCTACAACA
ATCTCCTAGATGGAGTAAGCGGCATAAGTGAGATAGAGACCTTCGACTGCACTC
AGTTTCCCACGAGAATTGCCGGAGAGATCAAGTCTTTTTCCACAGATGGGTGGGT
GGCCCCAAAGCTCTCCAAGAGGATGGACAAGTTCATGCTTTACTTGTTGACTGCT
GGCAAGAAAGCATTAGCAGATGCTGGAATTACCGAGGATGTGATGAAAGAGCTT
GATAAAAGAAAGTGTGGAGTTCTCATTGGCTCCGGAATGGGCGGCATGAAGTTG
TTCAACGATTCCATTGAAGCTCTGAGGGTTTCATATAAGAAGATGAATCCCTTTT
GTGTACCTTTTGCTACCACAAATATGGGATCAGCTATGCTTGCAATGGACTTGGG
ATGGATGGGTCCTAACTACTCGATATCGACTGCCTGTGCAACAAGTAATTTCTGT
ATACTGAATGCTGCAAACCACATAATCAGAGGCGAAGCTGACATGATGCTTTGT
GGTGGTTCGGATGCGGTCATTATACCTATTGGTTTGGGAGGTTTTGTGGCGTGCC
GAGCTTTGTCACAGAGGAATAGTGACCCTACCAAAGCTTCGAGACCATGGGATA
GTAATCGTGATGGATTTGTAATGGGCGAAGGAGCTGGAGTGTTACTTCTCGAGGA
GTTAGAGCATGCAAAGAAAAGAGGTGCAACCATTTATGCGGAATTTTTAGGGGG
CAGCTTCACTTGCGATGCCTACCACATGACCGAGCCTCACCCTGATGGAGCTGGA
GTGATCCTCTGCATAGAGAAGGCTTTGGCACAGTCCGGAGTCTCGAGGGAAGAC
GTCAATTACATAAATGCGCATGCAACTTCTACTCCTGCTGGAGATATCAAGGAAT
ACCAAGCTCTCGCCCACTGTTTCGGCCAAAACAGTGAGCTGAGAGTGAATTCCAC
TAAATCGATGATCGGTCATCTTCTTGGTGCAGCTGGTGGTGTAGAAGCTGTTACT
GTAATTCAGGCGATAAGGACTGGGTGGATTCATCCAAATCTTAATTTGGAAGACC
CGGACGAAGCCGTGGATGCAAAATTTCTCGTGGGACCTAAGAAGGAGAGATTGA
ATGTCAAGGTCGGTTTGTCCAATTCATTTGGGTTCGGTGGGCATAACTCATCCAT
ACTCTTCGCCCCTTACAATTAG
SEQ ID NO: 32
C. painteri KASIV CDS
ATGGCGGCCTCCTCTTGCATGGTTGCGTCCCCGTTCTGTACGTGGCTCGTATCCGC
ATGCATGTCTACTTCATTCGACAACGACCCACGTTCCCTTTCCCACAAGCGGCTC
CGCCTCTCCCGTCGCCGGAGGCCTCTCTCCTCTCATTGCTCCCTCCGCGGATCCAC
TCCCCAATGCCTCGACCCTTGCAATCAGCACTGCTTCCTCGGGGATAACGGATTC
GCTTCCCTCATCGGATCCAAGCCTCCCCGTTCCAATCTCGGCCACCTGAGGCTCG
GCCGCACTTCCCATTCCGGGGAGGTCATGGCTGTGGCACAGGAAGTCTCCACAA
ATAAGAAACATGCTACCAAGCAAAGGCGAGTAGTTGTGACAGGTATGGGCGTGG
TGACTCCTCTAGGCCATGACCCCGATGTTTACTACAACAATCTCCTAGAAGGAGT
AAGTGGCATCAGTGAGATAGAGAACTTCGACTGCTCTCAGCTTCCCACGAGAATT
GCCGGAGAGATCAAGTCTTTTTCCACAGATGGGTTGGTGGCCCCGAAGCTCTCCA
AGAGGATGGACAAGTTCATGCTTTACATCCTGACTGCAGGCAAGAAAGCATTAG
CAGATGGTGGAATCACTGAAGATGTGATGAAAGAGCTCGATAAAAGAAAGTGTG
GAGTTCTCATTGGCTCCGGATTGGGCGGTATGAAGGTATTCAGCGACTCCGTTGA
AGCTCTGAGGATTTCATATAAGAAGATCAGTCCCTTTTGTGTACCTTTTTCTACCA
CAAATATGGGATCCGCTATGCTTGCAATGGACTTGGGATGGATGGGCCCTAACTA
TTCGATATCAACTGCCTGTGCAACAAGTAACTTCTGTATACTGAATGCTGCGAAC
CACATAACCAAAGGCGAAGCTGACATGATGCTTTGTGGTGGCTCGGATGCGGCC
ATTTTACCTATTGGTATGGGAGGTTTCGTGGCATGCCGAGCTTTGTCACAGAGGA
ATAATGACCCTACCAAAGCTTCGAGACCATGGGACAGTAATCGTGATGGATTTGT
GATGGGAGAAGGAGCTGGAGTGTTACTTCTCGAGGAGTTAGAGCATGCAAAGAA
AAGAGGTGCAACCATTTATGCGGAATTTCTAGGTGGGAGTTTCACTTGCGATGCC
TACCACATGACCGAGCCTCACCCTGATGGAGCTGGAGTGATCCTCTGCATAGAGA
AGGCCTTGGCTCAGTCCGGAGTCTCGAGGGAAGAAGTAAATTACATAAATGCGC
ATGCAACTTCCACTCCTGCTGGAGATATCAAGGAATACCAAGCTCTCGCCCATTG
TTTCGGCCAAAACAGTGAGTTAAGAGTGAATTCCACCAAATCGATGATCGGTCAC
CTTCTTGGAGGAGCTGGTGGCGTAGAAGCAGTTACAGTAGTTCAGGCAATAAGG
ACTGGATGGATCCATCCAAATATTAATTTGGAAGACCCGGACAAAGGCGTGGAT
GCAAAACTGCTCGTCGGCCCTAAGAAGGAGAAACTGAAGGTCAAGGTCGGTTTG
TCCAATTCATTTGGGTTCGGCGGCCATAACTCATCCATACTCTTTGCCCCATGCAA
TTAG
SEQ ID NO: 33
C. avigera KASIVa CDS
ATGGCGGCCGCCGCTTCCATGGTTGCGTCCCCATTCTGTACGTGGCTCGTAGCCG
CTTGCATGTCCACTTCCGTCGACAAAGACCCACGTTCGCCGTCTATCAAGCGTCT
CCCCCGCCGGAAGAGGATTCATTCCCAATGCTCCCTCCGCGGATCCACCTTCCAA
TGCCTCGTCACCTCATACAACGACCCTTGCGAACAATACCGCTCATCCGCCTCCC
TTAGCTTCCTCGGGGATAACGGATTCGCATCCCTTTTCGGATCCAAGCCATTCCG
GTCCAATCGCGGCCACCGGAGGCTCGGCCGTGCTTCCCATTCCGGGGAGGCCATG
GCCGTGGCACTGCAACCTGCACAGGAAGTTGGCACGAAGAAGAAACCTGTTATC
AAGCAAAGGCGAGTAGTTGTTACAGGAATGGGCGTGGTGACTCCTCTAGGCCAT
GAACCTGATGTTTACTACAACAATCTCCTAGACGGAGTAAGCGGCATAAGTGAG
ATAGAGACCTTCGACTGCACTCAGTTTCCCACGAGAATTGCCGGAGAGATCAAGT
CTTTTTCCACAGATGGGTGGGTGGCTCCAAAGCTCTCTAAGAGGATGGACAAGTT
CATGCTTTACTTGTTGACTGCTGGCAAGAAAGCATTGGCAGATGGTGGAATCACC
GATGATGTGATGAAAGAGCTTGATAAAAGAAAGTGTGGAGTTCTCATTGGCTCC
GGATTGGGCGGTATGAAGGTATTTAGCGAGTCCATTGAAGCTCTGAGGACTTCAT
ATAAGAAGATCAGTCCCTTTTGTGTACCTTTTTCTACCACGAATATGGGATCCGCT
ATTCTTGCAATGGACTTGGGATGGATGGGCCCTAACTATTCGATATCGACTGCCT
GTGCAACAAGTAACTTCTGTATACTGAATGCTGCGAACCACATAACCAAAGGCG
AAGCAGACATGATGCTTTGTGGTGGCTCGGATTCGGTCATTTTACCTATTGGTAT
GGGAGGTTTCGTAGCATGCCGAGCTTTGTCACAGAGGAATAATGACCCTACCAA
AGCTTCGAGACCATGGGACAGTAATCGTGATGGATTTGTGATGGGAGAAGGTGC
TGGAGTTTTACTTCTCGAGGAGTTAGAGCATGCAAAGAAAAGAGGCGCAACCAT
TTATGCGGAATTTCTTGGTGGGAGTTTCACTTGCGATGCCTACCACATGACCGAG
CCTCACCCTGAAGGAGCTGGAGTGATCCTCTGCATAGAGAAGGCCTTGGCTCAGT
CCGGAGTCTCGAGGGAAGACGTAAATTACATAAATGCGCATGCAACTTCCACTC
CCGCTGGAGATATCAAAGAATACCAAGCTCTCGCCCACTGTTTCGGCCAAAACA
GTGAGTTAAGAGTGAATTCCACCAAGTCGATGATCGGTCACCTTCTTGGAGGAGC
CGGTGGCGTAGAAGCAGTTACAGTCGTTCAGGCAATAAGGACTGGATGGATCCA
TCCAAATATTAATTTGGACGACCCGGACGAAGGCGTGGATGCAAAACTGCTCGT
CGGCCCTAAGAAGGAGAAACTGAAGGTCAAGGTCGGTTTGTCCAATTCATTCGG
GTTCGGCGGCCATAACTCATCCATACTCTTTGCCCCATGCAATTAG
SEQ ID NO: 34
C. ignea KASIVa CDS
ATGGCGGCGGCCGCTTCCATGTTTACGTCCCCACTCTGTACGTGGCTCGTAGCCT
CTTGCATGTCGACTTCCTTCGACAACGACCCACGTTCGCCGTCCGTCAAGCGTCT
CCCCCGCCGGAGGAGGATTCTCTCCCAATGCTCCCTCCGCGGATCCACCTCCCAA
TGCCTCGTCACCTCATACATCGACCCTTGCAATAAGTACTGCTCCTCCGCCTCCCT
TAGCTTCCTCGGGGATAACGGATTCGCATCCCTTTTCGGATCTAAGCCATTCCGG
TCCAATCGCGGCCACCGGAGGCTCGGCCGTGCTTCCCATTCCGGGGAGGCCATGG
CTGTGGCTCTGCAACCTGCACAGGAAGTCACCACGAAGAAGAAACCTGTGATCA
AGCAAAGGCGAGTAGTTGTTACAGGAATGGGCGTGGTGACTCCTCTAGGCCATG
AACCTGATGTTTACTACAACAATCTCCTAGATGGAGTAAGCGGCATAAGTGAGAT
AGAGACCTTCGACTGCACTCAGTTTCCCACGAGAATCGCCGGAGAGATCAAGTCT
TTTTCCACAGATGGGTGGGTGGCCCCAAAGCTCTCCAAGAGGATGGACAAGTTC
ATGCTTTACTTGTTGACTGCTGGCAAGAAAGCATTAGCAGATGGTGGAATCACCG
ATGATGTGATGAAAGAGCTTGATAAAAGAAAGTGTGGGGTTCTCATTGGCTCTG
GAATGGGCGGCATGAAGTTGTTCAACGATTCCATTGAAGCTCTGAGGATTTCATA
TAAAAAGATGAATCCCTTTTGTGTACCTTTTGCTACCACAAATATGGGATCAGCT
ATGCTTGCAATGGACTTGGGATGGATGGGTCCTAACTACTCGATATCAACTGCCT
GTGCAACAAGTAATTTCTGTATACTGAATGCTTCAAACCACATAGTCAGAGGCGA
AGCTGACATGATGCTTTGTGGTGGCTCGGATGCGGTTATTATACCTATTGGTTTG
GGAGGTTTTGTGGCGTGCCGAGCTTTGTCACAGAGGAATAATGACCCTACCAAA
GCTTCGAGGCCATGGGATAGTAATCGTGATGGATTTGTAATGGGCGAAGGAGCT
GGAGTGTTACTTCTCGAGGAGTTAGAGCATGCAAAGAAAAGAGGTGCAACCATT
TATGCGGAATTTTTAGGGGGCAGTTTCACTTGCGATGCCTACCACATGACCGAGC
CTCACCCTGAAGGAGCTGGAGTGATCCTCTGCATAGAGAAGGCCTTGGCTCAGG
CCGGAGTCTCTAAAGAAGATGTAAATTACATAAATGCGCATGCAACTTCTACTCC
TGCTGGAGATATCAAGGAATACCAAGCTCTCGCCCAATGTTTCGGCCAAAACAGT
GAGCTGAGAGTGAATTCCACTAAATCGATGATCGGTCATCTTCTTGGAGCAGCTG
GTGGTGTAGAAGCAGTTACTGTGGTTCAGGCGATAAGGACTGGGTGGATCCATC
CAAATCTTAATTTGGAAGACCCGGACAAAGCCGTGGATGCAAAGTTGCTCGTGG
GACCTAAGAAGGAGAGACTGAATGTCAAGGTCGGTTTGTCCAATTCATTTGGGTT
CGGTGGGCATAATTCGTCCATACTCTTCGCCCCTTACAATTAG
SEQ ID NO: 35
C. avigera KASIa CDS
ATGCAATCCCTCCATTCCCCTGCCCTCCGGGCCTCCCCTCTCGACCCTCTCCGACT
CAAATCCTCCGCCAATGGCCCCTCTTCCACCGCCGCTTTCCGTCCCCTCCGCCGCG
CCACCCTCCCCAACATTCGGGCCGCCTCCCCCACCGTCTCCGCCCCCAAGCGCGA
GACCGACCCCAAGAAGCGTGTCGTCATCACCGGCATGGGCCTCGTCTCCGTCTTC
GGCTCCGATGTCGACGCTTATTACGAAAAGCTCCTCTCCGGCGAGAGCGGGATCA
GCTTAATCGACCGCTTCGACGCTTCCAAGTTCCCCACGAGGTTCGGCGGCCAGAT
CCGGGGATTCAACGCCACGGGATACATCGACGGCAAAAACGACAGGAGGCTCGA
CGATTGCCTCCGCTACTGCATTGTCGCCGGGAAGAAGGCTCTCGAAAATTCCGAT
CTCGGCGGCGATAGTCTCTCAAAGATTGATAAGGAGAGAGCTGGAGTGCTAGTT
GGAACTGGCATGGGTGGCCTAACCGTCTTCTCTGACGGGGTTCAGAATCTAATCG
AGAAAGGTCACCGGAAGATCTCCCCGTTTTTCATTCCATATGCCATTACAAACAT
GGGGTCTGCCCTGCTTGCCATCGATTTGGGTCTGATGGGCCCAAATTATTCGATTT
CAACTGCATGTGCTACTTCCAACTACTGCTTTTATGCTGCTGCTAATCATATCCGC
CGAGGCGAGGCTGACCTCATGATTGCTGGAGGAACTGAGGCTGCAATCATTCCA
ATTGGGTTAGGAGGATTCGTTGCTTGCAGGGCTTTATCTCAAAGGAATGATGACC
CTCAGACTGCCTCAAGGCCGTGGGATAAGGACCGTGATGGTTTTGTGATGGGTGA
AGGGGCTGGAGTATTGGTTATGGAGAGCTTAGAACATGCAATGAAACGAGGAGC
GCCGATTATTGCAGAATATTTGGGAGGTGCAGTCAACTGTGATGCTTATCATATG
ACTGATCCAAGGGCTGATGGGCTTGGTGTCTCCTCGTGCATTGAGAGCAGTCTCG
AAGATGCCGGGGTCTCACCTGAAGAGGTCAATTACATAAATGCTCATGCGACTTC
TACTCTTGCTGGGGATCTTGCCGAGATAAATGCCATCAAGAAGGTTTTCAAGAAC
ACCAAGGATATCAAAATCAATGCAACTAAGTCGATGATTGGACACTGTCTTGGA
GCATCAGGGGGTCTTGAAGCCATTGCGACAATTAAGGGAATAACCACTGGCTGG
CTTCATCCCAGCATAAACCAATTCAATCCCGAGCCATCAGTGGAATTTGACACTG
TTGCCAACAAGAAGCAGCAACATGAAGTCAATGTTGCTATCTCAAATTCATTCGG
ATTCGGAGGCCACAACTCAGTTGTAGCTTTCTCAGCTTTCAAGCCATGA
SEQ ID NO: 36
C. pulcherrima KASI CDS
ATGCATTCCCTCCAGTCACCCTCCCTTCGGGCCTCCCCGCTCGACCCCTTCCGCCC
CAAATCATCCACCGTCCGCCCCCTCCACCGAGCATCAATTCCCAACGTCCGGGCC
GCTTCCCCCACCGTCTCCGCTCCCAAGCGCGAGACCGACCCCAAGAAGCGCGTCG
TGATCACCGGAATGGGCCTTGTCTCCGTTTTCGGCTCCGACGTCGATGCGTACTA
CGACAAGCTCCTGTCAGGCGAGAGCGGGATCGGCCCAATCGACCGCTTCGACGC
CTCCAAGTTCCCCACCAGGTTCGGCGGCCAGATTCGTGGCTTCAACTCCATGGGA
TACATTGACGGCAAAAACGACAGGCGGCTTGATGATTGCCTTCGCTACTGCATTG
TCGCCGGGAAGAAGTCTCTTGAGGACGCCGATCTCGGTGCCGACCGCCTCTCCAA
GATCGACAAGGAGAGAGCCGGAGTGCTGGTTGGGACAGGAATGGGTGGTCTGAC
TGTCTTCTCTGACGGGGTTCAATCTCTTATCGAGAAGGGTCACCGGAAAATCACC
CCTTTCTTCATCCCCTATGCCATTACAAACATGGGGTCTGCCCTGCTCGCTATTGA
ACTCGGTCTGATGGGCCCAAACTATTCAATTTCCACTGCATGTGCCACTTCCAAC
TACTGCTTCCATGCTGCTGCTAATCATATCCGCCGTGGTGAGGCTGATCTTATGAT
TGCTGGAGGCACTGAGGCCGCAATCATTCCAATTGGGTTGGGAGGCTTTGTGGCT
TGCAGGGCTCTGTCTCAAAGGAACGATGACCCTCAGACTGCCTCTAGGCCCTGGG
ATAAAGACCGTGATGGTTTTGTGATGGGTGAAGGTGCTGGAGTGTTGGTGCTGGA
GAGCTTGGAACATGCAATGAAACGAGGAGCACCTATTATTGCAGAGTATTTGGG
AGGTGCAATCAACTGTGATGCTTATCACATGACTGACCCAAGGGCTGATGGTCTC
GGTGTCTCCTCTTGCATTGAGAGTAGCCTTGAAGATGCTGGCGTCTCACCTGAAG
AGGTCAATTACATAAATGCTCATGCGACTTCTACTCTAGCTGGGGATCTCGCCGA
GATAAATGCCATCAAGAAGGTTTTCAAGAACACAAAGGATATCAAAATTAATGC
AACTAAGTCAATGATCGGACACTGTCTTGGAGCCTCTGGAGGTCTTGAAGCTATA
GCGACTATTAAGGGAATAAACACCGGCTGGCTTCATCCCAGCATTAATCAATTCA
ATCCTGAGCCATCCGTGGAGTTCGACACTGTTGCCAACAAGAAGCAGCAACACG
AAGTTAATGTTGCGATCTCGAATTCATTTGGATTCGGAGGCCACAACTCAGTCGT
GGCTTTCTCGGCTTTCAAGCCATGA
SEQ ID NO: 37
C. aviga mitochondrial KAS CDS
ATGGTGTTTCTTCCTTGGCGAAAAATGCTCTGTCCATCTCAATACCGTTTTTTGCG
GCCCTTATCTTCATCTACAACTTTTGATCCTCGTAGGGTTGTTGTTACAGGCCTGG
GTATGGTGACTCCATTAGGATGCGGGGTGAACACCACATGGAAACAACTCATAG
AGGGGAAATGTGGGATAAGAGCAATATCCCTTGAAGACCTAAAGATGGATGCTT
TTGATATTGATACTCAGGCCTATGTATTTGATCAGCTGACCTCGAAGGTCGCTGC
CACCGTGCCCACCGGAGTGAATCCCGGAGAATTTAATGAAGATTTATGGTTCAAT
CAGAAGGAGCACCGTGCTATTGCAAGGTTCATAGCTTATGCACTCTGTGCAGCTG
ATGAAGCTCTTAAAGATGCAAATTGGGAACCTACTGAACCTGAAGAGAGAGAAA
TGACGGGTGTCTCCATTGGTGGAGGGACTGGAAGCATTAGCGATGTATTAGATGC
TGGTCGGATGATTTGTGAGAAGAAATTGCGTCGCCTAAGTCCATTCTTCATTCCA
CGCATATTGATAAATATGGCCTCTGGTCATGTGAGCATGAAATATGGTTTCCAGG
GACCCAACCATGCTGCTGTGACAGCTTGTGCAACAGGGGCTCATTCGATAGGTGA
TGCTGCAAGGATGATACAGTTTGGAGATGCAGATGTCATGGTCGCTGGAGGCAC
AGAATCTAGCATAGACGCCTTATCCATTGCAGGATTTTGCAGGTCAAGGGCTCTT
ACAACAAAGTATAATTCTTGCCCACAAGAAGCTTCACGACCCTTTGATACCGATA
GAGATGGGTTTGTAATAGGTGAAGGGTCTGGCGTCTTGGTATTGGAGGAACTAG
ATCATGCAAGAAAACGTGGTGCAAAGATGTATGCCGAGTTCTGTGGATATGGAA
TGTCTGGTGATGCGCATCATATAACCCAACCTCATAGCGATGGAAGAGGTGCCAT
TTTAGCAATGACCCGTGCATTGAAGCAGTCAAATCTACATCCGGATCAGGTGGAT
TATGTAAATGCTCACGCTACGTCTACTTCTTTAGGTGATGCAATTGAAGCTAAGG
CGATTAAAACAGTTTTCTCGGATCATGCGATGTCAGGTTCGCTCGCCCTTTCCTCC
ACCAAGGGAGCTATTGGGCATCTCCTCGGAGCAGCGGGTGCTGTGGAAGCCATT
TTCTCCATTCTGGCTATAAAAAACGGACTTGCGCCTTTGACGCTAAATGTCGCAA
GACCAGACCCTGTGTTTACCGAGCGGTTTGTGCCTTTGACTGCTTCAAAAGAGAT
GCATGTAAGGGCGGCGTTGTCAAACTCTTTTGGCTTTGGAGGTACAAATACTACA
CTTCTTTTCACTTCACCTCCTCAAAACTAA
SEQ ID NO: 38
Cuphea palustris KAS IV codon optimized for Prototheca with cloning sequence and tags. Nucleotide sequence of the C. palustris KASIV expression vector (D3145 and D3295, pSZ4312). The 5′ and 3′ homology arms enabling targeted integration into the pLOOP locus are noted with lowercase; the PmHXT1-2 promoter is noted in uppercase italic which drives expression of the ScMelibiase selection marker noted with lowercase italic followed by the PmPGK 3′UTR terminator highlighted in uppercase. The PmACP promoter (noted in bold text) drives the expression of the codon optimized Cpal KASIV (noted with lowercase bold text) and is terminated with the CvNR 3′UTR noted in underlined, lower case bold. Restriction cloning sites and spacer DNA fragments are noted as underlined, uppercase plain lettering.
SEQ ID NO: 39
aacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggc
gatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcggg
cggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagc
ttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacga
gcaaatcctgacactagaagggctgactcgcccggcacggctgaattaca
caggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtatttt
gttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgc
gacgaaggtggtgccaccactgtgccagccagtcctggcggctcccaggg
ccccgatcaagagccaggacatccaaactacccacagcatcaacgccccg
gcctatactcgaaccccacttgcactctgcaatggtatgggaaccacggg
gcagtcttgtgtgggtcgcgcctatcgcggtcggcgaagaccgggaaGGT
ACCCCGCTCCCGTCTGGTCCTCACGTTCGTGTACGGCCTGGATCCCGGAA
AGGGCGGATGCACGTGGTGTTGCCCCGCCATTGGCGCCCACGTTTCAAAG
TCCCCGGCCAGAAATGCACAGGACCGGCCCGGCTCGCACAGGCCATGACG
AATGCCCAGATTTCGACAGCAAAACAATCTGGAATAATCGCAACCATTCG
CGTTTTGAACGAAACGAAAAGACGCTGTTTAGCACGTTTCCGATATCGTG
GGGGCCGAAGCATGATTGGGGGGAGGAAAGCGTGGCCCCAAGGTAGCCCA
TTCTGTGCCACACGCCGACGAGGACCAATCCCCGGCATCAGCCTTCATCG
ACGGCTGCGCCGCACATATAAAGCCGGACGCCTTCCCGACACGTTCAAAC
AGTTTTATTTCCTCCACTTCCTGAATCAAACAAATCTTCAAGGAAGATCC
TGCTCTTGAGCAACTAGTatgttcgcgttctacttcctgacggcctgcat
ctccctgaagggcgtgttcggcgtctccccctcctacaacggcctgggcc
tgacgccccagatgggctgggacaactggaacacgttcgcctgcgacgtc
tccgagcagctgctgctggacacggccgaccgcatctccgacctgggcct
gaaggacatgggctacaagtacatcatcctggacgactgctggtcctccg
gccgcgactccgacggcttcctggtcgccgacgagcagaagttccccaac
ggcatgggccacgtcgccgaccacctgcacaacaactccttcctgttcgg
catgtactcctccgcgggcgagtacacgtgcgccggctaccccggctccc
tgggccgcgaggaggaggacgcccagttcttcgcgaacaaccgcgtggac
tacctgaagtacgacaactgctacaacaagggccagttcggcacgcccga
gatctcctaccaccgctacaaggccatgtccgacgccctgaacaagacgg
gccgccccatcttctactccctgtgcaactggggccaggacctgaccttc
tactggggctccggcatcgcgaactcctggcgcatgtccggcgacgtcac
ggcggagttcacgcgccccgactcccgctgcccctgcgacggcgacgagt
acgactgcaagtacgccggcttccactgctccatcatgaacatcctgaac
aaggccgcccccatgggccagaacgcgggcgtcggcggctggaacgacct
ggacaacctggaggtcggcgtcggcaacctgacggacgacgaggagaagg
cgcacttctccatgtgggccatggtgaagtcccccctgatcatcggcgcg
aacgtgaacaacctgaaggcctcctcctactccatctactcccaggcgtc
cgtcatcgccatcaaccaggactccaacggcatccccgccacgcgcgtct
ggcgctactacgtgtccgacacggacgagtacggccagggcgagatccag
atgtggtccggccccctggacaacggcgaccaggtcgtggcgctgctgaa
cggcggctccgtgtcccgccccatgaacacgaccctggaggagatcttct
tcgactccaacctgggctccaagaagctgacctccacctgggacatctac
gacctgtgggcgaaccgcgtcgacaactccacggcgtccgccatcctggg
ccgcaacaagaccgccaccggcatcctgtacaacgccaccgagcagtcct
acaaggacggcctgtccaagaacgacacccgcctgttcggccagaagatc
ggctccctgtcccccaacgcgatcctgaacacgaccgtccccgcccacgg
catcgcgttctaccgcctgcgcccctcctcctgaTACAACTTATTACGTA
TTCTGACCGGCGCTGATGTGGCGCGGACGCCGTCGTACTCTTTCAGACTT
TACTCTTGAGGAATTGAACCTTTCTCGCTTGCTGGCATGTAAACATTGGC
GCAATTAATTGTGTGATGAAGAAAGGGTGGCACAAGATGGATCGCGAATG
TACGAGATCGACAACGATGGTGATTGTTATGAGGGGCCAAACCTGGCTCA
ATCTTGTCGCATGTCCGGCGCAATGTGATCCAGCGGCGTGACTCTCGCAA
CCTGGTAGTGTGTGCGCACCGGGTCGCTTTGATTAAAACTGATCGCATTG
CCATCCCGTCAACTCACAAGCCTACTCTAGCTCCCATTGCGCACTCGGGC
GCCCGGCTCGATCAATGTTCTGAGCGGAGGGCGAAGCGTCAGGAAATCGT
CTCGGCAGCTGGAAGCGCATGGAATGCGGAGCGGAGATCGAATCAGGATC
CCGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGT
CGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTG
GTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGC
GAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTCGAAACGTTCACA
GCCTAGGGATATCGCCTGCTCAAGCGGGCGCTCAACATGCAGAGCGTCAG
CGAGACGGGCTGTGGCGATCGCGAGACGGACGAGGCCGCCTCTGCCCTGT
TTGAACTGAGCGTCAGCGCTGGCTAAGGGGAGGGAGACTCATCCCCAGGC
TCGCGCCAGGGCTCTGATCCCGTCTCGGGCGGTGATCGGCGCGCATGACT
ACGACCCAACGACGTACGAGACTGATGTCGGTCCCGACGAGGAGCGCCGC
GAGGCACTCCCGGGCCACCGACCATGTTTACACCGACCGAAAGCACTCGC
TCGTATCCATTCCGTGCGCCCGCACATGCATCATCTTTTGGTACCGACTT
CGGTCTTGTTTTACCCCTACGACCTGCCTTCCAAGGTGTGAGCAACTCGC
CCGGACATGACCGAGGGTGATCATCCGGATCCCCAGGCCCCAGCAGCCCC
TGCCAGAATGGCTCGCGCTTTCCAGCCTGCAGGCCCGTCTCCCAGGTCGA
CGCAACCTACATGACCACCCCAATCTGTCCCAGACCCCAAACACCCTCCT
TCCCTGCTTCTCTGTGATCGCTGATCAGCAACACATatggcttccgcggc
attcaccatgtcggcgtgccccgcgatgactggcagggcccctggggcac
gtcgctccggacggccagtcgccacccgcctgaggggctccaccttccag
tgcctggtgacctcctacatcgacccctgcaaccagttctcctcctccgc
ctccctgtccttcctgggcgacaacggcttcgcctccctgttcggctcca
agcccttccgctccaaccgcggccaccgccgcctgggccgcgcctcccac
tccggcgaggccatggccgtggccctggagcccgcccaggaggtggccac
caagaagaagcccctggtgaagcagcgccgcgtggtggtgaccggcatgg
gcgtggtgacccccctgggccacgagcccgacgtgtactacaacaacctg
ctggacggcgtgtccggcatctccgagatcgaggccttcgactgcaccca
gttccccacccgcatcgccggcgagatcaagtccttctccaccgacggct
gggtggcccccaagctgtccaagcgcatggacaagttcatgctgtacctg
ctgaccgccggcaagaaggccctggccgacggcggcatcaccgacgacgt
gatgaaggagctggacaagcgcaagtgcggcgtgctgatcggctccggcc
tgggcggcatgaagctgttctccgactccatcgaggccctgcgcatctcc
tacaagaagatgaaccccttctgcgtgcccttcgccaccaccaacatggg
ctccgccatgctggccatggacctgggctggatgggccccaactactcca
tctccaccgcctgcgccacctccaacttctgcatcctgaactccgccaac
cacatcgtgcgcggcgaggccgacatgatgctgtgcggcggctccgacgc
cgtgatcatccccatcggcctgggcggcttcgtggcctgccgcgccctgt
cccagcgcaacaacgaccccaccaaggcctcccgcccctgggactccaac
cgcgacggcttcgtgatgggcgagggcgccggcgtgctgctgctggagga
gctggagcacgccaagaagcgcggcgccaccatctacgccgagttcctgg
gcggctccttcacctgcgacgcctaccacatgaccgagccccaccccgag
ggcgccggcgtgatcctgtgcatcgagaaggccctggcccaggccggcgt
gtcccgcgaggacgtgaactacatcaacgcccacgccacctccacccccg
ccggcgacatcaaggagtaccaggccctggcccactgcttcggccagaac
tccgagctgcgcgtgaactccaccaagtccatgatcggccacctgatcgg
cgccgccggcggcgtggaggccgtgaccgtggtgcaggccatccgcaccg
gctggatccaccccaacctgaacctggaggaccccgacaaggccgtggac
gccaaggtgctggtgggccccaagaaggagcgcctgaacgtgaaggtggg
cctgtccaactccttcggcttcggcggccacaactcctccatcctgttcg
ccccctacaacaccatgtacccctacgacgtgcccgactacgcctgaTAT
CGAGgcagcagcagctcggatagtatcgacacactctggacgctggtcgt
gtgatggactgttgccgccacacttgctgccttgacctgtgaatatccct
gccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgct
tttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcat
ccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacg
ctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcg
cacagccttggtttgggctccgcctgtattctcctggtactgcaacctgt
aaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacaca
aatggaAAGCTTGAGCTCagcggcgacggtcctgctaccgtacgacgttg
ggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagc
gcggctcaaggatacttgaactcctggattgatatcggtccaataatgga
tggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggat
gcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcg
gtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgtt
ctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccg
tgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggc
aatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagagg
caacacactccgcttctta
Cuphea palustris KAS IV codon optimized for
Prototheca
SEQ ID NO: 40
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccttccagtgcctggtgacctcctacatcgacccctgcaaccag
ttctcctcctccgcctccctgtccttcctgggcgacaacggcttcgcctc
cctgttcggctccaagcccttccgctccaaccgcggccaccgccgcctgg
gccgcgcctcccactccggcgaggccatggccgtggccctggagcccgcc
caggaggtggccaccaagaagaagcccctggtgaagcagcgccgcgtggt
ggtgaccggcatgggcgtggtgacccccctgggccacgagcccgacgtgt
actacaacaacctgctggacggcgtgtccggcatctccgagatcgaggcc
ttcgactgcacccagttccccacccgcatcgccggcgagatcaagtcctt
ctccaccgacggctgggtggcccccaagctgtccaagcgcatggacaagt
tcatgctgtacctgctgaccgccggcaagaaggccctggccgacggcggc
atcaccgacgacgtgatgaaggagctggacaagcgcaagtgcggcgtgct
gatcggctccggcctgggcggcatgaagctgttctccgactccatcgagg
ccctgcgcatctcctacaagaagatgaaccccttctgcgtgcccttcgcc
accaccaacatgggctccgccatgctggccatggacctgggctggatggg
ccccaactactccatctccaccgcctgcgccacctccaacttctgcatcc
tgaactccgccaaccacatcgtgcgcggcgaggccgacatgatgctgtgc
ggcggctccgacgccgtgatcatccccatcggcctgggcggcttcgtggc
ctgccgcgccctgtcccagcgcaacaacgaccccaccaaggcctcccgcc
cctgggactccaaccgcgacggcttcgtgatgggcgagggcgccggcgtg
ctgctgctggaggagctggagcacgccaagaagcgcggcgccaccatcta
cgccgagttcctgggcggctccttcacctgcgacgcctaccacatgaccg
agccccaccccgagggcgccggcgtgatcctgtgcatcgagaaggccctg
gcccaggccggcgtgtcccgcgaggacgtgaactacatcaacgcccacgc
cacctccacccccgccggcgacatcaaggagtaccaggccctggcccact
gcttcggccagaactccgagctgcgcgtgaactccaccaagtccatgatc
ggccacctgatcggcgccgccggcggcgtggaggccgtgaccgtggtgca
ggccatccgcaccggctggatccaccccaacctgaacctggaggaccccg
acaaggccgtggacgccaaggtgctggtgggccccaagaaggagcgcctg
aacgtgaaggtgggcctgtccaactccttcggcttcggcggccacaactc
ctccatcctgttcgccccctacaacaccatgtacccctacgacgtgcccg
actacgcctga
C. camphora KASIV codon optimized for Prototheca. Nucleotide sequence from the C. camphora KASIV (D3147, pSZ4338) expression vector. Only the codon optimized C. camphora KASIV sequence is shown, the promoter, 3′UTR, selection marker and targeting arms are the same as in SEQ ID NO: 38.
SEQ ID NO: 41
atggccatgatggccggctcctgctccaacctggtgatcggcaaccgcga
gctgggcggcaacggcccctccctgctgcactacaacggcctgcgccccc
tggagaacatccagaccgcctccgccgtgaagaagcccaacggcctgttc
gcctcctccaccgcccgcaagtccaaggccgtgcgcgccatggtgctgcc
caccgtgaccgcccccaagcgcgagaaggaccccaagaagcgcatcgtga
tcaccggcatgggcctggtgtccgtgttcggcaacgacatcgacaccttc
tactccaagctgctggagggcgagtccggcatcggccccatcgaccgctt
cgacgcctcctccttctccgtgcgcttcgccggccagatccacaacttct
cctccaagggctacatcgacggcaagaacgaccgccgcctggacgactgc
tggcgctactgcctggtggccggccgccgcgccctggaggacgccaacct
gggccccgaggtgctggagaagatggaccgctcccgcatcggcgtgctga
tcggcaccggcatgggcggcctgtccgccttctccaacggcgtggagtcc
ctgatccagaagggctacaagaagatcacccccttcttcatcccctactc
catcaccaacatgggctccgccctgctggccatcgacaccggcgtgatgg
gccccaactactccatctccaccgcctgcgccaccgccaactactgcttc
cacgccgccgccaaccacatccgccgcggcgaggccgagatcatggtgac
cggcggcaccgaggccgccgtgtccgccaccggcgtgggcggcttcatcg
cctgccgcgccctgtcccaccgcaacgacgagccccagaccgcctcccgc
ccctgggacaaggaccgcgacggcttcgtgatgggcgagggcgccggcgt
gctggtgatggagtccctgcaccacgcccgcaagcgcggcgccaacatca
tcgccgagtacctgggcggcgccgtgacctgcgacgcccaccacatgacc
gacccccgcgccgacggcctgggcgtgtcctcctgcatcaccaagtccct
ggaggacgccggcgtgtcccccgaggaggtgaactacgtgaacgcccacg
ccacctccaccctggccggcgacctggccgaggtgaacgccatcaagaag
gtgttcaaggacacctccgagatgaagatgaacggcaccaagtccatgat
cggccactgcctgggcgccgccggcggcctggaggccatcgccaccatca
aggccatcaacaccggctggctgcaccccaccatcaaccagttcaacatc
gagcccgccgtgaccatcgacaccgtgcccaacgtgaagaagaagcacga
catccacgtgggcatctccaactccttcggcttcggcggccacaactccg
tggtggtgttcgcccccttcatgcccaccatgtacccctacgacgtgccc
gactacgcctga
C. camphora KASI (D3148, pSZ4339) codon
optimized for Prototheca
SEQ ID NO: 42
atgcagatcctgcagaccccctcctcctcctcctcctccctgcgcatgtc
ctccatggagtccctgtccctgacccccaagtccctgcccctgaagaccc
tgctgcccctgcgcccccgccccaagaacctgtcccgccgcaagtcccag
aacccccgccccatctcctcctcctcctcccccgagcgcgagaccgaccc
caagaagcgcgtggtgatcaccggcatgggcctggtgtccgtgttcggca
acgacgtggacgcctactacgaccgcctgctgtccggcgagtccggcatc
gcccccatcgaccgcttcgacgcctccaagttccccacccgcttcgccgg
ccagatccgcggcttcacctccgacggctacatcgacggcaagaacgacc
gccgcctggacgactgcctgcgctactgcatcgtgtccggcaagaaggcc
ctggagaacgccggcctgggcccccacctgatggacggcaagatcgacaa
ggagcgcgccggcgtgctggtgggcaccggcatgggcggcctgaccgtgt
tctccaacggcgtgcagaccctgcacgagaagggctaccgcaagatgacc
cccttcttcatcccctacgccatcaccaacatgggctccgccctgctggc
catcgagctgggcttcatgggccccaactactccatctccaccgcctgcg
ccacctccaactactgcttctacgccgccgccaaccacatccgccgcggc
gaggccgacctgatgctggccggcggcaccgaggccgccatcatccccat
cggcctgggcggcttcgtggcctgccgcgccctgtcccagcgcaacgacg
acccccagaccgcctcccgcccctgggacaaggaccgcgacggcttcgtg
atgggcgagggcgccggcgtgctggtgatggagtccctggagcacgccat
gaagcgcgacgcccccatcatcgccgagtacctgggcggcgccgtgaact
gcgacgcctaccacatgaccgacccccgcgccgacggcctgggcgtgtcc
acctgcatcgagcgctccctggaggacgccggcgtggcccccgaggaggt
gaactacatcaacgcccacgccacctccaccctggccggcgacctggccg
aggtgaacgccatcaagaaggtgttcaccaacacctccgagatcaagatc
aacgccaccaagtccatgatcggccactgcctgggcgccgccggcggcct
ggaggccatcgccaccatcaaggccatcaacaccggctggctgcacccct
ccatcaaccagttcaaccccgagccctccgtggagttcgacaccgtggcc
aacaagaagcagcagcacgaggtgaacgtggccatctccaactccttcgg
cttcggcggccacaactccgtggtggtgttctccgccttcaagcccacca
tgtacccctacgacgtgcccgactacgcctga
U. californica KASI
U. californica KASI (D3150, pSZ4341) codon
optimized for Prototheca
SEQ ID NO: 43
atggagtccctgtccctgacccccaagtccctgcccctgaagaccctgct
gcccttccgcccccgccccaagaacctgtcccgccgcaagtcccagaacc
ccaagcccatctcctcctcctcctcccccgagcgcgagaccgaccccaag
aagcgcgtggtgatcaccggcatgggcctggtgtccgtgttcggcaacga
cgtggacgcctactacgaccgcctgctgtccggcgagtccggcatcgccc
ccatcgaccgcttcgacgcctccaagttccccacccgcttcgccggccag
atccgcggcttcacctccgacggctacatcgacggcaagaacgaccgccg
cctggacgactgcctgcgctactgcatcgtgtccggcaagaaggccctgg
agaacgccggcctgggccccgacctgatggacggcaagatcgacaaggag
cgcgccggcgtgctggtgggcaccggcatgggcggcctgaccgtgttctc
caacggcgtgcagaccctgcacgagaagggctaccgcaagatgaccccct
tcttcatcccctacgccatcaccaacatgggctccgccctgctggccatc
gacctgggcttcatgggccccaactactccatctccaccgcctgcgccac
ctccaactactgcttctacgccgccgccaaccacatccgccgcggcgagg
ccgacgtgatgctggccggcggcaccgaggccgccatcatccccatcggc
ctgggcggcttcgtggcctgccgcgccctgtcccagcgcaacgacgaccc
ccagaccgcctcccgcccctgggacaaggaccgcgacggcttcgtgatgg
gcgagggcgccggcgtgctggtgatggagtccctggagcacgccatgaag
cgcgacgcccccatcatcgccgagtacctgggcggcgccgtgaactgcga
cgcctaccacatgaccgacccccgcgccgacggcctgggcgtgtccacct
gcatcgagcgctccctggaggacgccggcgtggcccccgaggaggtgaac
tacatcaacgcccacgccacctccaccctggccggcgacctggccgaggt
gaacgccatcaagaaggtgttcaccaacacctccgagatcaagatcaacg
ccaccaagtccatgatcggccactgcctgggcgccgccggcggcctggag
gccatcgccaccatcaaggccatcaacaccggctggctgcacccctccat
caaccagttcaaccccgagccctccgtggagttcgacaccgtggccaaca
agaagcagcagcacgaggtgaacgtggccatctccaactccttcggcttc
ggcggccacaactccgtggtggtgttctccgccttcaagcccaccatgta
cccctacgacgtgcccgactacgcctga
U. californica KASIV (D3152, pSZ4343) codon
optimized for Prototheca
SEQ ID NO: 44
atgacccagaccctgatctgcccctcctccatggagaccctgtccctgac
caagcagtcccacttccgcctgcgcctgcccacccccccccacatccgcc
gcggcggcggccaccgccaccccccccccttcatctccgcctccgccgcc
ccccgccgcgagaccgaccccaagaagcgcgtggtgatcaccggcatggg
cctggtgtccgtgttcggcaccaacgtggacgtgtactacgaccgcctgc
tggccggcgagtccggcgtgggcaccatcgaccgcttcgacgcctccatg
ttccccacccgcttcggcggccagatccgccgcttcacctccgagggcta
catcgacggcaagaacgaccgccgcctggacgactacctgcgctactgcc
tggtgtccggcaagaaggccatcgagtccgccggcttcgacctgcacaac
atcaccaacaagatcgacaaggagcgcgccggcatcctggtgggctccgg
catgggcggcctgaaggtgttctccgacggcgtggagtccctgatcgaga
agggctaccgcaagatctcccccttcttcatcccctacatgatccccaac
atgggctccgccctgctgggcatcgacctgggcttcatgggccccaacta
ctccatctccaccgcctgcgccacctccaactactgcatctacgccgccg
ccaaccacatccgccagggcgacgccgacctgatggtggccggcggcacc
gaggcccccatcatccccatcggcctgggcggcttcgtggcctgccgcgc
cctgtccacccgcaacgacgacccccagaccgcctcccgcccctgggaca
tcgaccgcgacggcttcgtgatgggcgagggcgccggcatcctggtgctg
gagtccctggagcacgccatgaagcgcgacgcccccatcctggccgagta
cctgggcggcgccgtgaactgcgacgcccaccacatgaccgacccccgcg
ccgacggcctgggcgtgtccacctgcatcgagtcctccctggaggacgcc
ggcgtggccgccgaggaggtgaactacatcaacgcccacgccacctccac
ccccaccggcgacctggccgagatgaaggccatcaagaacgtgttccgca
acacctccgagatcaagatcaacgccaccaagtccatgatcggccactgc
ctgggcgcctccggcggcctggaggccatcgccaccctgaaggccatcac
caccggctggctgcaccccaccatcaaccagttcaaccccgagccctccg
tggacttcgacaccgtggccaagaagaagaagcagcacgaggtgaacgtg
gccatctccaactccttcggcttcggcggccacaactccgtgctggtgtt
ctccgccttcaagcccaccatgtacccctacgacgtgcccgactacg
cctga
C. wrightii KASAI (D3153, pSZ4379) codon
optimized for Prototheca
SEQ ID NO: 45
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggt
acgtattccagtgcctggtggccagctgcatcgacccctgcgaccagtac
cgcagcagcgccagcctgagcttcctgggcgacaacggcttcgccagcct
gttcggcagcaagcccttcatgagcaaccgcggccaccgccgcctgcgcc
gcgccagccacagcggcgaggccatggccgtggccctgcagcccgcccag
gaggccggcaccaagaagaagcccgtgatcaagcagcgccgcgtggtggt
gaccggcatgggcgtggtgacccccctgggccacgagcccgacgtgttct
acaacaacctgctggacggcgtgagcggcatcagcgagatcgagaccttc
gactgcacccagttccccacccgcatcgccggcgagatcaagagcttcag
caccgacggctgggtggcccccaagctgagcaagcgcatggacaagttca
tgctgtacctgctgaccgccggcaagaaggccctggccgacggcggcatc
accgacgaggtgatgaaggagctggacaagcgcaagtgcggcgtgctgat
cggcagcggcatgggcggcatgaaggtgttcaacgacgccatcgaggccc
tgcgcgtgagctacaagaagatgaaccccttctgcgtgcccttcgccacc
accaacatgggcagcgccatgctggccatggacctgggctggatgggccc
caactacagcatcagcaccgcctgcgccaccagcaacttctgcatcctga
acgccgccaaccacatcatccgcggcgaggccgacatgatgctgtgcggc
ggcagcgacgccgtgatcatccccatcggcctgggcggcttcgtggcctg
ccgcgccctgagccagcgcaacagcgaccccaccaaggccagccgcccct
gggacagcaaccgcgacggcttcgtgatgggcgagggcgccggcgtgctg
ctgctggaggagctggagcacgccaagaagcgcggcgccaccatctacgc
cgagttcctgggcggcagcttcacctgcgacgcctaccacatgaccgagc
cccaccccgagggcgccggcgtgatcctgtgcatcgagaaggccctggcc
caggccggcgtgagcaaggaggacgtgaactacatcaacgcccacgccac
cagcaccagcgccggcgacatcaaggagtaccaggccctggcccgctgct
tcggccagaacagcgagctgcgcgtgaacagcaccaagagcatgatcggc
cacctgctgggcgccgccggcggcgtggaggccgtgaccgtggtgcaggc
catccgcaccggctggattcaccccaacctgaacctggaggaccccgaca
aggccgtggacgccaagctgctggtgggccccaagaaggagcgcctgaac
gtgaaggtgggcctgagcaacagcttcggcttcggcggccacaacagcag
catcctgttcgccccctgcaacgtgtga
C. avigera KASIVb (D3287, pSZ4453) codon
optimized for Prototheca
SEQ ID NO: 46
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccttccagtgctacatcggcgacaacggcttcggctccaagccc
ccccgctccaaccgcggccacctgcgcctgggccgcacctcccactccgg
cgaggtgatggccgtggccatgcagtccgcccaggaggtgtccaccaagg
agaagcccgccaccaagcagcgccgcgtggtggtgaccggcatgggcgtg
gtgaccgccctgggccacgaccccgacgtgtactacaacaacctgctgga
cggcgtgtccggcatctccgagatcgagaacttcgactgctcccagctgc
ccacccgcatcgccggcgagatcaagtccttctccgccgacggctgggtg
gcccccaagttctcccgccgcatggacaagttcatgctgtacatcctgac
cgccggcaagaaggccctggtggacggcggcatcaccgaggacgtgatga
aggagctggacaagcgcaagtgcggcgtgctgatcggctccggcctgggc
ggcatgaaggtgttctccgagtccatcgaggccctgcgcacctcctacaa
gaagatctcccccttctgcgtgcccttctccaccaccaacatgggctccg
ccatcctggccatggacctgggctggatgggccccaactactccatctcc
accgcctgcgccacctccaacttctgcatcctgaacgccgccaaccacat
caccaagggcgaggccgacatgatgctgtgcggcggctccgactccgtga
tcctgcccatcggcatgggcggcttcgtggcctgccgcgccctgtcccag
cgcaacaacgaccccaccaaggcctcccgcccctgggactccaaccgcga
cggcttcgtgatgggcgagggcgccggcgtgctgctgctggaggagctgg
agcacgccaagaagcgcggcgccaccatctacgccgagttcctgggcggc
tccttcacctgcgacgcctaccacatgaccgagccccaccccgagggcgc
cggcgtgatcctgtgcatcgagaaggccctggcccagtccggcgtgtccc
gcgaggacgtgaactacatcaacgcccacgccacctccacccccgccggc
gacatcaaggagtaccaggccctggcccactgcttcggccagaactccga
gctgcgcgtgaactccaccaagtccatgatcggccacctgctgggcggcg
ccggcggcgtggaggccgtgaccgtggtgcaggccatccgcaccggctgg
atccaccccaacatcaacctggacgaccccgacgagggcgtggacgccaa
gctgctggtgggccccaagaaggagaagctgaaggtgaaggtgggcctgt
ccaactccttcggcttcggcggccacaactcctccatcctgttcgccccc
tgcaacaccatgtacccctacgacgtgcccgactacgcctga
C. paucipetala KASIVb codon optimized
for Prototheca
SEQ ID NO: 47
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccttccagtgcctgggcgacatcggcttcgcctccctgatcggc
tccaagcccccccgctccaaccgcaaccaccgccgcctgggccgcacctc
ccactccggcgaggtgatggccgtggccatgcagcccgcccacgaggcct
ccaccaagaacaagcccgtgaccaagcagcgccgcgtggtggtgaccggc
atgggcgtggccacccccctgggccacgaccccgacgtgtactacaacaa
cctgctggacggcgtgtccggcatctcccagatcgagaacttcgactgca
cccagttccccacccgcatcgccggcgagatcaagtccttctccaccgag
ggctacgtgatccccaagttcgccaagcgcatggacaagttcatgctgta
cctgctgaccgccggcaagaaggccctggaggacggcggcatcaccgagg
acgtgatgaaggagctggacaagcgcaagtgcggcgtgctgatcggctcc
ggcatgggcggcatgaagatcatcaacgactccatcgccgccctgaacgt
gtcctacaagaagatgacccccttctgcgtgcccttctccaccaccaaca
tgggctccgccatgctggccatcgacctgggctggatgggccccaactac
tccatctccaccgcctgcgccacctccaactactgcatcctgaacgccgc
caaccacatcgtgcgcggcgaggccgacatgatgctgtgcggcggctccg
acgccgtgatcatccccgtgggcctgggcggcttcgtggcctgccgcgcc
ctgtcccagcgcaacaacgaccccaccaaggcctcccgcccctgggactc
caaccgcgacggcttcgtgatgggcgagggcgccggcgtgctgctgctgg
aggagctggagcacgccaagaagcgcggcgccaccatctacgccgagttc
ctgggcggctccttcacctgcgacgcctaccacatgaccgagccccaccc
cgacggcgccggcgtgatcctgtgcatcgagaaggccctggcccagtccg
gcgtgtcccgcgaggacgtgaactacatcaacgcccacgccacctccacc
cccgccggcgacatcaaggagtaccaggccctggcccactgcttcggcca
gaactccgagctgcgcgtgaactccaccaagtccatgatcggccacctgc
tgggcgccgccggcggcgtggaggccgtgaccgtggtgcaggccatccgc
accggctggatccaccccaacatcaacctggagaaccccgacgaggccgt
ggacgccaagctgctggtgggccccaagaaggagaagctgaaggtgaagg
tgggcctgtccaactccttcggcttcggcggccacaactcctccatcctg
ttcgccccctacaacaccatgtacccctacgacgtgcccgactacgc
ctga
C. ignea KASIVb (D3289, pSZ4455) codon
optimized for Prototheca
SEQ ID NO: 48
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccacctcccagtgcctggtgacctcctacatcgacccctgcaacaag
tactgctcctccgcctccctgtccttcctgggcgacaacggcttcgcctc
cctgttcggctccaagcccttccgctccaaccgcggccaccgccgcctgg
gccgcgcctcccactccggcgaggccatggccgtggccctgcagcccgcc
caggaggtgaccaccaagaagaagcccgtgatcaagcagcgccgcgtggt
ggtgaccggcatgggcgtggtgacccccctgggccacgagcccgacgtgt
actacaacaacctgctggacggcgtgtccggcatctccgagatcgagacc
ttcgactgcacccagttccccacccgcatcgccggcgagatcaagtcctt
ctccaccgacggctgggtggcccccaagctgtccaagcgcatggacaagt
tcatgctgtacctgctgaccgccggcaagaaggccctggccgacggcggc
atcaccgacgacgtgatgaaggagctggacaagcgcaagtgcggcgtgct
gatcggctccggcatgggcggcatgaagctgttcaacgactccatcgagg
ccctgcgcatctcctacaagaagatgaaccccttctgcgtgcccttcgcc
accaccaacatgggctccgccatgctggccatggacctgggctggatggg
ccccaactactccatctccaccgcctgcgccacctccaacttctgcatcc
tgaacgcctccaaccacatcgtgcgcggcgaggccgacatgatgctgtgc
ggcggctccgactccgtgaccgtgcccctgggcgtgggcggcttcgtggc
ctgccgcgccctgtcccagcgcaacaacgaccccaccaaggcctcccgcc
cctgggactccaaccgcgacggcttcgtgatgggcgagggcgccggcgtg
ctgctgctggaggagctggagcacgccaagaagcgcggcgccaccatcta
cgccgagttcctgggcggctccttcacctccgacgcctaccacatgaccg
agccccaccccgagggcgccggcgtgatcctgtgcatcgagaaggccctg
gcccagtccggcgtgtcccgcgaggacgtgaactacatcaacgcccacgc
cacctccacccccgccggcgacatcaaggagtaccaggccctggcccgct
gcttcggccagaactccgagctgcgcgtgaactccaccaagtccatgatc
ggccacctgctgggcgccgccggcggcgtggaggccgtggccgtgatcca
ggccatccgcaccggctggatccaccccaacatcaacctggaggaccccg
acgaggccgtggaccccaagctgctggtgggccccaagaaggagaagctg
aaggtgaaggtggccctgtccaactccttcggcttcggcggccacaactc
ctccatcctgttcgccccctgcaacaccatgtacccctacgacgtgcccg
actacgcctga
Cuphea procumbens KASIV (D3290, pSZ4456)
codon optimized for Prototheca
SEQ ID NO: 49
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccttccagtgcctggtgacctcccacaacgacccctgcaaccag
tactgctcctccgcctccctgtccttcctgggcgacaacggcttcggctc
caagcccttccgctccaaccgcggccaccgccgcctgggccgcgcctccc
actccggcgaggccatggccgtggccctgcagcccgcccaggaggtggcc
accaagaagaagcccgccatgaagcagcgccgcgtggtggtgaccggcat
gggcgtggtgacccccctgggccacgagcccgacgtgtactacaacaacc
tgctggacggcgtgtccggcatctccgagatcgagaccttcgactgcacc
cagttccccacccgcatcgccggcgagatcaagtccttctccaccgacgg
ctgggtggcccccaagctgtccaagcgcatggacaagttcatgctgtacc
tgctgaccgccggcaagaaggccctggccgacggcggcatcaccgacgac
gtgatgaaggagctggacaagcgcaagtgcggcgtgctgatcggctccgg
catgggcggcatgaagctgttcaacgactccatcgaggccctgcgcgtgt
cctacaagaagatgaaccccttctgcgtgcccttcgccaccaccaacatg
ggctccgccatgctggccatggacctgggctggatgggccccaactactc
catctccaccgcctgcgccacctccaacttctgcatcctgaacgccgcca
accacatcgtgcgcggcgaggccgacatgatgctgtgcggcggctccgac
gccgtgatcatccccatcggcctgggcggcttcgtggcctgccgcgccct
gtcccagcgcaacaacgaccccaccaaggcctcccgcccctgggactcca
accgcgacggcttcgtgatgggcgagggcgccggcgtgctgctgctggag
gagctggagcacgccaagaagcgcggcgccaccatctacgccgagttcct
gggcggctccttcacctgcgacgcctaccacatgaccgagccccaccccg
agggcgccggcgtgatcctgtgcatcgagaaggccctggcccagtccggc
gtgtcccgcgaggacgtgaactacatcaacgcccacgccacctccacccc
cgccggcgacatcaaggagtaccaggccctggcccactgcttcggccaga
actccgagctgcgcgtgaactccaccaagtccatgatcggccacctgctg
ggcgccgccggcggcgtggaggccgtgaccgtgatccaggccatccgcac
cggctggatccaccccaacctgaacctggaggaccccgacaaggccgtgg
acgccaagttcctggtgggccccaagaaggagcgcctgaacgtgaaggtg
ggcctgtccaactccttcggcttcggcggccacaactcctccatcctgtt
cgccccctgcaacaccatgtacccctacgacgtgcccgactacgcctga
C paucipetala KASIVa (D3291, pSZ4457) codon
optimized for Prototheca
SEQ ID NO: 50
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccttccagtgcctggtgaactcccacatcgacccctgcaaccag
aacgtgtcctccgcctccctgtccttcctgggcgacaacggcttcggctc
caaccccttccgctccaaccgcggccaccgccgcctgggccgcgcctccc
actccggcgaggccatggccgtggccctgcagcccgcccaggaggtggcc
accaagaagaagcccgccatcaagcagcgccgcgtggtggtgaccggcat
gggcgtggtgacccccctgggccacgagcccgacgtgttctacaacaacc
tgctggacggcgtgtccggcatctccgagatcgagaccttcgactgcacc
cagttccccacccgcatcgccggcgagatcaagtccttctccaccgacgg
ctgggtggcccccaagctgtccaagcgcatggacaagttcatgctgtacc
tgctgaccgccggcaagaaggccctggccgacgccggcatcaccgaggac
gtgatgaaggagctggacaagcgcaagtgcggcgtgctgatcggctccgg
catgggcggcatgaagctgttcaacgactccatcgaggccctgcgcgtgt
cctacaagaagatgaaccccttctgcgtgcccttcgccaccaccaacatg
ggctccgccatgctggccatggacctgggctggatgggccccaactactc
catctccaccgcctgcgccacctccaacttctgcatcctgaacgccgcca
accacatcatccgcggcgaggccgacatgatgctgtgcggcggctccgac
gccgtgatcatccccatcggcctgggcggcttcgtggcctgccgcgccct
gtcccagcgcaactccgaccccaccaaggcctcccgcccctgggactcca
accgcgacggcttcgtgatgggcgagggcgccggcgtgctgctgctggag
gagctggagcacgccaagaagcgcggcgccaccatctacgccgagttcct
gggcggctccttcacctgcgacgcctaccacatgaccgagccccaccccg
acggcgccggcgtgatcctgtgcatcgagaaggccctggcccagtccggc
gtgtcccgcgaggacgtgaactacatcaacgcccacgccacctccacccc
cgccggcgacatcaaggagtaccaggccctggcccactgcttcggccaga
actccgagctgcgcgtgaactccaccaagtccatgatcggccacctgctg
ggcgccgccggcggcgtggaggccgtgaccgtgatccaggccatccgcac
cggctggatccaccccaacctgaacctggaggaccccgacgaggccgtgg
acgccaagttcctggtgggccccaagaaggagcgcctgaacgtgaaggtg
ggcctgtccaactccttcggcttcggcggccacaactcctccatcctgtt
cgccccctacaacaccatgtacccctacgacgtgcccgactacgcctga
Cuphea painteri KASIV (D3292, pSZ4458) codon
optimized for Prototheca
SEQ ID NO: 51
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccccccagtgcctggacccctgcaaccagcactgcttcctgggc
gacaacggcttcgcctccctgatcggctccaagcccccccgctccaacct
gggccacctgcgcctgggccgcacctcccactccggcgaggtgatggccg
tggcccaggaggtgtccaccaacaagaagcacgccaccaagcagcgccgc
gtggtggtgaccggcatgggcgtggtgacccccctgggccacgaccccga
cgtgtactacaacaacctgctggagggcgtgtccggcatctccgagatcg
agaacttcgactgctcccagctgcccacccgcatcgccggcgagatcaag
tccttctccaccgacggcctggtggcccccaagctgtccaagcgcatgga
caagttcatgctgtacatcctgaccgccggcaagaaggccctggccgacg
gcggcatcaccgaggacgtgatgaaggagctggacaagcgcaagtgcggc
gtgctgatcggctccggcctgggcggcatgaaggtgttctccgactccgt
ggaggccctgcgcatctcctacaagaagatctcccccttctgcgtgccct
tctccaccaccaacatgggctccgccatgctggccatggacctgggctgg
atgggccccaactactccatctccaccgcctgcgccacctccaacttctg
catcctgaacgccgccaaccacatcaccaagggcgaggccgacatgatgc
tgtgcggcggctccgacgccgccatcctgcccatcggcatgggcggcttc
gtggcctgccgcgccctgtcccagcgcaacaacgaccccaccaaggcctc
ccgcccctgggactccaaccgcgacggcttcgtgatgggcgagggcgccg
gcgtgctgctgctggaggagctggagcacgccaagaagcgcggcgccacc
atctacgccgagttcctgggcggctccttcacctgcgacgcctaccacat
gaccgagccccaccccgacggcgccggcgtgatcctgtgcatcgagaagg
ccctggcccagtccggcgtgtcccgcgaggaggtgaactacatcaacgcc
cacgccacctccacccccgccggcgacatcaaggagtaccaggccctggc
ccactgcttcggccagaactccgagctgcgcgtgaactccaccaagtcca
tgatcggccacctgctgggcggcgccggcggcgtggaggccgtgaccgtg
gtgcaggccatccgcaccggctggatccaccccaacatcaacctggagga
ccccgacaagggcgtggacgccaagctgctggtgggccccaagaaggaga
agctgaaggtgaaggtgggcctgtccaactccttcggcttcggcggccac
aactcctccatcctgttcgccccctgcaacaccatgtacccctacgacgt
gcccgactacgcctga
C. avigera KASIVa (D3293, pSZ4459) codon
optimized for Prototheca
SEQ ID NO: 52
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccaccttccagtgcctggtgacctcctacaacgacccctgcgagcag
taccgctcctccgcctccctgtccttcctgggcgacaacggcttcgcctc
cctgttcggctccaagcccttccgctccaaccgcggccaccgccgcctgg
gccgcgcctcccactccggcgaggccatggccgtggccctgcagcccgcc
caggaggtgggcaccaagaagaagcccgtgatcaagcagcgccgcgtggt
ggtgaccggcatgggcgtggtgacccccctgggccacgagcccgacgtgt
actacaacaacctgctggacggcgtgtccggcatctccgagatcgagacc
ttcgactgcacccagttccccacccgcatcgccggcgagatcaagtcctt
ctccaccgacggctgggtggcccccaagctgtccaagcgcatggacaagt
tcatgctgtacctgctgaccgccggcaagaaggccctggccgacggcggc
atcaccgacgacgtgatgaaggagctggacaagcgcaagtgcggcgtgct
gatcggctccggcctgggcggcatgaaggtgttctccgagtccatcgagg
ccctgcgcacctcctacaagaagatctcccccttctgcgtgcccttctcc
accaccaacatgggctccgccatcctggccatggacctgggctggatggg
ccccaactactccatctccaccgcctgcgccacctccaacttctgcatcc
tgaacgccgccaaccacatcaccaagggcgaggccgacatgatgctgtgc
ggcggctccgactccgtgatcctgcccatcggcatgggcggcttcgtggc
ctgccgcgccctgtcccagcgcaacaacgaccccaccaaggcctcccgcc
cctgggactccaaccgcgacggcttcgtgatgggcgagggcgccggcgtg
ctgctgctggaggagctggagcacgccaagaagcgcggcgccaccatcta
cgccgagttcctgggcggctccttcacctgcgacgcctaccacatgaccg
agccccaccccgagggcgccggcgtgatcctgtgcatcgagaaggccctg
gcccagtccggcgtgtcccgcgaggacgtgaactacatcaacgcccacgc
cacctccacccccgccggcgacatcaaggagtaccaggccctggcccact
gcttcggccagaactccgagctgcgcgtgaactccaccaagtccatgatc
ggccacctgctgggcggcgccggcggcgtggaggccgtgaccgtggtgca
ggccatccgcaccggctggatccaccccaacatcaacctggacgaccccg
acgagggcgtggacgccaagctgctggtgggccccaagaaggagaagctg
aaggtgaaggtgggcctgtccaactccttcggcttcggcggccacaactc
ctccatcctgttcgccccctgcaacaccatgtacccctacgacgtgcccg
actacgcctga
C ignea KASIVa (D3294, pSZ4460) codon
optimized for Prototheca
SEQ ID NO: 53
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gctccacctcccagtgcctggtgacctcctacatcgacccctgcaacaag
tactgctcctccgcctccctgtccttcctgggcgacaacggcttcgcctc
cctgttcggctccaagcccttccgctccaaccgcggccaccgccgcctgg
gccgcgcctcccactccggcgaggccatggccgtggccctgcagcccgcc
caggaggtgaccaccaagaagaagcccgtgatcaagcagcgccgcgtggt
ggtgaccggcatgggcgtggtgacccccctgggccacgagcccgacgtgt
actacaacaacctgctggacggcgtgtccggcatctccgagatcgagacc
ttcgactgcacccagttccccacccgcatcgccggcgagatcaagtcctt
ctccaccgacggctgggtggcccccaagctgtccaagcgcatggacaagt
tcatgctgtacctgctgaccgccggcaagaaggccctggccgacggcggc
atcaccgacgacgtgatgaaggagctggacaagcgcaagtgcggcgtgct
gatcggctccggcatgggcggcatgaagctgttcaacgactccatcgagg
ccctgcgcatctcctacaagaagatgaaccccttctgcgtgcccttcgcc
accaccaacatgggctccgccatgctggccatggacctgggctggatggg
ccccaactactccatctccaccgcctgcgccacctccaacttctgcatcc
tgaacgcctccaaccacatcgtgcgcggcgaggccgacatgatgctgtgc
ggcggctccgacgccgtgatcatccccatcggcctgggcggcttcgtggc
ctgccgcgccctgtcccagcgcaacaacgaccccaccaaggcctcccgcc
cctgggactccaaccgcgacggcttcgtgatgggcgagggcgccggcgtg
ctgctgctggaggagctggagcacgccaagaagcgcggcgccaccatcta
cgccgagttcctgggcggctccttcacctgcgacgcctaccacatgaccg
agccccaccccgagggcgccggcgtgatcctgtgcatcgagaaggccctg
gcccaggccggcgtgtccaaggaggacgtgaactacatcaacgcccacgc
cacctccacccccgccggcgacatcaaggagtaccaggccctggcccagt
gcttcggccagaactccgagctgcgcgtgaactccaccaagtccatgatc
ggccacctgctgggcgccgccggcggcgtggaggccgtgaccgtggtgca
ggccatccgcaccggctggatccaccccaacctgaacctggaggaccccg
acaaggccgtggacgccaagctgctggtgggccccaagaaggagcgcctg
aacgtgaaggtgggcctgtccaactccttcggcttcggcggccacaactc
ctccatcctgttcgccccctacaacaccatgtacccctacgacgtgcccg
actacgcctga
C. avigera KASIa (D3342, pSZ4511) codon
optimized for Prototheca
SEQ ID NO: 54
atgcagtccctgcactcccccgccctgcgcgcctcccccctggaccccct
gcgcctgaagtcctccgccaacggcccctcctccaccgccgccttccgcc
ccctgcgccgcgccaccctgcccaacatccgcgccgcctcccccaccgtg
tccgcccccaagcgcgagaccgaccccaagaagcgcgtggtgatcaccgg
catgggcctggtgtccgtgttcggctccgacgtggacgcctactacgaga
agctgctgtccggcgagtccggcatctccctgatcgaccgcttcgacgcc
tccaagttccccacccgcttcggcggccagatccgcggcttcaacgccac
cggctacatcgacggcaagaacgaccgccgcctggacgactgcctgcgct
actgcatcgtggccggcaagaaggccctggagaactccgacctgggcggc
gactccctgtccaagatcgacaaggagcgcgccggcgtgctggtgggcac
cggcatgggcggcctgaccgtgttctccgacggcgtgcagaacctgatcg
agaagggccaccgcaagatctcccccttcttcatcccctacgccatcacc
aacatgggctccgccctgctggccatcgacctgggcctgatgggccccaa
ctactccatctccaccgcctgcgccacctccaactactgcttctacgccg
ccgccaaccacatccgccgcggcgaggccgacctgatgatcgccggcggc
accgaggccgccatcatccccatcggcctgggcggcttcgtggcctgccg
cgccctgtcccagcgcaacgacgacccccagaccgcctcccgcccctggg
acaaggaccgcgacggcttcgtgatgggcgagggcgccggcgtgctggtg
atggagtccctggagcacgccatgaagcgcggcgcccccatcatcgccga
gtacctgggcggcgccgtgaactgcgacgcctaccacatgaccgaccccc
gcgccgacggcctgggcgtgtcctcctgcatcgagtcctccctggaggac
gccggcgtgtcccccgaggaggtgaactacatcaacgcccacgccacctc
caccctggccggcgacctggccgagatcaacgccatcaagaaggtgttca
agaacaccaaggacatcaagatcaacgccaccaagtccatgatcggccac
tgcctgggcgcctccggcggcctggaggccatcgccaccatcaagggcat
caccaccggctggctgcacccctccatcaaccagttcaaccccgagccct
ccgtggagttcgacaccgtggccaacaagaagcagcagcacgaggtgaac
gtggccatctccaactccttcggcttcggcggccacaactccgtggtggc
cttctccgccttcaagcccaccatgtacccctacgacgtgcccgactacg
cctga
C. pulcherrima KASI (D3343, pSZ4512) codon
optimized for Prototheca
SEQ ID NO: 55
atgcactccctgcagtccccctccctgcgcgcctcccccctggacccctt
ccgccccaagtcctccaccgtgcgccccctgcaccgcgcctccatcccca
acgtgcgcgccgcctcccccaccgtgtccgcccccaagcgcgagaccgac
cccaagaagcgcgtggtgatcaccggcatgggcctggtgtccgtgttcgg
ctccgacgtggacgcctactacgacaagctgctgtccggcgagtccggca
tcggccccatcgaccgcttcgacgcctccaagttccccacccgcttcggc
ggccagatccgcggcttcaactccatgggctacatcgacggcaagaacga
ccgccgcctggacgactgcctgcgctactgcatcgtggccggcaagaagt
ccctggaggacgccgacctgggcgccgaccgcctgtccaagatcgacaag
gagcgcgccggcgtgctggtgggcaccggcatgggcggcctgaccgtgtt
ctccgacggcgtgcagtccctgatcgagaagggccaccgcaagatcaccc
ccttcttcatcccctacgccatcaccaacatgggctccgccctgctggcc
atcgagctgggcctgatgggccccaactactccatctccaccgcctgcgc
cacctccaactactgcttccacgccgccgccaaccacatccgccgcggcg
aggccgacctgatgatcgccggcggcaccgaggccgccatcatccccatc
ggcctgggcggcttcgtggcctgccgcgccctgtcccagcgcaacgacga
cccccagaccgcctcccgcccctgggacaaggaccgcgacggcttcgtga
tgggcgagggcgccggcgtgctggtgctggagtccctggagcacgccatg
aagcgcggcgcccccatcatcgccgagtacctgggcggcgccatcaactg
cgacgcctaccacatgaccgacccccgcgccgacggcctgggcgtgtcct
cctgcatcgagtcctccctggaggacgccggcgtgtcccccgaggaggtg
aactacatcaacgcccacgccacctccaccctggccggcgacctggccga
gatcaacgccatcaagaaggtgttcaagaacaccaaggacatcaagatca
acgccaccaagtccatgatcggccactgcctgggcgcctccggcggcctg
gaggccatcgccaccatcaagggcatcaacaccggctggctgcacccctc
catcaaccagttcaaccccgagccctccgtggagttcgacaccgtggcca
acaagaagcagcagcacgaggtgaacgtggccatctccaactccttcggc
ttcggcggccacaactccgtggtggccttctccgccttcaagcccaccat
gtacccctacgacgtgcccgactacgcctga
C. avigera mitochondrial KAS (D3344, pSZ4513)
codon optimized for Prototheca
SEQ ID NO: 56
atggtgttcctgccctggcgcaagatgctgtgcccctcccagtaccgctt
cctgcgccccctgtcctcctccaccaccttcgacccccgccgcgtggtgg
tgaccggcctgggcatggtgacccccctgggctgcggcgtgaacaccacc
tggaagcagctgatcgagggcaagtgcggcatccgcgccatctccctgga
ggacctgaagatggacgccttcgacatcgacacccaggcctacgtgttcg
accagctgacctccaaggtggccgccaccgtgcccaccggcgtgaacccc
ggcgagttcaacgaggacctgtggttcaaccagaaggagcaccgcgccat
cgcccgcttcatcgcctacgccctgtgcgccgccgacgaggccctgaagg
acgccaactgggagcccaccgagcccgaggagcgcgagatgaccggcgtg
tccatcggcggcggcaccggctccatctccgacgtgctggacgccggccg
catgatctgcgagaagaagctgcgccgcctgtcccccttcttcatccccc
gcatcctgatcaacatggcctccggccacgtgtccatgaagtacggcttc
cagggccccaaccacgccgccgtgaccgcctgcgccaccggcgcccactc
catcggcgacgccgcccgcatgatccagttcggcgacgccgacgtgatgg
tggccggcggcaccgagtcctccatcgacgccctgtccatcgccggcttc
tgccgctcccgcgccctgaccaccaagtacaactcctgcccccaggaggc
ctcccgccccttcgacaccgaccgcgacggcttcgtgatcggcgagggct
ccggcgtgctggtgctggaggagctggaccacgcccgcaagcgcggcgcc
aagatgtacgccgagttctgcggctacggcatgtccggcgacgcccacca
catcacccagccccactccgacggccgcggcgccatcctggccatgaccc
gcgccctgaagcagtccaacctgcaccccgaccaggtggactacgtgaac
gcccacgccacctccacctccctgggcgacgccatcgaggccaaggccat
caagaccgtgttctccgaccacgccatgtccggctccctggccctgtcct
ccaccaagggcgccatcggccacctgctgggcgccgccggcgccgtggag
gccatcttctccatcctggccatcaagaacggcctggcccccctgaccct
gaacgtggcccgccccgaccccgtgttcaccgagcgcttcgtgcccctga
ccgcctccaaggagatgcacgtgcgcgccgccctgtccaactccttcggc
ttcggcggcaccaacaccaccctgctgttcacctcccccccccagaacac
catgtacccctacgacgtgcccgactacgcctga
C. avigera KASIII (D3345, pSZ4514) Codon
optimized for Prototheca.
SEQ ID NO: 57
atggccaacgcctacggcttcgtgggctcctccgtgcccaccgtgggccg
cgccgcccagttccagcagatgggctccggcttctgctccgtggacttca
tctccaagcgcgtgttctgctgctccgccgtgcagggcgccgacaagccc
gcctccggcgactcccgcgccgagtaccgcaccccccgcctggtgtcccg
cggctgcaagctgatcggctccggctccgccatccccaccctgcaggtgt
ccaacgacgacctggccaagatcgtggacaccaacgacgagtggatctcc
gtgcgcaccggcatccgcaaccgccgcgtgctgaccggcaaggactccct
gaccaacctggccaccgaggccgcccgcaaggccctggagatggcccagg
tggacgccgaggacgtggacatggtgctgatgtgcacctccacccccgag
gacctgttcggctccgccccccagatccagaaggccctgggctgcaagaa
gaaccccctgtcctacgacatcaccgccgcctgctccggcttcgtgctgg
gcctggtgtccgccgcctgccacatccgcggcggcggcttcaacaacgtg
ctggtgatcggcgccgactccctgtcccgctacgtggactggaccgaccg
cggcacctgcatcctgttcggcgacgccgccggcgccgtgctggtgcagt
cctgcgacgccgaggaggacggcctgttcgccttcgacctgcactccgac
ggcgacggccagcgccacctgcgcgccgtgatcaccgagaacgagaccga
ccacgccgtgggcaccaacggctccgtgtccgacttccccccccgccgct
cctcctactcctgcatccagatgaacggcaaggaggtgttccgcttcgcc
tgccgctccgtgccccagtccatcgagctggccctgggcaaggccggcct
gaacggctccaacatcgactggctgctgctgcaccaggccaaccagcgca
tcatcgacgccgtggccacccgcctggaggtgccccaggagcgcgtgatc
tccaacctggccaactacggcaacacctccgccgcctccatccccctggc
cctggacgaggccgtgcgcggcggcaaggtgaagcccggccacctgatcg
ccaccgccggcttcggcgccggcctgacctggggctccgccatcgtgcgc
tggggcaccatgtacccctacgacgtgcccgactacgcctga
C. hookeriana FATB2 (“Ch FATB2”)
SEQ ID NO: 58
MVAAAASSAFFPVPAPGASPKPGKFGNWPSSLSPSFKPKSIPNGGFQVKA
NDSAHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLHQLPDWSRLLTAIT
TVFVKSKRPDMHDRKSKRPDMLVDSFGLESTVQDGLVFRQSFSIRSYEIG
TDRTASIETLMNHLQETSLNHCKSTGILLDGFGRTLEMCKRDLIWVVIKM
QIKVNRYPAWGDTVEINTRFSRLGKIGMGRDWLISDCNTGEILVRATSAY
AMMNQKTRRLSKLPYEVHQEIVPLFVDSPVIEDSDLKVHKFKVKTGDSIQ
KGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLALEYRREC
GRDSVLESVTAMDPSKVGVRSQYQHLLRLEDGTAIVNGATEWRPKNAGAN
GAISTGKTSNGNSVS
23S rRNA for UTEX 1439, UTEX 1441, UTEX 1435,
UTEX 1437 Prototheca moriformis
SEQ ID NO: 59
TGTTGAAGAATGAGCCGGCGACTTAAAATAAATGGCAGGCTAAGAGAATT
AATAACTCGAAACCTAAGCGAAAGCAAGTCTTAATAGGGCGCTAATTTAA
CAAAACATTAAATAAAATCTAAAGTCATTTATTTTAGACCCGAACCTGAG
TGATCTAACCATGGTCAGGATGAAACTTGGGTGACACCAAGTGGAAGTCC
GAACCGACCGATGTTGAAAAATCGGCGGATGAACTGTGGTTAGTGGTGAA
ATACCAGTCGAACTCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGC
GCAGCAATATATCTCGTCTATCTAGGGGTAAAGCACTGTTTCGGTGCGGG
CTATGAAAATGGTACCAAATCGTGGCAAACTCTGAATACTAGAAATGACG
ATATATTAGTGAGACTATGGGGGATAAGCTCCATAGTCGAGAGGGAAACA
GCCCAGACCACCAGTTAAGGCCCCAAAATGATAATGAAGTGGTAAAGGAG
GTGAAAATGCAAATACAACCAGGAGGTTGGCTTAGAAGCAGCCATCCTTT
AAAGAGTGCGTAATAGCTCACTG
Amino acid sequence of the C. hookeriana KASIV
(D3668, pSZ4756). The algal transit
peptide is underlined.
SEQ ID NO: 60
MASAAFTMSACPAMTGRAPGARRSGRPVATRLRGSTFQCLDPCNQQRFLG
DNGFASLFGSKPLRSNRGHLRLGRTSHSGEVMAVAMQPAQEVSTNKKPAT
KQRRVVVTGMGVVTPLGHDPDVYYNNLLDGISGISEIENFDCSQFPTRIA
GEIKSFSTDGWVAPKFSERMDKFMLYMLTAGKKALADGGITEDAMKELNK
RKCGVLIGSGLGGMKVFSDSIEALRTSYKKISPFCVPFSTTNMGSAILAM
DLGWMGPNYSISTACATSNFCILNAANHIIKGEADMMLCGGSDAAVLPVG
LGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAKK
RGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIEKALAQSGVSREDVN
YINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGGAGGVE
AVAVVQAIRTGWIHPNINLEDPDEGVDAKLLVGPKKEKLKVKVGLSNSFG
FGGHNSSILFAPCN
Nucleotide sequence of the C. hookeriana KASIV (D3668, pSZ4756) expression vector. The 5′ and 3′ homology arms enabling targeted integration into the SAD2-1 locus are noted with lowercase. The endogenous SAD2-1 promoter (present within the 5′ homology targeting arm) drives the expression of the codon optimized Ch KASIV (noted with lowercase bold text) and is terminated with the PmHSP90 3′UTR noted in underlined, lower case bold. The PmHXT1-2 promoter is noted in uppercase italic which drives expression of the ScMelibiase selection marker noted with lowercase italic followed by the PmPGK 3′UTR terminator highlighted in uppercase. Restriction cloning sites and spacer DNA fragments are noted as underlined, uppercase plain lettering.
SEQ ID NO: 61
gccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtga
tccaccgggtgaacgtagtcctcgacggaaacatctggttcgggcctcct
gcttgcactcccgcccatgccgacaacctttctgctgttaccacgaccca
caatgcaacgcgacacgaccgtgtgggactgatcggttcactgcacctgc
atgcaattgtcacaagcgcttactccaattgtattcgtttgttttctggg
agcagttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtg
gcctgggtgtttcgtcgaaaggccagcaaccctaaatcgcaggcgatccg
gagattgggatctgatccgagtttggaccagatccgccccgatgcggcac
gggaactgcatcgactcggcgcggaacccagctttcgtaaatgccagatt
ggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcga
gcgtatttggcgggcgtgctaccagggttgcatacattgcccatttctgt
ctggaccgctttactggcgcagagggtgagttgatggggttggcaggcat
cgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattca
atagtcggatgggcgacggtagaattgggtgtggcgctcgcgtgcatgcc
tcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccat
cttgctaacgctcccgactctcccgaccgcgcgcaggatagactcttgtt
caaccaatcgacaGGTACCatggcttccgcggcattcaccatgtcggcgt
gccccgcgatgactggcagggcccctggggcacgtcgctccggacggcca
gtcgccacccgcctgaggggcagcaccttccagtgcctggacccctgcaa
ccagcagcgcttcctgggcgacaacggcttcgcgtcgctgttcggctcca
agcccctgcgcagcaaccgcggccacctgcgcctgggccgcacctcgcac
tccggcgaggtgatggccgtcgcgatgcagcccgcccaggaggtgagcac
caacaagaagcccgcgaccaagcagcgccgcgtggtcgtgaccggcatgg
gcgtcgtgacccccctgggccacgaccccgacgtgtattataacaacctg
ctggacggcatctcgggcatctccgagatcgagaacttcgactgcagcca
gttccccacccgcatcgccggcgagatcaagtcgttctccaccgacggct
gggtcgcgcccaagttcagcgagcgcatggacaagttcatgctgtatatg
ctgaccgccggcaagaaggcgctggccgacggcggcatcaccgaggacgc
gatgaaggagctgaacaagcgcaagtgcggcgtgctgatcggctcgggcc
tgggcggcatgaaggtcttctccgacagcatcgaggccctgcgcacctcg
tataagaagatctcccccttctgcgtgcccttcagcaccaccaacatggg
ctcggcgatcctggcgatggacctgggctggatgggccccaactattcca
tcagcaccgcgtgcgccacctcgaacttctgcatcctgaacgcggccaac
cacatcatcaagggcgaggcggacatgatgctgtgcggcggctccgacgc
cgcggtgctgcccgtcggcctgggcggcttcgtggcctgccgcgcgctga
gccagcgcaacaacgaccccaccaaggcctcgcgcccctgggactccaac
cgcgacggcttcgtcatgggcgagggcgcgggcgtgctgctgctggagga
gctggagcacgccaagaagcgcggcgcgaccatctatgccgagttcctgg
gcggcagcttcacctgcgacgcgtatcacatgaccgagccccaccccgag
ggcgccggcgtcatcctgtgcatcgagaaggcgctggcccagtcgggcgt
gtcccgcgaggacgtgaactatatcaacgcgcacgccaccagcacccccg
cgggcgacatcaaggagtatcaggccctggcgcactgcttcggccagaac
tcggagctgcgcgtcaactccaccaagagcatgatcggccacctgctggg
cggcgccggcggcgtggaggcggtcgccgtggtccaggcgatccgcaccg
gctggatccaccccaacatcaacctggaggaccccgacgagggcgtggac
gccaagctgctggtcggccccaagaaggagaagctgaaggtgaaggtcgg
cctgtcgaactccttcggcttcggcggccacaacagctcgatcctgttcg
cgccctgcaactgaCTCGAGacagacgaccttggcaggcgtcgggtaggg
aggtggtggtgatggcgtctcgatgccatcgcacgcatccaacgaccgta
tacgcatcgtccaatgaccgtcggtgtcctctctgcctccgttttgtgag
atgtctcaggcttggtgcatcctcgggtggccagccacgttgcgcgtcgt
gctgcttgcctctcttgcgcctctgtggtactggaaaatatcatcgaggc
ccgtttttttgctcccatttcctttccgctacatcttgaaagcaaacgac
aaacgaagcagcaagcaaagagcacgaggacggtgaacaagtctgtcacc
tgtatacatctatttccccgcgggtgcacctactctctctcctgccccgg
cagagtcagctgccttacgtgacCCTAGGTGCGGTGAGAATCGAAAATGC
ATCGTTTCTAGGTTCGGAGACGGTCAATTCCCTGCTCCGGCGAATCTGTC
GGTCAAGCTGGCCAGTGGACAATGTTGCTATGGCAGCCCGCGCACATGGG
CCTCCCGACGCGGCCATCAGGAGCCCAAACAGCGTGTCAGGGTATGTGAA
ACTCAAGAGGTCCCTGCTGGGCACTCCGGCCCCACTCCGGGGGCGGGACG
CCAGGCATTCGCGGTCGGTCCCGCGCGACGAGCGAAATGATGATTCGGTT
ACGAGACCAGGACGTCGTCGAGGTCGAGAGGCAGCCTCGGACACGTCTCG
CTAGGGCAACGCCCCGAGTCCCCGCGAGGGCCGTAAACATTGTTTCTGGG
TGTCGGAGTGGGCATTTTGGGCCCGATCCAATCGCCTCATGCCGCTCTCG
TCTGGTCCTCACGTTCGCGTACGGCCTGGATCCCGGAAAGGGCGGATGCA
CGTGGTGTTGCCCCGCCATTGGCGCCCACGTTTCAAAGTCCCCGGCCAGA
AATGCACAGGACCGGCCCGGCTCGCACAGGCCATGCTGAACGCCCAGATT
TCGACAGCAACACCATCTAGAATAATCGCAACCATCCGCGTTTTGAACGA
AACGAAACGGCGCTGTTTAGCATGTTTCCGACATCGTGGGGGCCGAAGCA
TGCTCCGGGGGGAGGAAAGCGTGGCACAGCGGTAGCCCATTCTGTGCCAC
ACGCCGACGAGGACCAATCCCCGGCATCAGCCTTCATCGACGGCTGCGCC
GCACATATAAAGCCGGACGCCTAACCGGTTTCGTGGTTATGACTAGTatg
ttcgcgttctacttcctgacggcctgcatctccctgaagggcgtgttcgg
cgtctccccctcctacaacggcctgggcctgacgccccagatgggctggg
acaactggaacacgttcgcctgcgacgtctccgagcagctgctgctggac
acggccgaccgcatctccgacctgggcctgaaggacatgggctacaagta
catcatcctggacgactgctggtcctccggccgcgactccgacggcttcc
tggtcgccgacgagcagaagttccccaacggcatgggccacgtcgccgac
cacctgcacaacaactccttcctgttcggcatgtactcctccgcgggcga
gtacacgtgcgccggctaccccggctccctgggccgcgaggaggaggacg
cccagttcttcgcgaacaaccgcgtggactacctgaagtacgacaactgc
tacaacaagggccagttcggcacgcccgagatctcctaccaccgctacaa
ggccatgtccgacgccctgaacaagacgggccgccccatcttctactccc
tgtgcaactggggccaggacctgaccttctactggggctccggcatcgcg
aactcctggcgcatgtccggcgacgtcacggcggagttcacgcgccccga
ctcccgctgcccctgcgacggcgacgagtacgactgcaagtacgccggct
tccactgctccatcatgaacatcctgaacaaggccgcccccatgggccag
aacgcgggcgtcggcggctggaacgacctggacaacctggaggtcggcgt
cggcaacctgacggacgacgaggagaaggcgcacttctccatgtgggcca
tggtgaagtcccccctgatcatcggcgcgaacgtgaacaacctgaaggcc
tcctcctactccatctactcccaggcgtccgtcatcgccatcaaccagga
ctccaacggcatccccgccacgcgcgtctggcgctactacgtgtccgaca
cggacgagtacggccagggcgagatccagatgtggtccggccccctggac
aacggcgaccaggtcgtggcgctgctgaacggcggctccgtgtcccgccc
catgaacacgaccctggaggagatcttcttcgactccaacctgggctcca
agaagctgacctccacctgggacatctacgacctgtgggcgaaccgcgtc
gacaactccacggcgtccgccatcctgggccgcaacaagaccgccaccgg
catcctgtacaacgccaccgagcagtcctacaaggacggcctgtccaaga
acgacacccgcctgttcggccagaagatcggctccctgtcccccaacgcg
atcctgaacacgaccgtccccgcccacggcatcgcgttctaccgcctgcg
cccctcctcctgATACAACTTATTACGTATTCTGACCGGCGCTGATGTGG
CGCGGACGCCGTCGTACTCTTTCAGACTTTACTCTTGAGGAATTGAACCT
TTCTCGCTTGCTGGCATGTAAACATTGGCGCAATTAATTGTGTGATGAAG
AAAGGGTGGCACAAGATGGATCGCGAATGTACGAGATCGACAACGATGGT
GATTGTTATGAGGGGCCAAACCTGGCTCAATCTTGTCGCATGTCCGGCGC
AATGTGATCCAGCGGCGTGACTCTCGCAACCTGGTAGTGTGTGCGCACCG
GGTCGCTTTGATTAAAACTGATCGCATTGCCATCCCGTCAACTCACAAGC
CTACTCTAGCTCCCATTGCGCACTCGGGCGCCCGGCTCGATCAATGTTCT
GAGCGGAGGGCGAAGCGTCAGGAAATCGTCTCGGCAGCTGGAAGCGCATG
GAATGCGGAGCGGAGATCGAATCAGATATCAAGCTCCATCGAGCTCcagc
cacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgag
caagatctgcgggctgatcgccagcgacgagggccggcacgagatcgcct
acacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtc
gccgcctacgccaacatgatgcgcaagcagatcaccatgcccgcgcacct
catggacgacatgggccacggcgaggccaacccgggccgcaacctcttcg
ccgacttctccgcggtcgccgagaagatcgacgtctacgacgccgaggac
tactgccgcatcctggagcacctcaacgcgcgctggaaggtggacgagcg
ccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgc
cccagcgcttccggaaactcgccgagaagaccgccgccaagcgcaagcgc
gtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcat
ggtctagggagcgacgagtgtgcgtgcggggctggcgggagtgggacgcc
ctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctac
gcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtg
gctgccgggatatagatccggccgcacatcaaagggcccctccgccagag
aagaagctcctttcccagcagactcct
Nucleotide sequence of the C. hookeriana KASIV
CDS codon optimized for P. moriformis.
SEQ ID NO: 62
atggcttccgcggcattcaccatgtcggcgtgccccgcgatgactggcag
ggcccctggggcacgtcgctccggacggccagtcgccacccgcctgaggg
gcagcaccttccagtgcctggacccctgcaaccagcagcgcttcctgggc
gacaacggcttcgcgtcgctgttcggctccaagcccctgcgcagcaaccg
cggccacctgcgcctgggccgcacctcgcactccggcgaggtgatggccg
tcgcgatgcagcccgcccaggaggtgagcaccaacaagaagcccgcgacc
aagcagcgccgcgtggtcgtgaccggcatgggcgtcgtgacccccctggg
ccacgaccccgacgtgtattataacaacctgctggacggcatctcgggca
tctccgagatcgagaacttcgactgcagccagttccccacccgcatcgcc
ggcgagatcaagtcgttctccaccgacggctgggtcgcgcccaagttcag
cgagcgcatggacaagttcatgctgtatatgctgaccgccggcaagaagg
cgctggccgacggcggcatcaccgaggacgcgatgaaggagctgaacaag
cgcaagtgcggcgtgctgatcggctcgggcctgggcggcatgaaggtctt
ctccgacagcatcgaggccctgcgcacctcgtataagaagatctccccct
tctgcgtgcccttcagcaccaccaacatgggctcggcgatcctggcgatg
gacctgggctggatgggccccaactattccatcagcaccgcgtgcgccac
ctcgaacttctgcatcctgaacgcggccaaccacatcatcaagggcgagg
cggacatgatgctgtgcggcggctccgacgccgcggtgctgcccgtcggc
ctgggcggcttcgtggcctgccgcgcgctgagccagcgcaacaacgaccc
caccaaggcctcgcgcccctgggactccaaccgcgacggcttcgtcatgg
gcgagggcgcgggcgtgctgctgctggaggagctggagcacgccaagaag
cgcggcgcgaccatctatgccgagttcctgggcggcagcttcacctgcga
cgcgtatcacatgaccgagccccaccccgagggcgccggcgtcatcctgt
gcatcgagaaggcgctggcccagtcgggcgtgtcccgcgaggacgtgaac
tatatcaacgcgcacgccaccagcacccccgcgggcgacatcaaggagta
tcaggccctggcgcactgcttcggccagaactcggagctgcgcgtcaact
ccaccaagagcatgatcggccacctgctgggcggcgccggcggcgtggag
gcggtcgccgtggtccaggcgatccgcaccggctggatccaccccaacat
caacctggaggaccccgacgagggcgtggacgccaagctgctggtcggcc
ccaagaaggagaagctgaaggtgaaggtcggcctgtcgaactccttcggc
ttcggcggccacaacagctcgatcctgttcgcgccctgcaactga
Amino acid sequence of the C. aequipetala
KASIV. The algal transit peptide is
underlined.
C aeque KASIV
SEQ ID NO: 63
MAAAASMVASPLCTWLVAACMSTSFDNDPRSPSIKRIPRRRRILSQSSLR
GSTFQCLVTSYIDPCNQFSSSASLSFLGDNGFASLFGSKPFRSIRGHRRL
GRASHSGEAMAVALEPAQEVATKKKPVVKQRRVVVTGMGVVTPLGHEPDV
YYNNLLDGVSGISEIETFDCNQFPTRIAGEIKSFSTDGWVAPKLSKRMDK
FMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGLGGMKLFSDSIE
ALRISYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCI
LNSANHIVRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTKASR
PWDSNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMT
EPHPEGAGVILCIEKALAQAGVSREDVNYINAHATSTPAGDIKEYQALAH
CFGHNSELRVNSTKSMIGHLIGAAGGVEAVTVVQAIRTGWIHPNLNLEDP
DKAVDAKLLVGPKKERLNVKVGLSNSFGFGGHNSSILFAPYN
Amino acid sequence of the C. glassostoma KASIV.
The algal transit peptide is underlined.
S07_Cg_Locus_4548_Transcript_4/9_translation
SEQ ID NO: 64
MAAAASSQLCTWLVAACMSTSFDNNPRSPSIKRLPRRRRVLSHCSLRGST
FQCLVTSYIDPCNQYCSSASLSFLGDNGFTPLIGSKPFRSNRGHPRLGRA
SHSGEAMAVALQPAQEVATKKKPAMKQRRVVVTGMGVVTPLGHEPDVYYN
NLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDGWVAPKLSKRMDKFML
YLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGMGGMKLFNDSIEALR
VSYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCILNA
ANHIVRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTKASRPWD
SNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPH
PEGAGVILCIEKALAQAGVSREDVNYINAHATSTPAGDIKEYQALAHCFG
QNSELRVNSTKSMIGHLLGAAGGVEAVTVIQAIRTGWIHPNLNLDDPDKA
VDAKFLVGPKKERLNVKVGLSNSFGFGGHNSSILFAPYN
Amino acid sequence of the C. hookeriana KASIV.
The algal transit peptide is
underlined.
S26_ChookKASIV_trinity_43853 - translation
SEQ ID NO: 65
MAASSCMVGSPFCTWLVSACMSTSFDNDPRSLSHKRLRLSRRRRTLSSHC
SLRGSTPQCLDPCNQHCFLGDNGFASLFGSKPPRSDLGHLRLGRTSHSGE
VMAVAQEVSTNKKPATKQRRVVVTGMGVVTPLGHDPDVYYNNLLDGVSGI
SEIETFDCTQFPTRIAGEIKSFSTDGLVAPKLSKRMDKFMLYILTAGKKA
LADGGITEDVMKELDKRKCGVLIGSGLGGMKVFSDSVEALRISYKKISPF
CVPFSTTNMGSAILAMDLGWMGPNYSISTACATSNFCILNAANHITKGEA
DMMLCGGSDAAILPIGMGGFVACRALSQRNNDPTKASRPWDSNRDGFVMG
EGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILC
IEKALAQAGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSELRVNS
TKSMIGHLIGAAGGVEAVTVIQAIRTGWIHPNLNLENPDKAVDAKLLVGP
KKERLDVKVGLSNSFGFGGHNSSILFAPYN
Amino acid sequence of the C. glassostoma KASIV.
The algal transit peptide is underlined
S07_Cg_Locus_3059_Transcript_2/2_translation
SEQ ID NO: 66
MAAASSMVASSFSTSLVAACMSTSFDNDPRFLSHKRIRLSLRRGSTFQCL
GDNGFASLIGSKPPRSNHGHRRLGRTSHSGEAMAVAMQPAQEASTKNKHV
TKQRRVVVTGMGVVTPLGHDPDVYYNNLLDGVSGISEIENFDCSQFPTRI
AGEIKSFSTEGYVIPKFAKRMDKFMLYLLTAGKKALEDGGITEDVMKELD
KRKCGVLIGSGMGGMKIINDSIAALNVSYKKMTPFCVPFSTTNMGSAMLA
IDLGWMGPNYSISTACATSNYCILNAANHIIRGEANMMLCGGSDAVVIPV
GLGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAK
KRGATIYAEFLGGSFTCDAYHMTEPHPDGAGVILCIEKALAQSGVSREDV
NYINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGAAGGV
EAVSVVQAIRTGWIHPNINLEDPDEAVDAKLLVGPKKEKLKVKVGLSNSF
GFGGHNSSILFAPCN
Amino acid sequence of the C. carthagenesis KASIV.
The algal transit peptide is underlined
S05_CcrKASIV_17190_Seq_7/7_translation
SEQ ID NO: 67
MAAAAAFASPFCTWLVAACMSSASRHDPLPSPSSKPRLRRKILFQCAGRG
SSAGSGSSFHSLVTSYLGCLEPCHEYYTSSSSLGFSSLFGSTPGRTSRRQ
RRLHRASHSGEAMAVALQPAQEVTTKKKPSIKQRRVVVTGMGVVTPLGHD
PDVFYNNLLDGASGISEIETFDCAQFPTRIAGEIKSFSTDGWVAPKLSKR
MDKFMLYMLTAGKKALADGGISEDVMKELDKRKCGVLIGSAMGGMKVFND
AIEALRISYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSN
FCILNAANHITRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTK
ASRPWDSNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAY
HMTEPHPKGAGVILCIERALAQSGVSREDVNYINAHATSTPAGDIKEYQA
LAHCFGQNSELRVNSTKSMIGHLLGAAGGVEAVTVVQAIRTGWVHPNINL
ENPDEGVDAKLLVGPKKEKLKVKVGLSNSFGFGGHNSSILFAPYN
Amino acid sequence of the C. carthagenesis KASIV.
The algal transit peptide is underlined
S05_CcrKASIV_17190_Seq_6/7_translation
SEQ ID NO: 68
MAAAASVVASPFCTWLVAACMSASFDNEPRSLSPKRRRSLSRSSSASLRF
LGGNGFASLFGSDPLRPNRGHRRLRHASHSGEAMAVALQPAQEVSTKKKP
VTKQRRVVVTGMGVVTPLGHDPDVYYNNLLDGVSGISEIETFDCTQFPTR
IAGEIKSFSTDGWVAPKLSKRMDKFMLYMLTAGKKALADGGITEEVMKEL
DKRKCGVLIGSGMGGMKLFNDSIEALRISYKKMNPFCVPFATTNMGSAML
AMDLGWMGPNYSISTACATSNFCILNAANHITRGEADMMLCGGSDAVIIP
IGLGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHA
KKRGATIYAEFLGGSFTCDAYHMTEPHPKGAGVILCIERALAQSGVSRED
VNYINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGAAGG
VEAVTVVQAIRTGWVHPNINLENPDEGVDAKLLVGPKKEKLKVKVGLSNS
FGFGGHNSSILFAPYN
Amino acid sequence of the C. pulcherrima KASIV.
The algal transit peptide is underlined
pSZ2181 - CpulcKASIV
SEQ ID NO: 69
MPAASSLLASPLCTWLLAACMSTSFHPSDPLPPSISSPRRRLSRRRILSQ
CAPLPSASSALRGSSFHTLVTSYLACFEPCHDYYTSASLFGSRPIRTTRR
HRRLNRASPSREAMAVALQPEQEVTTKKKPSIKQRRVVVTGMGVVTPLGH
DPDVFYNNLLDGTSGISEIETFDCAQFPTRIAGEIKSFSTDGWVAPKLSK
RMDKFMLYMLTAGKKALTDGGITEDVMKELDKRKCGVLIGSAMGGMKVFN
DAIEALRISYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATS
NFCIMNAANHIIRGEADVMLCGGSDAVIIPIGMGGFVACRALSQRNSDPT
KASRPWDSNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDA
YHMTEPHPDGAGVILCIEKALAQSGVSREDVNYINAHATSTPAGDIKEYQ
ALIHCFGQNRELKVNSTKSMIGHLLGAAGGVEAVSVVQAIRTGWIHPNLN
LENPDEGVDTKLLVGPKKERLNVKVGLSNSFGFGGHNSSILFAPYI
Clade 1 KASIV consensus C8 and C10
SEQ ID NO: 70
MAAASCMVASPFCTWLVAACMSTSXDNDPRSLSHKRLRLSRRRRTLSSHC
SLRGSTFQCLDPCNQHCFLGDNGFASLFGSKPPRSNRGHLRLGRTSHSGE
VMAVAXQXAQEVSTNKKPATKQRRVVVTGMGVVTPLGHDPDVYYNNLLDG
VSGISEIENFDCSQFPTRIAGEIKSFSTDGWVAPKLSKRMDKFMLYILTA
GKKALADGGITEDVMKELDKRKCGVLIGSGLGGMKVFSDSIEALRTSYKK
ISPFCVPFSTTNMGSAILAMDLGWMGPNYSISTACATSNFCILNAANHIT
KGEADMMLCGGSDAAILPIGMGGFVACRALSQRNNDPTKASRPWDSNRDG
FVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMTEPHPEGAG
VILCIEKALAQSGVSREDVNYINAHATSTPAGDIKEYQALAHCFGQNSEL
RVNSTKSMIGHLLGGAGGVEAVTVVQAIRTGWIHPNINLEDPDEGVDAKL
LVGPKKEKLKVKVGLSNSFGFGGHNSSILFAPCN
Clade 2 KASIV consensus C10 only
SEQ ID NO: 71
MAAAASMXXSPLCTWLVAACMSTSFDNDPRSPSIKRLPRRRRVLSQCSLR
GSTFQCLVTSYIDPCNQYCSSASLSFLGDNGFASLFGSKPFRSNRGHRRL
GRASHSGEAMAVALQPAQEVATKKKPVIKQRRVVVTGMGVVTPLGHEPDV
YYNNLLDGVSGISEIETFDCTQFPTRIAGEIKSFSTDGWVAPKLSKRMDK
FMLYLLTAGKKALADGGITDDVMKELDKRKCGVLIGSGMGGMKLFNDSIE
ALRXSYKKMNPFCVPFATTNMGSAMLAMDLGWMGPNYSISTACATSNFCI
LNAANHIVRGEADMMLCGGSDAVIIPIGLGGFVACRALSQRNNDPTKASR
PWDSNRDGFVMGEGAGVLLLEELEHAKKRGATIYAEFLGGSFTCDAYHMT
EPHPEGAGVILCIEKALAQAGVSREDVNYINAHATSTPAGDIKEYQALAH
CFGQNSELRVNSTKSMIGHLLGAAGGVEAVTVXQAIRTGWIHPNLNLEDP
DKAVDAKLLVGPKKERLNVKVGLSNSFGFGGHNSSILFAPYNV
Clade 1 KASIV consensus mature protein
SEQ ID NO: 72
KQRRVVVTGMGVVTPLGHDPDVYYNNLLDGVSGISEIENFDCSQFPTRIA
GEIKSFSTDGWVAPKLSKRMDKFMLYILTAGKKALADGGITEDVMKELDK
RKCGVLIGSGLGGMKVFSDSIEALRTSYKKISPFCVPFSTTNMGSAILAM
DLGWMGPNYSISTACATSNFCILNAANHITKGEADMMLCGGSDAAILPIG
MGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAKK
RGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIEKALAQSGVSREDVN
YINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGGAGGVE
AVTVVQAIRTGWIHPNINLEDPDEGVDAKLLVGPKKEKLKVKVGLSNSFG
FGGHNSSILFAPCN
Clade 2 KASIV consensus mature protein
KQRRVVVTGMGVVTPLGHEPDVYYNNLLDGVSGISEIETFDCTQFPTRIA
GEIKSFSTDGWVAPKLSKRMDKFMLYLLTAGKKALADGGITDDVMKELDK
RKCGVLIGSGMGGMKLFNDSIEALRXSYKKMNPFCVPFATTNMGSAMLAM
DLGWMGPNYSISTACATSNFCILNAANHIVRGEADMMLCGGSDAVIIPIG
LGGFVACRALSQRNNDPTKASRPWDSNRDGFVMGEGAGVLLLEELEHAKK
RGATIYAEFLGGSFTCDAYHMTEPHPEGAGVILCIEKALAQAGVSREDVN
YINAHATSTPAGDIKEYQALAHCFGQNSELRVNSTKSMIGHLLGAAGGVE
AVTVXQAIRTGWIHPNLNLEDPDKAVDAKLLVGPKKERLNVKVGLSNSFG
FGGHNSSILFAPYNV
