EP2971024A1 - Thioestérases et cellules pour la production d'huiles à façon - Google Patents

Thioestérases et cellules pour la production d'huiles à façon

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Publication number
EP2971024A1
EP2971024A1 EP14769502.7A EP14769502A EP2971024A1 EP 2971024 A1 EP2971024 A1 EP 2971024A1 EP 14769502 A EP14769502 A EP 14769502A EP 2971024 A1 EP2971024 A1 EP 2971024A1
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
oil
amino acid
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14769502.7A
Other languages
German (de)
English (en)
Other versions
EP2971024A4 (fr
Inventor
George N. RUDENKO
Jason Casolari
Scott Franklin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Corbion Biotech Inc
Original Assignee
Solazyme Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US13/837,996 external-priority patent/US9290749B2/en
Application filed by Solazyme Inc filed Critical Solazyme Inc
Publication of EP2971024A1 publication Critical patent/EP2971024A1/fr
Publication of EP2971024A4 publication Critical patent/EP2971024A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02014—Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Type II fatty acid biosynthesis typically involves extension of a growing acyl-ACP (acyl-carrier protein) chain by two carbon units followed by cleavage by an acyl-ACP thioesterase.
  • acyl-ACP acyl-carrier protein
  • acyl-ACP thioesterases In plants, two main classes of acyl-ACP thioesterases have been identified: (i) those encoded by genes of the FatA class, which tend to hydro lyze oleoyl-ACP into oleate (an 18: 1 fatty acid) and ACP, and (ii) those encoded by genes of the FatB class, which liberate C8-C16 fatty acids from corresponding acyl-ACP molecules.
  • invention(s) contemplated herein may include, but need not be limited to, any one or more of the following embodiments:
  • Embodiment 1 A nucleic acid construct including a regulatory element and a FatB gene expressing an active acyl-ACP thioesterase operable to produce an altered fatty acid profile in an oil produced by a cell expressing the nucleic acid construct, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 5 of Table la, the sequence having at least 94.6% sequence identity with each of SEQ ID NOs: 88, 82, 85, and 103, and optionally wherein the fatty acid of the oil is enriched in C8 and CIO fatty acids.
  • Embodiment 2 A nucleic acid construct including a regulatory element and a FatB gene expressing an active acyl-ACP thioesterase operable to produce an altered fatty acid profile in an oil produced by a cell expressing the nucleic acid construct, wherein the FatB gene expresses a protein having an amino acid sequence falling within one of clades 1-12 of Table la.
  • Embodiment 3 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade
  • Embodiment 4 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade
  • Embodiment 5 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade
  • Embodiment 6 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 79, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
  • Embodiment 7 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade
  • Embodiment 8 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade
  • Embodiment 9 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade
  • Embodiment 10 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 9 of Table la, the sequence having at least 83.8% sequence identity with each of SEQ ID NOs: 187-189, and optionally wherein the fatty acid of the oil is enriched in C12 and C14 fatty acids.
  • Embodiment 11 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 10 of Table la, the sequence having at least 95.9% sequence identity with each of SEQ ID NOs: 147, 149, 146, 150, 152, 151, 148, 154, 156, 155, 157, 108, 75, 190, 191, and 192, and optionally wherein the fatty acid of the oil is enriched in C14 and C16 fatty acids.
  • Embodiment 12 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 11 of Table la, the sequence having at least 88.7% sequence identity with SEQ ID NO: 121, and optionally wherein the fatty acid of the oil is enriched in C14 and C16 fatty acids.
  • Embodiment 13 The nucleic acid construct of embodiment 2, wherein the FatB gene expresses a protein having an amino acid sequence falling within clade 12 of Table la, the sequence having at least 72.8% sequence identity with each of SEQ ID NOs: 129 and 186, and optionally wherein the fatty acid of the oil is enriched in C16 fatty acids.
  • Embodiment 14 An isolated nucleic acid or recombinant DNA construct including a nucleic acid, wherein the nucleic acid has at least 80%> sequence identity to any of SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107, 109 or any equivalent sequences by virtue of the degeneracy of the genetic code.
  • Embodiment 15 An isolated nucleic acid sequence encoding a protein or a host cell expressing a protein having at least 80% sequence identity to any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, 77, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 108, 110-192 or a fragment thereof having acyl-ACP thioesterase activity.
  • Embodiment 16 The isolated nucleic acid of embodiment 15, wherein, the protein has acyl-ACP thioesterase activity operable to alter the fatty acid profile of an oil produced by a recombinant cell including that sequence.
  • Embodiment 17 A method of producing a recombinant cell that produces an altered fatty acid profile, the method including transforming the cell with a nucleic acid according to any of embodiments 1-3.
  • Embodiment 18 A host cell produced by the method of embodiment
  • Embodiment 19 The host cell of embodiment 18, wherein the host cell is selected from a plant cell, a microbial cell, and a microalgal cell.
  • Embodiment 20 A method for producing an oil or oil-derived product, the method including cultivating a host cell of embodiment 5 or 6, and extracting oil produced thereby, optionally wherein the cultivation is heterotrophic growth on sugar.
  • Embodiment 21 The method of embodiment 20, further including producing a fatty acid, fuel, chemical, or other oil-derived product from the oil.
  • Embodiment 22 An oil produced by the method of embodiment 20, optionally having a fatty acid profile including at least 20% C8, CIO, C12, C14 or C16 fatty acids.
  • Embodiment 23 An oil-derived product produced by the method of embodiment 21.
  • Embodiment 24 The oil of embodiment 23, wherein the oil is produced by a microalgae and optionally, lacks C24-alpha sterols.
  • 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 "natural oil” or “natural 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.
  • the natural oil or natural 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.
  • the terms oil and fat are used interchangeably, except where otherwise noted.
  • 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.
  • 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.
  • natural oil and natural 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 natural oil can also be a "noninteresterified natural oil", which means that the natural 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 R A 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
  • 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.
  • sequence comparison to determine percent nucleotide or amino acid identity
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm 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.
  • BLAST 2 Sequences Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50;
  • a “profile” is the distribution of particular species or triglycerides or fatty acyl groups within the oil.
  • a “fatty acid profile” is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone.
  • Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID).
  • FAME fatty acid methyl ester
  • FAME gas chromatography
  • FID flame ionization detection
  • 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.
  • 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.
  • 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.
  • 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.
  • RNAi interfering RNA
  • 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.
  • 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.
  • a recombinant nucleic acid 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.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
  • Embodiments of the present invention relate to the use of FatB genes isolated from plants, which can be expressed in a host cell in order to alter the fatty acid profile of an oil produced by the recombinant cell.
  • the microalga Prototheca moriformis
  • the genes are useful in a wide variety of host cells.
  • the genes can be expressed in bacteria, other microalgae, or higher plants.
  • the genes can be expressed in higher plants according to the methods of US Patent Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495;
  • the fatty acids can be further converted to triglycerides, fatty aldehydes, fatty alcohols and other oleochemicals either synthetically or biosynthetically.
  • triglycerides are produced by a host cell expressing a novel FatB gene.
  • a triglyceride-containing natural oil can be recovered from the host cell.
  • the natural oil can be refined, degummed, bleached and/or deodorized.
  • the oil in its natural or processed form, can be used for foods, chemicals, fuels, cosmetics, plastics, and other uses.
  • the FatB gene may not be novel, but the expression of the gene in a microalga is novel.
  • the genes can be used in a variety of genetic constructs including plasmids or other vectors for expression or recombination in a host cell.
  • the genes can be codon optimized for expression in a target host cell.
  • the proteins produced by the genes can be used in vivo or in purified form.
  • the gene can be prepared in an expression vector comprising an operably linked promoter and 5 'UTR.
  • a suitably active plastid targeting peptide can be fused to the FATB gene, as in the examples below.
  • this transit peptide is replaced with a 38 amino acid sequence that is effective in the Prototheca moriformis host cell for transporting the enzyme to the plastids of those cells.
  • the invention contemplates deletions and fusion proteins in order to optimize enzyme activity in a given host cell.
  • a transit peptide from the host or related species may be used instead of that of the newly discovered plant genes described here.
  • a selectable marker gene may be included in the vector to assist in isolating a transformed cell.
  • selectable markers useful in microlagae include sucrose invertase and antibiotic resistance genes.
  • the gene sequences disclosed can also be used to prepare antisense, or inhibitory R A (e.g., RNAi or hairpin R A) to inhibit complementary genes in a plant or other organism.
  • inhibitory R A e.g., RNAi or hairpin R A
  • FatB genes found to be useful in producing desired fatty acid profiles in a cell are summarized below in Table 1. Nucleic acids or proteins having the sequence of SEQ ID NOS: 1-109 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: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107 or 109.
  • 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 3a.
  • Codon usage for Arabidopsis is shown in Table 3b; for example, the most preferred of codon for each amino acid can be selected. Codon tables for other organisms including microalgae and higher plants are known in the art.
  • the first and/or second most preferred Prototheca codons are employed for codon optimization.
  • 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.
  • protein or a nucleic acid encoding a protein having any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, 77, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 108, or 110-192.
  • the invention encompasses a fragment any of the above-described proteins or nucleic acids
  • the fragment includes a domain of an acyl-ACP thioesterase that mediates a particular function, e.g., a specificity-determining domain.
  • Illustrative fragments can be produced by C-terminal and/or N-terminal truncations and include at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%), 98%o, or 99%> of the full-length sequences disclosed herein.
  • 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: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105, 107 or 109) or full-length amino acid sequence (e.g., one of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64
  • the nucleic acids can be in isolated form, or part of a vector or other construct, chromosome or host cell. It has been found that is many cases the full length gene (and protein) is not needed; for example, deletion of some or all of the N- terminal hydrophobic domain (typically an 18 amino acid domain starting with LPDW) yields a still-functional gene. In addition, fusions of the specificity determining regions of the genes in Table 1 with catalytic domains of other acyl-ACP thioesterases can yield functional genes.
  • the invention encompasses functional fragments (e.g., specificity determining regions) of the disclosed nucleic acid or amino acids fused to heterologous acyl-ACP thioesterase nucleic acid or amino acid sequences, respectively.
  • Cuphea CvisFATB 1 published SEQ ID N/A SEQ ID viscosissima NO: 73 NO: 74
  • Cuphea CvisFATB2 published SEQ ID N/A SEQ ID viscosissima NO: 75 NO: 76
  • Cuphea CvisFATB3 published SEQ ID N/A SEQ ID viscosissima NO: 77 NO: 78
  • Consensus JcFATB2 Consensus SEQ ID None, SEQ ID sequence NO: 108 can be NO: 109 codon
  • a host cell e.g. plant or microalgal cell
  • a recombinant FATB protein falling into one of clades 1-12 of Table la.
  • These clades were determined by sequence alignment and observation of changes in fatty acid profile when expressed in Prototheca. See Example 5.
  • the FATB amino acid sequence can fall within x% amino acid sequence identity of each sequence in that clade listed in Table la, where x is a first second or third cutoff value, also listed in Table la.
  • Table la Groupings of Novel FatB genes into clades.
  • CwFATB3 (SEQ ID NO: 112) Increase C12/C 14 85.9 98.9 99.5 CwFATB3a (SEQ ID NO: 113) fatty acids
  • ChtFATB2e (SEQ ID NO: 142)
  • ChtFATB2h (SEQ ID NO: 145)
  • ChtFATB2f (SEQ ID NO: 143)
  • ChtFATB2g (SEQ ID NO: 144)
  • ChtFATB2a (SEQ ID NO: 139)
  • ChtFATB2c (SEQ ID NO: 140)
  • ChtFATB2b (SEQ ID NO: 138)
  • ChtFATB2d (SEQ ID NO: 141)
  • CcrFATB2c (SEQ ID NO: 187) Increase C12/C 14 83.8 90 95 CcrFATB2 (SEQ ID NO: 188) fatty acids
  • ChtFATB3b (SEQ ID NO: 147) Increase C14/C16 95.9 98 99 ChtFATB3d (SEQ ID NO: 149)
  • ChtFATB3a (SEQ ID NO: 146)
  • ChtFATB3e (SEQ ID NO: 150)
  • ChtFATB3g (SEQ ID NO: 152)
  • ChtFATB3f (SEQ ID NO: 151)
  • ChtFATB3c (SEQ ID NO: 148)
  • ChsFATB2 (SEQ ID NO: 154)
  • ChsFATB2c (SEQ ID NO: 156)
  • ChsFATB2b (SEQ ID NO: 155)
  • ChsFATB2d (SEQ ID NO: 157)
  • JcFATB2/SzFATB2 (SEQ ID NO: 108)
  • CcFATB3 (SEQ ID NO: 129) Increase C16 fatty 72.8 85 90 UcFATB3 (SEQ ID NO: 186) acids
  • GCA 66 (0.07) AAC 201 (0.96) GCT 101(0.11)
  • TGC 105 (0.90) CCC 267 (0.49) Asp GAT 43(0.12) Gin CAG 226(0.82)
  • CAC 154 (0.79) TCC 173 (0.31) lie ATA 4 (0.01) ACG 184 (0.38)
  • GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val)
  • GUC V 0.2115.0 ( 40) GCC A 0.2018.0 ( 48) GAC D 0.3721.0 ( 56) GGC G 0.20 18.0 ( 48) GUAV0.1410.1 ( 27) GCA A 0.3329.6 ( 79) GAA E 0.4118.3 ( 49) GGAG0.35 31.4 ( 84)
  • GUAV0.15 9.9 (308605) GCAA0.2717.5 (543180) GAA E 0.5234.3 (1068012) GGA G 0.3724.2 (751489) GUG V 0.2617.4 (539873) GCG A 0.14 9.0 (280804) GAG E 0.4832.2 (1002594) GGG G0.1610.2 (316620) 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 Fatb genes in a plant are given in 5,850,022; 5,723,761; 5,639,790; 5,807,893;
  • 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.
  • microalgal cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae.
  • heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae.
  • oleaginous microalgae are provided in Published PCT Patent Applications
  • WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/150411 including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs.
  • the oleaginous cells can be, for example, capable of producing 25, 30, 40, 50, 60, 70, 80, 85, or about 90% oil by cell weight, ⁇ 5%.
  • the oils produced can be low in DHA or EPA fatty acids.
  • 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.
  • microalgal cells 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).
  • a sugar e.g., glucose, fructose and/or sucrose.
  • the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock.
  • the cells can metabolize xylose from cellulosic feedstocks.
  • 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.
  • 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
  • the oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes.
  • some embodiments feature natural 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 oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939.
  • the raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, as animal feed, for human nutrition, or for fertilizer.
  • a fatty acid profile of a triglyceride also referred to as a
  • triacylglyceride or "TAG" cell oil
  • TAG triacylglyceride
  • TAG cell oil
  • 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 513C is an expression of the ratio of
  • the stable carbon isotope value 513C (0/00) of the oils can be related to the 513C value of the feedstock used.
  • the oils are derived from oleaginous organisms
  • the 513C (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.
  • the microalga can be, without limitation, fall 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 was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and ⁇ -sitosterol, when detected by GC-MS.
  • all sterols produced by Chlorella have C24P stereochemistry.
  • the molecules detected as campesterol, stigmasterol, and ⁇ -sitosterol are actually 22,23- dihydrobrassicasterol, proferasterol and clionasterol, respectively.
  • the oils produced by the microalgae described above can be distinguished from plant oils by the presence of sterols with C24 stereochemistry and the absence of C24a stereochemistry in the sterols present.
  • the oils produced may contain 22, 23 -dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in ⁇ -sitosterol, and/or contain poriferasterol while lacking stigmasterol.
  • oils may contain significant amounts of ⁇ 7 - poriferasterol.
  • 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 include pigments (e.g. chlorophyll), dl3C values and sensory analysis (e.g. taste, odor, and mouth feel).
  • pigments e.g. chlorophyll
  • dl3C values e.g. dl3C values
  • sensory analysis e.g. taste, odor, and mouth feel.
  • Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter.
  • Campesterol, b-sitosterol, and stigamsterol are common plant sterols, with b-sitosterol being a principle plant sterol.
  • b-sitosterol was found to be in greatest abundance in an analysis of certain seed oils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74% in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Cell and Molecular Biology 5:71-79, 2006).
  • 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.
  • ergosterol was found to be the most abundant of all the sterols, accounting for about 50% or more of the total sterols. The amount of ergosterol is greater than that of campesterol, ⁇ -sitosterol, and stigmasterol combined. Ergosterol is steroid commonly found in fungus and not commonly found in plants, and its presence particularly in significant amounts serves as a useful marker for non-plant oils. Secondly, the oil was found to contain brassicasterol. With the exception of rapeseed oil, brassicasterol is not commonly found in plant based oils. Thirdly, less than 2% ⁇ -sitosterol was found to be present.
  • ⁇ -sitosterol is a prominent plant sterol not commonly found in microalgae, and its presence particularly in significant amounts serves as a useful marker for oils of plant origin.
  • Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of ⁇ -sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol : ⁇ - sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.
  • the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% ⁇ - sitosterol. In other embodiments the oil is free from ⁇ -sitosterol.
  • the oil is free from one or more of ⁇ -sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from ⁇ -sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.
  • 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.
  • the 24-ethylcholest-5-en-3-ol is clionasterol.
  • 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.
  • 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.
  • the 24-methylcholest-5-en-3-ol is 22, 23-dihydrobrassicasterol.
  • 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.
  • 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.
  • the 5, 22-cholestadien-24-ethyl-3- ol is poriferasterol.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ratio of ergosterol to brassicasterol is at least 5: 1, 10: 1, 15: 1, or 20: 1.
  • the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% ⁇ -sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% ⁇ -sitosterol. In some embodiments, the oil content further comprises brassicasterol. [0070] Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes.
  • C27 sterols Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols.
  • the sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols b-sitosterol and stigmasterol.
  • 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 primary sterols in the microalgal oils provided herein are sterols other than b-sitosterol and stigmasterol.
  • C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.
  • 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.
  • 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, CIO, C12, C14 or C16 fatty acids.
  • the level of myristate (C14:0) in the oil is greater than 30%.
  • 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, etc).
  • the oils, triglycerides, fatty acids from the oils may be subjected to C-H activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic
  • 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.
  • a residual biomass from heterotrophic algae can be used in such products.
  • Seeds of oleaginous plants were obtained from local grocery stores or requested through USDA ARS National Plant Germplasm System (NPGS) from North Central Regional Plant Introduction Station (NCRIS) or USDA ARS North Central Soil Conservation Research Laboratory (Morris, MI). Dry seeds were homogenized in liquid nitrogen to powder, resuspended in cold extraction buffer containing 6-8M Urea and 3M LiCl and left on ice for a few hours to overnight at 4 °C. The seed homogenate was passed through NucleoSpin Filters (Macherey-Nagel) by centrifugation at 20,000g for 20 minutes in the refrigerated microcentrifuge (4 °C).
  • RNA pellets were resuspended in the buffer containing 20 mM Tris HC1, pH7.5, 0.5% SDS, 100 mM NaCl, 25 mM EDTA, 2% PVPP) and RNA was subsequently extracted once with Phenol-Chloroform-Isoamyl Alcohol (25:24: 1, v/v) and once with chloroform. RNA was finally precipitated with isopropyl alcohol (0.7 Vol.) in the presence of 150 mM of Na Acetate, pH5.2, washed with 80% ethanol by centrifugation, and dried. RNA samples were treated with Turbo DNAse (Lifetech) and purified further using RNeasy kits (Qiagen) following manufacturers' protocols. The resulting purified RNA samples were converted to pair-end cDNA libraries and subjected to next-generation sequencing (2xl00bp) using Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed
  • transcriptomes using Trinity or Oases packages. Putative thioesterase-containg cDNA contigs were identified by mining transcriptomes for sequences with homology to known thioesterases. These in silico identified putative thioesterase cDNAs have been further verified by direct reverse transcription PCR analysis using seed RNA and primer pairs targeting full-length thioesterase cDNAs. The resulting amplified products were cloned and sequenced de novo to confirm authenticity of identified thioesterase genes.
  • ChtFATB3a SEQ ID NO: 146
  • ChtFATB3f SEQ ID NO: 151
  • ChtFATB3g SEQ ID NO: 152
  • Example 1 R A was extracted from dried plant seeds and submitted for paired-end sequencing using the Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using Trinity or Oases packages and putative thioesterase-containing cDNA contigs were identified by mining transcriptomes for sequences with homology to known thioesterases.
  • Cinnamomum camphora, Cuphea hyssopifolia, Cuphea PSR23, Cuphea wrightii, Cuphea heterophylla, and Cuphea viscosissima were synthesized in a codon- optimized form to reflect Prototheca moriformis (UTEX 1435) codon usage.
  • 27 genes synthesized 24 were identified by our transcriptome sequencing efforts and the 3 genes from Cuphea viscosissima, were from published sequences in GenBank.
  • Strain A Prototheca moriformis, derived from UTEX 1435 by classical mutation and screening for high oil production
  • the construct pSZ2760 encoding Cinnamomum camphora (Cc) FATBlb is shown as an example, but identical methods were used to generate each of the remaining 26 constructs encoding the different respective thioesterases.
  • Construct pSZ2760 can be written as
  • Bold, lowercase sequences at the 5 ' and 3 ' end of the construct represent genomic DNA from UTEX 1435 that target integration to the 6S locus via homologous recombination. Proceeding in the 5 ' to 3 ' direction, the selection cassette has the C reinhardtii ⁇ -tubulin promoter driving expression of the S. cerevisiae gene SUC2 (conferring the ability to grow on sucrose) and the Chlorella vulgaris Nitrate Reductase (NR) gene 3 ' UTR.
  • the promoter is indicated by lowercase, boxed text.
  • the initiator ATG and terminator TGA for ScSUC2 are indicated by bold, uppercase italics, while the coding region is indicated with lowercase italics.
  • the 3 ' UTR is indicated by lowercase underlined text.
  • the spacer region between the two cassettes is indicated by upper case text.
  • Cinnamomum camphora is driven by the Prototheca moriformis endogenous AMT3 promoter, and has the Chlorella vulgaris Nitrate Reductase (NR) gene 3 ' UTR.
  • the AMT3 promoter is indicated by lowercase, boxed text.
  • the initiator ATG and terminator TGA for the CcFATBlb gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics and the spacer region is indicated by upper case text.
  • the 3 ' UTR is indicated by lowercase underlined text. The final construct was sequenced to ensure correct reading frame and targeting sequences.
  • CcFATBlb from pSZ2760 in Table 6 were transformed into Strain A, and selected for the ability to grow on sucrose. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described. After cultivating on sucrose under low nitrogen conditions to accumulate oil, fatty acid profiles were determined by FAME-GC. The top performer from each transformation, as judged by the ability to produce the highest level of midchain fatty acids, is shown in Table 4.
  • CcFATBlb causes an increase in myristate levels from 2% of total fatty acids in the parent, Strain A, to -15% in the D 1670- 13 primary trans formant.
  • Other examples include CcFATB4, which exhibits an increase in laurate levels from 0% in Strain A to -33%, and ChsFATB3, which exhibits an increase in myristate levels to -34%.
  • constructs such as the deduced amino acid sequence of the encoded acyl-ACP thioesterase, the native CDS coding sequence, the Prototheca moriformis codon- optimized coding sequence, and the nature of the sequence variants examined, is provided as SEQ ID NOS: 1-78.
  • the nine putative Acyl-ACP FatB Thioesterases from the species Cuphea calcarata, Cuphea painter, Cuphea hookeriana, Cuphea avigera var.
  • pulcherrima Cuphea paucipetala, Cuphea procumbens, and Cuphea ignea were synthesized in a codon-optimized form to reflect UTEX 1435 codon usage.
  • the new Acyl-ACP FatB thioesterases were synthesized with a modified transit peptide from Chlorella protothecoides (Cp) in place of the native transit peptide.
  • the modified transit peptide derived from the CpSADl gene, "CpSADltp_trimmed” was synthesized as an in- frame, N-terminal fusion to the FatB acyl-ACP thioesterases in place of the native transit peptide; the resulting sequences are listed below.
  • the novel FatB genes were cloned into Prototheca moriformis as described above. Constructs encoding heterologous FatB genes were transformed into strain S6165 (a descendant of S3150/Strain A) and selected for the ability to grow on sucrose. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described. The results for the nine novel FatB acyl-ACP thioesterases are displayed in the table immediately below.
  • CigneaFATBl which exhibits 8% C10:0 and 1% C12:0 fatty acid levels
  • CcalcFATBl which exhibits 18% C14:0 and 12% C12:0 levels
  • CaFATBl which exhibits 22% C8:0 and 9% C10:0 fatty acid levels.
  • CaFATBl which exhibits high C8:0 and C10:0 levels, is of particular interest.
  • CaFATBl arose from two separate contigs that were assembled from the Cupha avigera var. pulcherrima transcriptome, S17_Cavig_trinity_7406 and
  • CpuFATB3 The coding sequence of CpuFATB3 is 100% identical to the CaFATBl gene we identified and contains one nucleotide difference in the RNA sequence outside the predicted coding region. Tjellstrom et al. (2013) showed that CpuFATB3 produces an average of 4.8%> C8:0 when expressed in Arabidopsis, and further requires deletion of two acyl-ACP synthetases, AAE15/16, to produce an average of 9.2% C8:0 with a maximum level of -12% C8.0.
  • the CaFATBl gene we identified was codon-optimized for expression in UTEX1435 and generated as a CpSADltp-trimmed transit peptide fusion before introduction into S6165.
  • the CpSADltp_trimmed:CaFATBl gene produces an average C8:0 level of 14% and a maximum level of 22% C8:0 without requiring the deletion of endogenous acyl-ACP synthetases. [0093] Table 7. Amino Acid Sequences of Additional Novel FatB Acyl-ACP
  • CprocFATBl (Cuphea procumbens FATB1) SEO ID NO: 172
  • CprocFATB2 (Cuphea procumbens FATB2) SEO ID NO: 173
  • CprocFATB3 (Cuphea procumbens FATB3) SEO ID NO: 174
  • ChookFATB4 (Cuphea hookeriana FATB4) SEO ID NO: 177
  • CaFATBl (Cuphea avigera var. pulcherrima FATB1) SEO ID NO: 178
  • CpauFATBl (Cuphea paucipetala FATB1) SEO ID NO: 179
  • CprocFATBl (Cuphea procumbens FATB1) SEO ID NO: 180
  • CprocFATB2 (Cuphea procumbens FATB2) SEO ID NO: 181
  • CprocFATB3 (Cuphea procumbens FATB3) SEO ID NO: 182
  • thioesterases in UTEX1435, S3150 we identified several thioesterases with increased CI 0:0 and CI 6:0 activity above the background midchain levels found in the strain. We reasoned that a consensus sequence could be obtained for an idealized C10:0 thioesterase and C16:0 thioesterase from aligning the best- performing C10:0 and C16:0 thioesterases.
  • a consensus C10:0 specific thioesterase sequence was generated using the C palustris FatBl (CpFATBl), C. PSR23 FatB3 (CuPSR23FATB3), C viscosissima FatB 1 (CvisFATB 1 ), C glossostoma FatB 1
  • CgFATBl C. carthagenensis FatB2
  • CcrFATB2 C. carthagenensis FatB2 sequences as inputs resulting in a C10:0 specific consensus sequence termed JcFATBl/SzFATBl .
  • a consensus CI 6:0 specific thioesterase sequence was generated using the C. heterophylla FatB3a (ChtFATB3a), C carthagenensis FatBl (CcrFATBl), C viscosissima FatB2
  • CvisFATB2 C hookeriana FatBl (ChFATBl; AAC48990), C hyssopifolia FatB2 (ChsFATB2), C calophylla FatB2 (CcalFATB2; ABB71581), C hookeriana FatBl-1 (ChFATBl-1; AAC72882), C lanceolata FatBl (C1FATB1; CAC 19933), and C. wrightii FatB4a (CwFATB4a) sequences as inputs resulting in a CI 6:0 specific consensus sequence termed JcFATB2/SzFATB2.
  • the resulting consensus sequences were synthesized, cloned into a vector identical to that used to test other FatB thioesterases, and introduced into S3150 as described above.
  • the consensus amino acid sequences are given as SEQ ID NOs. 106 and 107; the nucleic acid sequences were based on these amino acid sequences using codon optimization for Prototheca moriformis.
  • the trans formants were selected, cultivated and the oil was extracted and analyzed by FAME-GC-FID. The fatty acid profiles obtained are given in the table below.
  • Cinnamomum camphora (Cc) FATBlb variant M25L, M322R, AT367-D368 amino acid sequence MATTSLASAFCSMKAVMLARDGRGLKPRSSDLQLRAGNAQTSLKMINGTKFSYTESLKKLPD WSMLFAVITTIFSAAEKQWTNLEWKPKPNPPQLLDDHFGPHGLVFRRTFAIRSYEVGPDRSTSI VAVMNHLQEAALNHAKSVGILGDGFGTTLEMSKRDLIWVVKRTHVAVERYPAWGDTVEVE CWVGASGNNGRRHDFLVRDCKTGEILTRCTSLSVMMNTRTRRLSKIPEEVRGEIGPAFIDNVA VKDEEIKKPQKLNDSTADYIQGGLTPRWNDLDINQHVNNIKYVDWILETVPDSIFESHHISSFTI EYRRECTRDSVLQSLTTVSGGSSEAGLVCEHLLQLEGGSEVLRAKTEWRPKLSFRGISVIPAES
  • Cinnamomum camphora (Cc) FATBlb variant M25L, M322R, AT367-D368 coding DNA sequence
  • Cinnamomum camphora (Cc) FATBlb variant M25L, M322R, AT367-D368 coding DNA sequence codon optimized for Prototheca moriformis
  • Cinnamomum camphora (Cc) FATB4 amino acid sequence
  • Cinnamomum camphora (Cc) FATB3 amino acid sequence
  • Cuphea hyssopifolia (Chs) FATB1 amino acid sequence
  • Cuphea hyssopifolia (Chs) FATB1 coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea hyssopifolia (Chs) FATB2 amino acid sequence
  • Cuphea hyssopifolia (Chs) FATB2b +a.a.248-259 variant amino acid sequence
  • Cuphea hyssopifolia (Chs) FATB2b+a.a.248-259 variant coding DNA sequence
  • Cuphea hyssopifolia (Chs) FATB3 amino acid sequence
  • Cuphea hyssopifolia (Chs) FATB3 coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea hyssopifolia (Chs) FATB3b (V204I,C239F, E243D, M25 IV variant) amino acid sequence
  • Cuphea hyssopifolia (Chs) FATB3b (V204I,C239F, E243D, M25 IV variant) coding DNA sequence
  • Cuphea hyssopifolia (Chs) FATB3b (V204I,C239F, E243D, M25 IV variant) coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea heterophylla (Cht) FATBlb (P16S, T20P, G94S, G105W, S293F, L305F variant) amino acid sequence
  • Cuphea heterophylla (Cht) FATBlb(P16S, T20P, G94S, G105W, S293F, L305F variant) coding DNA sequence
  • Cuphea heterophylla (Cht) FATBlb (P16S, T20P, G94S, G105W, S293F, L305F variant) coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea heterophylla (Cht) FATB2a (S17P, P21 S, T28N, L30P, S33L, G76D, S78P, G137W variant) amino acid sequence
  • Cuphea heterophylla (Cht) FATB2a (S17P, P21 S, T28N, L30P, S33L, G76D, S78P, G137W variant) coding DNA sequence
  • Cuphea heterophylla (Cht) FATB2a (S17P, P21 S, T28N, L30P, S33L, G76D, S78P, G137W variant) coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea heterophylla (Cht) FATB2d (S21P, T28N, L30P, S33L, G76D, R97L, H124L, W127L, I132S, K258N, C303R, E309G, K334T, T386A variant) amino acid sequence
  • Cuphea heterophylla (Cht) FATB2d (S21P, T28N, L30P, S33L, G76D, R97L, H124L, W127L, I132S, K258N, C303R, E309G, K334T, T386A variant) coding DNA sequence
  • Cuphea heterophylla (Cht) FATB2d (S21P, T28N, L30P, S33L, G76D, R97L, H124L, W127L, I132S, K258N, C303R, E309G, K334T, T386A variant) coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea heterophylla (Cht) FATB2e (G76D, R97L, H124L, I132S, G152S, H165L, T211N, K258N, C303R, E309G, K334T, T386A variant) amino acid sequence
  • Cuphea heterophylla (Cht) FATB2e (G76D, R97L, H124L, I132S, G152S, H165L, T211N, K258N, C303R, E309G, K334T, T386A variant) coding DNA sequence
  • Cuphea heterophylla (Cht) FATB2e (G76D, R97L, H124L, I132S, G152S, H165L, T211N, K258N, C303R, E309G, K334T, T386A variant) coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea heterophylla (Cht) FATB2f (R97L, H124L, I132S, G152S, H165L, T211N variant) amino acid sequence
  • Cuphea heterophylla (Cht) FATB2f (R97L, H124L, I132S, G152S, H165L, T21 IN variant) coding DNA sequence
  • Cuphea heterophylla (Cht) FATB2f (R97L, H124L, I132S, G152S, H165L, T21 IN variant) coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea heterophylla (Cht) FATB2g (A6T, A16V, S17P, G76D, R97L, H124L, I132S, S 143I, G152S, A157T, H165L, T21 IN, G414A variant) amino acid sequence
  • Cuphea heterophylla (Cht) FATB2g (A6T, A16V, S17P, G76D, R97L, H124L, I132S, S143I, G152S, A157T, H165L, T21 IN, G414A variant) coding DNA sequence
  • Cuphea heterophylla (Cht) FATB2g (A6T, A16V, S17P, G76D, R97L, H124L, I132S, S 143I, G152S, A157T, H165L, T21 IN, G414A variant) coding DNA sequence codon optimized for Prototheca moriformis
  • CM Cuphea heterophylla
  • Cuphea avigera var. pulcherrima (Ca) FATB1 amino acid sequence
  • Cuphea avigera var. pulcherrima (Ca) FATB1 coding DNA sequence
  • Cuphea avigera var. pulcherrima (Ca) FATB1 coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea procumbens (Cproc) FATB1 amino acid sequence
  • Cuphea procumbens (Cproc) FATB1 coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea procumbens (Cproc) FATB2 amino acid sequence
  • Cuphea procumbens (Cproc) FATB2 coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea procumbens (Cproc) FATB3 amino acid sequence MVAAAASSAFFPAPAPGSSPKPGKSGNWPSSLSPSFKSKSIPYGRFQVKANASAHPKANGSAV NLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLSAITTVFVAAEKQWTMLDRKSKRPDMLVD SVGLKNIVRDGLVSRQSFLIRSYEIGADRTASIETLMNHLQETSINHCKSLGLLNDGFGRTPGM CKNDLIWVLTKMQIMVNRYPAWGDTVEINTWFSQSGKIGMGSDWLISDCNTGEILIRATSVW AMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDRKLHKFDVKTGDSIRKGLTPRWNDLD VNQHV VKYIGWILESTPPEVLETQELCSLTLEYRRECGRESVLESLTAVDPSGEGGYGSQFQ HLLRLEDGGEIVKGRTEWRPKNAGINGVLPT
  • Cuphea procumbens (Cproc) FATB3 coding DNA sequence codon optimized for Prototheca moriformis
  • Cuphea ignea (Cignea) FATB1 coding DNA sequence codon optimized for Prototheca moriformis
  • CaFATBl (Cuphea avigera var. pulcherrima FATB1)
  • CaFATBl (Cuphea avigera var. pulcherrima FATB1)
  • DTSSSPPPRAFLNQLPDWSMLLSAITTVFVAAEKQWTMLDRKSKRPDMLVDSVGLKNIVRDG LVSRQSFLIRSYEIGADRTASIETLMNHLQETSINHCKSLGLLNDGFGRTPGMCKNDLIWVLTK MQIMVNRYPAWGDTVEINTWFSQSGKIGMGSDWLISDCNTGEILIRATSVWAMMNQKTRRFS RLPYEVRQELTPHFVDSPHVIEDNDRKLHKFDVKTGDSIRKGLTPRWNDLDVNQHVS VKYI GWILESMPIEVLEAQELCSLTVEYRRECGMDSVLESVTAVDPSEDGGRSQYNHLLRLEDGTDV VKGRTEWRPKNAETNGAISPGNTSNGNSIS SEQ ID NO: 181
  • Cuphea carthagenensis CCrFATB2c (V138L variant of FATB2)

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Abstract

La présente invention concerne des gènes de thioestérases acyl-ACP végétales du type FATB et des protéines codées par lesdits gènes. Ces gènes sont utiles pour construire des cellules hôtes de recombinaison présentant des profils d'acide gras modifiés. L'invention concerne également des cellules hôtes de microalgue oléagineuse comportant les nouveaux gènes ou les gènes FATB identifiés précédemment. Les cellules microalgales selon l'invention produisent des triglycérides présentant des profils d'acide gras utiles.
EP14769502.7A 2013-03-15 2014-03-13 Thioestérases et cellules pour la production d'huiles à façon Withdrawn EP2971024A4 (fr)

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US201361791861P 2013-03-15 2013-03-15
US13/837,996 US9290749B2 (en) 2013-03-15 2013-03-15 Thioesterases and cells for production of tailored oils
US201361917217P 2013-12-17 2013-12-17
PCT/US2014/026644 WO2014151904A1 (fr) 2013-03-15 2014-03-13 Thioestérases et cellules pour la production d'huiles à façon

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JP (1) JP2016518112A (fr)
KR (1) KR20150128770A (fr)
CN (1) CN105143458A (fr)
AU (2) AU2014236763B2 (fr)
BR (1) BR112015023192A8 (fr)
CA (1) CA2904395A1 (fr)
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US9816079B2 (en) 2013-01-29 2017-11-14 Terravia Holdings, Inc. Variant thioesterases and methods of use
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US9290749B2 (en) 2013-03-15 2016-03-22 Solazyme, Inc. Thioesterases and cells for production of tailored oils
US9783836B2 (en) 2013-03-15 2017-10-10 Terravia Holdings, Inc. Thioesterases and cells for production of tailored oils
US9765368B2 (en) 2014-07-24 2017-09-19 Terravia Holdings, Inc. Variant thioesterases and methods of use
US10125382B2 (en) 2014-09-18 2018-11-13 Corbion Biotech, Inc. Acyl-ACP thioesterases and mutants thereof
US20180142218A1 (en) 2016-10-05 2018-05-24 Terravia Holdings, Inc. Novel acyltransferases, variant thioesterases, and uses thereof
MY205555A (en) 2017-04-03 2024-10-25 Genomatica Inc Thioesterase variants having improved activity for the production of medium-chain fatty acid derivatives
EP4090735A1 (fr) 2020-01-16 2022-11-23 Corbion Biotech, Inc. Variants de bêta-cétoacyl-acp synthase ii

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MX2015011507A (es) 2016-04-07
AU2014236763B2 (en) 2018-08-23
KR20150128770A (ko) 2015-11-18
WO2014151904A1 (fr) 2014-09-25
JP2016518112A (ja) 2016-06-23
CN105143458A (zh) 2015-12-09
AU2018267601A1 (en) 2018-12-06
AU2014236763A1 (en) 2015-10-01
CA2904395A1 (fr) 2014-09-25
EP2971024A4 (fr) 2016-11-16
BR112015023192A2 (pt) 2017-11-21
BR112015023192A8 (pt) 2018-01-02

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