HK1191975B - Accumulation of omega-7 fatty acids in plant seeds - Google Patents

Description

Accumulation of OMEGA-7 fatty acids in plant seeds

The invention was made under CRADA (C-05-11) between Dow Agrosciences LLC and Brookhaven sciences associates, LLC, which operates for the U.S. department of energy. The government has certain rights in the invention.

Priority requirement

The present application claims benefit OF filing date OF "acumulation OF N-7FATTY ACIDS IN PLANT SEEDS" from U.S. provisional patent application serial No. 61/358,318 filed 24/6/2010.

Technical Field

In a particular embodiment, the invention relates to a method of developing a⁹-16:0 ACP desaturase function novel mutant Ricinus (Ricinus) delta⁹An 18:0 ACP desaturase, designated Com 25. Another embodiment relates to metabolic engineering methods to manipulate metabolic branch points in plants, for example, to redirect carbon into omega-7 fatty acids. In certain embodiments, the invention relates to methods for expressing Com25 as part of a metabolic engineering strategy to redirect carbon into omega-7 fatty acids in plant seeds.

Background

It is estimated that more than 1,000 fatty acid structures may exist in nature. Millar et al, (2000) trends plasma Sci 5(3): 95-101. Many of these fatty acids were synthesized by derivatizing fatty acids with a panel of prototype desaturase variants. The first of these variant desaturases to be isolated is the Ricinus (Ricinus) oleic hydroxylase from the castor endosperm, the enzyme responsible for ricinoleic acid synthesis. Van de Loo et al, (1995) Proc.Natl.Acad.Sci.USA 92(15): 6743-6747. This is followed by the genes encoding Vernonia (Vernonia) linoleate epoxidase and Crepis paniculata (Crepis) oleic acid ethynylase (actinonase). Lee et al, (1998) Science280(5365): 915-18. The isolation of these genes led to the idea that their heterologous expression in oil crop plants could promote the accumulation of the corresponding unusual fatty acids. Broun et al, (1997) Plant Journal 13: 201-10. However, the resulting accumulation of unusual fatty acids is always lower than that present in the plant of natural origin from which the gene was isolated. Napier, J.A, (2007) Annu.Rev.plant biol.58: 295-319.

The specific activity profile of the enzyme variant desaturases that have been isolated from tissues that accumulate unusual fatty acids is consistent with the role of producing the corresponding unusual fatty acids. However, they exhibit very poor specific activity compared to all stearoyl-ACP desaturases reported to date and have proven ineffective in producing altered fatty acid phenotypes upon heterologous expression. Cahoon et al, (1994) prog. lipid Res.33: 155-63. For example, seed-specific expression of castor hydroxylase under the control of a strong seed-specific promoter in the model plant Arabidopsis (Arabidopsis) results in the accumulation of only about 17% ricinoleic acid, much less than about 90% of that present in castor seeds. Broun and Somerville (1997) Plant physiol.113: 933-42. Similar disappointing results have been reported for epoxy and acetylenic fatty acids, which have been reported to accumulate 15 and 25% after heterologous expression of cyclooxygenase (epoxygenase) and acetylenase, respectively, in Arabidopsis thaliana. Lee et al, (1998) Science280(5365): 915-18. In addition to exhibiting poor activity, variant desaturases tend to form insoluble aggregates upon purification. Lower stability and poor catalytic rates are shared by many newly evolved enzymes that appear as gene duplication events in which stability and/or turnover selection are released during which mutations accumulate, ultimately leading to a functional change. Govindarajan and Goldstein (1998) Proc. Natl. Acad. Sci. USA 95:5545-49, Goldstein (2001) in Protein Folding, Evolution and Design (Broglia, R.A., Shakhnovch, E.I. and Tiana, eds.) CXLIV, I.O.S.Press, Amsterdam.

Many potential explanations for the accumulation of low levels of targeted fatty acids have been advanced. Napier, J.A, (2007) Annu.Rev.plant biol.58: 295-319. Demonstration suggests that specialized enzymes may play a key role in incorporating unusual fatty acids into triacylglycerols. For example, the accumulation of laurate in transgenic canola (Brassicanapus) seeds increased from 50% to 60% after co-expression of coconut lysophosphatidic acid acyltransferase and the medium chain thioesterase from the bay tree of california (Calfornia bay). Knutzon et al (1999) plant Physiol.120(3): 739-46. Recently, co-expression of ricinus communis type 2 acyl-CoA diacylglycerol acyltransferase (RcDGAT2) and ricinus hydroxylase has accumulated ricinoleic acid from about 17% to about 30%. Burgal et al, (2008) Plant Biotechnol. J.6(8): 819-31.

It must still be reported that high levels of unusual (unusual) fatty acids, equivalent to those present in naturally occurring species, accumulate in transgenic plants. Because unusual fatty acids are highly desirable in many industries and applications, there is a need to better express them in transgenic plants designed for their production.

Summary of the invention

Disclosed herein are nucleotide sequences and amino acid sequences thereof encoding a novel variant desaturase designated Com 25.

Also disclosed are methods of expressing Com25 in a plant cell to utilize Com25 enzyme versus WT ricin delta⁹Enhanced desaturase activity of 18:0 desaturases, making of unusual fatty acids in plant seedsThe percentage composition increases. In some embodiments, the method comprises expressing Com25 in arabidopsis. In certain embodiments, the increased unusual fatty acid in the plant seed is an omega-7 fatty acid. In these embodiments, the omega-7 fatty acid can be 16:1 delta⁹-and/or 18: 1. delta¹¹。

Also provided are methods of expressing Com25 in a plant cell, wherein the plant cell is impaired in plastid and extraplastid fatty acid elongation such that the percentage composition of unusual fatty acids in plant seeds is increased. In some embodiments, the method comprises expressing Com25 in arabidopsis. In certain embodiments, the increased unusual fatty acid in the plant seed is an omega-7 fatty acid. In these embodiments, the omega-7 fatty acid can be 16:1 delta⁹-and/or 18: 1. delta¹¹。

Other methods of expressing Com25 in a plant cell are provided, wherein KASII is inhibited in the plant cell such that the percentage composition of unusual fatty acids in plant seeds is increased. In some embodiments, the method comprises expressing Com25 in arabidopsis. In certain embodiments, the increased unusual fatty acid in the plant seed is an omega-7 fatty acid. In these embodiments, the omega-7 fatty acid can be 16:1 delta⁹-and/or 18: 1. delta¹¹。

Still other methods of expressing Com25 in a plant cell are provided, wherein KASII and plastid and extraplastid fatty acid elongation are inhibited in the plant cell such that the percentage composition of unusual fatty acids in plant seeds is increased. In some embodiments, the method comprises expressing Com25 in arabidopsis. In certain embodiments, the increased unusual fatty acid in the plant seed is an omega-7 fatty acid. In these embodiments, the omega-7 fatty acid can be 16:1 delta⁹-and/or 18: 1. delta¹¹。

The foregoing and other features will become more apparent from the following detailed description of several embodiments and the accompanying drawings referred to therein.

Brief Description of Drawings

FIG. 1 depicts a schematic of fatty acid synthesis and modification in Arabidopsis plastids and endoplasmic reticulum. The response mediated by the 16:0 desaturase is indicated as 1: delta⁹-16:0 ACP desaturase; 2: delta in vitro⁹-16:0 ACP desaturase. Boxes for omega-7 FA, i.e., 16:1 Δ⁹And 18: 1. delta¹¹。

FIG. 2 shows a representative gas chromatographic separation of FAME after expression of Com25 in various backgrounds of Arabidopsis. Panels a and B, WT; c and D, fab 1; e and F, fab1/fae 1. Panels A, C and E, untransformed; b, D and F, transformed with Phas: Com 25. Mark FAME peaks: 16:0(1),16:1 Delta⁹(2),16:2(3),18:0(4),18:1Δ⁹(5),18:1Δ¹¹(6),18:2(7),20:0(8),20:1Δ¹¹(6),18:2(7),20:0(8),20:1Δ¹¹(9),18:3+20:1Δ¹³(10) And 22:1 (11).

FIG. 3 shows the relationship between 16:0 vs. omega-7 accumulation (in mol%) in host seeds.

FIG. 4 shows a representative gas chromatographic separation of FAME after expression of Com25 in various backgrounds of Arabidopsis. Small graph A: the best Fab1/fae1, Phas Com25, Fab1-HPAS, An Δ 9DS and Ln Δ 9DS transformation systems; small graph B: cat's claw (Doxantha) seed. The peak names are as described in figure 2.

FIG. 5 is a schematic arrangement of DNA elements in an embodiment of a specific construct of the present invention.

Detailed Description

I. Brief summary of several embodiments

Disclosed herein are encoding enzymes delta⁹A nucleotide molecule of a desaturase enzyme comprising a nucleotide sequence at least 60% identical to SEQ ID No. 1. The nucleic acid molecule may further comprise a gene regulatory element. In some embodiments, the gene regulatory element may be a phaseolin promoter.

Also disclosed is an enzyme⁹A desaturase comprising an amino acid sequence at least 80% identical to SEQ ID NO: 2. An enzyme Δ of the invention in which the amino acid sequence is at least 80% identical to SEQ ID NO 2⁹The desaturase may further comprise a serine at a position similar to position 114 of SEQ ID No. 2; arginine at a position analogous to position 117 in SEQ ID NO. 2; cysteine at position 118 in SEQ ID NO. 2 and leucine at position 179 in SEQ ID NO. 2; and/or threonine at a position analogous to position 188 in SEQ ID NO. 2.

The nucleic acid molecules and enzymes Δ of the invention may be expressed in plant material, cells, tissues, or whole plants⁹Desaturase enzymes increase the amount of unusual fatty acids in the plant material, cell, tissue, or whole plant relative to the amount observed in wild-type plants of the same species. An alternative embodiment of the invention includes a method for increasing the amount of unusual fatty acids in a plant material, cell, tissue, or whole plant comprising transforming a plant material, cell, tissue, or whole plant with a nucleic acid molecule of SEQ ID NO. 1 such that the amount of unusual fatty acids in the plant material, cell, tissue, or whole plant is increased.

In a preferred embodiment, the plant material, cell, tissue, or whole plant transformed by the disclosed methods further comprises one or more means for increasing the level of 16:0-ACP in the plant material, cell, tissue, or whole plant (means). In certain embodiments, the means for increasing the level of 16:0-ACP in a plant material, cell, tissue, or whole plant may be: expressing an extraplastidial desaturase; inhibition of KASII, for example, by introducing a mutation in the fab1 gene; and/or reducing elongation of 16:0 fatty acids, for example by introducing a mutation in the fae1 gene.

The methods disclosed herein can be performed, for example, on plants of the genus arabidopsis, or plant material derived from plants. A particular embodiment relates to a method for creating or regenerating a genetically engineered plant comprising an increased amount of unusual fatty acids in the plant compared to a wild type plant of the same species, comprising transforming plant material with a nucleic acid molecule of SEQ ID NO 1; and culturing the transformed plant material to obtain the plant. Also disclosed are plants, plant materials, plant cells, and seeds obtained by the foregoing methods.

Abbreviation II

x:yΔ^zFatty acids containing x carbons and y double bonds at position z from the carboxy terminus

ACP acyl carrier proteins

COA coenzyme A

KASII beta-ketoacyl-ACP synthase II

FA fatty acid

FAS fatty acid synthesis

FAME fatty acid methyl ester

WT wild type

Term of

Fatty acid: as used herein, the term "fatty acid" refers to long chain aliphatic acids (alkanoic acids) of varying chain lengths from about C12 to C22, although both longer and shorter chain length acids are known. The structure of the fatty acids is given by the symbols x: y.DELTA^zWhere "x" is the total number of carbon (C) atoms in a particular fatty acid and "y" is the number of double bonds in the carbon chain as counted from the carboxy terminus of the acid in the "z" position.

Unusual fatty acids: for the purposes of the present invention, unusual fatty acids are those whose synthesis in the natural system is initiated by the enzyme variant desaturase modification of the FAS intermediate.

Metabolic pathway: the term "metabolic pathway" refers to a series of chemical reactions present within a cell that are catalyzed by an enzyme to effect conversion of a metabolite or initiation of another metabolic pathway. A metabolic pathway may involve several or many steps, and may compete with different metabolic pathways for a particular reaction substrate. Similarly, a product of one metabolic pathway may be a substrate of another metabolic pathway.

Metabolic engineering: for the purposes of the present invention, "metabolic engineering" refers to the design of a rational strategy to alter one or more metabolic pathways in a cell such that stepwise modification of the initial substrate to a product with the desired precise chemical structure is achieved within the overall scheme of the overall metabolic pathway running in the cell.

Desaturase enzymes: as used herein, the term "desaturase" refers to a polypeptide that can desaturate (i.e., introduce a double bond) one or more fatty acids to produce a fatty acid or precursor of interest. Plant soluble fatty acid desaturase enzymes introduce double bond regiospecificity into saturated acyl-ACP substrates. This reaction involves the activation of molecular oxygen through a two-electron reducing diiron center coordinated by a four-helix bundle forming the core of the desaturase construct. Of particular interest herein is Δ⁹Desaturase enzymes.

Δ⁹-18:0¹ACP desaturases are required by all plants to maintain membrane fluidity. Although this enzyme primarily desaturates stearoyl-ACP, it is also active to a minor extent in the case of palmitoyl-ACP.

Variant desaturases: as used herein, the term "variant desaturase" encompasses those desaturases that have a specific activity profile consistent with a role in the production of unusual fatty acids. Variant desaturases can be isolated from an organism or engineered via directed evolution programs.

Progeny plants: for the purposes of the present invention, "progeny plant" refers to any plant, or plant material obtained therefrom, that can be obtained by plant breeding methods. Plant breeding methods are well known in the art and include natural breeding, artificial breeding, selective breeding involving analysis of DNA molecular markers, transgenics, and commercial breeding.

Plant material: as used herein, the term "plant material" refers to any cell or tissue obtained from a plant.

Nucleic acid molecule (A): polymeric forms of nucleotides, which may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the foregoing. A nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of any type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of ordinary skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (moieties) (e.g., peptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators (alkylators), and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplex, triplex, hairpin, circular, and padlock conformations.

Operatively connected: the first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. In recombinant production, operably linked nucleic acid sequences are generally contiguous and, where necessary to join two protein coding regions, in the same reading frame. However, the nucleic acids need not be contiguous for operable linkage.

An adjusting element: as used herein, the term "regulatory element" refers to a nucleic acid molecule having gene regulatory activity; i.e., a nucleic acid molecule having the ability to affect transcription or translation of an operably linked transcribable nucleic acid molecule. Regulatory elements such as promoters, leaders, introns and transcription termination regions are non-coding nucleic acid molecules with gene regulatory activity that play a part in the overall gene expression of living cells. Thus, isolated regulatory elements that function in plants can be used to modify plant phenotypes via molecular engineering techniques. "regulatory element" means a series of nucleotides that determine whether, when, and at what level a particular gene is expressed. The regulatory DNA sequences interact specifically with regulatory proteins or other proteins.

As used herein, the term "gene regulatory activity" refers to a nucleic acid molecule capable of affecting transcription or translation of an operably linked nucleic acid molecule. An isolated nucleic acid molecule having gene regulatory activity can provide temporal or spatial expression or modulate the level and rate of expression of an operably linked nucleic acid molecule. An isolated nucleic acid molecule having gene regulatory activity can include a promoter, intron, leader, or 3' transcription termination region.

A promoter: as used herein, the term "promoter" refers to a nucleic acid molecule that involves recognition and binding of RNA polymerase II or other proteins such as transcription factors (trans-acting protein factors that regulate transcription) to initiate transcription of an operably linked gene. The promoter may itself contain sub-elements such as cis-elements or enhancer domains that affect the transcription of an operably linked gene. A "plant promoter" is a native or non-native promoter that is functional in a plant cell. Plant promoters may be used as 5' regulatory elements to regulate the expression of one or more operably linked genes. Plant promoters may be defined in their temporal, spatial, or developmental expression patterns. The nucleic acid molecules described herein may comprise a nucleic acid sequence comprising a promoter.

Sequence identity: the similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is usually expressed in terms of percent identity; the higher the percentage, the more similar the two sequences. Methods for aligning and comparing sequences are described in detail below.

Analogous positions in the amino acid sequence: nucleic acid and amino acid sequences can be aligned by the methods described in the following paragraphs. In an alignment, a position in one sequence is in a "similar position" to a position in an aligned sequence if the sequences are identical within the consensus sequence.

Methods for aligning and comparing sequences are well known in the art. Various programs and alignment algorithms are described in: smith and Waterman, Adv.appl.math.2:482,1981, Needleman and Wunsch, J.mol.biol.48:443,1970, Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444,1988, Higgins and Sharp, Gene 73: 237-. Altschul et al, J.mol.biol.215: 403-.

The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) is available on the internet (in BLAST. NCBI. nlm. nih. gov/BLAST. cgi), which is used in conjunction with sequence analysis programs such as blastp and blastn. A description of how to determine sequence identity using this program is available on the internet via NCBI in blast.

For comparison of amino acid sequences, the "BLAST 2 sequence" function (bl2seq) of the BLAST program was employed using the default parameters. Certain parameters may be adjusted within the judgment of those skilled in the art, for example to provide a mismatch penalty or a match reward.

Transformed: as used herein, the term "transformed" refers to a cell, tissue, organ, or organism that has received an introduction of a foreign nucleic acid molecule, such as a construct. The introduced nucleic acid molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced polynucleotide molecule is inherited by subsequent progeny. "transgenic" or "transformed" cells or organisms also include progeny of the cells or organisms and progeny that have been generated from breeding programs that employ such transgenic plants as parents, for example in crosses, and exhibit altered phenotypes resulting from the presence of foreign nucleic acid molecules.

Systemic metabolic engineering methods for accumulation of unusual fatty acids in host cells, tissues, or organisms

A. Overview

One embodiment of the invention includes metabolic engineering of omega-7 Fatty Acids (FA) (which are formed from palmitoleic acid (16: 1. DELTA.) -in, for example, plant seeds⁹) And vaccenic acid (18: 1. delta¹¹) Constitutes) a systematic approach to accumulation. To illustrate the method for intercepting the newly synthesized fatty acid stream in plastids, Com25 was expressed under the control of a seed-specific phaseolin promoter, a source of directed evolution program to enhance ricinus communis delta⁹16:0 ACP desaturase with 16: 0-desaturase Activity of 18: 0-desaturase. Any seed-specific promoter may be used in the embodiments disclosed herein. This method increased accumulation of omega-7 FA from less than 2% in Wild Type (WT) to about 14% in Com25 transformants.

In other exemplary methods, expression of Com25 in the fab1/fae1 double mutant (which is impaired in plastid and extraplastid fatty acid elongation, respectively) resulted in increased accumulation of omega-7 FA to about 50%. Furthermore, the introduction of additional Com25 under the control of LTP170 promoter increased omega-7 FA accumulation to about 58%, suggesting that the desaturase activity limitation that may result from its low turnover rate has been overcome. The phaseolin: Com25 construct was expressed in a series of KASII deficient backgrounds, and a proportional increase in omega-7 FA content with 16:0 content of up to about 30% was observed, with an overall omega-7 FA accumulation of up to about 55%. Interestingly, the transgene accumulating 56% omega-7 FA still contains about 19%16:0, more than twice that of WT plants. The expression of an in vitro 16:0 desaturase was investigated to intercept the in-transit flux of 16:0 to triacylglycerols. Coexpression of plastidic and extraplastidial desaturases and inhibition of KASII in the fab1/fae1 double mutant background resulted in an increase in accumulation of omega-7 FA from about 2% in the WT to about 71% in the best engineered line, equal to that present in the catclaw seed.

Omega-7 FA was chosen as the target because its synthesis in the natural system is initiated by the enzyme variant desaturase modification of the FAs intermediate, as is the synthesis of other, unusual FAs. Cahoon et al, (1997) Plant mol.biol.33:1105-10; and Cahoon et al, (1998) Plant physiol.117(2): 593-8. In addition, omega-7 FA has potential commercial applications as a polymer feedstock, while having physical properties similar to naturally occurring unsaturated fatty acids.

By comparing a previously unreported⁹Introduction of the 16: 0-Acyl Carrier Protein (ACP) desaturase Com25 into the model plant Arabidopsis thaliana initiated metabolic engineering studies under the control of a seed-specific promoter. Methods to redirect carbon flux into omega-7 FA were explored by selecting mutant backgrounds containing elevated 16:0 levels and by co-expressing constructs designed to divert carbon flux into target fatty acids by affecting competition for substrates. The enzyme extra-plastidial desaturase was expressed to desaturate residual 16:0 after export from the plastids.

Co-expression of plastidic and extra-plastidial desaturases and inhibition of KASII in the fab1/fae1 background resulted in an increase in accumulation of omega-7 FA from less than 2% up to about 71% in the WT, which is higher than that present in the milkweed (Asclepias) and equal to that present in the catclaw flower seed.

16:0-ACP, the precursor of omega-7 fatty acids, at the first branch point of fatty acid biosynthesis, which is competed by FatB thioesterases and KASII elongases (elongase); and introduction of a 16:0 ACP desaturase makes this three-way competitive. Inhibition of KASII and FATB is an effective way to reduce substrate competition and increase omega-7 accumulation. The increase in omega-7 FA accumulation was saturable in the host line at about 30% since above this level desaturases were limiting. Increasing the dosage of Com25 by expressing a second copy under the control of a seed-specific promoter further increased the accumulation of omega-7 fatty acids. However, seeds with high omega-7 FA accumulation also contained levels of 16:0 ranging from about 20%, presenting an opportunity to desaturate the extra-plastid 16: 0. Expression of both extra-plastidial desaturases increased omega-7 FA accumulation, resulting in an approximately 50% reduction in 16:0 in mature seeds.

As described in more detail below, systemic metabolic engineering may be a successful strategy for engineering unusual fatty acid accumulation levels comparable to those observed in natural sources, as the best fab1/fae1/Com25/Ln Δ 9D and An Δ 9D lines accumulate 71% ω -7FA, substantially (substitially) at higher levels than in milkweed and equal to the levels present in the catclaw flower seed. B. Nucleic acids

The nucleic acid sequences in some embodiments of the invention show increased percent identity when aligned with SEQ ID No. 1. Particular nucleic acid sequences within these and other embodiments may comprise sequences having, for example, at least 60%,65%,70%,75%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%96%,97%,98% or 100% identity to SEQ id No. 2. It is understood by those of ordinary skill in the art that nucleic acid molecules can be modified without substantially changing the amino acid sequence of the encoded polypeptide, e.g., according to codon degeneracy with permissible nucleotide substitutions.

In some embodiments, the nucleic acid molecule of the invention comprises a promoter. The promoter may be selected based on the cell type that will be subjected to insertion of the vector construct. Promoters that function in bacteria, yeast and plants are well known in the art. Promoters may also be selected based on their regulatory characteristics. Examples of such features include enhanced transcriptional activity, inducibility, tissue specificity, and developmental stage specificity. Inducible, constitutively active, temporally regulated, and spatially regulated promoters of viral or synthetic origin have been described in plants (see, for example, Poszkowski et al, (1989) EMBO J.3:2719; Odell et al, (1985) Nature 313:810; Chau et al, (1989) Science 244: 174-81).

Constitutive promoters often used include, for example, the CaMV 35S promoter, the enhanced CaMV 35S promoter, the figwort mosaic virus promoter, the mannopine synthase promoter, the nopaline synthase promoter, and the octopine synthase promoter.

Useful inducible promoters include, for example, promoters induced by salicylic acid or polyacrylic acid induced by application of safeners (substituted benzenesulfonamide herbicides), heat shock promoters, nitrate inducible promoters derived from spinach nitrate reductase transcribable nucleic acid molecule sequences, hormone inducible promoters, and light inducible promoters associated with the small subunit of RuBP carboxylase and LHCP family.

Examples of useful tissue-specific, developmentally-regulated promoters include the beta-conglycinin 7S alpha promoter and seed-specific promoters. Plant functional promoters useful for preferential expression in seed plastids include those from proteins involved in fatty acid biosynthesis in oilseeds and from plant storage proteins. Examples of such promoters include the 5' regulatory region from transcribable nucleic acid molecule sequences such as phaseolin, napin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and oleosin. Another exemplary tissue specific promoter is a seed tissue specific lectin promoter.

Other useful promoters include nopaline synthase, mannopine synthase, and octopine synthase promoters, which are carried on tumor inducing plasmids of Agrobacterium tumefaciens (Agrobacterium tumefaciens); cauliflower mosaic virus (CaMV)19S and 35S promoters; an enhanced CaMV 35S promoter; the figwort mosaic virus 35S promoter; a light-inducible promoter from the small subunit of ribulose-1, 5-bisphosphate carboxylase (ssRUBISCO); the EIF-4A promoter from tobacco (Mandel et al, (1995) Plant mol.biol.29: 995-1004); a corn sucrose synthase; a corn alcohol dehydrogenase I; corn light harvesting composite (lighting composition); a corn heat shock protein; chitinase promoter from arabidopsis; LTP (lipid transfer protein) promoter; petunia chalcone isomerase; glycine-rich protein 1 of beans; potato patatin; a ubiquitin promoter; and actin promoter. Preferably, useful promoters are seed-selective, tissue-selective, or inducible. Seed-specific regulation is discussed in e.g. EP 0255378.

C. Amino acid sequence

Amino acid sequences according to some embodiments of the invention show increased percent identity when aligned with SEQ ID No. 2. Particular amino acid sequences within these and other embodiments may comprise sequences having, for example, at least 70%,75%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%96%,97%,98% or 100% identity to SEQ ID No. 2. In many embodiments, amino acid sequences having the foregoing sequence identities when aligned with SEQ ID NO:2 encode peptides having Δ 9-18:0 ACP desaturase enzyme activity.

D. Change Com 25: 5 mutations

Aspects of the invention concern novel genetically engineered desaturases derived from parent ricinic desaturase enzymes. In a specific embodiment, the genetically engineered desaturase is Com 25. Com25 differs from the parent ricinic desaturase in the following 5 amino acid position: M114S, T117R, L118C, P179L, and G188T (numbered according to mature castor desaturase PDB entry 1 AFR). In other embodiments, the genetically engineered desaturase may comprise one or more of these 5 mutations in Com 25. For example, a genetically engineered desaturase can differ from a parent ricinic desaturase at the following positions: m114, T117, L118, P179, G188, M114 and T117, M114 and L118, M114 and P179, M114 and G188, T117 and L118, T117 and P179, T117 and G188, L118 and P179, L118 and G188, P179 and G188, M114, T117 and L118, M114, T117 and P179, M114, T1117 and G188, M114, L118 and P179, M114, L118 and G188, M114, P179 and G188, T117, L118 and P179, T117, L118 and G188, T117, P179 and G188; or L118C, P179L and G188T.

E. A host comprising elevated 16:0 fatty acid levels.

In preferred embodiments, a host cell or material transformed with Com25 may exhibit elevated 16:0 fatty acid levels. Host cells can exhibit elevated 16:0 fatty acid levels, for example, by causing 16:0-ACP metabolism to be reduced in those host cells. Other methods of increasing the level of 16:0 fatty acids in the host cell may be used, and one skilled in the art may select such methods by applying judgment. Examples of methods for increasing the level of 16:0 fatty acids in a host cell include, but are not limited to: 1) expressing an in vitro desaturase in a host cell; 2) inhibition of KASII in a host cell, for example, by introducing a mutation in the fab1 gene; and 3) reduce 16:0 fatty acid elongation, e.g., by introducing a mutation in the fae1 gene.

F. Method for genetic transformation of plant material

The invention also relates to methods of producing transformed cells comprising one or more nucleic acid molecules comprising a nucleic acid sequence that is at least 60% identical to SEQ id No. 1. Such nucleic acid molecules may also comprise, for example, non-coding regulatory elements, such as promoters. Other sequences may also be introduced into the cell along with non-coding regulatory elements and transcribable nucleic acid molecule sequences. These other sequences may include a 3 'transcription terminator, a 3' polyadenylation signal, other untranslated sequences, transport or targeting sequences, selectable markers, enhancers, and operators.

Generally, the transformation process comprises the following steps: selecting a suitable host cell, transforming the host cell with the recombinant vector, and obtaining the transformed host cell.

Techniques for introducing DNA into cells are well known to those skilled in the art. In general, these methods can be divided into 5 classes: (1) chemical Methods (Graham and Van der Eb (1973) Virology54(2):536-9; Zatlouka et al, (1992) Ann.N.Y.Acad.Sci.660:136-53), (2) physical Methods such as microinjection (Capechi (1980) Cell22(2):479-88), electroporation (Wong and Neumann, Biochim.Biophys.Res.Commun. (1982)107(2):584-7; Fromm et al, (1985) Proc.Natl.Acad.Sci.USA 82(17):5824-8; U.S. Pat. No.5,384,253), and particle acceleration (Johnson and Tang (1994) Cell biol.43(A):353-65; Fynan et al, (1993) Proc. Acad.90.90: 82; Warole et al. (1982) 23: 82; Waglen et al, (1986) receptor (1984) 2; Reg.90: 82; Reg.52-23; Reg.90: 82; Reg.23; Reg.90: 82; Reg.3; Reg.23; Reg.3; Reg.90; Reg.3: 18; Reg.3: 82; Reg.3; Reg.90; 1985), (1992) natl, Acad, Sci, USA 89(13): 6099-; and (5) bacteria-mediated mechanisms, such as with Agrobacterium. Alternatively, the nucleic acid may be introduced directly into the pollen by direct injection into the reproductive organs of the Plant (Zhou et al, (1983) Methods in Enzymology 101:433; Hess (1987) Intern. Rev. Cytol.107:367; Luo et al, (1988) Plant mol. biol. reporter 6:165; Pena et al, (1987) Nature 325: 274). Other transformation methods include, for example, protoplast transformation, as exemplified in U.S. Pat. No.5,508,184. Nucleic acid molecules can also be injected into immature embryos (Neuhaus et al, (1987) Theor. appl. Genet.75: 30).

The most commonly used methods for transforming plant cells are: agrobacterium-mediated DNA transfer methods (Fraley et al, (1983) Proc. nat. Acad. Sci. USA 80:4803) (as exemplified in U.S. Pat. No.5,824,877; U.S. Pat. No.5,591,616; U.S. Pat. No.5,981,840; and U.S. Pat. No.6,384,301) and biolistic or microprojectile bombardment-mediated methods (i.e., gene guns) (such as those described in U.S. Pat. No.5,550,318; U.S. Pat. No.5,538,880; U.S. Pat. No.6,160,208; U.S. Pat. No.6,399,861; and U.S. Pat. No.6,403,865). In general, nuclear transformation is desirable, but where specific transformation of plastids is desired, such as chloroplasts or amyloplasts, plant plastids can be transformed with microparticle-mediated delivery of desired nucleic acid molecules to certain plant species, such as arabidopsis, tobacco, potato, and brassica species.

Agrobacterium-mediated transformation is achieved via the use of genetically engineered Agrobacterium belonging to the genus Agrobacterium. Several agrobacterium species mediate the transfer of specific DNA, referred to as "T-DNA," which can be genetically engineered to carry any desired portion of DNA into many plant species. The major events that carry out the T-DNA mediated pathogenesis process are: induction of virulence genes, and processing and transfer of TDNA. This procedure is the subject of many reviews (Ream (1989) Ann. Rev. Phytopathol.27: 583. about.618; Howard and Citovsky (1990) Bioassays 12:103-8; Kado (1991) Crit. Rev. plant Sci.10:1-32; Zambryski (1992) Annual Rev. plant Physiol.plant mol.biol.43:465-90; Gelvin (1993) In Transgenic Plants, Kung and Wu eds, Academic Press, San Diego, pp.49-87; Binns and Howitz (1994) In bacterial Pathos. Plants and Animals, Benlin: Sprag, pp.119-38; Hoygen. about.1994: Phytopho. about.26. and Cell: 5832; Cell: Zuk.32; Cell: Zuk.3: 583. about.32; Cell: 1995: Zymyski & gt, 1995).

To select or score transformed plant cells (regardless of the method of transformation), the DNA introduced into the cells may contain genes that function in regenerable plant tissue to produce compounds that confer resistance to compounds that are otherwise toxic to the plant tissue. Genes of interest for use as selection, screening, or scoring criteria include, but are not limited to, GUS, Green Fluorescent Protein (GFP), luciferase, and antibiotic or herbicide tolerance genes. Examples of antibiotic resistance genes include those conferring resistance to penicillin, kanamycin (and neomycin, G418, bleomycin); methotrexate (and trimethoprim); chloramphenicol; and tetracycline resistance genes. For example, glyphosate (glyphosate) resistance may be conferred by a herbicide resistance gene. Della-Cioppa et al, (1987) Bio/Technology 5: 579-84. Other selection devices may also be implemented, including but not limited to tolerance to phosphinothricin, bialaphos, and the forward selection mechanism, Joersbro et al, (1998) mol. breed.4:111-7, and are considered to be within the scope of the present invention.

Transformed cell mature plants identified by selection or screening and cultured in a suitable medium to support regeneration may then be allowed to mature.

The presently disclosed methods can be used with any transformable plant cell or tissue. As used herein, transformable cells and tissues include, but are not limited to, those cells or tissues that are capable of further proliferation to produce plants. Those skilled in the art recognize that many plant cells or tissues are transformable in that they can form differentiated plants upon insertion of exogenous DNA and appropriate culture conditions. Tissues suitable for these purposes may include, but are not limited to, immature embryos, scutellum (scutellar) tissue, suspension cell cultures, immature inflorescences, shoot meristems, node explants, callus tissue, hypocotyl tissue, cotyledons, roots, and leaves.

Regeneration, development, and culture of plants from transformed plant protoplasts or explants is known in the art. Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, (eds.) (academic Press, Inc., San Diego, Calif.; Horsch et al, (1985) Science227: 1229-31. This regeneration and growth method generally comprises the following steps: transformed cells are selected and those cells are cultured to the stage of normal embryonic development through the rooted plantlet stage. Similarly, transgenic embryos and seeds are regenerated. In this method, the transformants are generally cultured in the presence of a selection medium which selects for successfully transformed cells and induces regeneration of plant shoots (shoots). Fraley et al, (1993) Proc. Natl. Acad. Sci. USA 80: 4803. These shoots are usually obtained within 2 to 4 months. Thereafter, the resulting transgenic rooted shoots are planted in a suitable plant growth medium such as soil. Cells that survive exposure to the selective agent, or cells that have scored positive in the screening assay, can be cultured in a medium that supports plant regeneration. The shoots can then be transferred to a suitable root induction medium containing a selective agent and an antibiotic that prevents bacterial growth. Many shoots will form roots. These are then transplanted into soil or other media to allow for continued development of roots. Generally, the methods as outlined above will vary depending on the particular plant line employed, and therefore, the details of the methods are within the discretion of those skilled in the art.

The regenerated transgenic plant may be self-pollinated to provide a homozygous transgenic plant. Alternatively, pollen obtained from regenerated transgenic plants can be crossed with an inbred line of a non-transgenic plant, preferably an agronomically important species. In contrast, pollen from non-transgenic plants can be used to pollinate regenerated transgenic plants.

The transgenic plant may transmit the transformed nucleic acid sequence to its progeny. Preferably, the transgenic plant is homozygous for the transformed nucleic acid sequence and, after and as a result of sexual reproduction, inherits said sequence to all its progeny. Progeny can be cultured from seeds produced by the transgenic plant. These additional (additional) plants can then be self-pollinated to generate a true plant breeding line.

Progeny from these plants can be evaluated for gene expression, and so forth. Gene expression can be detected by several common methods such as Western blotting, Northern blotting, immunoprecipitation, and ELISA (enzyme-linked immunosorbent assay). Transformed plants can also be analyzed for the presence of the introduced DNA and the expression levels and/or fatty acid profiles conferred by the nucleic acid molecules and amino acid molecules of the invention. Those skilled in the art are aware of the many methods available for analyzing transformed plants. For example, methods for plant analysis include, but are not limited to, Southern or Northern blots, PCR-based methods, biochemical assays, phenotypic screening, field evaluation, and immunodiagnostic assays.

Methods for the specific transformation of dicotyledonous plants are well known to those skilled in the art. Regeneration of transformed plants using these methods has been described for many crops, including but not limited to members of the arabidopsis genus, cotton (Gossypium hirsutum), soybean (glycine emax), peanut (Arachis hypogaea), and members of the brassica genus. Cotton (U.S. Pat. No.5,004,863; U.S. Pat. No.5,159,135; U.S. Pat. No.5,518,908); soybean (U.S. Pat. No.5,569,834; U.S. Pat. No.5,416,011; McCabe, et al, (1988) Biotechnology 6:923; Christou, et al, (1988) Plant physiol.87: 671-4; Brassica (U.S. Pat. No.5,463,174); peanut (Cheng, et al, (1996) Plant Cell Rep.15:653-7; McKently, et al, (1995) Plant Rep.14: 699-.

Methods for transforming monocots are also well known in the art. Many crops, including but not limited to barley (Hordeum vulgare); maize (Zea mays); oats (Avenasativa); orchard grass (Dactylis megrata); rice (Oryza sativa), including indian and japanese varieties; sorghum (Sorghum bicolor); sugar cane (sugarcane (Saccharumsp)); festuca arundinacea (Festuca arundinacea)); grassland grass (turfgrass) species (e.g., stolonifera (agrostistolonifera), Poa pratensis (Poa pratensis), blumea conifera (stenotrophatum); wheat (wheat aestivum); alfalfa (alfalfa) describes transformation and plant regeneration using these methods. It will be apparent to those skilled in the art that a number of transformation methods can be used and modified to produce stable transgenic plants for a number of target crops of interest.

Any plant can be selected for use in the presently disclosed methods. Preferred plants modified according to the invention include Arabidopsis thaliana (Arabidopsis thaliana) thaliana, borage (Borago) species), brassica campestris (Canola), Ricinus communis (Ricinus communis), cacao (Theobroma cacao), maize (maize), cotton (Gossypium) species, Crambe species, Cuphea species, flax (flax) species, atriplexus species, lespedeza (Lesquerella) and spermaceti (Limnanthes) species, linala, eclipta alba (xerophthalmus) species, Oenothera (Oenothera) species, Oenothera species, olives (oliv) (oleracea) species, palmetto (aeis) species, Arachis arachidis (arachidis), Arachis sativa (arachidis), soybean (soybean) species, sunflower (soybean) species, soybean (soybean) species, soybean (soybean species, soybean, Tobacco (Nicotiana species), Vernonia (Vernonia) species, wheat (Triticum species), barley (Hordeum species), rice (Oryza species), oats (Avena species), Sorghum (Sorghum species) (Sorghum spp.), rye (Secale species) or other members of the grass family (Gramineae).

It will be apparent to those skilled in the art that a number of transformation methods can be used and modified to produce stable transgenic plants for a number of target crops of interest.

G. Transgenic seed

In some embodiments of the invention, the transgenic seed comprises a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID No. 2. In these and other embodiments, the transgenic seed comprises a nucleic acid sequence that is at least 60% identical to SEQ ID NO. 1. In certain embodiments, the seeds of the invention exhibit elevated unusual fatty acids, e.g., omega-7 fatty acids, such as 16:1 Δ⁹-and/or 18: 1. delta¹¹And (4) horizontal. Seeds can be harvested from fertile transgenic plants and used to grow progeny of transformed plants of the invention, including hybrid plant lines comprising a nucleic acid sequence according to the invention and another gene or nucleic acid construct of interest.

The following examples are provided to illustrate certain specific features and/or embodiments. These examples should not be construed as limiting the invention to the specific features or embodiments described.

Examples

Example I: materials and methods

Plant growth and transformation

Arabidopsis (Arabidopsis) plants are grown in soilUnder continuous exposure to 300 micro-einstein light (1 micro-einstein =1mol light) at E7/2^TMCultured in a controlled environment growth chamber (Conviron). Plants were transformed with Agrobacterium tumefaciens strain GV3101 according to the method of Clough and Bent (1998) Plant J.16(6): 735-43. We passed the use of an X5LED from a camera with a 25A red filter^TMFluorescent identification of individual T carrying transgenes following Green illumination of flashlight (Inova)₁Seed (Stuitje et al (2003) plant Biotechnol. J.1(4): 301-9). Pidkowich et al, (2007) Proc.Natl.Acad.Sci.USA 104(11): 4742-7). Filters FITC 535 and FITC 515 were used, respectively, using a filter equipped with Olympus U-LH100HG^TMWILD for lighting system^TMM3Z dissecting microscope distinguishes between seeds carrying Zs-green and Ds-red markers. Seed-specific expression is achieved by placing the construct under the control of the phaseolin seed storage protein promoter or the LTP170 promoter. Slightom et al (1983) Proc. Natl. Acad. Sci. USA80(7): 1897-. Source of Com25

Com25 is castor delta⁹A variant of an 18:0 ACP desaturase derived from a combinatorial saturation mutagenesis/selection procedure designed to identify variants with improved activity against acyl chains less than 18C in length. Whittle and Shanklin (2001) J.biol.chem.276(24): 21500-5. Com25 differs from the parent ricinic desaturase at the following 5 amino acid positions: M114S, T117R, L118C, P179L and G188T (numbered according to mature castor desaturase PDB entry 1 AFR).

Plastid constructs

Phas: Com 25. The entire reading frame of the ricinic variant Com25 engineered to contain its authentic transit peptide and flanked by 5 'PacI and 3' XhoI restriction sites was cloned into the corresponding site of plasmid pDs-Red-Phas (Pidkowich et al, (2007) proc.natl.acad.sci.usa 104(11):4742-7) (with Ds-Red marker) to create Phas: Com25 (fig. 5).

Phas: Com25, LTP170: Com 25. The LTP170 promoter was amplified from Arabidopsis genomic DNA using primers P17-5 'BamHI (GGGATCCCCGGGTTGACATTTTTA CCTTTTT; SEQ ID NO:3) and P17-3' PacI (GGTTAATTAAGTCTTCAAACTCTAGGA; SEQ ID NO:4), subcloned into pGEMT-Easy, after which the BamHI-PacI fragment was isolated and cloned into the corresponding site of plasmid pDs-Red-Ph as: Com25 (described above) to create pDs-Red-LTP170: Com 25. A fragment containing Com25 and the phaseolin terminator was excised using BamHI and EcoRV and cloned into the BamHI and SmaI restriction sites within vector pDs-Red-LTP170-Com25 to create Phas: Com25/LTP170: Com25 (FIG. 5).

Phas Fab 1-HPAS. This construct was created in two steps; first, Phas: FatB-HP was constructed, after which the antisense portion of the FatB gene was inserted to replace the portion of the Fad2 intron that separates the sense and antisense portions of the FatB gene containing the hairpin. To achieve this, a 150bp Arabidopsis FatB3 ' UTR was amplified from genomic DNA in both sense (using primer FatB-hps-5 ' PstIGGGCTGCAGAAACAAGTTTTCGGCCACAACC C; SEQ ID NO:5 and FatB-hps-3 ' XhoICCCCTCGAGACATCAGAATTCGTAATGAT; SEQ ID NO:6) and antisense (using primer FatB-hpa-5 ' NheIGGGGCTAGCTAGCAAGTTTCGGCCACCACACCC; SEQ ID NO:7 and FatB-hpa-3 ' PacICCCTTAAATTAAACAATTCGTGAAT; SEQ ID NO:8) orientations. These fragments were restricted with PstI/XhoI and NheI/PacI and used to replace the 5' UTR sense and antisense portions of FabI at their equivalent sites in pGEM-T-Easy-HTM3(Pidkowich et al, (2007) Proc. Natl. Acad. Sci. USA 104(11):4742-7) to create the intermediate plasmid pGEM-T-Easy-HTM 4. To create a 300bp antisense portion of the FatB coding region, the fragment was amplified with the primers FatB-Exon-5 'Sp-Bam (CCACTAGTGGATCCACCTCTGCTACGTCGTCATT; SEQ ID NO:9) and FatB-Exon-3' Bg-Sal (GGAGATCTGTCGACGTAGTTATAGCAGCAAGA AG; SEQ ID NO:10), and the Fad 2-intron after restriction with BglII and SpeI was replaced with a BamHI and SalI restricted fragment to create pGEM-T-Easy-HTM 5.

The assembled HPAS fragment was excised by using PacI and XhoI and cloned into pZs-Green-Phas: Com25 (plasmid pDs-Red-Phas: Com25 described above, in which the Green fluorescent protein marker pCVMV: Zs-Green 25 had been used^TM(Clonetech) replaced the fluorescent marker pCVMV: Ds-Red) in the equivalent site to create plasmid Phas: FatB-HPAS (FIG. 5).

Phas: AnD9d, Phas: LnD 9D. Two fungal acyl-CoA D9 desaturases were combined in plasmid pDAB7318, where both genes were driven by a seed-specific Phas promoter from kidney bean (Phaseolus vulgaris). The first gene in the construct was an acyl-CoA Δ 9-desaturase from Aspergillus nidulans (Aspergillus nidulans) that was redesigned and synthesized for optimal expression in plants (U.S. patent application 20080260933a1) and fused to the 3 'untranslated region and the 3' MAR of the phaseolin gene from phaseolus vulgaris. The second desaturase gene in this construct is an acyl-CoA Δ 9-desaturase from lumenal cerebellum nodorum (Leptosphaeria nodorum), which is also redesigned and synthesized for plant expression, and fused to the untranslated region of agrobacterium tumefaciens ORF 233'. Sequencing of items by Micrococcus nodorum (Broad Institute of Harvard and MIT) by homology search against Septoria nodorum (S.nodorum)(http://www.broad.mit.edu))The published genomic sequences identify this desaturase. It was shown by the ole1 mutant of the complementing Saccharomyces cerevisiae (Saccharomyces cerevisiae) that it has a preference for desaturation of palmitate. Phas: Fab1-HPAS-Phas: Com 25. To simplify the gene stacking experiments, plasmid Phas: Fab1/HPAS-Phas: Com25 was constructed to combine Com25 expression with KASII inhibition. To accomplish this, the phaseolin terminator was isolated from Phas: Com25 and cloned into the intermediate vector pBL (which has the EcoRV-EcoRV fragment containing the phaseolin promoter driving Com 25) to create pBL-Phas: Com 25-PhasTer. This Com25 expression cassette was excised using flanking EcoRI-EcoRI restriction sites and cloned into the corresponding sites within Phas: Fab1-HPAS to create Phas: Fab1-HPAS-Phas-Com 25. See fig. 5.

Fatty acid synthesis

To analyze the fatty acids of a single seed, Fatty Acid Methyl Esters (FAME) were prepared by incubating the seeds with 0.2M trimethylthiohydroxide in methanol. Butte et al, (1982) anal. Lett.15(10): 841-50. To analyze bulk seed similarly, FAME was prepared by placing the seed at 0.5mLBCl₃In 80 ℃ temperature 1 h incubation, with 1mL hexaneTaking, then in N₂And (5) drying. By HP6890^TMGas chromatography-flame ionization detectors (Agilent Technologies), or HP5890 equipped with a 60mx 250 μ M SP-2390 capillary column (Supelco)^TMThe FAME was analyzed by gas chromatography-mass spectrometer (Hewlett-Packard). The furnace temperature was increased from 100 ℃ to 240 ℃ at a flow rate of 1.1 mL/min at a rate of 15 ℃ per minute during the analysis. By HP5973^TMMass spectrometry was performed using a mass selective detector (Hewlett-Packard). We determined the double-stranded position of the monounsaturated FAME by dimethyl sulfide derivatization. Yamamoto et al (1991) chem. and Phys. lipids 60(1): 39-50.

Example II: com25 expression in WT Arabidopsis thaliana

Several plants, including milkweed (Hopkins and Chisholm (1961) Can.J.Biochem.Physiol.39:829-35) and Cat claw (Chisholm and Hopkins (1965) J.Am.Oilchem.Soc.42:49-50) have been reported to accumulate omega-7 FA in their seeds. Genes encoding the enzyme desaturase responsible for palmitate synthesis (palmiteleate) have been isolated. The activity of the corresponding recombinant desaturase enzymes (Cahoon et al, (1997) Plant mol. biol.33:1105-10; Cahoon et al, (1998) Plant physiol.117(2):593-8) was as low as that reported for the prototype stearoyl-ACP desaturase. Whittle and Shanklin (2001) J.biol.chem.276(24): 21500-5. We compared the effect of expressing several variants of the enzyme milkweed and buttercup desaturase and ricinus desaturase, including desaturase from ricinus variant 5.2 and Com25, derived from Com25 designed to enhance ricinus delta in arabidopsis thaliana⁹Directed evolution experiments on the 16: 0-desaturase activity of 18: 0-desaturases (Whittle and Shanklin (2001) J.biol.chem.276(24): 21500-5). In these experiments, Com25 outperforms other desaturase enzymes (Bondauk et al, (2007) Plant Breeding126:186-94) and 16: 1. delta. in WT Arabidopsis thaliana⁹And their extension products 18: 1. delta¹¹Increase to a level of about 2% and about 12%, respectively, which is barely detectable in untransformed plants; a total of about 14% omega-7 fatty acids were produced in the Com25 transformants. FIG. 2A; fig. 2B.

Table 1 shows that although Com25 compares the substrate for 16:0-ACP to that for ricinus communis WT (2.8 min)^-1) Has greatly improved k_cat(11.1min^-1) However, it did not reach the report for the castor variant 5.2 (25.3 min-1). Whittle and Shanklin (2001) J.biol.chem.276(24): 21500-5. However, K for Com25 versus 16:0-ACP_mK of (0.12. mu.M) to 5.2 for the ricinus variant_m(0.55) 4.6 times lower and K is higher than that of castor WT_m(5.0) 42 times lower. com25 specificity factor (specificity factor) 91. mu.M in the case of 16:0-ACP substrate^-1·min^-1About twice as high as the specificity factor for castor variant 5.2 and 163 times as high as the specificity factor for castor WT. Indeed, the specificity factor for Com25 in the case of 16:0-ACP is equal to that of ricinus communis WT in the case of its native 18:0-ACP substrate (92. mu.M)^-1·min^-1). K to 16:0-ACP improved by Com2 over ricinus variant 5.2_mSuggesting that it competes more efficiently for the substrate with FatB and KASII, providing information on despite its K_catLower, an explanation for why its expression promotes greater accumulation of omega-7 FA than the ricinic variant 5.2. Kinetic parameters of ricinic desaturase and variants thereof under various substrate conditions.

TABLE 1

Example IV: expression of Com25 in a host containing increased 16:0 levels

FIG. 1. if acted upon by FATB, palmitoyl thioesterase, 16:0 free fatty acids are released from plastids to the cytoplasm where it is esterified to CoA, followed by transesterification to phospholipids of the intima system, or β -ketoacyl-ACP synthase II (KASII) prolongs most of the 16:0-ACP to 18:0-ACP, which is then subject to Δ⁹Desaturation of stearoyl-ACP desaturase to produce oleoyl-AAnd (6) CP. FATA, an oleoyl-ACP thioesterase, releases oleic acid, which leaves the plastid and, like palmitate, is activated to CoA-thioester and transferred to phospholipids. In ER, oleate may be extended to 10: 1. delta. by the action of Fatty Acid Elongases (FAE) I¹¹Or sequentially desaturated by the action of FAD2 and FAD3 to produce linolenic acid or linolenic acid, respectively.

16:0-ACP is the earliest metabolite in the FA synthetic pathway that can be desaturated to 16: 1. delta. by its desaturation⁹ACP for omega-7 production. To achieve this, the expression of plastid Δ under the control of a seed-specific promoter was explored⁹Feasibility of a 16:0 ACP-specific desaturase (see FIG. 1 (reaction 1)).

As described above, β -ketoacyl-ACP synthase II (KAS II) extends 16:0-ACP to 18: 0-ACP. thus, lines with reduced KASII activity were sought that would contain elevated 16:0 substrate levels FIG. 2 shows representative GC traces of seed FA methyl ester despite numerous mutagenesis screens, reporting only one mutant fab1(James and Dooner (1990) the or. apple Genet.80:241-45) that exhibits elevated 16:0 levels in leaves and seeds, containing about 21% 16:0 compared to about 10% in WT, as shown in FIG. 2C, and. biochemical evidence shown in Table 2 shows that fab1 damage in KASII because its activity is reduced in mutants Carlsson et al, (2002) Plant J.29(6): 761-70. express Com 539 2 in fab1 16: 1. DELTA.1⁹And 18: 1. delta¹¹The accumulation of (a) increased to about 23% and about 16%, respectively, resulting in a total of about 39% omega-7 FA. FIG. 2D; and table 2. This large increase in ω -7FA after expression of Com25 in the fab1-1 background correlated with an increase in overall 16:0 accumulation in mature seeds and was likely derived from a decrease in competition for 16:0-ACP substrates by KASII.

We also combined the fab1 mutation with fae1 because its defect in the in vitro elongation of C18 to C20 fatty acids further increased the amount of 16:0 fatty acids and simplified the analysis. The untransformed double mutant contains about 9% omega-7 fatty acids, presumably reflected in a.DELTA.in the presence of increased levels of the 16:0-ACP substrate⁹The 18:0 ACP desaturase increases desaturation of 16:0 ACP. FIG. 2E; and table 2. Expression of Com25 in fab1/fae1 resulted in 16: 1. delta⁹And 18: 1. delta¹¹Increasing to about 26% and about 23%, respectively, resulted in an increase to about 50% of omega-7 FA. FIG. 2F; and table 2.

From the above results, increased accumulation of 16:1 in fab1 and fab1/fae1 seeds correlated with 16:0 increase, and therefore, we sought lines expressing Com25 in which 16:0 levels were higher than those of fab1/fae1 double mutants. Two such mutants have recently been reported, of which Fab1 is inhibited, one by Hairpin (HP) RNAi (Pidkowich et al, (2007) Proc. Natl. Acad. Sci. USA 104(11):4742-7), and the other by a novel inhibition method called hairpin-antisense (HPAS) RNAi (Nguyen and Shanklin (2009) Journal of the American Oil chemists society 86: 41-9). These lines contained strongly elevated levels of 16:0 accumulation in seeds of 42% and 46%, respectively. Fig. 3. Conversion with Com25 produced a further increase of about 5% in omega-7 FA in both cases. Table 2. Thus, an increase in 16:0 accumulation at low levels of 16:0 predicts increased Com25 desaturation, as evidenced by a proportional increase in ω -7FA accumulation, but this response is clearly saturable slightly above 30% because there is no difference between ω -7FA accumulation expression in the 42% or 46%16:0 accumulating host. Fig. 3. Without intending to be limited to any particular theory, it is possible that factors other than substrate, i.e., desaturase abundance and/or reductant availability, become limiting in these transgenes.

Recently, a T-DNA knockout allele fab1-2 was described that has elevated 16:0 levels in heterozygotes; however, homozygotes were shown to be embryo lethal. Pidkowich et al, (2007) Proc.Natl.Acad.Sci.USA 104(11): 4742-7). We hypothesized that the lethal phenotype results from the reduction of unsaturated fatty acids, and theorize that expression of Com25 in this line could confer viability. In contrast, the fab1/fae1 double mutant was viable in homozygous conditions and distinguishable from WT in terms of growth and development. Therefore, we performed subsequent experiments using the fab1/fae1 double mutant as experimental host.

Example V: increasing the dose of the Com25 gene resulted in increased omega-7 accumulation

Prototype castor delta⁹The-18: 0 ACP desaturase had a 42min profile^-1K of (a)_cat(Whittle and Shanklin (2001) J.biol.chem.276(24): 21500-5); alignment of them delta⁹-16:0 ACP desaturase reported k_catSeveral times higher. Cahoon et al, (1997) Plant mol.biol.33:1105-10, and Cahoon et al, (1998) Plant physiol.117(2): 593-8. While this turnover is comparable to that of similar Fe-dependent oxidation reactions such as cytochrome P450s, these rates are lower (in many cases, by orders of magnitude) than many metabolic enzymes. The low turnover rate of desaturases requires high levels of protein expression to be responsible for desaturating a large proportion of carbon stored in seeds, suggesting the possibility that the abundance of the enzyme desaturase can limit omega-7 accumulation. To test this hypothesis, Com25 was engineered under the control of the seed-specific LTP170 promoter, which controls expression of seed storage proteins, and this was co-expressed with the phaseolin driven Com25 construct described above. Co-expression of Com25 under the control of phaseolin and LTP170 promoter in the fab1/fae1 background resulted in an increase in omega-7 FA accumulation from about 50% to about 58%; 16: 1. delta⁹Increase (about 6%) was greater than 18: 1. delta¹¹Increase in (about 2%). This increase in ω -7FA accumulation was moderate, suggesting that Com25 may not be limiting in seeds expressing both Com25 constructs.

TABLE 2

Example VI: expression of the in vitro Δ 9-16:0 desaturase increased ω -7FA accumulation. As previously discussed, the use of background arabidopsis thaliana that accumulate high levels of 16:0 correlates with omega-7 FA formation following expression of a 16:0 ACP desaturase, but significant amounts of 16:0 remain in the plastid and accumulate in the oilseeds. See table 2. Thus, two methods of reducing this 16:0 accumulation in seed oil are contemplated. One strategy is to reduce the activity of the enzyme sulfatase FATB cleaving 16:0 from 16:0-ACP (figure 1). Inhibition of FATB via HPAS-RNAi reduced 16:0 accumulation by about 3%, with an increase of omega-7 FA by about 6%. See table 2. The feasibility of further reducing seed 16:0 accumulation beyond what was observed by inhibiting FATB by desaturating 16:0 after export from plastids was explored.

Free fatty acids released from the plastid are esterified en route to CoA by the acyl Co-a synthase to accumulate as triacylglycerols. Shockkey et al, (2003) Plant physiol.132(2): 1065-76. These cytosolic fatty acyl Co-a and phospholipid-linked FAs represent a potentially available substrate pool for extra-plastidial desaturases. Expression of the in vitro fungi Aspergillus nidulans (An) and Leptosphaeria nodorum (Ln) desaturases, alone or in combination, were evaluated in reducing 16:0 levels in Arabidopsis. Co-expression of two desaturases from Ln and An under the control of the phaseolin promoter yielded promising results in the reduction of 16:0 in WT Arabidopsis thaliana. Thus, expression of the Ln and An constructs was tested in KASII HPAS-RNAi suppression lines along with expression of a single copy of Com 25. Expression of Ln Δ 9D and An Δ 9 desaturases resulted in about half the 16:0 conversion to 16:1 Δ⁹Resulting in a decrease in 16:0 accumulation in the seeds from about 19% to about 11% (roughly the level seen in WT seeds), 16: 1. delta⁹The response increased from about 27% to 43%. 18: 1. delta¹¹The levels of (A) remained the same in the host fab1/fae1/Com25 line and in this line transformed with Ln Δ 9D and An Δ 9 desaturase, respectively (about 25% and about 23%), indicating that the fae1 mutant is almost completely deficient in 16:1 Δ 9⁹Prolonging the activity. This strategy of co-expressing both plastidial and extra-plastidial desaturases resulted in a mean accumulation of approximately 67% omega-7 FA, with individual plants showing greater than 71%.

Claims (14)

1. A method for increasing the amount of unusual fatty acids in plant material, the method comprising: transforming plant material comprising an extraplastidial desaturase with a nucleic acid molecule,

the nucleic acid molecule comprises:

a nucleotide sequence at least 60% identical to SEQ ID NO 1; and

the phaseolin promoter or LTP170 promoter,

wherein the nucleotide sequence encodes a Δ 9 desaturase having at least 90% identity to SEQ ID No. 2;

so as to increase the amount of unusual fatty acids in the plant material,

wherein the in vitro desaturase is An Ln Δ 9D or An Δ 9D desaturase.

2. The method of claim 1, further comprising transforming plant material with additional nucleic acid molecules as described in claim 1.

3. The method of claim 1, wherein the plant material comprises a means for increasing the level of 16:0-ACP in the plant material selected from the group consisting of: inhibiting KASII; and reducing elongation of 16:0 fatty acids in the plant material.

4. The method of claim 3, wherein inhibiting KASII is achieved by introducing a mutation in the fab1 gene.

5. The method of claim 3 or 4, wherein reducing elongation of 16:0 fatty acids in the plant material is achieved by introducing a mutation in the fae1 gene.

6. The method of claim 1, wherein the plant material is selected from the genera consisting of: arabidopsis (Arabidopsis), borage (Borago), Ricinus (Ricinus), Theobroma (Theobroma), maize (Zea), cotton (Gossypium), Crambe (Crambe), Cuphea (Cuphea), Linum (Linum), Bidens (Lesquerella), spermum (Limnanthes), eclipta (Tropaeolus), Oenothera (Oenothera), Oleanophora (Olea), Elaeis (Elaeis), Arachis (Arachi), Carthamus (Carthamus), Glycine (Glycine), Helianthus (Helianthus), Nicotiana (Nicotiana), Vernonia (Vernonia), Triticum (Triticum), Hordeum (Hordeum), Oryza (Oryza), Avena (Sorghum), Sorghum (Averme), or other members of the genus Gracilaria.

7. The method of claim 1, wherein the plant material is selected from the group consisting of Canola (Canola), Linola, rapeseed (rapeseed), and wild soybean (Soja).

8. The method of claim 1, wherein the plant material comprises two means for increasing the level of 16:0-ACP in the plant material.

9. The method of claim 8, wherein the first means for increasing levels of 16:0-ACP in the plant material is a mutation in the fab1 gene, and wherein the second means for increasing levels of 16:0-ACP in the plant material is a mutation in the fae1 gene.

10. A method for creating a genetically engineered plant comprising an increased amount of unusual fatty acids in the plant compared to a wild type plant, the method comprising:

transforming plant material comprising an extraplastidial desaturase with a nucleic acid molecule;

the nucleic acid molecule comprises:

a nucleotide sequence at least 60% identical to SEQ ID NO 1, and

the phaseolin promoter or LTP170 promoter,

wherein the nucleotide sequence encodes a Δ 9 desaturase having at least 90% identity to SEQ ID No. 2; and are

Culturing the transformed plant material to obtain a plant,

wherein the in vitro desaturase is An Ln Δ 9D or An Δ 9D desaturase.

11. The method of claim 10, wherein the plant material is selected from the genera consisting of: arabidopsis, borage, ricinus, theobroma, maize, gossypium, crambe, cuphea, flax, atriplex, sperm oil grass, eclipta, evening primrose, trifolium, elaeis, arachis, echeveria, soybean, sunflower, tobacco, vernonia, wheat, barley, rice, oat, sorghum, rye, or other members of the gramineae family.

12. The method of claim 10, wherein the plant material is selected from the group consisting of canola, Linola, rapeseed, and wild soybean.

13. The method of claim 1, wherein the nucleotide sequence is operably linked to a phaseolin promoter or an LTP170 promoter.

14. The method of claim 10, wherein the nucleotide sequence is operably linked to a phaseolin promoter or an LTP170 promoter.