WO2004007744A2 - Mise au point de la synthase $g(b)-cetoacyl acp pour une nouvelle specificite de substrat - Google Patents

Mise au point de la synthase $g(b)-cetoacyl acp pour une nouvelle specificite de substrat Download PDF

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Publication number
WO2004007744A2
WO2004007744A2 PCT/US2000/022359 US0022359W WO2004007744A2 WO 2004007744 A2 WO2004007744 A2 WO 2004007744A2 US 0022359 W US0022359 W US 0022359W WO 2004007744 A2 WO2004007744 A2 WO 2004007744A2
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ketoacyl
amino acid
kas
engineered
acp
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PCT/US2000/022359
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English (en)
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Dehesh Kaytayoon
Dale Val
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Calgene, Llc
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Priority to PCT/US2000/022359 priority Critical patent/WO2004007744A2/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
    • 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)

Definitions

  • the present invention is directed to proteins, nucleic acid sequences and constructs, and methods related thereto.
  • Fatty acids are organic acids having a hydrocarbon chain of from about 4 to 24 carbons. Many different kinds of fatty acids are known which differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX: Y, where "X" indicates number of carbons and "Y" indicates number of double bonds.
  • fatty acids in plants begins in the plastid with the reaction between acetyl-CoA and malonyl-ACP to produce acetoacetyl-ACP catalyzed by the enzyme, ⁇ -ketoacyl-ACP synthase III.
  • Elongation of acetyl-ACP to 16- and 18- carbon fatty acids involves the following cycle of reactions: condensation with a two-carbon unit from malonyl-ACP to form a ⁇ -ketoacyl-ACP ( ⁇ -ketoacyl-ACP synthase), reduction of the keto-function to an alcohol ( ⁇ -ketoacyl-ACP reductase), dehydration to form an enoyl-ACP ( ⁇ -hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl- ACP (enoyl-ACP reductase).
  • ⁇ -ketoacyl-ACP synthase I catalyzes elongation up to palmitoyl-ACP (C16:0)
  • ⁇ -ketoacyl-ACP synthase U catalyzes the final elongation to stearoyl-ACP (C18:0).
  • the longest chain fatty acids produced by the FAS are typically 18 carbons long. Additional biochemical steps in the cell produce specific fatty acid constituents, for example through desaturation and elongation.
  • ⁇ -ketoacyl synthases condensing enzymes, comprise a structurally and functionally related family that play critical roles in the biosynthesis of a variety of natural products, including fatty acids, and the polyketide precursors leading to antibiotics, toxins, and other secondary metabolites, ⁇ -ketoacyl synthases catalyze carbon-carbon bond forming reactions by condenisng a variety of acyl chain precursors with an elongating carbon source, usually malonyl or methyl malonyl moieties, that are covalently attached through a thioester linkage to an acyl carrier protein.
  • an elongating carbon source usually malonyl or methyl malonyl moieties
  • Condensing enzymes can be part of multienzyme complexes, domains of large, multifunctional polypeptide chains as the mammalian fatty acid synthase, or single enzymes as the ⁇ -ketoacyl synthases in plants and most bacteria.
  • Condensing enzymes have been identified with properties subject to exploitation in the areas of plant oil modification, polyketide engineering, and ultimately design anti- cancer and anti-tuberculosis agents.
  • Cerulinin a mycotoxin produced by the fungus Cephalosporium caerulens, acts as a potent inhibitor of KAS by covalent modification of the active cysteine thiol.
  • Conderising enzymes from many other pathways and sources have all been shown to be inactivated by this antibiotic with the exception of the synthase from C.
  • the present invention is directed to ⁇ -ketoacyl ACP synthase (KAS), and in particular to engineered KAS polypeptides and polynucleotides encoding engineered KAS proteins having a modified substrate specificity with respect to the native (also referred to herein as wild-type) KAS protein.
  • KAS ⁇ -ketoacyl ACP synthase
  • engineered polypeptides and polynucleotides of the present invention include those derived from plant and bacterial sources.
  • polynucleotides encoding engineered polypeptides particularly, polynucleotides that encode a KAS protein with a modified substrate specificity with respect to the native KAS protein, are provided.
  • the invention relates to oligonucleotides derived from the engineered KAS proteins and oligonucleotides which include partial or complete engineered KAS encoding sequences. It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for transcription or transcription and translation (expression) of an engineered KAS protein having an altered substrate specificity with respect to the native KAS protein.
  • constructs are provided which are capable of transcription or transcription and translation in host cells.
  • Particularly preferred constructs are those capable of transcription or transcription and translation in plant cells.
  • methods are provided for production of engineered KAS proteins having a modified substrate specificity with respect to the native KAS in a host cell or progeny thereof.
  • host cells are transformed or transfected with a DNA construct which can be used for transcription or transcription and translation of an engineered KAS.
  • the recombinant cells which contain engineered KAS are also part of the present invention.
  • the present invention relates to methods of using the engineered polynucleotide and polypeptide sequences of the present invention to modify the fatty acid composition in a host cell, as well as to modify the composition and/or structure of triglyceride molecules, particularly in seed oil of oilseed crops.
  • Plant cells having such a modified triglyceride content are also contemplated herein.
  • the modified plants, seeds and oils obtained by the expression of the plant engineered KAS proteins are also considered part of the invention.
  • Figure 1 provides the coordinates of the crystal structure of the E. coli KAS protein.
  • the second column provides the amino acid residue type (three letter abbreviation),
  • the third column provides the subunit in which the amino acid is located, the forth column provides the amino acid position in the protein sequence base don the mature unprocessed protein, columns seven through nine provide the x, y and z coordinates, respectively, of the three dimensional location of the respective atom in the crystal structure.
  • Figure 2 provides the profile of the crystal structure of the E. coli KAS- cerulenin complex.
  • the second column provides the amino acid residue type (three letter abbreviation),
  • the third column provides the subunit in which the amino acid is located, the forth column provides the amino acid position in the protein sequence base don the mature unprocessed protein, columns seven through nine provide the x, y and z coordinates, respectively, of the three dimensional location of the respective atom in the crystal structure.
  • Figure 3 provides the effects of KAS LT mutations on the fatty acid composition of E. coli.
  • Figure 4 shows that mutations I108F, I108L and A193M all cause significant reduction in the activity of KAS II on 8:0-ACP as compared to 6:0-ACP (38, 31 and 12 fold reductions respectively), without significantly reducing the activity on 6:0-ACP.
  • Figure 5 shows that the combined mutations at 1108 and A193 have the effect of reducing the activity of KAS II on 6:0- ACP substrates.
  • Figure 6 shows that the combined effect of two or more mutations had a greater effect on the activity with acyl-ACPs 8:0 and longer (14:0) substrates.
  • Figure 7 shows the complete list of mutations that were generated.
  • Figure 8 provides the structure of the Cpu KAS I homodimer
  • Figure 9 provides the structure of the Cpu KAS IV homodimer
  • Figure 10 provides the structure of the Cpu KAS II Cpu KAS IN heterodimer.
  • Figure 11 provides the sequence differences in the hydrophobic pocket of the E. coli KAS ⁇ and C. pu KASIV.
  • Figure 12 provides an amino acid sequence alignment of KAS protein sequences from plant (Arabidopsis, Brassica, Cuphea hookeriana and pullcherima, Hordeum, Riccinus), bacterial (E. coli, streptococcus, tuberculosis), mammalian (rat, mouse) and others (C.elegans).
  • Figure 13 provides a bar graph representing the results of fatty acid analysis of seeds from transformed Arabidopsis lines containing pCG ⁇ 11058, pCGN11062, pCGN11041, or nontransformed control lines (AT002-44).
  • bars represent, from left to right, C12:0, C14:0, C16:0, C16:l, C18:0, C18:l (delta 9), C18:l (delta 11), C18:2, C18:3, C20:0, C20:l (delta 11), C20:l (delta 13), C20:2, C20:3, C22:0, C22:l, C22:2, C22:3, C24:0, and C24:l fatty acids.
  • Figure 14 provides the nucleotide sequence of the plastid targeting sequence from Cuphea hookeriana KASII-7. DETAILED DESCRIPTION OF THE INVENTION
  • engineered nucleotide sequences are provided which are capable of coding sequences of amino acids, such as, a protein, polypeptide or peptide.
  • the engineered nucleotide sequences encode ⁇ -ketoacyl-ACP synthase (KAS) proteins with a modified substrate specificity compared to the native KAS protein (also referred to herein as the wild-type KAS protein) under enzyme reaction conditions.
  • KAS ⁇ -ketoacyl-ACP synthase
  • engineered ⁇ -ketoacyl- ACP synthase also referred to as engineered KAS
  • the engineered nucleic acid sequences find use in the preparation of constructs to direct their expression in a host cell.
  • engineered nucleic acid sequences find use in the preparation of plant expression constructs to alter the fatty acid composition of a plant cell.
  • enzyme reactive conditions is meant that any necessary conditions are available in an environment (for example, such factors as temperature, pH, lack of inhibiting substances) which will permit the enzyme to function.
  • An engineered ⁇ -ketoacyl-ACP synthase nucleic acid sequence of this invention includes any nucleic acid sequence coding a ⁇ -ketoacyl-ACP synthase having altered substrate specificity relative to the native KAS in a host cell, includign but not limited to, in vivo, or in a cell-like environment, for example, in vitro.
  • substrate specificity is meant an alteration in the acyl-ACP substrates elongated by the KAS enzyme or an alteration in the elongator molecule used by the KAS to elongate the acyl-ACP relative to the native or unaltered KAS protein.
  • An alteration in the acyl- ACP substrate elongated by the KAS enzymes includes, but is not limited to, elongation of an acyl-ACP substrate not elongated by the wild-type KAS, the inability to elongate an acyl-ACP substrate elongated by the wild-type KAS, and a preference for elongating acyl-ACP substrates not normally preferred by the wild-type KAS.
  • An alteration in the elongator molecule used by the engineered KAS for the elongation of the acyl-ACP substrate includes, but is not limited to, methyl-malonyl ACP for the production of branched chain fatty acids.
  • a first aspect of the present invention relates to engineered ⁇ -ketoacyl-ACP synthase polypeptides.
  • engineered KAS LT polypeptides are provided.
  • Preferred peptides include those found in the hydrophobic fatty acid/cerulenin binding pocket of the KAS protein.
  • Such polypeptides include the engineered polypeptides set forth in the Sequence Listing, as well as polypeptides and fragments thereof, particularly those polypeptides which exhibit a modified substrate specificity with respect to the wild- type KAS polypeptide.
  • Particularly preferred polypeptides include those having engineered amino acid residues 105 to 120, 130-140, 190-200 and 340- 400.
  • polypeptides include those having engineered amino acid residues I108A, I108F, I108G, I108L, LI 11 A, II 14A, F133 A, V134A, V134G, I138A, I138G, A162G, A193G, A193I, A193M, L197A, F202L, F202I, F202G, L342A, and L342G.
  • Amino acid positions refer to the amino acid residue position in the active or processed protein.
  • Engineered ⁇ -ketoacyl-ACP synthases can be prepared by random (via chemical mutagenesis or DNA shuffling) or specific mutagenesis of a ⁇ -ketoacyl-ACP synthase encoding sequence to provide for one or more amino acid substitutions in the translated amino acid sequence.
  • an engineered ⁇ -ketoacyl-ACP synthase can be prepared by domain swapping between related ⁇ -ketoacyl-ACP synthases, wherein extensive regions of the native ⁇ -ketoacyl-ACP synthase encoding sequence are replaced with the corresponding region from a different ⁇ -ketoacyl-ACP synthase.
  • Altered substrate specificities of an engineered ⁇ -ketoacyl-ACP synthase can be reflected by the elongation of an acyl-ACP substrates of particular chain length fatty acyl-ACP groups which are not elongated by the native ⁇ -ketoacyl-ACP synthase enzyme.
  • altered substrate specificities can be reflected by the in ability to elongate an acyl-ACP substrate of particular chain length fatty acyl-ACP groups which are not normally preferred by the native ⁇ -ketoacyl-ACP synthase enzyme.
  • the newly recognized acyl-ACP substrate can differ from native substrates of the enzyme in various ways, such as by having a shorter or longer carbon chain length (usually reflected by the addition or deletion of one or more 2-carbon units), as well as by degrees of unsaturation.
  • Another aspect of the present invention relates to engineered ⁇ -ketoacyl-ACP synthase polynucleotides.
  • engineered ⁇ -ketoacyl-ACP synthase II polynucleotides are provided.
  • the polynucleotide sequences of the present invention include engineered polynucleotides that encode the polypeptides of the invention having a deduced amino acid sequence selected from the group of sequences set forth in the Sequence Listing.
  • the invention provides a polynucleotide sequence identical over its entire length to each coding sequence as set forth in the Sequence Listing.
  • the invention also provides the coding sequence for the mature polypeptide or a fragment thereof, as well as the coding sequence for the mature engineered polypeptide or a fragment thereof in a reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, pro-, or prepro- protein sequence.
  • the polynucleotide can also include non-coding sequences, including for example, but not limited to, non- coding 5' and 3' sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize rnRNA, introns, polyadenylation signals, and additional coding sequence that encodes additional amino acids.
  • non-coding sequences including for example, but not limited to, non- coding 5' and 3' sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize rnRNA, introns, polyadenylation signals, and additional coding sequence that encodes additional amino acids.
  • a marker sequence can be included to facilitate the purification of the fused polypeptide.
  • Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression.
  • analysis of the KAS II cerulinin crystal structure complex is performed using modeling software to produce a profile of the complex, as well as the KAS U protein alone. Based on comparisons of the two profiles, amino acid residues are identified, which when mutagenized, alter the fatty acyl substrate specificities.
  • engineering of the nucleic acid sequence to modify the amino acid sequence in particular regions of the KAS protein effectively modify the substrate specificity of the engineered KAS.
  • Particular ranges for the engineering of the protein include amino acid residues 105 to 120, 130-140, 190-200 and 340-345.
  • engineering of residues 108, 111, 114, 133, 193 and 197 can alter the length of the fatty acids synthesized by the engineered KAS II protein. More particularly, engineering of residues 108, 111, 114, 133, 193 and 197 with variously sized hydrophobic residues will alter the length of the fatty acids synthesized by the engineered KAS II protein. Furthermore, engineering the amino acid residue at position 400 can also have an effect on the substrate specificity.
  • the acyl-ACP substrate specificity of b-ketoacyl-ACP synthases may be modified by various amino acid changes to the protein sequence, such as amino acid substitutions, insertions or deletions in the mature protein portion of the b-ketoacyl-ACP synthases.
  • Modified substrate specificity can be detected by expression of the engineered b-ketoacyl-ACP synthase s in E. coli and assaying to detect enzyme activity or by using purified protein in in vitro assays.
  • Modified substrate specificity can be indicted by a shift in acyl-ACP substrate preference such that the engineered b-ketoacyl-ACP synthase is newly capable of utilizing a substrate not recognized by the native b-ketoacyl-ACP synthase .
  • the newly recognized substrate can vary from substrates of the native enzyme by carbon chain length and/or degree of saturation of the fatty acyl portion of the substrate.
  • modified substrate specificity can be reflected by a shift in the relative b- ketoacyl-ACP synthase activity on two or more substrates of the native b-ketoacyl-ACP synthase such that an engineered b-ketoacyl-ACP synthase exhibits a different order of preference for the acyl-ACP substrates.
  • KAS proteins with an altered elongator molecule preference.
  • KAS protein by widening the hydrophobic fatty acid binding different elongator molecules, other than Malonyl-ACP, can be utilized by the KAS protein.
  • Methyl-malonyl-ACP can be utilized by the engineered KAS resulting in the synthesis of branched chained fatty acid.
  • the mutations that lengthen the pocket may to some degree also widen it, in addition mutations A193G, I108G, L342A or G, V134A or G,F202L,I or G may well cause widening of the pocket sufficiently to allow Methyl-malonyl-ACP to be accepted as an elongator.
  • alterations in the nucleic acid sequence of the E. coli KAS II are prepared for the production of shorter chain length fatty acids.
  • I108A, LI 11 A, I114A, F133A, L197A, and combinations thereof are prepared for increasing the length of fatty acids produced by the host cell.
  • nucleotide sequences or polynucleotides, in recombinant DNA constructs to direct the transcription or transcription and translation (expression) of the engineered KAS sequences of the present invention in a host plant cell.
  • the expression constructs generally comprise a promoter functional in a host plant cell operably linked to a nucleic acid sequence encoding a engineered KAS of the present invention and a transcriptional termination region functional in a host plant cell.
  • Chloroplast and plastid specific promoters are also envisioned.
  • One set of promoters are constitutive promoters such as the CaMV35S or
  • FMN35S promoters that yield high levels of expression in most plant organs. Enhanced or duplicated versions ofthe CaMN35S and FMN35S promoters are useful in the practice of this invention (Odell, et al. (1985) Nature 313:810-812; Rogers, U.S. Patent Number 5,378, 619). In addition, it may also be preferred to bring about expression of the engineered KAS in specific tissues of the plant, such as leaf, stem, root, tuber, seed, fruit, etc., and the promoter chosen should have the desired tissue and developmental specificity.
  • nucleic acid sequences of the present invention from transcription initiation regions which are preferentially expressed in a plant seed tissue.
  • seed preferential transcription initiation sequences include those sequences derived from sequences encoding plant storage protein genes or from genes involved in fatty acid biosynthesis in oilseeds.
  • promoters include the 5' regulatory regions from such genes as napin (Kridl et al, Seed Sci. Res. i:209:219 (1991)), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, soybean ⁇ ' subunit of ⁇ -conglycinin (soy 7s, (Chen et al, Proc. Natl. Acad. Sci., 83:8560-8564 (1986))) and oleosin.
  • CTP chloroplast transit peptides
  • PTP plastid transit peptides
  • the expression construct will additionally contain a gene encoding a transit peptide to direct the protein of interest to the plastid.
  • the chloroplast transit peptides may be derived from the gene of interest, or may be derived from a heterologous sequence having a CTP. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) Plant Physiol.
  • Additional transit peptides for the translocation of the engineered KAS protein to the endoplasmic reticulum (ER), or vacuole may also find use in the constructs of the present invention.
  • additional constructs can be employed containing the nucleic acid sequence which provides for the suppression of the host cell's endogenous KAS protein. Where antisense inhibition of a host cells native KAS protein is desired, the entire wild-type KAS sequence is not required.
  • Transcript termination regions may be provided by the DNA sequence encoding the wild-type KAS or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region.
  • the transcript termination region which is naturally associated with the transcript initiation region.
  • any convenient transcript termination region which is capable of terminating transcription in a plant cell may be employed in the constructs of the present invention.
  • constructs may be prepared to direct the expression of the engineered KAS sequences directly from the host plant cell plastid.
  • constructs and methods are known in the art and are generally described, for example, in Svab, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917 and in U.S. Patent Number 5,693,507.
  • a plant cell, tissue, organ, or plant into which the recombinant DNA constructs containing the expression constructs have been introduced is considered transformed, transfected, or transgenic.
  • a transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a engineered KAS nucleic acid sequence.
  • Plant expression or transcription constructs having an engineered KAS as the DNA sequence of interest for increased or decreased expression thereof may be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Most especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed (Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences may be required. Importantly, this invention is applicable to dicotyledyons and monocotyledons species alike and will be readily applicable to new and/or improved transformation and regulation techniques.
  • engineered KAS constructs in plants which have been genetically engineered to produce a particular fatty acid in the plant seed oil, where TAG in the seeds of nonengineered plants of the engineered species, do not naturally contain that particular fatty acid.
  • the engineered KAS constructs of the present invention can also be used to provide a means for the production of plants having resistance to plant pathogens.
  • Engineered KAS constructs providing for an increased production of particular fatty acids involved in the biosynthesis of pathogen response signals or inhibitors For example, engineered KAS constructs providing for the increased production of C:8 fatty acids allows for the production of transgenic plants having an increased tolerance to fungal pathogens.
  • the gene sequences may be synthesized, either completely or in part, especially where it is desirable to provide plant-preferred sequences.
  • all or a portion of the desired structural gene may be synthesized using codons preferred by a selected host.
  • Host-preferred codons may be determined, for example, from the codons used most frequently in the proteins expressed in a desired host species.
  • the desired engineered KAS nucleic acid sequence may be manipulated in a variety of ways. Where the sequence involves non-coding flanking regions, the flanking regions may be subjected to resection, mutagenesis, etc. Thus, transitions, trans versions, deletions, and insertions may be performed on the naturally occurring sequence. In addition, all or part ofthe sequence may be synthesized.
  • one or more codons may be modified to provide for a modified amino acid sequence, or one or more codon mutations may be introduced to provide for a convenient restriction site or other purpose involved with construction or expression.
  • the structural gene may be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like.
  • nucleic acid or amino acid sequences encoding an engineered KAS of this invention may be combined with other non-native, or “heterologous", sequences in a variety of ways.
  • heterologous sequences is meant any sequence which is not naturally found joined to the engineered KAS, including, for example, combinations of nucleic acid sequences from the same plant which are not naturally found joined together.
  • the DNA sequence encoding an engineered KAS of this invention may be employed in conjunction with all or part of the gene sequences normally associated with the wild-type KAS.
  • a DNA sequence encoding engineered KAS is combined in a DNA construct having, in the 5' to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding engineered KAS and a transcription and translation termination region.
  • Potential host cells include both prokaryotic and eukaryotic cells.
  • a host cell may be unicellular or found in a multicellular differentiated or undifferentiated organism depending upon the intended use.
  • Cells of this invention may be distinguished by having an engineered KAS foreign to the wild-type cell present therein, for example, by having a recombinant nucleic acid construct encoding an engineered KAS therein.
  • the methods used for the transformation of the host plant cell are not critical to the present invention.
  • the transformation of the plant is preferably permanent, i.e. by integration of the introduced expression constructs into the host plant genome, so that the introduced constructs are passed onto successive plant generations.
  • the skilled artisan will recognize that a wide variety of transformation techniques exist in the art, and new techniques are continually becoming available. Any technique that is suitable for the target host plant can be employed within the scope of the present invention.
  • the constructs can be introduced in a variety of forms including, but not limited to as a strand of DNA, in a plasmid, or in an artificial chromosome.
  • the introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to calcium-phosphate-DNA co- precipitation, electroporation, microinjection, Agrobacte ⁇ um infection, liposomes or microprojectile transformation.
  • techniques including, but not limited to calcium-phosphate-DNA co- precipitation, electroporation, microinjection, Agrobacte ⁇ um infection, liposomes or microprojectile transformation.
  • the skilled artisan can refer to the literature for details and select suitable techniques for use in the methods of the present invention.
  • included with the DNA construct will be a structural gene having the necessary regulatory regions for expression in a host and providing for selection of transformant cells.
  • the gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
  • a cytotoxic agent e.g. antibiotic, heavy metal, toxin, etc.
  • complementation providing prototrophy to an auxotrophic host, viral immunity or the like.
  • one or more markers may be employed, where different conditions for selection are used for the different hosts.
  • a vector may be used which may be introduced into the Agrobacterium host for homologous recombination with T-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host.
  • the Ti- or Ri-plasmid containing the T-DNA for recombination may be armed (capable of causing gall formation) or disarmed (incapable of causing gall formation), the latter being permissible, so long as the vir genes are present in the transformed Agrobacterium host.
  • the armed plasmid can give a mixture of normal plant cells and gall.
  • the expression or transcription construct bordered by the T-DNA border region(s) will be inserted into a broad host range vector capable of replication in E. coli and Agrobacterium, there being broad host range vectors described in the literature. Commonly used is pRK2 or derivatives thereof. See, for example, Ditta, et al, (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and ⁇ PA 0 120 515, which are incorporated herein by reference.
  • markers which allow for selection of transformed Agrobacterium and transformed plant cells.
  • a number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, kanamycin, the aminoglycoside G418, hygromycin, or the like.
  • the particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.
  • explants For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of vegetable oils.
  • any means for producing a plant comprising a construct having a DNA sequence encoding the engineered KAS of the present invention, and at least one other construct having another DNA sequence encoding an enzyme are encompassed by the present invention.
  • the expression construct can be used to transform a plant at the same time as the second construct either by inclusion of both expression constructs in a single transformation vector or by using separate vectors, each of which express desired genes.
  • the second construct can be introduced into a plant which has already been transformed with the engineered KAS expression construct, or alternatively, transformed plants, one expressing the engineered KAS construct and one expressing the second construct, can be crossed to bring the constructs together in the same plant.
  • the invention also relates to vectors that include a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell free translation systems can be employed to produce such protein using RNAs derived from the DNA constructs of the invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the present invention.
  • Introduction of a polynucleotide into a host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).
  • Such methods include, but are not limited to, calcium phosphate transfection, DEAE dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading ballistic introduction and infection.
  • bacterial cells such as streptococci, staphylococci, enterococci, E. coli, streptomyces, and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • plant cells as described above.
  • vectors include, but are not limited to, chromosomal, episomal, and virus derived vectors, for example vectors from bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, such as SB40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations of such viruses, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector which is suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host can be used for expression.
  • the appropriate DNA sequence can be inserted into the chosen expression by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, Molecular Cloning, A Laboratory Manual, (supra).
  • Appropriate secretion signals can be incorporated into the expressed polypeptide to allow the secretion of the protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment.
  • polypeptides of the present invention can be recovered and purified from recombinant cell cultures by any of a number of well known methods, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. It is most preferable to use high performance liquid chromatography (HPLC) for purification. Any of the well known techniques for protein refolding can be used to regenerate an active confirmation if the polypeptide is denatured during isolation and/or purification.
  • HPLC high performance liquid chromatography
  • the engineered KAS polynucleotides and polypeptides of the present invention find use in a variety of applications.
  • the engineered KAS polynucleotides and polypeptides as well as the constructs containing such engineered KAS polynucleotides and polypeptides find use in the alteration of fatty acid composition. Furthermore, the engineered KAS polynucleotides and polypeptides of the present invention find use in the production of particular fatty acid components. For example, an engineered KAS having a preference for elongating 6, 8, 10, and 12 carbon acyl-ACP substrates can be used in the production of medium chain fatty acids.
  • Such engineered KAS polynucleotides and polypeptides can also be used with additional sequences for the production of medium chain fatty acids, including, but not limited to, medium chain specific thioesterases (see for example USPN 5,512,482).
  • the present invention further provides methods for the engineering of polyketides and for the identification of molecules useful in cancer therapy, immunosuppressants, anti-parasite, and antibiotic production.
  • the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including inhibitory compounds, capable of binding to the active site or substrate binding site of KAS, in whole or in part.
  • a first approach enabled by this invention is to use the structure coordinates of KAS to design compounds that bind to the enzyme and alter the physical properties of the compounds in different ways, e.g., solubility.
  • this invention enables the design of compounds that act as competitive inhibitors of the KAS enzyme by binding to, all or a portion of, the active site of KAS.
  • This invention also enables the design of compounds that act as uncompetitive inhibitors of the KAS enzyme. These inhibitors may bind to, all or a portion of, the substrate binding site of KAS already bound to its substrate and may be more potent and less non-specific than known competitive inhibitors that compete only for the KAS active site.
  • non- competitive inhibitors that bind to and inhibit KAS whether or not it is bound to another chemical entity may be designed using the structure coordinates of KAS of this invention.
  • reversible and irreversible inhibitors can also be designed.
  • a second design approach is to probe KAS with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate ICE inhibitors and the enzyme. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of where each type of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their KAS inhibitor activity. Travis, J., Science, 262, p. 1374 (1993).
  • This invention also enables the development of compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to KAS, with KAS.
  • the reaction intermediates of KAS can also be deduced from the reaction product in co- complex with KAS.
  • Such information is useful to design improved analogues of known KAS inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the KAS enzyme and KAS-inhibitor co-complex. This provides a novel route for designing KAS inhibitors with both high specificity and stability.
  • Another approach made possible and enabled by this invention is to screen computationally small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to the KAS enzyme.
  • the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy.
  • the KASII-cerulenin complex was prepared as described previously (Edwards, et al. (1997) EE ⁇ S Lett. 402:62-66). Crystals of the complex were grown by the hanging drop method. Droplets consisting of equal amounts of protein solution (6 mg ml "1 , 21 protein, 0.3 MNaCl, 25 mMTris, pH 8.0, 5 mMimidazole, and 10% v/v glycerol) and reservoir solution were equilibrated against 26% w/v polyethylene glycol 8000 and 0.1% v/v 2-mercaptoethanol in water. Data from two crystals were collected at 298 K at the synchrotron in MAX-lab, beamline 1711, in Lund.
  • the crystals are very radiation-sensitive, but cannot be frozen in a cryostream. Due to non-isomorphism, data of only two crystals could be merged.
  • the crystals of the complex have space group P3 ⁇ 21 with similar cell dimensions as the native enzyme.
  • the coordinates of the native enzyme (Huang, et al. (1998) EMBO J. 17:1183-1191) were used to calculate initial electron density maps with SIGMAA (Read (1986) Acta Crystallogr. 42:140-149). All data were used in the refinement; no sigma cutoff was applied.
  • the overall structure of the KAS dimer is unchanged upon binding of cerulenin; the root mean square deviations for the 411 C ⁇ atoms of the subunit is 0.23 A between the two structures. These differences are mainly localized in the active site, in particular in the loop comprising residues 398X01.
  • the main differences in structure between the native enzyme and the cerulenin complex are in the conformation of the side chains of Phe-400 (which was anticipated already from the native structure) and of He- 108, which have completely new rotamer conformations, and in the positions of the side chains of Cys-163, His-340, and Leu-342, which also have moved substantially.
  • the inhibitor amide carbonyl oxygen is within hydrogen bond distance to the N ⁇ atoms of the side chains of His-340 and His-303, while the amide NH group does not make any close interactions. It is, however, not possible from the structure to exclude the opposite conformation and interactions for the amide group.
  • the hydroxyl group at C3 forms a hydrogen bond to the main chain NH of Phe-400.
  • the carbonyl oxygen at C4 does not form any polar interactions, in fact, it is located in a very hydrophobic pocket formed by side chains Phe-400, Phe- 202, and Val- 134 from the other subunit in the dimer.
  • the binding site for the hydrophobic part of the inhibitor is also lined with hydrophobic residues: Ala-162, Gly- 107, Leu-342, Phe-202, Leu-Ill, Ile-108, Ala-193, Gly-198; and from the second subunit in the dimer, Ile-138, Val-134, and Phe-133.
  • the two double bonds with trans configuration give the hydrophobic tail a shape that fits to the hydrophobic groove once residue Ile-108 has changed rotamer.
  • binding of tetrahydrocerulenin would cost entropy, and as expected it shows more than 2 orders of magnitude less inhibitory activity (DAgnolo, et ⁇ .(1973) Biochim. Biophys.
  • the structure of the cerulenin complex can be considered to mimic the intermediate formed upon reaction of KAS with the acyl-ACP. In such a complex the hydrophobic cavity would harbor the hydrocarbon tail of the acyl intermediate.
  • acyl hydrophobic tails will not be restricted by two double bonds (as in the case of cerulenin), and this will allow longer acyl chains to be buried in this pocket. Inspection of the active site cavity suggests that it would not be possible to harbor a linear acyl chain longer than 14 carbon atoms without structural changes. Such conformational changes must occur since KASII is able to elongate 16:1 to 18:1 (Garwin, et al. (1980) J. Biol. Chem. 255:3263-3265).
  • the structure of the E.coli KAS II-cerulenin complex was analyzed using the Swiss Pdb Viewer (SPN) modeling program, and by stereo viewing of printouts of the structure in different orientations.
  • SPN Swiss Pdb Viewer
  • each of the hydrophobic residues surrounding the bound cerulenin residue were changed to all the possible larger hydrophobic residues, and each of the rotamers for the mutant amino acids were examined for steric clashes (SPN rotamer score) with adjacent amino acids and the bound cerulenin molecule.
  • the identified amino acids were targeted for mutagenesis for decreasing the fatty acid chain length specificity of the KAS II protein.
  • the candidate chain length shortening mutations chosen were those that made the least steric clashes with neighboring amino acids while having the most clashes with the end 1 to 6 carbons of cerulenin.
  • the structure of the E.coli KAS LT / cerulenin complex was studied as described above and the hydrophobic amino acid residues near the end of the cerulenin binding "pocket" were identified. These amino acids were identified for mutagenesis for the increase in fatty acid chain length recognition. The large hydrophobic residues positioned beyond the end of the cerulenin potentially preventing longer fatty acids from occupying this pocket were chosen for mutagenesis to smaller (alanine) residues.
  • PCR site-directed mutagenesis was performed using the Quick-ChangeTM site- directed mutagenesis kit (Stratagene) following the manufacturers protocol. For the preparation of the specific mutations listed in Table 1, the following oligonucleotide primers were used in the reactions.
  • AGCCAATTGC (SEQ ID NO: 10)
  • E. coli Expression Constructs A series of constructs are prepared to direct the expression of the engineered
  • the construct pCGN10440 contains the I108F mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10441 contains the I108L mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN 10442 contains the A 1931 mutant expressed from the pQ ⁇ 30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10443 contains the I108F, A193I mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10444 contains the I108L, A193I mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10445 contains the A193M mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10446 contains the I108F, A193M mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10447 contains the I108L, A193M mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10448 contains the LI 11 A mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10449 contains the F133A mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10450 contains the LI 11 A, F133A mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10451 contains the I108A, L11A, I114A mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10452 contains the F133A, L197A mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10453 contains the I108A, L11A, I114A, F133A, L197A mutant expressed from the ⁇ QE30 (Qiagen) vector for expression in a host E. coli cell.
  • the construct pCGN10454 contains the L197A mutant expressed from the pQE30 (Qiagen) vector for expression in a host E. coli cell.
  • KAS sequences in plant host cells both alone and in combination with additional sequences encoding proteins involved in fatty acid biosynthesis.
  • a plasmid containing the napin cassette derived from pCGN3223 (described in USPN 5,639,790, the entirety of which is incorporated herein by reference) was modified to make it more useful for cloning large DNA fragments containing multiple restriction sites, and to allow the cloning of multiple napin fusion genes into plant binary transformation vectors.
  • An adapter comprised of the self annealed oligonucleotide of sequence CGCGATTTAA ATGGCGCGCCCTGC AGGCGGCCGCCTGC AGGGCGCGCC ATT
  • TAAAT (SEQ ID NO: ) was ligated into the cloning vector pBC SK+ (Stratagene) after digestion with the restriction endonuclease BssHII to construct vector pCGN7765.
  • Plamids ⁇ CGN3223 and pCGN7765 were digested with Notl and ligated together.
  • the resultant vector, pCGN7770 contains the pCGN7765 backbone with the napin seed specific expression cassette from pCGN3223.
  • pCGN5139 A binary vector for plant transformation, pCGN5139, was constructed from pCGN1558 (McBride and Summerfelt, (1990) Plant Molecular Biology, 14:269-276).
  • the polylinker of pCGN1558 was replaced as a HindIII/Asp718 fragment with a polylinker containing unique restriction endonuclease sites, Ascl, Pad, Xbal, Swal, BamHI, and Notl.
  • the Asp718 and HindHI restriction endonuclease sites are retained in pCGN5139.
  • a binary vector, pCGN8642 was constructed to allow for the rapid cloning of various expression cassettes into the vector for use in plant transformation.
  • the construct contains a multiple cloning region located between the right and left borders of the Agrobacterium transfer DNA.
  • the construct also contains the Tn5 gene expressed from the 35S promoter between the multiple cloning site and the left border for selection of transformed plants on kanamycin.
  • a 354 bp BglH fragment containing the Cuphea hookeriana KASII-7 plastid targeting sequence (Figure 14) (SEQ ID NO: ) was cloned into the S ⁇ mHI site of the various pQE30 constructs containing the E. coli KASI ⁇ (FabF) wild type or mutant KAS sequences.
  • the resultant chimeric KAS II targeting sequence/ FabF encoding sequence were cloned as HindHU Sail fragments into filled-in Sal ⁇ /Xho ⁇ sites of the napin expression cassette, pCGN7770.
  • the resulting napin/KAS cassettes were cloned as Notl fragments into the Notl sites of various plant binary constructs as described below.
  • a napin cassette containing the coding sequence of the Cuphea hookeriana FatB2 protein (described in PCT Publication WO 98/46776, the entirety of which is incorporated herein by reference) was cloned as a Notl fragment into the Notl site of pCG ⁇ 8642 to create pCGNl 1000.
  • a napin cassette containing the coding sequence of the Garm FatAl protein (described in PCT Publication WO 97/12047, the entirety of which is incorporated herein by reference) was cloned into the Notl site of pCG ⁇ 8642 to create pCGN11003.
  • a napin cassette containing the native (wild-type) E. coli KAS II coding sequence was cloned into the Notl site of pCG ⁇ l 1003 to create pCG ⁇ l 1040.
  • a napin cassette containing the native (wild-type) E. coli KAS II coding sequence was cloned into the Notl site of pCG ⁇ l 1003 to create pCG ⁇ l 1040.
  • a napin cassette containing the native (wild-type) E. coli KAS II coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGN11041.
  • a napin cassette containing the native (wild-type) E. coli KAS II coding sequence was cloned into the Notl site of pCG ⁇ l 1000 to create pCG ⁇ l 1042.
  • a napin cassette containing the LI 11 A KAS II mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1003 to create pCG ⁇ l 1045.
  • a napin cassette containing the L111A KAS LT mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGNl 1046.
  • a napin cassette containing the F133A KAS II mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1003 to create pCG ⁇ l 1049.
  • a napin cassette containing the F133A KAS II mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1003 to create pCG ⁇ l 1050.
  • a napin cassette containing the LI 11 A, F133A KAS LT double mutant coding sequence was cloned into the Notl site of pCG ⁇ 11003 to create pCGN11053.
  • a napin cassette containing the LI 11 A, F133A KAS II double mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGNl 1054.
  • a napin cassette containing the I108A, LI 11 A, II 14A KAS II triple mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1003 to create pCG ⁇ l 1057.
  • a napin cassette containing the I108A, LI 11 A, I114A KAS II triple mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGN11058.
  • a napin cassette containing the I108A, LI 11 A, I114A, F133A, L197A KAS II multiple mutant coding sequence was cloned into the Notl site of pCG ⁇ 11003 to create pCGN11061.
  • a napin cassette containing the I108A, LI 11 A, I114A, F133A, L197A KAS LT mulitple mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGN11062.
  • a napin cassette containing the I108F KAS II mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1000 to create pCG ⁇ l 1065.
  • a napin cassette containing the I108F KAS II mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGNl 1066.
  • a napin cassette containing the I108F, A193I KAS U double mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1000 to create pCG ⁇ l 1069.
  • a napin cassette containing the I108F, A193I KAS II double mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGNl 1070.
  • a napin cassette containing the A193M KAS LT mutant coding sequence was cloned into the Notl site of pCG ⁇ l 1000 to create pCG ⁇ l 1073.
  • a napin cassette containing the A193M KAS II mutant coding sequence was cloned into the Notl site of pCG ⁇ 8642 to create pCGN11074.
  • Figure 7 shows the complete list of mutations that were generated in E.coli KAS LT using the Stratagene Quick-ChangeTM site-directed mutagenesis kit, and confirmed by DNA sequencing.
  • the mutant KAS II genes cloned behind an IPTG inducible T5 promoter (pQ ⁇ 30 vector, Qiagen) were transformed into E.coli strain M15/pREP4.
  • the effect of the expression of these KAS II mutants on the fatty acid composition of E.coli is shown in Figure 3.
  • E.coli M15/pREP4 strains containing no vector (-Vec), vector without insert (+Nec), or vectors expression wild-type KAS I or II or single or multiple engineered forms of KASII were grown to mid-log phase in LB media at
  • KAS II/Ll 11A produced the highest level of 18:0 (13%) while KAS II/Ll 11 A-F133A accumulated the highest levels of 20:0 and 20:1 (2 and 4% respectively).
  • Mutations I108A and I114A appeared to decrease the long chain fatty acid accumulation due to L111A and F133A.
  • the KAS II mutants prepared to shorten the maximum fatty acids were analyzed in vitro for the ability to utilize various chain length acyl-ACP substrates.
  • results of the in vitro assays ( Figures 4, 5, and 6) demonstrates that the mutants I108F, I108L, A193M, and A193I have a reduced ability to utilize C8-ACP and longer substrates for condensation.
  • these mutations are able to utilize C6-ACP substrates for elongation to produce C8 fatty acids.
  • at least one mutation, A193M had an increased ability to utilize C6-ACP substrates compared to the wild- type KAS for elongation.
  • A193I only causes a 1.8 fold decrease in activity on 8:0- ACP as compared to 6:0- ACP.
  • Figure 5 shows that the combined mutations at 1108 and A193 have the effect of reducing the activity of KAS II on 6:0-ACP somewhat, but figure 6 shows that the combined effect was much greater effect on the activity with acyl-ACPs 8:0 and longer (14:0). Consequently the double mutants are even more specific for the synthesis of 8 carbon fatty acids.
  • the most specific is KAS II I108F/A193 KAS II which is 90X more active on 6:0-ACP than it is on 8.O-ACP suggesting that it is now an enzyme highly specific for the synthesis of fatty acids only up to 8 carbons in length.
  • Example 5 Structural Comparisons of a Plant Medium-Chain specific KAS with E.coli KAS H
  • the modeled structure of a plant medium-chain (8:0, 10:0) specific KAS [Cuphea. pulcherrima, (Cpu) KASIN] was compared with the crystal structure of E.coli KAS II.
  • Figure 8 shows that Cpu KAS I is predicted to share essentially the same folding pattern as E.coli KAS II with the exception of a few loop regions, as might be expected given the structural similarity between KAS enzymes.
  • Cpu KAS IV also has a similar structure (Figure 9).
  • the general structure for the KAS family of proteins follows the ⁇ - ⁇ - ⁇ - ⁇ - ⁇ folding pattern. Indeed at the amino acid sequence level, all but 7 of the 55 highly conserved residues among KAS enzymes are identical (87% identity). However there is only 60% identity in hydrophobic fatty acid binding pocket region with 8 of the 20 amino acids being different consistent with this region of the protein being responsible for the differences in the enzymes specificity. Furthermore the model shows no stearic hinderance in the formation of KASI and KASIV heterodimer ( Figure 10). In addition, amino acid sequence comparisons between plant, mammalian, bacterial
  • Example 3B The expression constructs described in Example 3B above were used to transform Arabidopsis thaliana (Columbia) and or Columbia mutants fabl,fael-l, and / ⁇ el-2.
  • Seeds from transformed Arabidopsis lines were analyzed for fatty acid composition and are provided in Table 2 below and shown in Figure 13.
  • Fatty acid methyl esters (FAME) extracted in hexane were resolved by gas chromatography (GC) on a Hewlett Packard model 6890 GC.
  • T2 pooled seeds from transgenic Arabidopsis lines containing pCGN 11041 (11041-AT002-9) expressing the native E. coli KAS II protein in the seed tissue demonstrated nearly the same fatty acid composition as the nontransformed control Arabidopsis plants (AT002-44).
  • T2 pooled seeds from transgenic Arabidopsis var Columbia containing the construct pCGNl 1058 demonstrated the ability to synthesize longer carbon chain fatty acids compared to the nontransformed control plants as well as transgenic plants containing the wild-type E. coli KAS II protein.
  • Particular increases in the production of 18:1 ell, 20:1 cl3, 24:0 and 24:1 are observed in transgenic plants containing ⁇ CGN11058.
  • Increases of 18:1 ell, 20:1 cl3, 24:0 and 24:1 of 2 to 3 fold are obtained compared to nontransformed control plants.
  • T2 pooled seeds from transgenic Arabidopsis var Columbia containing the construct pCGN11062 also demonstrated the ability to synthesize longer chain fatty acids compared to nontransformed control plants and transgenic plants containing the wild-type E. coli KAS II protein construct.
  • T2 pooled seeds of 11062 transgenic lines were found to have a 3 to 4 fold increase in 22: 1 as well as increased amounts of 20:2, 20:3 and 22:3, consistent with the presence of a KAS II protein being present in the plastid.

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Abstract

L'invention porte sur des procédés de modification de la spécificité d'un substrat de la synthase bêta-cétoacyl-ACP, ainsi que sur les synthases bêta-cétoacyl-ACP mises au point ou produites. L'invention porte également sur des séquences d'ADN et des produits de recombinaison de l'expression des synthases bêta-cétoacyl-ACP mises au point, ainsi que sur les nouvelles synthases bêta-cétoacyl-ACP produites à partir de celles-ci. Ces séquences d'ADN peuvent être utilisées dans l'expression des synthases bêta-cétoacyl-ACP mises au point des cellules hôtes, notamment des cellules de graines des cultures d'oléagineux en vue de modifier la composition de l'acide gras.
PCT/US2000/022359 2000-07-31 2000-07-31 Mise au point de la synthase $g(b)-cetoacyl acp pour une nouvelle specificite de substrat WO2004007744A2 (fr)

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