WO2012030628A1 - Esters de cire provenant du crambe - Google Patents

Esters de cire provenant du crambe Download PDF

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Publication number
WO2012030628A1
WO2012030628A1 PCT/US2011/049231 US2011049231W WO2012030628A1 WO 2012030628 A1 WO2012030628 A1 WO 2012030628A1 US 2011049231 W US2011049231 W US 2011049231W WO 2012030628 A1 WO2012030628 A1 WO 2012030628A1
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Prior art keywords
seed
plant
transgenic
wax
crambe
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PCT/US2011/049231
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English (en)
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Dean E. Engler
Oliver J. Ratcliffe
Joshua I. Armstrong
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Mendel Biotechnology, Inc.
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Publication of WO2012030628A1 publication Critical patent/WO2012030628A1/fr

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    • 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
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0105—Long-chain-fatty-acyl-CoA reductase (1.2.1.50)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01075—Long-chain-alcohol O-fatty-acyltransferase (2.3.1.75)

Definitions

  • the present invention relates to plant genomics and plant improvement.
  • plant oils are used for food.
  • plant oils are used for production of detergents, lubricants, plasticizers, slip agents, inks, coatings, paints, nylons, hydraulic fluids, cosmetics and a wide variety of other applications.
  • Most plant oils are triacylglycerols in which three fatty acids with chain lengths from 12-24 carbons long are attached via ester linkages to the three hydroxyl groups of glycerol.
  • castor oil in which the fatty acids contain a mid-chain h ydroxyl group, all of the fatty acids in commercial plant lipids are simple straight chains with 0 to 3 alkenyl groups
  • the single exceptional plant-derived oil is jojoba (Simmondsia chinensis) oil.
  • Jojoba is a slow- growing desert shrub that produces a large bean containing a liquid wax composed of wax esters.
  • a wax ester molecule can be considered as two fatty acid molecules that have been joined by a single ester linkage.
  • the physical properties of wax esters are significantly different than triacylglycerols; most significantly, wax esters are much more stable under high temperature and pressure than triacylglycerols, which suggests that they would be useful compounds when used as a component of industrial lubricants.
  • the only widely used sources of biologically -derived wax esters in industry have been whale oil and Jojoba.
  • sperm-whale oil was a common ingredient in high-quality lubricants. It was used extensively in vehicle differentials and transmissions, in hydraulic fluids that need a low coefficient of friction, and in cutting and drawing oils.
  • jojoba oil is expensive, primarily because jojoba plants grow slowly and do not produce seeds for many years. Additionally, the plants are not well-suited to mechanical harvest, so the cost of harvest is relatively high. Attempts to organize the large-scale production of jojoba have been unsuccessful because of the large initial outlay and long times until production. As a result, jojoba oil sells at a strong premium to other types of plant derived oils such as soybean oil. Three enzymes have been identified that enable Jojoba to convert very long chain fatty acids into wax esters (Lassner, et al. 1999. In: J. Janick (ed.), Perspectives on new crops and new uses. ASHS Press, Alexandria, VA. p. 220- 224).
  • One enzyme is a reductase that converts fatty acyl-CoAs to a long chain fatty alcohol (fatty acid reductase: FAR).
  • FAR fatty acid reductase
  • the other enzyme a synthase, condenses the fatty alcohol with a second molecule of acyl-CoA to produce the wax ester (wax synthase: WS).
  • the Jojoba FAR has a preference for very long chain acyl-CoA substrates, working predominantly on acyl-CoA chains with a length of 20 carbons or longer.
  • acyl-CoA chains greater than 18 carbons for the activity of FAR, and these molecules may be present naturally in the seed oil of a plant, or they can be created by introduction of a gene or genes such as the Lunaria 3-keto-acyl-CoA synthase (KCS; fatty acid elongase) gene which enhance the production of long chain acyl-CoA molecules.
  • KCS Lunaria 3-keto-acyl-CoA synthase
  • the FAR and WS genes from Jojoba and the KCS gene from Lunaria were expressed in transgenic Brassica or Arabidopsis plants, up to about 70% (by weight) of the resulting oil was composed of wax esters (Lardizabal et al., 2000 Plant Physiol. 122: 645-655).
  • any acyl-CoA greater than CI 8: 1 present in the oil of a developing seed is a good substrate for the Jojoba FAR gene, these elongating chains will be converted into their respective fatty alcohols as they form, thus resulting in 3 predominant fatty alcohols populations; C20: l, C22: l, and C24:l .
  • the relative amounts of each of these alcohols, and the relative amount of the very long chain fatty acids which correspond in length, would be expected to be determined by the activity of the enzymes controlling the chain length elongating function.
  • Jojoba oil is described as containing a mixture of wax ester molecules with chain lengths of 36 to 46 carbons.
  • Crambe abyssinica
  • Crambe is also attractive because it produces high quantities of very long chain fatty acids, and a method for its transformation has now been developed (see, for example, PCT patent publication WO2009067398).
  • Crambe is currently grown on limited acreage; however Crambe oil is used exclusively for industrial purposes, and is not considered a human food source.
  • Camelina sativa seed oil is not a good precursor for the production of wax esters because it is low in erucic acid (only 1-3%) and high in the double and triple unsaturated C18 species linoleic acid and alpha-linolenic acid. Diversion of oil precursors away from the desired single desaturated species erucic acid and nervonic acid would be expected to result in low amounts of wax esters following
  • the present patent application makes use of successful Crambe transformation methods (see PCT patent application WO2009067398) and nucleic acid constructs that encode a wax synthase, a fatty acid reductase, and, optionally, a fatty acid elongase to produce wax esters in Crambe.
  • the present description provides a transgenic seed that has been transformed with at least one recombinant polynucleotide, and in a preferred embodiment, two recombinant polynucleotides, or three recombinant polynucleotides.
  • the recombinant polynucleotide(s) generally comprise(s) at least one promoter capable of functioning in a cell of a plant seed, a nucleic acid sequence encoding a wax synthase, a nucleic acid sequence encoding a fatty acid reductase, and, optionally, a nucleic acid sequence encoding a fatty acid elongase.
  • the lipid content of the seed will consist of at least at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%,
  • the seed is produced by a plant of the genus Crambe.
  • Crambe species that may be transformed with sequences encoding a wax synthase, a fatty acid reductase, and, optionally, a fatty acid elongase, by the methods described herein, or by other means, where the seed of the transformed Crambe plants produces wax esters as a result of said transformation include, but may not be limited to: C. abyssinica, C. cordifolia, C. hispanica, C. koktebelica, C. kotschyana, C. maritima, C. orientalis, and C. tatarica.
  • the Crambe species may also be transformed with a 3-keto-acyl- CoA synthase (also referred to herein as KCS, or "fatty acid elongase").
  • the wax synthase comprises SEQ ID NO: 2.
  • the fatty acid reductase comprises SEQ ID NO: 4.
  • the KCS is encoded by a DNA sequence derived from Lunaria annua, and in a preferred embodiment, the KCS polypeptide comprises SEQ ID NO: 6.
  • the promoter is preferentially expressed in a cell of a seed, such as a seed of the transgenic plant.
  • a transgenic seed produced by a Crambe plant is viable (that is, the seed is capable of germinating and producing a mature plant).
  • the instant description provides for a transgenic seed that is capable of being grown into a transgenic plant, where the transgenic plant may produce and be used as a source of progeny transgenic seeds that comprise a seed oil comprising wax esters, up to 100% wax esters, with the non-wax ester portion of the seed oil comprising triacylglycerols.
  • the lipid content of the seed oil may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%
  • Methods for producing transformed Crambe plants where the plants produce wax esters in seeds, including, for example, greater levels of wax esters than a control plant, are also provided.
  • Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the instant description. The traits associated with the use of the sequences are included in the Examples.
  • FIG. 1 Map of P29858 (prNapin::FAR::prNapin::WS::prNapin::KCS::prNOS::NptII::
  • Adrl2 SEQ ID NO: 7: Binary plasmid P29858 contains the jojoba wax synthase gene (WS; NCBI: AF149919), the jojoba fatty acid reductase gene (FAR; NCBI: AF149917), and the Lunaria 3-keto-acyl- CoA synthase (KCS; fatty acid elongase; NCBI: EU871787) gene, all of which are expressed from repeated Napin cassettes.
  • This construct also contains the NPTII selectable marker gene expressed from a NOS promoter and an Adrl2 terminator.
  • FIG. 1 Map of P29859 (prNapin::FAR::prSphas::WS::prNOS::NptII::Adrl2; SEQ ID NO: 8): Binary plasmid P29859 contains the jojoba WS and FAR genes expressed from Napin cassettes and 7Sa' cassettes. The construct also contains the NPTII selectable marker gene expressed from a NOS promoter and an Adrl2 terminator.
  • binary plasmid P29860 contains the jojoba WS and FAR genes, but these are expressed from SpHAS cassettes and 7Sa' cassettes.
  • the construct also contains the NPTII selectable marker gene expressed from a NOS promoter and an Adrl2 terminator.
  • the present description relates to polynucleotides and polypeptides for modifying pheno types of plants, particularly by increasing the content of wax esters (WEs) in plant seeds of the genus Crambe, and methods for producing and using WEs from Crambe seeds.
  • various information sources are referred to and/or are specifically incorporated.
  • the information sources include scientific journal articles, patent documents, textbooks, and World Wide Web page addresses. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of "incorporation by reference” is noted.
  • the contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the instant description.
  • a host cell includes a plurality of such host cells
  • a reference to “a stress” is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.
  • WE Wood Ester
  • R 1 COOR 2 typically contains at least 38 carbon atoms in total and where and R 2 are each hydrocarbon chains joined by an ester linkage.
  • Polynucleotide is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides.
  • a polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof.
  • a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof.
  • the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker, a transcriptional activation or repression domain, or the like.
  • the polynucleotide can be single-stranded or double-stranded DNA or RNA.
  • the polynucleotide optionally comprises modified bases or a modified backbone.
  • the polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like.
  • the polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the polynucleotide can comprise a sequence in either sense or antisense orientations. "Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single - stranded.
  • a "recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity.
  • the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acids.
  • isolated polynucleotide is a polynucleotide, whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not.
  • an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.
  • Gene refers to the partial or complete coding sequence of a gene, its complement, and its 5' or 3' untranslated regions.
  • a gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide.
  • a gene may be isolated, partially isolated, or found within an organism's genome.
  • a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.
  • genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and that may be used to determine the limits of the genetically active unit (Rieger et al. 1976. Glossarv of Genetics and Cvtogenetics: Classical and Molecular, 4th ed., Springer Verlag, Berlin).
  • a gene generally includes regions preceding ("leaders”; upstream) and following ("trailers”; downstream) the coding region.
  • a gene may also include intervening, non-coding sequences, referred to as "introns", located between individual coding segments, referred to as "exons". Most genes have an associated promoter region, a regulatory sequence 5' of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.
  • a “promoter” or “promoter region” refers to an RNA polymerase binding site on a segment of DNA, generally found upstream or 5' relative to a coding sequence under the regulatory control of the promoter.
  • the promoter will generally comprise response elements that are recognized by transcription factors. Transcription factors bind to the promoter sequences, recruiting RNA polymerase, which synthesizes RNA from the coding region. Dissimilarities in promoter sequences account for different efficiencies of transcription initiation and hence different relative expression levels of different genes.
  • polypeptide is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues.
  • the polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
  • Protein refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
  • a “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide.
  • a “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art.
  • the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.
  • Homology refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.
  • Identity or similarity refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison.
  • the phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences.
  • Sequence similarity refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value between 0% and 100%. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison.
  • a degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences.
  • a degree of identity of polypeptide sequences is a function of the number of identical amino acids at corresponding positions shared by the polypeptide sequences.
  • a degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences.
  • “Complementary” refers to the natural hydrogen bonding by base pairing between purines and pyrrolidines.
  • sequence A-C-G-T (5' -> 3') forms hydrogen bonds with its complements A-C-G-T (5' -> 3') or A-C-G-U (5' -> 3').
  • Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or "completely complementary” if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization and amplification reactions.
  • “Fully complementary” refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.
  • polypeptides provided in the Sequence Listing have a novel activity, such as, for example, enzymatic activity.
  • all conservative amino acid substitutions for example, one basic amino acid substituted for another basic amino acid
  • Most mutations, conservative or non-conservative, made to a protein but outside of a conserved domain required for function and protein activity will not affect the activity of the protein to any great extent.
  • variable refers to molecules with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference (native) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide of amino acid sequence.
  • polynucleotide variants differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide.
  • Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations may result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.
  • a variant of a gene promoter listed in the Sequence Listing that is, one having a sequence that differs from one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence.
  • plant includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, guard cells, egg cells, and the like), and progeny of same.
  • shoot vegetative organs/structures for example, leaves, stems and tubers
  • roots for example, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules)
  • seed including embryo, endosperm, and seed coat
  • fruit the mature ovary
  • plant tissue for example, vascular tissue, ground tissue, and the like
  • cells for example, guard cells, egg cells, and the like
  • the class of plants that can be used in the instant method is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae (see, for example, Daly et al. 2001. Plant Physiol. 127: 1328-1333; Ku et al., 2000. Proc. Natl. Acad. Sci. USA 97: 9121-9126; and see also Tudge, 2000. In The Variety of Life, Oxford University Press, New York, NY pp. 547-606).
  • control plant refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant.
  • a control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the instant description that is expressed in the transgenic or genetically modified plant being evaluated.
  • a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested.
  • control plants include, for example, genetically unaltered or non-transgenic plants such as wild-type plants of the same species, or non-transformed plants, plants that do not overexpress a particular polypeptide of interest, plants that have mutations in one or more loci, or transgenic plant lines that comprise an empty expression vector.
  • a "transgenic plant” refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar.
  • the genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty.
  • the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
  • a transgenic plant may contain a nucleic acid construct (e.g., an expression vector or cassette).
  • the nucleic acid construct typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to an inducible regulatory sequence, such as a promoter, that allows for the controlled expression of polypeptide.
  • the nucleic acid construct can be introduced into a plant by transformation or by breeding after transformation of a parent plant.
  • a plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • Wild type or wild-type, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which expression of a polypeptide, such as a transcription factor polypeptide, is altered, e.g., in that it has been overexpressed or ectopically expressed.
  • a polypeptide such as a transcription factor polypeptide
  • a “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring tolerance to a form of stress, such as water deficit or water deprivation, or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as extent of wilting, turgor, hyperosmotic stress tolerance or in a preferred embodiment, yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
  • Trait modification refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the instant description relative to a plant not doing so, such as a wild-type plant.
  • the trait modification can be evaluated quantitatively.
  • the trait modification can entail at least about a 2% increase or decrease, or an even greater difference, in an observed trait as compared with a control or wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to control or wild- type plants.
  • Ectopic expression or altered expression in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species.
  • the pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species.
  • the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant.
  • the term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression.
  • the resulting expression pattern can be transient or stable, constitutive or inducible.
  • the term "ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of chemical modification of the polypeptides.
  • overexpression refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more proteins are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also occur when expression in a particular cell-type, groups of cell-types (tissues) or in specific whole organs is increased relative to the level normally found in those cells (e.g., in non-transgenic plants of the same species), or in comparison to the average expression level in all other tissues in that plant. Thus, overexpression may occur throughout a plant or in a specific sub-group of cells or in a specific tissue or organ, depending on the promoter used. See, for example, US patent 7,365,186, or US patent 7,619,133.
  • Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to a polypeptide that can confer an improved trait, for example, increased stress tolerance or improved yield. Overexpression may also occur in plant cells where endogenous expression of the present proteins that confer an improved trait, for example, improved stress tolerance, or functionally equivalent molecules, normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the protein that confers the improved trait in the plant, cell or tissue.
  • a “nucleic acid construct” may comprise a polypeptide-encoding sequence operably linked (that is, under regulatory control of) to appropriate inducible, cell-specific, tissue-specific, cell-enhanced, tissue-enhanced, condition-enhanced, developmental, or constitutive regulatory sequences that allow for the controlled expression of the polypeptide.
  • the expression vector or cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant.
  • a plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, for example, a plant explant, to produce a recombinant plant (for example, a recombinant plant cell comprising the nucleic acid construct) as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • a plant part such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, for example, a plant explant, to produce a recombinant plant (for example, a recombinant plant cell comprising the nucleic acid construct) as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • WEs derived from plants may be of significant value (either directly or following chemical modification) for a number of reasons, including, but not limited to, the following: cosmetics, pharmaceutical carriers, anti-foam agents, plastic modifiers, polishes, candle making, treatment of papers, insulations, etc, manufacture of varnishes, rubbers, adhesives, inks, lubricants, specialized lubricants (e.g. for use in high temperature and/or high pressure applications), cutting oils, quenchants, and synthons.
  • Crambe oil is naturally rich in long chain mono-unsaturated fatty acids, which are ideal precursors for biological synthesis of wax esters.
  • Crambe is currently grown as a source of industrial oils and therefore all the procedures and processes that are necessary to supply a consumer with an industrial oil from Crambe already exists (harvest, delivery methods, seed crushing facilities, etc).
  • the production costs of wax esters from canola are considerably lower than from jojoba, which opens up the possibility of new applications for these oils, where the high-costs of jojoba oil currently cannot compete with similar molecules that are more cheaply produced from petroleum-based sources.
  • Recombinant polynucleotides that encode wax synthases that may be used in the instant description include, but are not limited to, SEQ ID NO: 1.
  • the wax synthases that may thus be used to produce WEs thus include SEQ ID NO: 2, which is encoded by SEQ ID NO: 1
  • Recombinant polynucleotides that encode fatty acid reductases that may be used in the instant description include, but are not limited to, SEQ ID NO: 3.
  • the fatty acid reductases that may thus be used to produce WEs thus include SEQ ID NO: 4, which is encoded by SEQ ID NO: 3.
  • Recombinant polynucleotides that encode fatty acid elongases that may be used in the instant description include, but are not limited to, SEQ ID NO: 5.
  • the fatty acid elongases that may thus be used to produce WEs thus include SEQ ID NO: 6, which is encoded by SEQ ID NO: 5.
  • the instant description provides the production of WEs in plant seeds as a result of the overexpression of at least one wax synthase and at least one fatty acid reductase in a plant, and, optionally, at least one fatty acid elongase.
  • Recombinant polynucleotides including nucleic acid constructs, expression vectors, or expression cassettes, where the recombinant polynucleotides encode wax synthases, fatty acid reductases and, optionally, fatty acid elongases, may be constructed with means well known in the art, of, for example, with the methods provided in Example I. These recombinant polynucleotides may be introduced into plants for the purpose of conferring increased wax ester production in seeds. In a preferred embodiment, the seeds are derived from a transgenic Crambe plant. Nucleic acid sequences encoding any of various wax synthases and fatty acid reductases may be recombined into the recombinant polynucleotides.
  • sequences encoding the wax synthase, the fatty acid reductase and the fatty acid elongase may be comprised within one, two or three discrete recombinant polynucleotides, such as in a one-, two- or three-component system where one, two or three nucleic acid vectors, respectively, encode the two to three enzymes.
  • the lipid content of the transgenic seeds of the instant description produced by plants of the genus Crambe may comprise at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least
  • first, second and optionally the third nucleic acid sequences are integrated into the host Crambe plant seed cell in a stable manner
  • the plant produces a seed with more than 0% to 99% by weight of its lipid content consisting of wax esters.
  • first, second and optionally the third nucleic acid sequences are integrated into the host Crambe plant seed cell in a stable manner
  • first, second and optionally the third nucleic acid sequences are integrated into the host Crambe plant seed cell in a stable manner
  • the plant produces a seed with more than 0% to 99% by weight of its lipid content consisting of wax esters.
  • Polynucleotides that are sequentially similar to those provided in the Sequence Listing may be made that have some alterations in the nucleotide sequence and yet retain the function of the listed sequences.
  • the sequences will typically share at least about at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, from any of the above values up to but not including 100%, or about 100% sequence identity to the polynucleotides provided in the Sequence Listing, including any of SEQ ID NOs: 1, 3, 5, 7, 8, or 9.
  • the sequences will typically share at least about at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
  • Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). MEGALIGN can create alignments between two or more sequences using different methods, for example, the clustal method (see, for example, Higgins and Sharp. 1988. Gene 73: 237-244). The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, WI), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1 (see USPN 6,262,333).
  • HSPs high scoring sequence pairs
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff. 1989. Proc. Natl. Acad. Set USA 89: 10915-10919).
  • sequence identity refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter "off (see, for example, internet website at www.ncbi.nlm.nih.gov/).
  • Example I Transformation of Crambe somatic tissues by cocultivation with Agrobacterium tumefaciens.
  • Seeds of Crambe abyssinica varieties Meyer and Bel Ann were sterilized by immersion in 20% bleach for 10 minutes followed by three rinses in sterile distilled water. All manipulations after the sterilization steps were performed in an aseptic manner in a laminar air flow cabinet. The sterilized seeds were planted onto solid Basal Medium (MSO) and incubated in the dark at 25°C for four days, during which time the seeds germinated and produced elongated (etiolated) hypocotyls. Five explants from each hypocotyl were collected by slicing approximately 1 mm thick disks from the region just below the cotyledon attachment point. Care was taken to avoid inclusion of the apical meristem in these explants.
  • MSO solid Basal Medium
  • Agrobacterium strain LSLJ4571 is strain LBA4404 (Hoekema) which carries the binary plasmid pSLJ4571
  • the transformed shoots were then transferred onto solidified basal medium for growth and rooting. If rooting does not occur spontaneously, the shoots can be treated with an auxin-containing medium such as an indole 3-butyric acid (IBA)- containing medium to induce them to root. Rooted shoots were transplanted into soil and grown to maturity. The transformation efficiency of this process was greater than 1%, and transformation efficiencies of at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, can be produced.
  • IBA indole 3-butyric acid
  • Example II Improvement of regeneration of Crambe somatic tissue by selection for improved genetic sub-populations.
  • Etiolated hypocotyls from 91 Crambe seeds of the variety Meyer were prepared as described in Example I. Each of these hypocotyls was individually assayed for its relative capacity for regeneration by cutting into about 20 segments and transfer to REG medium. The number of regenerating shoots from each hypocotyl was recorded. The apical meristems from some of these seedlings were grown into mature plants by transfer to MSO medium, and when rooted, transfer to soil and growth into mature plants. These mature plants were self -pollinated and the resulting seeds were collected. A numbering system was used such that the seeds from each mature plant and the original hypocotyl associated with it could be identified.
  • hypocotyl regeneration assays showed that 61 of the 91 (67%) hypocotyls from Meyer seedlings have little or no capacity for regeneration, whereas only 13 (14%) of these hypocotyls regenerated reasonably well (as defined by more than 5 shoots emerging from callus produced on the 20 assayed hypocotyl disks).
  • This example is a demonstration of a method for producing seed lines (e.g. Line #71) with hypocotyls having increased regenerabihty relative to control seed lines (e.g., parental lines, or lines of plants not selected for increased regenerabihty), that is, the hypocotyl tissue is derived from a plant line selected for greater hypocotyl regenerabihty than that of a control plant line.
  • the method can be repeated, for example, by using selfed seed lines with increased hypocotyl regenerabihty as starting material.
  • Each iteration of the method, that is, each generation and selection for regenerabihty would result in seed lines with further increases in hypocotyl regenerabihty relative to a parental or control plant.
  • Example III Improved transformation of Crambe somatic tissue using genetic sub-populations selected for improved regeneration.
  • Seedlings produced as a result of self-pollination of plant #71 described in Example II were used in a transformation experiment according to the protocol of Example I.
  • hypocotyls from 100 seedlings from Line #71 were cocultivated. Twenty four transformed calli were produced and 10 of them regenerated shoots. All calli and regenerated shoots expressed the GUS gene, indicating that no escapes were produced.
  • Previous experiments using hypocotyls from non-selected Meyer seedlings produced transformed calli at similar frequencies, but in these experiments, two or fewer transformed shoots were produced. No escapes were produced in these previous experiments.
  • Example IV Transformation of Crambe embryogenic callus by cocultivation with Agrobacteriiim tumefaciens.
  • Crambe seeds of the varieties Meyer and Bel Ann were planted in soil, and grown to maturity in soil. Water and fertilizer were provided when needed with a solution of 0.4g/L of Peters fertilizer at each watering. Plants were grown at 25°C under continuous white light provided by 400 Watt HID fixtures (Voigt Lighting Industries, Inc). Approximately 6-8 weeks after planting, the plants had flowered, and clusters of immature flower buds were harvested. The clusters of immature flower buds were sterilized by immersion into approximately 100 ml of a 10% solution of bleach with one drop of Triton X-100 added and swirled for 10 minutes, and rinsed twice with sterile distilled water .
  • Transformation of the ecallus described above was initiated by cocultivation with Agrobacteriiim tumefaciens.
  • a mass of about 600 mg of ecallus was transferred into five ml of MinA media, and (500 ⁇ ) of the Agrobacteriiim tumefaciens strain LSLJ4571, which had been grown to saturation in MinA medium was added.
  • Agrobacteriiim strain LSLJ4571 is strain LBA4404 (ref) which carries the binary plasmid pSLJ4571.
  • the ecallus and bacterial cells were thoroughly mixed, and the inoculated ecalli were then transferred onto ECIGM medium supplemented with 100 ⁇ acetosyringone and cocultivated at 22°C in the dark for two days.
  • Selection for transformed Crambe cells and counterselection against the Agrobacteriiim was performed by transferring the cocultivated ecalli onto solid ECM medium supplemented with 25 mg/L Geneticin for selection and 150 mg/L Timenton for counterselection.
  • the ecalli were incubated in the same culture conditions as were indicated above for ecallus initiation and growth. These selection/counterselection cultures were transferred to fresh medium every 2-3 weeks. Following 25 days of incubation, most of the ecalli were brown, or black and appeared dead, but a minority of the ecalli had segments that appeared to be growing and thus resistant to the Geneticin.
  • a GUS assay demonstrated the presence of the GUS gene in some or all of the tissues assayed in all nine of these ecallus lines.
  • Example V Transformation of Crambe embryogenic callus by particle bombardment.
  • Transformation of the ecallus may also be accomplished by bombardment of the embryogenic callus by microparticles coated with a nucleic acid construct (i.e., a plasmid, vector, cassette or other DNA preparation) of choice.
  • a nucleic acid construct i.e., a plasmid, vector, cassette or other DNA preparation
  • the PDS-1000 Biolistics particle bombardment device may be used to deliver DNA to the target cells. The operation of this device is detailed in the operating instructions available from the manufacturer (Bio-Rad Laboratories, Hercules, Calif.).
  • DNA and particles of materials with large specific gravity are associated and the preparation is dried on plastic macrocarriers.
  • materials with large specific gravity e.g., tungsten, gold, palladium, or platinum
  • tungsten particles Prior to association with the transforming DNA, tungsten particles are prepared essentially as described in U.S. Pat. No. 5,990,387 to by Tomes et al.. Such particles are also known as microparticles or
  • microprojectiles Prior to each bombardment, the expendables are mounted in the device. Expendables include the macrocarrier with a dried DNA/particle preparation, a rupture disk, and a stopping screen. The material intended to be bombarded is positioned upon on target platform. The embryogenic callus may be pre-treated prior to introduction into the particle gun by contacting it with an osmotic conditioning agent. An osmotic conditioning agent or osmoticum may be beneficial to particle gun mediated transformation. While the precise mechanism has not been identified, a preferred explanation holds that plasmolyzed cells, a consequence of an osmotic conditioning treatment, are less apt to lyse when penetrated by a particle.
  • the chamber of the device is evacuated with a vacuum pump to near 28 mm Hg.
  • a small reservoir behind the rupture disk is then slowly filled with helium.
  • the rupture disk breaks and releases a burst of helium.
  • the helium burst pushes against the macrocarrier and accelerates it towards the stopping screen.
  • the stopping screen a metal mesh, abruptly stops the macrocarrier.
  • the DNA/particle preparation that is dried upon the macrocarrier is released from the macrocarrier and continues on a path to strike the target.
  • the chamber is equalized with the atmosphere, and the expendibles are removed.
  • the same or a different osmotic agent may be used as a post-particle gun transformation treatment. This example generally cites U.S. patent 7,057,089 to Collins and Marsh as to methods for operation of the PDS-1000 particle bombardment device.
  • the embryogenic callus is cultured in the presence of a selective agent to identify and/or select for transformed cells, and the transformed cells are grown and regenerated into whole plants as is described in Example IV.
  • Crambe lines have been transformed with nucleic acid constructs that encode WS and FAR using procedures described below and in PCT patent application WO2009067398 by Engler et al., which is herein incorporated by reference. These constructs include P29859 and P29860.
  • P29859 (SEQ ID NO: 8, Figure 2, prNapin::FAR::prSphas::WS::prNOS::NptII::Adrl2) contains the jojoba WS and FAR genes expressed from Napin cassettes and 7Sa' cassettes.
  • the construct also contains the NPTII selectable marker gene expressed from a NOS promoter and an Adrl2 terminator.
  • P29860 (SEQ ID NO: 9, Figure 3, prSphas::FAR::prNapin::WS::prNOS::NptII::Adrl2) is similar to P29859, but P29860 contains the jojoba WS and FAR genes expressed from spHAS (promoter of hyaluronan synthase from Streptococcus pyogenes) cassettes and 7Sa' cassettes. Like P29859, the construct also contains the NPTII selectable marker gene expressed from a NOS promoter and an Adrl2 terminator.
  • spHAS promoter of hyaluronan synthase from Streptococcus pyogenes
  • Transgenic Crambe lines were been generated by transforming Crambe plants with nucleic acid constructs that encode WS, FAR and KCS polypeptides, using procedures described below and in PCT patent application WO2009067398 by Engler et al.
  • PCT patent application WO2009067398 is herein incorporated by reference.
  • the construct P29858 (SEQ ID NO: 7, Figure 1,
  • prNapin::FAR::prNapin::WS::prNapin::KCS::prNOS::NptII: :Adrl2) is a plasmid that contains the jojoba WS gene, the jojoba FAR gene, and the Lunaria KCS gene, all expressed from repeated Napin cassettes. This construct also contains the NPTII selectable marker gene expressed from a NOS promoter and an Adrl2 terminator.
  • Mature seeds from Meyer line #71 (decedents from a plant selected on the basis of its highly regenerable hypocotyl phenotype from the Crambe abyssinica variety Meyer - see WO2009067398, example II) were sterilized by immersion in 20% bleach for 10 minutes followed by three rinses in sterile distilled water. All manipulations after the sterilization steps were performed in an aseptic manner in a laminar air flow cabinet. The sterilized seeds were planted onto solid Basal Medium (MSO) and incubated in the dark at 25 °C for four days, during which time the seeds germinated and produced elongated (etiolated) hypocotyls.
  • MSO Solid Basal Medium
  • explants from each hypocotyl were collected by slicing approximately 1 mm thick disks from the region just below the cotyledon attachment point. Care was taken to avoid inclusion of the apical meristem in these explants.
  • the explants were cocultivated with an Agrobacterium LBA4404 strain (Hoekema) carrying one of the plasmids P29858, P29859, or P29860 ( Figures 1, 2 and 3, respectively) by soaking them in a 10- fold dilution of a culture grown in Agrobacterium Growth Medium (MinA) to saturation.
  • MinA Agrobacterium Growth Medium
  • the explants were transferred onto a filter paper disks which overlaid Regeneration Medium (REG) supplemented with 100 ⁇ Acetosyringone, and allowed to cocultivate for three days at 25°C in darkness.
  • REG Regeneration Medium
  • These cocultivated explants were then transferred to REG medium lacking acetosyringone but supplemented with 50 mg/L Geneticin® (Phytotechnology Laboratories, Shawnee Mission, KS; and 150 mg/L Timenton (Phytotechnology Laboratories; and incubated at 25°C under white light with 12 hour photoperiods provided by cool white bulbs. Incubation in these conditions was continued with transfer to fresh media of the same composition every 2-3 weeks until transformed calli with shoots formed.
  • hypocotyl segments were originally cocultivated, and 150 calli formed which were resistant to the 50 PPM G418. Fifteen of these produced regenerating shoots, 6 of which appeared normal, and 4 of which were successfully established in a glasshouse. All 4 of these plants were confirmed to contain the WS gene by PCR.
  • W refers to a wax ester of a specific carbon chain length (e.g., W42 is a wax ester molecule with a chain length of 42 carbon atoms)
  • T refers to a triacylglycerol (TAG) of a specific carbon chain length (e.g., T66 has a chain length of 66 carbon atoms)
  • the Crambe seeds containing the Lunaria KCS gene in addition to the FAR and WS genes have significantly longer chain wax ester species than seeds containing only the FAR and WS genes (as evidenced by the wax analysis of seeds from plants transformed with P29858 compared to seeds from plants transformed with P29859 and P29860).
  • jojoba oil derived from jojoba plants may not be suitable for all applications.
  • the pour point the lowest temperature at which an oil will flow, is an important physical characteristic for lubricating oils, particularly for applications at low temperatures.
  • the pour point of jojoba oil is higher than that of oils derived from petroleum or some other plants, in the range of 12°C to 17° C (Miwa et al. 1979. . Am. Oil Chem. Soc. 56: 765-770;
  • pour point of oils including both jojoba and Crambe oils
  • pour point depressants which may reduce the size and cohesiveness of wax crystal structures and increasing flow at lower temperatures
  • Crambe oil with a reported viscosity at 100°C of 10.5 centistokes (cST; Erhan et al., 2006, supra), has a significantly lower viscosity at nearly the same temperature than jojoba oil (27 cST at 99°C; Heilweil, 1997, supra).
  • Crambe oil is an example of an oil with a lower viscosity, better fluidity, and therefore better lubricating ability near 100°C.
  • advantages of Crambe oil over jojoba oil include the former having a lower pour point and viscosity at higher temperatures that jojoba oil, and the former will likely possess better lubrication capability across a range of temperatures.
  • an advantage of producing wax esters in transgenic Crambe by introducing nucleotide sequences derived from jojoba is that unique oil compositions will be created. As these oil compositions may have intermediate concentrations of products found in Crambe or jojoba oils, the present transgenic Crambe plants will produce oils that have pour points and/or viscosities that may be lower than would be associated with jojoba oil produced in jojoba plants.
  • transgenic Crambe plants transformed with the sequences provided in the Sequence Listing, or functionally similar homologous sequences may be selected for their production of an oil with some superior or more optimized characteristics over jojoba oil, such as specific physical characteristics including a more useful pour point or viscosity, and better lubricating ability.
  • transgenic Crambe plants may also be selected for more useful or optimized acid value and oxidative stability than jojoba oil from jojoba plants.
  • the oils that may be derived from the present transgenic Crambe plants thus represent useful compositions that are unique and possibly superior with respect to jojoba oil, including, for example, a lower or more optimized pour point, and better viscosity, acid value, oxidative stability, or lubricating ability, or a combination of these characteristics.
  • Transgenic Crambe lines can be analyzed and selected for favorable combinations of these attributes.
  • Example IX Viable seed from transgenic Crambe expressing jojoba proteins, including wax synthase and fatty acid reductase
  • transgenic event #Z47034 (and the rest of the seedlots which were investigated for germinability) produced some viable seeds that germinated and gave rise to normal appearing plants. In light of this unanticipated result, it is expected that a transgenic seed with the same wax ester content or less as transgenic event #Z47034 may be viable and capable of being grown into transgenic plants where the latter transgenic plants may produce progeny transgenic seed.
  • the lipid content of the viable transgenic seed may thus comprise at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% by weight of one or more wax esters.
  • transformation of a non-food crop species such as a Crambe species, including but not limited to C. abyssinica, C. cordifolia, C. hispanica, C. koktebelica, C. kotschyana, C. maritima, C. orientalis, and C. tataric, with a nucleic acid sequence encoding a wax synthase, a nucleic acid sequence encoding a fatty acid reductase, and, optionally, a fatty acid elongase, can be useful for the production of wax esters in commercially useful quantities.
  • a non-food crop species such as a Crambe species, including but not limited to C. abyssinica, C. cordifolia, C. hispanica, C. koktebelica, C. kotschyana, C. maritima, C. orientalis, and C. tataric
  • a nucleic acid sequence encoding a wax synthase a nu
  • wax esters in commercially useful quantities can be achieved by the expression of SEQ ID NO: 2 and SEQ ID NO: 4, and optionally SEQ ID NO: 6, or structurally similar and functionally similar homologs, and their encoding polynucleotides, can be achieved in non-food plants such as Crambe species.
  • the nucleic acid sequence encoding the wax synthase, the nucleic acid sequence encoding a fatty acid reductase, and the nucleic acid sequence encoding a fatty acid elongase may be derived from a diverse range of species.
  • wax esters may be produced in non-jojoba plants in of similar or greater quantity than can be achieved in jojoba.
  • wax esters may also be produced in growing conditions or ranges that may be sub-optimal or impossible for growth of jojoba and hence for production of wax esters from jojoba.
  • Products including plant products such as seed oil, wax, one or more wax esters, and/or a seed oil comprising both wax and triacylglycerols, wherein the products are derived from transgenic seed produced by the methods described herein and the products are produced by processing of a tissue of the transgenic seed, are also contemplated in this application.
  • sequences provided in the sequence listing may be used to produce a nucleic acid construct for these transformations, although homologous sequences that are phylogenetically related and functionally similar can similarly be used to produce wax esters in transformed plants.
  • a non-jojoba plant line or plant cell has been transformed (and the latter plant host cell regenerated into a plant) and shown to produce wax esters
  • the transformed non-jojoba plant line may be crossed with itself, a plant from the same line, a non -transformed or wild-type plant line plant, or a transformed plant from a different line of plants.
  • Transformed seed comprising the nucleic acid constructs described herein or produced with phylogenetically related and functionally similar sequences are also contemplated in this application. It is also expected that the same methods may be applied to other plant species other than Crambe species.
  • Individual jojoba plants may yield 1- 5 kg (dry weight) of seeds a year (Buchanan and Duke,
  • transgenic Crambe plants may also yield similar or higher amounts of oil, including, for example, about 500 kg/ha, about 1000 kg/ha, about 1,500 kg/ha, about 2,000 kg/ha, about 2,500 kg/ha, about 3000 kg/ha, or greater amounts, depending to some extent on the plant lines selected and/or the growing conditions.

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  • Plant Pathology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des plantes transformées du genre Crambe qui produisent des esters de cire dans leurs graines. La présente invention concerne la transformation de plantes du genre Crambe au moyen d'au moins un polynucléotide recombinant et, de préférence, au moyen de deux ou trois polynucléotides recombinants. Ledit ou lesdits polynucléotides recombinants comprendront généralement au moins un promoteur capable de fonctionner dans une cellule d'une graine, une séquence d'acide nucléique codant pour une cire synthase, une séquence d'acide nucléique codant pour une acide gras réductase et, éventuellement, une séquence d'acide nucléique codant pour une acide gras élongase. Suite à l'expression de la cire synthase et de l'acide gras réductase dans la graine, la plante produira des esters de cire dans ses graines. Dans un autre mode de réalisation préféré, le promoteur s'exprime préférentiellement dans une cellule d'une graine, par exemple d'une graine de la plante transgénique.
PCT/US2011/049231 2010-09-02 2011-08-25 Esters de cire provenant du crambe WO2012030628A1 (fr)

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US61/379,627 2010-09-02

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US5679881A (en) * 1991-11-20 1997-10-21 Calgene, Inc. Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
US20030228668A1 (en) * 1994-06-23 2003-12-11 Lardizabal Kathryn Dennis Fatty acyl-CoA: fatty alcohol acyltransferases
US6828475B1 (en) * 1994-06-23 2004-12-07 Calgene Llc Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
US20100122377A1 (en) * 2006-11-21 2010-05-13 Vesna Katavic Lunaria annua,cardamine graeca and teesdalia nudicaulis fae genes and their use in producing nervonic and eicosenoic acids in seed oils
US20100199548A1 (en) * 2007-07-06 2010-08-12 Ls9, Inc. Systems and methods for the production of fatty esters
WO2011082253A2 (fr) * 2009-12-30 2011-07-07 Board Of Trustees Of Michigan State University Procédé de production d'acétyldiacylglycérols (ac-tag) par expression d'un gène d'acétyltransférase isolé à partir d'euonymus alatus (fusain ailé)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679881A (en) * 1991-11-20 1997-10-21 Calgene, Inc. Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
US20030228668A1 (en) * 1994-06-23 2003-12-11 Lardizabal Kathryn Dennis Fatty acyl-CoA: fatty alcohol acyltransferases
US6828475B1 (en) * 1994-06-23 2004-12-07 Calgene Llc Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
US20100122377A1 (en) * 2006-11-21 2010-05-13 Vesna Katavic Lunaria annua,cardamine graeca and teesdalia nudicaulis fae genes and their use in producing nervonic and eicosenoic acids in seed oils
US20100199548A1 (en) * 2007-07-06 2010-08-12 Ls9, Inc. Systems and methods for the production of fatty esters
WO2011082253A2 (fr) * 2009-12-30 2011-07-07 Board Of Trustees Of Michigan State University Procédé de production d'acétyldiacylglycérols (ac-tag) par expression d'un gène d'acétyltransférase isolé à partir d'euonymus alatus (fusain ailé)

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JETTER ET AL.: "Plant surface lipid biosynthetic pathways and their utility for metabolic engineering of waxes and hydrocarbon biofuels.", THE PLANT JOURNAL, vol. 54, no. ISS. 4, May 2008 (2008-05-01), pages 670 - 683, Retrieved from the Internet <URL:http://www.botany.ubc.calkunsUJournals/Biofuels.pdf> [retrieved on 20111226] *

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