WO2006028839A2 - Delta 6-desaturase genes and uses thereof - Google Patents
Delta 6-desaturase genes and uses thereof Download PDFInfo
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- WO2006028839A2 WO2006028839A2 PCT/US2005/030945 US2005030945W WO2006028839A2 WO 2006028839 A2 WO2006028839 A2 WO 2006028839A2 US 2005030945 W US2005030945 W US 2005030945W WO 2006028839 A2 WO2006028839 A2 WO 2006028839A2
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Definitions
- the subject invention relates to the identification and isolation of genes that encode an enzyme (i.e., ⁇ 6-desaturase) involved in the synthesis of polyunsaturated fatty acids and to uses thereof.
- ⁇ 6-desaturase catalyzes the conversion of, for example, linoleic acid (C18:2n-6) to ⁇ - linolenic acid (C18:3n-6) and ⁇ -linolenic acid (C18:3n-3) to stearidonic acid (C18:4n-3) .
- the converted products may then be utilized as substrates in the production of other polyunsaturated fatty acids (PUFAs) .
- the products or other polyunsaturated fatty acids may be added to pharmaceutical compositions, nutritional compositions, animal feeds as well as other products such as cosmetics.
- Desaturases are critical in the production of long-chain polyunsaturated fatty acids that have many important functions.
- polyunsaturated fatty acids PUFAs
- PUFAs polyunsaturated fatty acids
- They also serve as precursors to mammalian prostacyclins, eicosanoids, leukotrienes and prostaglandins.
- PUFAs are necessary for the proper development of the developing infant brain as well as for tissue formation and repair.
- attempts are being made to produce them, as well as intermediates leading to their production, in an efficient manner.
- a number of enzymes, most notably desaturases and elongases, are involved in PUFA biosynthesis (see Figure 1) .
- an elongase catalyzes the conversion of ⁇ - linolenic acid (GLA) to dihomo- ⁇ -linolenic acid (DGLA) and of stearidonic acid (C18:4n-3) to (n-3) -eicosatetraenoic acid (C20:4n-3) .
- Linoleic acid (LA, C18:2n-9,12 or C18:2n-6) is produced from oleic acid (C18:l- ⁇ 9) by a ⁇ l2-desaturase.
- GLA C18 :3n-6, 9, 12
- ⁇ 6- desaturase It must be noted that animals cannot desaturate beyond the ⁇ 9 position and therefore cannot convert oleic acid into linoleic acid.
- ⁇ -linolenic acid (ALA, C18:3n- 9,12,15) cannot be synthesized by mammals.
- ⁇ - linolenic acid can be converted to stearidonic acid (STA, C18 :4n-6, 9, 12, 15) by a ⁇ 6-desaturase (see PCT publication WO 96/13591 and The FASEB Journal, Abstracts, Part I, Abstract 3093, page A532 (Experimental Biology 98, San Francisco, CA, April 18-22, 1998) ; see also U.S. Patent No. 5,552,306) , followed by elongation to (n-3) -eicosatetraenoic acid (C20:4n- 8,11,14,17) in mammals and algae.
- STA stearidonic acid
- ⁇ 6-desaturase see PCT publication WO 96/13591 and The FASEB Journal, Abstracts, Part I, Abstract 3093, page A532 (Experimental Biology 98, San Francisco, CA, April 18-22, 1998) ; see also U.S. Patent No. 5,552,306) , followed by elongation to
- This polyunsaturated fatty acid i.e., C20 :4n-8, 11, 14, 17
- eicosapentaenoic acid EPA, C20 : 5n-5, 8, 11, 14, 17
- EPA can then, in turn, be converted to ⁇ 3- docosapentaenoic acid (C22:5n-3) by an elongase.
- Other eukaryotes, including fungi and plants, have enzymes which desaturate at carbon 12 (see PCT publication WO 94/11516 and U.S. Patent No. 5,443,974) and carbon 15 (see PCT publication WO 93/11245) .
- the major polyunsaturated fatty acids of animals therefore are either derived from diet and/or from desaturation and elongation of linoleic acid or ⁇ linolenic acid.
- genes involved in PUFA synthesis from species that naturally produce 'these fatty acids and to express these genes in a microbial, plant, or animal system which can be altered to provide production of commercial quantities of one or more PUFAs.
- the ⁇ 6-desaturase enzyme there is a definite need for the ⁇ 6-desaturase enzyme, the respective genes encoding this enzyme, as well as recombinant methods of producing this enzyme.
- oils containing levels of PUFAs beyond those naturally present as well as those enriched in novel PUFAs can only be made by isolation and expression of the ⁇ 6-desaturase genes.
- the present invention includes an isolated nucleotide sequence or fragment thereof encoding a polypeptide having desaturase activity, wherein said polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 and SEQ ID NO: 8.
- the present invention encompasses an isolated nucleic acid sequence or fragment thereof comprising, or complementary to, a nucleotide sequence having at least 90% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID N0:l, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO: 7.
- the nucleotide sequences described above encode a functionally active desaturase that utilizes a monounsaturated or polyunsaturated fatty acid as a substrate.
- the nucleotide sequences may be derived for example, from Delacroixia coronatus.
- the present invention also includes purified proteins and fragments thereof encoded by the above-referenced nucleotide sequences.
- the present invention also includes a purified polypeptide which desaturates polyunsaturated fatty acids at carbon 6 and has an amino acid sequence having at least 90% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 and SEQ ID NO:8.
- the present invention includes a method of producing a desaturase comprising the steps of: isolating a nucleotide sequence comprising or complementary to a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO: 8 ; constructing a vector comprising: i) the isolated nucleotide sequence operably linked to ii) a promoter; and introducing said vector into a host cell for a time and under conditions sufficient for expression of the desaturase.
- the host cell may be, for example, a eukaryotic cell or a prokaryotic cell.
- the prokaryotic cell may be, for example, E. coli, cyanobacteria or B. subtilis.
- the eukaryotic cell may be, for example, a mammalian cell, an insect cell, a plant cell or a fungal cell (e.g., a yeast cell such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp. , Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. or Pichia spp.) .
- yeast cell such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida spp. , Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces spp., Hansenula spp., Trichoderma spp. or Pichia spp.
- the present invention also includes a vector comprising: an isolated nucleotide sequence comprising or complementary to a nucleotide sequence encoding a polypeptide having an amino acid sequence having at least 90% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8, operably linked to a regulatory sequence (e.g., a promoter) .
- the invention also includes a host cell comprising this vector.
- the host cell may be, for example, a eukaryotic cell or a prokaryotic cell. Suitable eukaryotic cells and prokaryotic cells are as defined above.
- the present invention includes an isolated plant cell, plant or plant tissue comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid by the plant cell, plant or plant tissue.
- the polyunsaturated fatty acid may be, for example, selected from the group consisting of ⁇ -linolenic acid or stearidonic acid.
- the invention also includes one or more plant oils or acids expressed by the above plant cell, plant or plant tissue.
- the present invention also encompasses a transgenic plant comprising the above vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid in seeds of the transgenic plant.
- the present invention also includes a method ("first method") for producing a polyunsaturated fatty acid comprising the steps of: isolating a nucleic acid sequence comprising or complementary to a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 and SEQ ID NO: 8 or 90% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7; constructing a vector comprising the isolated nucleotide sequence; introducing the vector into a host cell for a time and under conditions sufficient for expression of ⁇ 6- desaturase; and exposing the expressed ⁇ 6-desaturase to a substrate polyunsaturated fatty acid in order to convert the substrate to a product polyunsaturated fatty acid.
- the substrate polyunsaturated fatty acid may be, for example, linolenic acid or ⁇ -linolenic acid
- the product polyunsaturated fatty acid may be, for example, ⁇ -linolenic acid or stearidonic acid, respectively.
- This method may further comprise the step of exposing the product polyunsaturated fatty acid to another enzyme (e.g., an elongase) in order to convert the product polyunsaturated fatty acid to another polyunsaturated fatty acid (i.e., "second" method) .
- another enzyme e.g., an elongase
- the product polyunsaturated fatty acid may be, for example, ⁇ -linolenic acid or stearidonic acid and the "another" polyunsaturated fatty acid may be, for example, dihomo- ⁇ -linolenic acid or eicosatetraenoic acid.
- the present invention includes a method of producing a polyunsaturated fatty acid comprising the steps of: exposing a substrate monounsaturated or polyunsaturated fatty acid to an enzyme (e.g., an elongase or desaturase) in order to convert the substrate to a product polyunsaturated fatty acid; and exposing the product polyunsaturated fatty acid to a ⁇ 6-desaturase comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID N0:4, SEQ ID NO:6 and SEQ ID NO:8, in order to convert the product polyunsaturated fatty acid to a final product polyunsaturated fatty acid.
- an enzyme e.g., an elongase or desaturase
- a ⁇ 6-desaturase comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID N0:4, SEQ ID NO:6 and SEQ ID NO:8, in order to convert the
- ⁇ 3-desaturase e.g., ⁇ 15-desaturase
- product polyunsaturated fatty acid e.g., ⁇ -linolenic acid
- product polyunsaturated fatty acid e.g., stearidonic acid
- a substrate monounsaturated fatty acid such as oleic acid may be exposed to a desaturase (e.g., ⁇ l2-desaturase) in order to convert the substrate to a product polyunsaturated fatty acid such as linoleic acid.
- the product polyunsaturated fatty acid may then be converted to the final product polyunsaturated fatty acid, ⁇ -linolenic acid by exposure to the ⁇ 6-desaturase of the present invention.
- the ⁇ 6-desaturase is utilized in the last step of the method in order to create the "final" desired product.
- the AA may then be exposed to an elongase in order to convert it to adrenic acid.
- the adrenic acid may be exposed to ⁇ 4-desaturase in order to convert it to ⁇ 6-docosapentaenoic acid (see Figure 1) .
- the method involves the utilization of a linoleic acid substrate and a series of desaturase and elongase enzymes, in addition to the ⁇ 6-desaturase, in order to arrive at the final product.
- a linoleic acid substrate and a series of desaturase and elongase enzymes, in addition to the ⁇ 6-desaturase, in order to arrive at the final product.
- Possible substrates include those shown in Figure 1, for example, linoleic acid and ⁇ -linolenic acid.
- the present invention also encompasses a composition comprising at least one polyunsaturated fatty acid selected from the group consisting of the "product" polyunsaturated fatty acid produced according to the methods described above and the "another" polyunsaturated fatty acid produced according to the methods described above.
- the product polyunsaturated fatty acid may be, for example, ⁇ -linolenic acid or stearidonic acid.
- the another polyunsaturated fatty acid may be, for example, dihomo- ⁇ -linolenic acid or eicosatetraenoic acid.
- the present invention encompasses a method of preventing or treating a condition caused by insufficient intake of polyunsaturated fatty acids comprising administering to the patient the composition above in an amount sufficient to effect prevention or treatment. Moreover, the present invention also includes a further method for producing a polyunsaturated fatty acid.
- This method comprises the steps of: a) isolating a nucleic acid sequence comprising or complementary to a nucleotide sequence: i) encoding a polypeptide comprising an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID N0:4, SEQ ID NO:6 and SEQ ID NO:8 or ii) having at least 90% identity to a nucleotide sequence selected from the group consisting of SEQ ID N0:l, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO: 7; b) constructing a vector comprising: i) the isolated nucleotide sequence, ii) an isolated nucleotide sequence encoding an elongase and iii) an isolated nucleotide sequence encoding a ⁇ 5-desaturase; c) introducing the vector into a host cell for a time and under conditions sufficient for expression of the ⁇ 6-desaturase
- the substrate polyunsaturated fatty acid may be linoleic acid
- the product polyunsaturated fatty acid may be ⁇ -linolenic acid
- the another polyunsaturated fatty acid may be dihomo- ⁇ - linolenic acid and the final product polyunsaturated fatty acid may be arachidonic acid.
- the substrate polyunsaturated fatty acid may be ⁇ -linolenic acid
- the product polyunsaturated fatty acid may be stearidonic acid
- the another polyunsaturated fatty acid may be eicosatetraenoic acid
- the final product polyunsaturated fatty acid may be eicosapentaenoic acid.
- the present invention includes an isolated nucleic acid sequence or fragment thereof which hybridizes, under moderate or high stringency conditions, to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO: 7.
- the present invention also encompasses an isolated nucleic acid or fragment thereof, which hybridizes, under moderate or high stringency conditions, to an isolated nucleic acid sequence encoding a polypeptide having desaturase activity, wherein the amino acid sequence of said polypeptide has at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO: 8.
- Figure 1 illustrates the fatty acid biosynthetic pathway and the role of ⁇ 6-desaturase in this pathway.
- Major pathway intermediates found in the total lipid profile of Delacroixia are boxed.
- Figure 2 illustrates the consensus nucleotide sequence of the putative desaturase from Delacroixia (SEQ ID NO:1), obtained by the alignment of three separate overlapping clones.
- Figure 3 illustrates the consensus amino acid sequence of the putative desaturase from Delacroixia (SEQ ID N0:2), obtained by the alignment of three separate overlapping clones .
- Figure 4 illustrates the GAP alignment of the translated amino acid sequence of Figure 3 (SEQ ID NO:2) and the Mortierella alpina ⁇ 6-desaturase (SEQ ID NO:40) .
- Figure 6 illustrates the nucleotide sequence of the putative desaturase gene from Delacroixia coronatus ATCC 28565, in construct pRDC ⁇ , with the designated start site underlined (SEQ ID NO:3) .
- Figure 7 illustrates the putative amino acid sequence of the ⁇ -desaturase gene (SEQ ID NO:4) from Delacroixia coronatus ATCC 28565, in construct pRDC8.
- Figure 8 illustrates the nucleotide sequence of the putative desaturase gene (SEQ ID NO:5) from Delacroixia coronatus ATCC 28565, in construct pRDCIO, with the designated start site underlined.
- Figure 9 illustrates the putative amino acid sequence of the ⁇ 6-desaturase gene (SEQ ID NO:6) from Delacroixia coronatus ATCC 28565, in construct pRDCIO .
- Figure 10 illustrates the nucleotide sequence of Del-D6 (SEQ ID N0:7), the ⁇ 6-desaturase from Delacroixia coronatus ATCC 28565, in construct pRDC12.
- Figure 11 illustrates the putative amino acid sequence of Del-D6 (SEQ ID NO: 8), the ⁇ 6-desaturase from Delacroixia coronatus ATCC 28565, in construct pRDC12.
- Figure 12 illustrates the alignment of the putative amino acid sequence encoded by Del-OS (in pRDC12) (see SEQ ID NO: 8) (i.e., the ⁇ -desaturase from Delacroixia coronatus ATCC 28565) and the Mortierella alpina ⁇ 6-desaturase (see SEQ ID NO:40) .
- Figure 13 illustrates the alignment of the putative amino acid sequence encoded by Del-D6 (in pRDC12) (SEQ ID NO: 8) with known ⁇ 6-desaturase sequences from Mortierella alpina (Accession #AAF08685) (SEQ ID NO:40) , Phaeodactylum tricornatum (Accession #AAL92563) (SEQ ID NO:37) , Rhizopus oryzae (Accession #AAS93682) (SEQ ID NO:38) , Pythium irregulare (Accession # AAL13310) (SEQ ID NO:39) , and Mucor circinelloides (Accession # BAB69055) (SEQ ID NO:45) .
- Identical residues are underlined and the conserved histidine- box motifs as well as the conserved cytochrome b 5 domain are boxed.
- the subject invention relates to the nucleotide and translated amino acid sequences of the ⁇ 6-desaturase genes derived from the fungus Delacroixia coronata. Furthermore, the subject invention also includes uses of the genes and of the enzymes encoded by this gene. For example, the genes and encoded, corresponding enzymes may be used in the production of polyunsaturated fatty acids such as, for instance, ⁇ - linolenic acid and stearidonic acid which may be added to pharmaceutical compositions, nutritional compositions and to other valuable products.
- Figure 2 illustrates the consensus nucleotide sequence (SEQ ID N0:l) of the putative ⁇ -desaturase from Delacroixia coronatus
- Figure 3 illustrates the consensus amino acid sequence (SEQ ID NO:2) of the putative ⁇ 6-desaturase from Delacroixia coronatus.
- the present invention also encompasses isolated nucleotide sequences (and the corresponding encoded proteins) having sequences comprising, corresponding to, identical to, or complementary to at least about 70%, preferably at least about 80%, and more preferably at least about 90% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7. (All integers (and portions thereof) between 70% and 100% are also considered to be within the scope of the present invention with respect to percent identity.) Such sequences may be derived from any source, either isolated from a natural source, or produced via a semi ⁇ synthetic route, or synthesized de novo. In particular, such sequences may be isolated or derived from sources other than described in the examples (e.g., bacteria, fungus, algae, C. elegans, mouse or human) .
- the present invention also encompasses fragments and derivatives of the nucleic acid sequences of the present invention (i.e., SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7) , as well as of the sequences derived from other sources, and having the above-described complementarity, identity or correspondence.
- Functional equivalents of the above full length sequences and fragments i.e., sequences having ⁇ 6-desaturase activity, as appropriate are also encompassed by the present invention.
- a "fragment" of a nucleotide sequence is defined as a contiguous sequence of approximately at least 6, preferably at least about 8, more preferably at least about 10 nucleotides, and even more preferably at least about 15 nucleotides corresponding to a region of the specified nucleotide sequence.
- the invention also includes a purified polypeptide which desaturates polyunsaturated fatty acids at the carbon 6 position and has at least about 70% amino acid similarity or identity, preferably at least about 80% amino acid similarity or identity and more preferably at least about 90% amino acid similarity or identity to the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6 or SEQ ID NO:8 of the above- noted proteins which are, in turn, encoded by the above- described nucleotide sequences.
- identity refers to the relatedness of two sequences on a nucleotide-by-nucleotide basis over a particular comparison window or segment.
- identity is defined as the degree of sameness, correspondence or equivalence between the same strands (either sense or antisense) of two DNA segments (or two amino acid sequences) .
- Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identical base or amino acid occurs in both sequences in order to yield the number of matched positions, dividing the number of such positions by the total number of positions in the segment being compared and multiplying the result by 100.
- Optimal alignment of sequences may be conducted by the algorithm of Smith Sc Waterman, Appl . Math. 2:482 (1981) , by the algorithm of Needleman & Wunsch, J. MoI. Biol.
- complementarity is defined as the degree of relatedness between two DNA segments. It is determined by measuring the ability of the sense strand of one DNA segment to hybridize with the anti- sense strand of the other DNA segment, under appropriate conditions, to form a double helix.
- a “complement” is defined as a sequence which pairs to a given sequence based upon the canonic base-pairing rules. For example, a sequence A-G-T in one nucleotide strand is "complementary" to T-C-A in the other strand.
- Similarity between two amino acid sequences is defined as the presence of a series of identical as well as conserved amino acid residues in both sequences. The higher the degree of similarity between two amino acid sequences, the higher the correspondence, sameness or equivalence of the two sequences.
- Encoded by refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 amino acids, more preferably at least 8 amino acids, and even more preferably at least 15 amino acids from a polypeptide encoded by the nucleic acid sequence.
- the present invention also encompasses an isolated nucleotide sequence which encodes a PUFA having desaturase activity (i.e., ⁇ 6-desaturase activity) and that is hybridizable, under moderately stringent conditions, to a nucleic acid having a nucleotide sequence comprising or complementary to the nucleotide sequences described above (see SEQ ID N0:l, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7) .
- a nucleic acid molecule is "hybridizable" to another nucleic acid molecule when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and ionic strength (see Sambrook et al., "Molecular Cloning: A Laboratory Manual, Second Edition (1989) , Cold Spring Harbor Laboratory Press,
- hybridization is generally used to mean hybridization of nucleic acids at appropriate conditions of stringency as would be readily evident to those skilled in the art depending upon the nature of the probe sequence and target sequences. Conditions of hybridization and washing are well known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature and/or ionic strength of the solution are readily accomplished. See, for example, Sambrook, J. et al . , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring harbor Press, Cold Spring harbor, N.Y., 1989, as noted above and incorporated herein by reference. (See also Short Protocols in Molecular Biology, ed. Ausubel et al . and Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,
- the choice of conditions is dictated by the length of the sequences being hybridized, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted.
- Low stringency conditions are preferred when partial hybridization between strands that have lesser degrees of complementarity is desired.
- high stringency conditions are preferred.
- the hybridization solution contains 6 X S.S.C, 0.01 M EDTA, 1 X Denhardt ' s solution and 0.5% SDS.
- Hybridization is carried out at about 68 degrees Celsius for about 3 to 4 hours for fragments of cloned DNA and for about 12 to about 16 hours for total eukaryotic DNA.
- moderate stringencies one may utilize filter pre-hybridizing and hybridizing with a solution of 3 X sodium chloride, sodium citrate (SSC) , 50% formamide (0.1 M of this buffer at pH 7.5) and 5 X Denhardt' s solution.
- T n is known to be a function of the G-C content and duplex length as well as the ionic strength of the solution.
- Hybridization requires that two nucleic acids contain complementary sequences. However, depending on the stringency of the hybridization, mismatches between bases may occur. As noted above, the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation. Such variables are well known in the art. More specifically, the greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al . , supra) . For hybridization with shorter nucleic acids, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al . , supra) .
- an "isolated nucleic acid fragment or sequence” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
- An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
- a "fragment" of a specified polynucleotide refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10 nucleotides, and even more preferably at least about 15 nucleotides, and most preferable at least about 25 nucleotides identical or complementary to a region of the specified nucleotide sequence.
- Nucleotides (usually found in their 5 ' -monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, "G” for guanylate or deoxyguanylate, "U” for uridylate, "T” for deoxythymidylate, "R” for purines (A or G) , "Y
- fragment or subfragment that is functionally equivalent and “functionally equivalent fragment or subfragment” are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme.
- the fragment or subfragment can be used in the design of chimeric constructs to produce the desired phenotype in a transformed plant. Chimeric constructs can be designed for use in co-suppression or antisense by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the appropriate orientation relative to a plant promoter sequence.
- nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype.
- modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences .
- Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
- Near gene refers to a gene as found in nature with its own regulatory sequences.
- chimeric construct refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
- a "foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric constructs.
- a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
- Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
- Regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences) , within, or downstream (3 ' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
- Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
- Promoter sequences can also be located within the transcribed portions of genes, and/or downstream of the transcribed sequences. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most host cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, Biochemistry of Plants 15:1-82
- DNA fragments of some variation may have identical promoter activity.
- An "intron” is an intervening sequence in a gene that does not encode a portion of the protein sequence. Thus, such sequences are transcribed into RNA but are then excised and are not translated. The term is also used for the excised RNA sequences.
- An “exon” is a portion of the gene sequence that is transcribed and is found in the mature messenger RNA derived from the gene, but is not necessarily a part of the sequence that encodes the final gene product.
- the "translation leader sequence” refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence.
- the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
- the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D. (1995) Molecular Biotechnology 3:225) .
- the "3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
- the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
- ⁇ RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
- RNA transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA.
- Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
- cDNA refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
- Sense RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro.
- Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary- transcript or mRNA and that blocks the expression of a target gene (U.S. Patent No. 5,107,065) .
- the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
- “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
- complementary and reverse complement are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.
- endogenous RNA refers to any RNA which is encoded by any nucleic acid sequence present in the genome of the host prior to transformation with the recombinant construct of the present invention, whether naturally- occurring or non-naturally occurring, i.e., introduced by recombinant means, mutagenesis, etc.
- non-naturally occurring means artificial, not consistent with what is normally found in nature.
- operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other.
- a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter) .
- Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
- the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3 ' to the target mRNA, or within the target mRNA, or a first complementary region is 5 ' and its complement is 3 ' to the target mRNA.
- expression refers to the production of a functional end-product. Expression of a gene involves transcription of the gene and translation of the mRNA into a precursor or mature protein. "Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
- Codon refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020) .
- “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
- Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and pro- peptides still present. Pre- and pro-peptides may be but are not limited to intracellular localization signals.
- “Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, resulting in genetically stable inheritance.
- “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance.
- Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic” organisms.
- the preferred method of cell transformation of rice, corn and other monocots is the use of particle-accelerated or "gene gun” transformation technology (Klein et al . , (1987) Nature (London) 327:70-73; U.S. Patent No.
- transformation refers to both stable transformation and transient transformation.
- Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook") .
- PCR Polymerase Chain Reaction
- PCR Polymerase Chain Reaction
- PCR a technique for the synthesis of large quantities of specific DNA segments, consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, CT) .
- the double stranded DNA is heat denatured, the two primers complementary to the 3 ' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature.
- One set of these three consecutive steps is referred to as a cycle.
- PCR Polymerase chain reaction
- Patent No. 4,683,194 The process utilizes sets of specific in vitro synthesized oligonucleotides to prime DNA synthesis.
- the design of the primers is dependent upon the sequences of DNA that are desired to be analyzed.
- the technique is carried out through many cycles (usually 20-50) of melting the template at high temperature, allowing the primers to anneal to complementary sequences within the template and then replicating the template with DNA polymerase.
- the products of PCR reactions are analyzed by separation in agarose gels followed by ethidium bromide staining and visualization with UV transillumination.
- radioactive dNTPs can be added to the PCR in order to incorporate label into the products. In this case the products of PCR are visualized by exposure of the gel to x-ray film.
- the added advantage of radiolabeling PCR products is that the levels of individual amplification products can be quantitated.
- recombinant construct refers to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art. Such construct may be itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention.
- the gene encoding the ⁇ 6-desaturase enzyme may then be introduced into either a prokaryotic or eukaryotic host cell through the use of a vector or construct.
- the vector for example, a bacteriophage, cosmid or plasmid, may comprise the nucleotide sequence encoding the ⁇ 6-desaturase enzyme, as well as any regulatory sequence
- the regulatory sequence (e.g., promoter) which is functional in the host cell and is able to elicit expression of the desaturase encoded by the nucleotide sequence.
- the regulatory sequence e.g., promoter
- the regulatory sequence is in operable association with, or operably linked to, the nucleotide sequence.
- a promoter is said to be "operably linked" with a coding sequence if the promoter affects transcription or expression of the coding sequence.
- Suitable promoters include, for example, those from genes encoding alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglycerate kinase, acid phosphatase, T7, TPI, lactase, metallothionein, cytomegalovirus immediate early, whey acidic protein, glucoamylase, and promoters activated in the presence of galactose, for example, GALl and GALlO.
- nucleotide sequences which encode other proteins, oligosaccharides, lipids, etc. may also be included within the vector as well as other regulatory sequences such as a polyadenylation signal (e.g., the poly-A signal of SV-40T- antigen, ovalalbumin or bovine growth hormone) .
- a polyadenylation signal e.g., the poly-A signal of SV-40T- antigen, ovalalbumin or bovine growth hormone
- a ⁇ 6-desaturase gene sequence of the present invention an elongase gene sequence and a ⁇ 5- desaturase gene sequence into the vector, one may co-express the encoded ⁇ 6-desaturase, the encoded elongase, as well as the encoded ⁇ 5-desaturase, respectively, in order to convert, for example, LA to ARA and ALA to EPA.
- the vector may then be introduced into the host cell of choice by methods known to those of ordinary skill in the art including, for example, transfection, transformation and electroporation (see Molecular Cloning: A Laboratory Manual, 2 nd ed. , Vol. 1-3, ed. Sambrook et al . , Cold Spring Harbor Laboratory Press (1989)) .
- the host cell is then cultured under suitable conditions permitting expression of the genes leading to the production of the desired PUFA, which is then recovered and purified.
- suitable prokaryotic host cells include, for example, bacteria such as Escherichia coli, Bacillus subtilis as well as Cyanobacteria such as Spirulina spp. (i.e., blue- green algae) .
- suitable eukaryotic host cells include, for example, mammalian cells, plant cells, yeast cells such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Lipomyces starkey, Candida spp. such as
- Yarrowia (Candida) lipolytica Kluyveromyces spp., Pichia spp., Trichoderma spp. or Hansenula spp.
- fungal cells such as filamentous fungal cells, for example, Aspergillus, Neurospora and Penicillium.
- Saccharomyces cerevisiae (baker's yeast) cells are utilized.
- Transient expression in a host cell can be accomplished in a transient or stable fashion.
- Transient expression can occur from introduced constructs which contain expression signals functional in the host cell, but which constructs do not replicate and rarely integrate in the host cell, or when the host cell is not proliferating.
- Transient expression also can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems frequently exhibit a low basal level of expression.
- Stable expression can be achieved by introduction of a construct that can integrate into the host genome or that autonomously replicates in the host cell .
- Stable expression of the gene of interest can be selected through the use of a selectable marker located on or transfected with the expression construct, followed by selection for cells expressing the marker.
- the site of the construct' s integration can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
- a transgenic mammal may also be used in order to express the enzyme of interest (i.e., ⁇ 6-desaturase) , and ultimately the PUFA(s) of interest. More specifically, once the above- described construct is created, it may be inserted into the pronucleus of an embryo. The embryo may then be implanted into a recipient female. Alternatively, a nuclear transfer method could also be utilized (Schnieke et al . , Science 278:2130-2133 (1997)) . Gestation and birth are then permitted (see, e.g., U.S. Patent No. 5,750,176 and U.S. Patent No. 5,700,671) .
- Milk, tissue or other fluid samples from the offspring should then contain altered levels of PUFAs, as compared to the levels normally found in the non-transgenic animal. Subsequent generations may be monitored for production of the altered or enhanced levels of PUFAs and thus incorporation of the gene encoding the desired desaturase enzyme into their genomes.
- the mammal utilized as the host may be selected from the group consisting of, for example, a mouse, a rat, a rabbit, a pig, a goat, a sheep, a horse and a cow. However, any mammal may be used provided it has the ability to incorporate DNA encoding the enzyme of interest into its genome.
- a desaturase polypeptide For expression of a desaturase polypeptide, functional transcriptional and translational initiation and termination regions are operably linked to the DNA encoding the desaturase polypeptide.
- Transcriptional and translational initiation and termination regions are derived from a variety of nonexclusive sources, including the DNA to be expressed, genes known or suspected to be capable of expression in the desired system, expression vectors, chemical synthesis, or from an endogenous locus in a host cell.
- Expression in a plant tissue and/or plant part presents certain efficiencies, particularly where the tissue or part is one which is harvested early, such as seed, leaves, fruits, flowers, roots, etc. Expression can be targeted to that location with the plant by utilizing specific regulatory sequence such as those of U.S. Patent Nos.
- the expressed protein can be an enzyme which produces a product which may be incorporated, either directly or upon further modifications, into a fluid fraction from the host plant.
- Expression of a desaturase gene, or antisense desaturase transcripts can alter the levels of specific PUFAs, or derivatives thereof, found in plant parts and/or plant tissues.
- the desaturase polypeptide coding region may be expressed either by itself or with other genes, in order to produce tissues and/or plant parts containing higher proportions of desired PUFAs or in which the PUFA composition more closely resembles that of human breast milk (Prieto et al . , PCT publication WO 95/24494) .
- the termination region may be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. A large number of termination regions are known to and have been found to be satisfactory in a variety of hosts from the same and different genera and species. The termination region usually is selected as a matter of convenience rather than because of any particular property.
- a plant e.g., Glycine max (soybean) , cotton, safflower, sunflower, palm, coconut, maize, nuts, beans, peas or Brassica napus (canola)
- plant tissue may also be utilized as a host or host cell, respectively, for expression of the desaturase enzyme which may, in turn, be utilized in the production of polyunsaturated fatty acids. More specifically, desired PUFAS can be expressed in seed. Methods of isolating seed oils are known in the art.
- seed oil components may be manipulated through the expression of the desaturase gene, as well as perhaps other desaturase genes and elongase genes, in order to provide seed oils that can be added to nutritional compositions, pharmaceutical compositions, animal feeds and cosmetics.
- a vector which comprises a DNA sequence encoding the desaturase operably linked to a promoter will be introduced into the plant tissue or plant for a time and under conditions sufficient for expression of the desaturase gene.
- the vector may also comprise one or more genes that encode other enzymes, for example, ⁇ 5-desaturase, elongase, ⁇ 12-desaturase, ⁇ l5-desaturase, ⁇ l7-desaturase, and/or ⁇ l9-desaturase.
- the plant tissue or plant may produce the relevant substrate (e.g., linoleic acid or ⁇ -linolenic acid) upon which the enzyme acts or a vector encoding enzymes which produce such substrates may be introduced into the plant tissue, plant cell or plant. In addition, substrate may be sprayed on plant tissues expressing the appropriate enzymes.
- PUFAs e.g., n- ⁇ unsaturated fatty acids such as ⁇ 6-docosapentaenoic acid, or n-3 fatty acids such as docosahexaenoic acid
- the invention also encompasses a transgenic plant comprising the above-described vector, wherein expression of the nucleotide sequence of the vector results in production of a polyunsaturated fatty acid in, for example, the seeds of the transgenic plant.
- This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil .
- the development or regeneration of plants containing the foreign, exogenous gene that encodes a protein of interest is well known in the art.
- the regenerated plants are self-pollinated to provide homozygous transgenic plants.
- a transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
- Transformation of monocotyledons using electroporation, particle bombardment, and Agrobacterium have also been reported. Transformation and plant regeneration have been achieved in asparagus (Bytebier et al. , Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)) ; barley (Wan and Lemaux, Plant Physiol 104:37 (1994)) ; Zea mays (Rhodes et al. , Science 240:204 (1988) , Gordon-Kamm et al. , Plant Cell 2:603-618 (1990), Fromm et al . , BiolTechnology 8:833 (1990), Koziel et al .
- Transient expression systems may be used to functionally dissect gene constructs (see generally, Maliga et al. , Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995)) . It is understood that any of the nucleic acid molecules of the present invention can be introduced into a plant cell in a permanent or transient manner in combination with other genetic elements such as vectors, promoters, enhancers etc.
- substrates which may be produced by the host cell either naturally or transgenically, as well as the enzymes which may be encoded by DNA sequences present in the vector, which is subsequently introduced into the host cell, are shown in Figure 1.
- the isolated desaturase genes and the desaturase enzymes encoded thereby have many uses .
- the gene and corresponding enzyme may be used indirectly or directly in the production of polyunsaturated fatty acids, for example, ⁇ -desaturase may be used in the production of ⁇ -linolenic acid or stearidonic acid.
- "Directly" is meant to encompass the situation where the enzyme directly converts the acid to another acid, the latter of which is utilized in a composition (e.g., the conversion of linoleic acid to ⁇ -linolenic acid) .
- “Indirectly” is meant to encompass the situation where an acid (e.g., ⁇ -linolenic acid) is converted to another acid (i.e., a pathway intermediate such as stearidonic acid) by the desaturase, and then the latter acid is converted to another acid by use of a desaturase or non-desaturase enzyme (e.g., stearidonic acid to eicosatetraenoic acid by an elongase) .
- a desaturase or non-desaturase enzyme e.g., stearidonic acid to eicosatetraenoic acid by an elongase
- the present invention includes "indirect" situations in which the PUFA is first converted to another polyunsaturated fatty acid by a non- ⁇ 6-desaturase enzyme (for example, an elongase or another desaturase) and then converted to a final product via ⁇ - desaturase.
- a non- ⁇ 6-desaturase enzyme for example, an elongase or another desaturase
- linoleic acid may be converted to ⁇ -linolenic acid by a desaturase (i.e., ⁇ 15-desaturase) , and then converted to stearidonic acid by a ⁇ 6-desaturase.
- polyunsaturated fatty acids i.e., those produced either directly or indirectly by activity of the ⁇ 6-desaturase enzyme
- the present invention includes nutritional compositions.
- Such compositions for purposes of the present invention, include any food or preparation for human consumption including for enteral or parenteral consumption, which when taken into the body (a) serve to nourish or build up tissues or supply energy and/or (b) maintain, restore or support adequate nutritional status or metabolic function.
- the nutritional composition of the present invention comprises at least one oil or acid produced directly or indirectly by use of the desaturase gene, in accordance with the present invention, and may either be in a solid or liquid form. Additionally, the composition may include edible macronutrients, vitamins and minerals in amounts desired for a particular use. The amount of such ingredients will vary depending on whether the composition is intended for use with normal, healthy infants, children or adults having specialized needs such as those which accompany certain metabolic conditions (e.g., metabolic disorders) .
- macronutrients which may be added to the composition include but are not limited to edible fats, carbohydrates and proteins. Examples of such edible fats include but are not limited to coconut oil, borage oil, fungal oil, black current oil, soy oil, and mono- and diglycerides.
- carbohydrates include but are not limited to glucose, edible lactose and hydrolyzed starch.
- proteins which may be utilized in the nutritional composition of the invention include but are not limited to soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.
- vitamins and minerals may be added to the nutritional compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.
- the components utilized in the nutritional compositions of the present invention will be of semi-purified or purified origin.
- semi-purified or purified is meant a material which has been prepared by purification of a natural material or by synthesis.
- nutritional compositions of the present invention include but are not limited to infant formulas, dietary supplements (e.g., adult nutritional products and oil) , dietary substitutes, and rehydration compositions.
- Nutritional compositions of particular interest include but are not limited to those utilized for enteral and parenteral supplementation for infants, specialized infant formulas, supplements for the elderly, and supplements for those with gastrointestinal difficulties and/or malabsorption.
- the nutritional composition of the present invention may also be added to food even when supplementation of the diet is not required.
- the composition may be added to food of any type including but not limited to margarines, modified butters, cheeses, milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats, fish and beverages.
- the nutritional composition is an enteral nutritional product, more preferably, an adult or pediatric enteral nutritional product .
- This composition may be administered to adults or children experiencing stress or having specialized needs due to chronic or acute disease states.
- the composition may comprise, in addition to polyunsaturated fatty acids produced in accordance with the present invention, macronutrients, vitamins and minerals as described above.
- the macronutrients may be present in amounts equivalent to those present in human milk or on an energy basis, i.e., on a per calorie basis.
- Methods for formulating liquid or solid enteral and parenteral nutritional formulas are well known in the art. (See also the Examples below.)
- the enteral formula for example, may be sterilized and subsequently utilized on a ready-to-feed (RTF) basis or stored in a concentrated liquid or powder.
- the powder can be prepared by spray drying the formula prepared as indicated above, and reconstituting it by rehydrating the concentrate.
- Adult and pediatric nutritional formulas are well known in the art and are commercially available (e.g., Similac®, Ensure®, Jevity® and Alimentum® from Ross Products Division, Abbott Laboratories, Columbus, Ohio) .
- An oil or acid produced in accordance with the present invention may be added to any of these formulas.
- the energy density of the nutritional compositions of the present invention when in liquid form, may range from about 0.6 Kcal to about 3 Kcal per ml.
- the nutritional supplements may contain from about 1.2 to more than 9 Kcals per gram, preferably about 3 to 7 Kcals per gm.
- the osmolality of a liquid product should be less than 700 mOsm and, more preferably, less than 660 mOsm.
- the nutritional formula may include macronutrients, vitamins, and minerals, as noted above, in addition to the PUFAs produced in accordance with the present invention.
- the presence of these additional components helps the individual ingest the minimum daily requirements of these elements.
- a pharmaceutical composition may also be supplemented with these elements .
- the nutritional composition comprises, in addition to antioxidants and at least one PUFA, a source of carbohydrate wherein at least 5 weight percent of the carbohydrate is indigestible oligosaccharide.
- the nutritional composition additionally comprises protein, taurine, and carnitine.
- the PUFAs produced in accordance with the present invention, or derivatives thereof, may be added to a dietary substitute or supplement, particularly an infant formula, for patients undergoing intravenous feeding or for preventing or treating malnutrition or other conditions or disease states.
- human breast milk has a fatty acid profile comprising from about 0.15% to about 0.36% as DHA, from about 0.03% to about 0.13% as EPA, from about 0.30% to about 0.88% as AA, from about 0.22% to about 0.67% as DGLA, and from about 0.27% to about 1.04% as GLA.
- fatty acids such as AA, EPA and/or docosahexaenoic acid (DHA)
- DHA docosahexaenoic acid
- a composition for use in a pharmacologic or food supplement, particularly a breast milk substitute or supplement will preferably comprise one or more of AA, DGLA and GLA. More preferably, the oil will comprise from about 0.3 to 30% AA, from about 0.2 to 30% DGLA, and/or from about 0.2 to about 30% GLA.
- Parenteral nutritional compositions comprising from about 2 to about 30 weight percent fatty acids calculated as triglycerides are encompassed by the present invention.
- the preferred composition has about 1 to about 25 weight percent of the total PUFA composition as GLA (U.S. Patent No.
- AA, DGLA and GLA can be adapted for a particular given end use.
- a composition which comprises one or more of AA, DGLA and GLA will be provided in a ratio of about 1:19:30 to about 6:1:0.2, respectively.
- the breast milk of animals can vary in ratios of AA:DGLA:GLA ranging from 1:19:30 to 6:1:0.2, which includes intermediate ratios which are preferably about 1:1:1, 1:2:1, 1:1:4.
- adjusting the rate and percent of conversion of a precursor substrate such as GLA and DGLA to AA can be used to precisely control the
- PUFA ratios For example, a 5% to 10% conversion rate of DGLA to AA can be used to produce an AA to DGLA ratio of about 1:19, whereas a conversion rate of about 75% TO 80% can be used to produce an AA to DGLA ratio of about 6:1. Therefore, whether in a cell culture system or in a host animal, regulating the timing, extent and specificity of desaturase expression, as well as the expression of other desaturases and elongases, can be used to modulate PUFA levels and ratios.
- the PUFAs produced in accordance with the present invention e.g., AA and EPA
- may then be combined with other PUFAs/acids e.g., GLA
- PUFA produced in accordance with the present invention or host cells containing them may also be used as animal food supplements to alter an animal's tissue or milk fatty acid composition to one more desirable for human or animal consumption.
- the present invention also encompasses a pharmaceutical composition comprising one or more of the acids and/or resulting oils produced using the desaturase genes, in accordance with the methods described herein. More specifically, such a pharmaceutical composition may comprise one or more of the acids and/or oils as well as a standard, well-known, non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle such as, for example, phosphate buffered saline, water, ethanol, polyols, vegetable oils, a wetting agent or an emulsion such as a water/oil emulsion.
- the composition may be in either a liquid or solid form.
- the composition may be in the form of a tablet, capsule, ingestible liquid or powder, injectible, or topical ointment or cream.
- Proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
- isotonic agents for example, sugars, sodium chloride and the like.
- the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfuming agents.
- Suspensions in addition to the active compounds, may comprise suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures of these substances.
- suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures of these substances.
- Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art.
- PUFAs produced in accordance with the present invention can be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate.
- Tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate.
- Capsules can be prepared by incorporating these excipients into a gelatin capsule along with antioxidants and the relevant PUFA(s) .
- the antioxidant and PUFA components should fit within the guidelines presented above.
- the PUFAs produced in accordance with the present invention or derivatives thereof may be incorporated into commercial formulations such as IntralipidsTM.
- the typical normal adult plasma fatty acid profile comprises 6.64 to 9.46% of AA, 1.45 to 3.11% of DGLA, and 0.02 to 0.08% of GLA.
- These PUFAs or their metabolic precursors can be administered alone or in combination with other PUFAs in order to achieve a normal fatty acid profile in a patient.
- the individual components of the formulations may be provided individually, in kit form, for single or multiple use.
- a typical dosage of a particular fatty acid is from 0.1 mg to 20 g (up to 100 g) daily and is preferably from 10 mg to 1, 2, 5 or 10 g daily.
- Possible routes of administration of the pharmaceutical compositions of the present invention include, for example, enteral (e.g., oral and rectal) and parenteral.
- a liquid preparation may be administered, for example, orally or rectally.
- a homogenous mixture can be completely dispersed in water, admixed under sterile conditions with physiologically acceptable diluents, preservatives, buffers or propellants in order to form a spray or inhalant .
- the route of administration will, of course, depend upon the desired effect. For example, if the composition is being utilized to treat rough, dry, or aging skin, to treat injured or burned skin, or to treat skin or hair affected by a disease or condition, it may perhaps be applied topically.
- the dosage of the composition to be administered to the patient may be determined by one of ordinary skill in the art and depends upon various factors such as weight of the patient, age of the patient, immune status of the patient, etc.
- the composition may be, for example, a solution, a dispersion, a suspension, an emulsion or a sterile powder which is then reconstituted.
- the present invention also includes the treatment of various disorders by use of the pharmaceutical and/or nutritional compositions described herein.
- the compositions of the present invention may be used to treat restenosis after angioplasty.
- symptoms of inflammation, rheumatoid arthritis, asthma and psoriasis may also be treated with the compositions of the invention.
- Evidence also indicates that PUFAs may be involved in calcium metabolism; thus, the compositions of the present invention may, perhaps, be utilized in the treatment or prevention of osteoporosis and of kidney or urinary tract stones.
- compositions of the present invention may also be used in the treatment of cancer.
- Malignant cells have been shown to have altered fatty acid compositions.
- compositions of the present invention may also be useful for treating cachexia associated with cancer.
- compositions of the present invention may also be used to treat diabetes (see U.S. Patent No. 4,826,877 and Horrobin et al . , Am. J. Clin. Nutr. Vol. 57 (Suppl.) 732S- 737S) . Altered fatty acid metabolism and composition have been demonstrated in diabetic animals.
- compositions of the present invention comprising PUFAs produced either directly or indirectly through the use of the desaturase enzymes, may also be used in the treatment of eczema, in the reduction of blood pressure, and in the improvement of mathematics examination scores.
- compositions of the present invention may be used in inhibition of platelet aggregation, induction of vasodilation, reduction in cholesterol levels, inhibition of proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al . , Adv. Exp. Med. Biol. Vol. 83, p.85-101,
- compositions of the present invention include use in the treatment of AIDS, multiple sclerosis, and inflammatory skin disorders, as well as for maintenance of general health. Additionally, the composition of the present invention may be utilized for cosmetic purposes. It may be added to pre-existing cosmetic compositions such that a mixture is formed or may be used as a sole composition.
- compositions may be utilized in connection with animals (i.e., domestic or non-domestic) , as well as humans, as animals experience many of the same needs and conditions as humans.
- animals i.e., domestic or non-domestic
- the oil or acids of the present invention may be utilized in animal feed supplements, animal feed substitutes, animal vitamins or in animal topical ointments .
- the PUFAs described in the Detailed Description may be utilized in various nutritional supplements, infant formulations, nutritional substitutes and other nutritional solutions.
- -Dual carbohydrates corn syrup and sucrose designed to enhance carbohydrate absorption and reduce the risk of exceeding the absorptive capacity of the damaged gut.
- Similac NeoCare is a nutritionally complete formula developed to provide premature infants with extra calories, protein, vitamins and minerals needed to promote catch-up growth and support development .
- -More calcium and phosphorus for improved bone mineralization.
- Ingredients -D Water, nonfat milk, hydrolyzed cornstarch, lactose, fractionated coconut oil (medium- chain triglycerides) , whey protein concentrate, soy oil, coconut oil, calcium phosphate tribasic, potassium citrate, magnesium chloride, sodium citrate, ascorbic acid, calcium carbonate, mono and diglycerides, soy lecithin, carrageenan, choline chloride, m-inositol, taurine, niacinamide, L-carnitine, alpha tocopheryl acetate, zinc sulfate, potassium chloride, calcium pantothenate, ferrous sulfate, cupric sulfate, riboflavin, vitamin A palmitate, thiamine chloride hydrochloride, pyridoxine hydrochloride, biotin, folic acid, manganese sulfate, phylloquinone, vitamin D3 , sodium selenite and cyanocobalamin.
- ENSURE is a low-residue liquid food designed primarily as an oral nutritional supplement to be used with or between meals or, in appropriate amounts, as a meal replacement.
- ENSURE is lactose- and gluten-free, and is suitable for use in modified diets, including low- cholesterol diets. Although it is primarily an oral supplement, it can be fed by tube.
- ENSURE BARS are complete, balanced nutrition for supplemental use between or with meals. They provide a delicious, nutrient-rich alternative to other snacks.
- ENSURE BARS contain ⁇ 1 g lactose/bar, and Chocolate Fudge Brownie flavor is gluten-free. (Honey Graham Crunch flavor contains gluten.)
- Honey Graham Crunch. High-Fructose Corn Syrup, Soy Protein Isolate, Brown Sugar, Honey, Maltodextrin (Corn) , Crisp Rice (Milled Rice, Sugar [Sucrose] , Salt [Sodium Chloride] and Malt) , Oat Bran, Partially Hydrogenated Cottonseed and Soy Oils, Soy Polysaccharide, Glycerine, Whey Protein Concentrate, Polydextrose, Fructose, Calcium Caseinate, Cocoa Powder, Artificial Flavors, Canola Oil, High-Oleic Safflower Oil, Nonfat Dry Milk, Whey Powder, Soy Lecithin and Corn Oil. Manufactured in a facility that processes nuts.
- Vitamins and Minerals Calcium Phosphate Tribasic, Potassium Phosphate Dibasic, Magnesium Oxide, Salt
- Chromium Chloride Potassium Iodide, Sodium Selenate, Sodium Molybdate, Phylloquinone, Vitamin D3 and Cyanocobalamin.
- Protein Honey Graham Crunch - The protein source is a blend of soy protein isolate and milk proteins.
- the fat source is a blend of partially hydrogenated cottonseed and soybean, canola, high oleic safflower, oils, and soy lecithin.
- Soy lecithin 4% Carbohydrate Honey Graham Crunch -
- the carbohydrate source is a combination of high-fructose corn syrup, brown sugar, maltodextrin, honey, crisp rice, glycerine, soy polysaccharide, and oat bran.
- ENSURE HIGH PROTEIN is a concentrated, high- protein liquid food designed for people who require additional calories, protein, vitamins, and minerals in their diets. It can be used as an oral nutritional supplement with or between meals or, in appropriate amounts, as a meal replacement.
- ENSURE HIGH PROTEIN is lactose- and gluten-free, and is suitable for use by people recovering from general surgery or hip fractures and by patients at risk for pressure ulcers.
- vanilla Supreme -D Water, Sugar (Sucrose) , Maltodextrin (Corn), Calcium and Sodium Caseinates, High-OIeic Safflower Oil, Soy Protein Isolate, Soy Oil, Canola Oil, Potassium Citrate, Calcium Phosphate Tribasic, Sodium
- the protein source is a blend of two high-biologic-value proteins: casein and soy.
- Soy protein isolate 15% Fat Soy protein isolate 15% Fat :
- the fat source is a blend of three oils: high-oleic safflower, canola, and soy.
- AHA Heart Association
- ENSURE HIGH PROTEIN contains a combination of maltodextrin and sucrose.
- the mild sweetness and flavor variety vanilla supreme, chocolate royal, wild berry, and banana
- ENSURE LIGHT is a low-fat liquid food designed for use as an oral nutritional supplement with or between meals.
- ENSURE LIGHT is lactose- and gluten-free, and is suitable for use in modified diets, including low- cholesterol diets.
- French Vanilla -D Water, Maltodextrin (Corn) , Sugar (Sucrose) , Calcium Caseinate, High-Oleic Safflower Oil, Canola Oil, Magnesium Chloride, Sodium Citrate, Potassium Citrate, Potassium Phosphate Dibasic, Magnesium Phosphate Dibasic, Natural and Artificial Flavor, Calcium Phosphate Tribasic, Cellulose Gel, Choline Chloride, Soy Lecithin, Carrageenan, Salt (Sodium Chloride) , Ascorbic Acid, Cellulose Gum, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Zinc Sulfate, Niacinamide, Manganese Sulfate, Calcium Pantothenate, Cupric Sulfate, Thiamine Chloride Hydrochloride, Vitamin A Palmitate, Pyridoxine Hydrochloride, Riboflavin, Chromium Chloride, Folic Acid, Sodium Molybdate, Bio
- the protein source is calcium caseinate.
- the fat source is a blend of two oils: high-oleic safflower and canola.
- the level of fat in ENSURE LIGHT meets American Heart Association (AHA) guidelines.
- AHA American Heart Association
- ENSURE LIGHT represent 13.5% of the total calories, with 1.4% of the fat being from saturated fatty acids and 2.6% from polyunsaturated fatty acids. These values are within the AHA guidelines of ⁇ 30% of total calories from fat, ⁇ 10% of the, calories from saturated fatty acids, and ⁇ 10% of total calories from polyunsaturated fatty acids.
- ENSURE LIGHT contains a combination of maltodextrin and sucrose.
- the chocolate flavor contains corn syrup as well.
- the mild sweetness and flavor variety (French vanilla, chocolate supreme, strawberry swirl) , plus VARI-
- An 8-fl-oz serving of ENSURE LIGHT provides at least 25% of the RDIs for 24 key vitamins and minerals.
- ENSURE PLUS is a high-calorie, low-residue liquid food for use when extra calories and nutrients, but a normal concentration of protein, are needed. It is designed primarily as an oral nutritional supplement to be used with or between meals or, in appropriate amounts, as a meal replacement. ENSURE PLUS is lactose- and gluten- free. Although it is primarily an oral nutritional supplement, it can be fed by tube .
- vanilla -D Water, Corn Syrup, Maltodextrin (Corn), Corn Oil, Sodium and Calcium Caseinates, Sugar (Sucrose) , Soy Protein Isolate, Magnesium Chloride, Potassium Citrate, Calcium Phosphate Tribasic, Soy Lecithin, Natural and Artificial Flavor, Sodium Citrate, Potassium Chloride, Choline Chloride, Ascorbic Acid, Carrageenan, Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Niacinamide, Calcium Pantothenate, Manganese Sulfate, Cupric
- Protein The protein source is a blend of two high-biologic- value proteins: casein and soy.
- the fat source is corn oil .
- ENSURE PLUS contains a combination of maltodextrin and sucrose.
- the mild sweetness and flavor variety vanilla, chocolate, strawberry, coffee, buffer pecan, and eggnog
- An 8-fl-oz serving of ENSURE PLUS provides at least 15% of the RDIs for 25 key Vitamins and minerals.
- Chocolate flavor contains 3.1 mg Caffeine/8 fl oz. Coffee flavor contains a trace amount of caffeine.
- ENSURE PLUS HN is a nutritionally complete high- calorie, high-nitrogen liquid food designed for people with higher calorie and protein needs or limited volume tolerance. It may be used for oral supplementation or for total nutritional support by tube. ENSURE PLUS HN is lactose- and gluten-free.
- vanilla -D Water, Maltodextrin (Corn), Sodium and Calcium Caseinates, Corn Oil, Sugar (Sucrose) , Soy- Protein Isolate, Magnesium Chloride, Potassium Citrate, Calcium Phosphate Tribasic, Soy Lecithin, Natural and Artificial Flavor, Sodium Citrate, Choline Chloride, Ascorbic Acid, Taurine, L-Carnitine, Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Niacinamide, Carrageenan, Calcium Pantothenate, Manganese Sulfate, Cupric Sulfate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Vitamin A Palmitate, Folic Acid, Biotin, Chromium Chloride, Sodium Molybdate, Potassium Iodide, Sodium Selenite, Phylloquinone, Cyanocobalamin and Vitamin D3.
- ENSURE POWDER (reconstituted with water) is a low-residue liquid food designed primarily as an oral nutritional supplement to be used with or between meals.
- ENSURE POWDER is lactose- and gluten-free, and is suitable for use in modified diets, including low- cholesterol diets.
- the protein source is a blend of two high-biologic-value proteins: casein and soy.
- Fat The fat source is corn oil.
- ENSURE POWDER contains a combination of corn syrup, maltodextrin, and sucrose.
- ENSURE PUDDING is a nutrient-dense supplement providing balanced nutrition in a nonliquid form to be used with or between meals. It is appropriate for consistency-modified diets (e.g., soft, pureed, or full liquid) or for people with swallowing impairments.
- ENSURE PUDDING is gluten-free.
- vanilla -D Nonfat Milk, Water, Sugar (Sucrose) , Partially Hydrogenated Soybean Oil, Modified Food Starch, Magnesium Sulfate, Sodium Stearoyl Lactylate, Sodium Phosphate Dibasic, Artificial Flavor, Ascorbic Acid, Zinc Sulfate, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Choline Chloride, Niacinamide, Manganese Sulfate, Calcium Pantothenate, FD&C Yellow #5, Potassium Citrate, Cupric Sulfate, Vitamin A Palmitate, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, FD&C Yellow #6, Folic Acid, Biotin, Phylloquinone, Vitamin D3 and Cyanocobalamin.
- the protein source is nonfat milk.
- the fat source is hydrogenated soybean oil .
- Carbohydrate contains a combination of sucrose and modified food starch.
- the mild sweetness and flavor variety vanilla, chocolate, butterscotch, and tapioca) help prevent flavor fatigue.
- the product contains 9.2 grams of lactose per serving.
- I. ENSURE® WITH FIBER Usage: ENSURE WITH FIBER is a fiber-containing, nutritionally complete liquid food designed for people who can benefit from increased dietary fiber and nutrients. ENSURE WITH FIBER is suitable for people who do not require a low-residue diet. It can be fed orally or by tube, and can be used as a nutritional supplement to a regular diet or, in appropriate amounts, as a meal replacement. ENSURE WITH FIBER is lactose- and gluten- free, and is suitable for use in modified diets, including low-cholesterol diets.
- vanilla -D Water; Maltodextrin (Corn) , Sugar (Sucrose) , Sodium and Calcium Caseinates, Oat Fiber, High-Oleic
- the protein source is a blend of two high-biologic- value proteins-casein and soy. Sodium and calcium caseinates 80% Soy protein isolate 20%
- the fat source is a blend of three oils: high-oleic safflower, canola, and corn.
- the level of fat in ENSURE WITH FIBER meets American Heart Association (AHA) guidelines.
- AHA American Heart Association
- the 6 grams of fat in ENSURE WITH FIBER represent 22% of the total calories, with 2.01 % of the fat being from saturated fatty acids and 6.7% from polyunsaturated fatty acids. These values are within the AHA guidelines of £ 30% of total calories from fat, ⁇ 10% of the calories from saturated fatty acids, and ⁇ _ 10% of total calories from polyunsaturated fatty acids .
- ENSURE WITH FIBER contains a combination of maltodextrin and sucrose.
- the mild sweetness and flavor variety vanilla, chocolate, and butter pecan
- the fiber blend used in ENSURE WITH FIBER consists of oat fiber and soy polysaccharide. This blend results in approximately 4 grams of total dietary fiber per 8-fl. oz can. The ratio of insoluble to soluble fiber is 95:5.
- Oxepa is a low-carbohydrate, calorically dense, enteral nutritional product designed for the dietary management of patients with or at risk for ARDS. It has a unique combination of ingredients, including a patented oil blend containing eicosapentaenoic acid (EPA from fish oil) , ⁇ -linolenic acid (GLA from borage oil) , and elevated antioxidant levels.
- EPA eicosapentaenoic acid
- GLA ⁇ -linolenic acid
- Caloric density is high at 1.5 Cal/tnL (355 Cal/8 fl oz) , to minimize the volume required to meet energy needs.
- the distribution of Calories in Oxepa is shown in Table A.
- Fat -Oxepa contains 22.2 g of fat per 8-fl oz serving (93.7 g/L) .
- the fat source is an oil blend of 31.8% canola oil, 25% medium-chain triglycerides (MCTs) , 20% borage oil, 20% fish oil, and 3.2 % soy lecithin.
- MCTs medium-chain triglycerides
- borage oil 20% borage oil
- fish oil 3.2 % soy lecithin.
- the typical fatty acid profile of Oxepa is shown in Table B.
- MCTs Medium-chain trigylcerides
- Fatty acids equal approximately 95% of total fat
- the carbohydrate content is 25.0 g per 8-fl-oz serving (105.5 g/L) .
- carbohydrate sources are 45% maltodextrin (a complex carbohydrate) and 55% sucrose (a simple sugar) , both of which are readily digested and absorbed.
- carbohydrate sources are 45% maltodextrin (a complex carbohydrate) and 55% sucrose (a simple sugar) , both of which are readily digested and absorbed.
- sucrose a simple sugar
- the high-fat and low-carbohydrate content of Oxepa is designed to minimize carbon dioxide (C02) production. High C02 levels can complicate weaning in ventilator- dependent patients.
- the low level of carbohydrate also may be useful for those patients who have developed stress-induced hyperglycemia.
- -Oxepa is lactose-free.
- Dietary carbohydrate the amino acids from protein, and the glycerol moiety of fats can be converted to glucose within the body. Throughout this process, the carbohydrate requirements of glucose-dependent tissues (such as the central nervous system and red blood cells) are met. However, a diet free of carbohydrates can lead to ketosis, excessive catabolism of tissue protein, and loss of fluid and electrolytes. These effects can be prevented by daily ingestion of 50 to 100 g of digestible carbohydrate, if caloric intake is adequate. The carbohydrate level in Oxepa is also sufficient to minimize gluconeogenesis, if energy needs are being met.
- -Oxepa contains 14.8 g of protein per 8-fl-oz serving
- -Oxepa provides enough protein to promote anabolism and the maintenance of lean body mass without precipitating respiratory problems .
- High protein intakes are a concern in patients with respiratory insufficiency. Although protein has little effect on CO 2 production, a high protein diet will increase ventilatory drive.
- the protein sources of Oxepa are 86.8% sodium caseinate and 13.2% calcium caseinate.
- -Oxepa is gluten-free.
- D. coronata The fatty acid composition analysis of the fungus Delacroixia coronata (D. coronata) (ATCC 28565) was investigated to determine the types and amounts of polyunsaturated fatty acids (PUFAs) it produced. This fungus was found to contain significant amounts (-19% of total lipid) of the PUFA arachidonic acid (ARA, C20:4n-6) (see Figure 1) . Thus, it was determined that D.
- PUFAs polyunsaturated fatty acids
- coronata probably contains a ⁇ 6-desaturase which converts linoleic acid (LA, C18:2n-6) to ⁇ -linolenic acid (GLA, C18:3n-6) and a ⁇ 5-desaturase which converts dihomo- ⁇ -linolenic acid (DGLA, C20:3n-6) to ARA.
- LA linoleic acid
- GLA ⁇ -linolenic acid
- DGLA dihomo- ⁇ -linolenic acid
- oligonucleotides that represent amino acid motifs that are conserved in known front-end desaturases .
- These primers could be then used in a PCR reaction to identify a gene fragment containing the conserved regions present in the putative desaturase genes from Delacroixia. Since the only fungal desaturases identified, at the time, were the ⁇ 5- and ⁇ 6-desaturase genes from Mortierella alpina (Genbank accession numbers AF067650, AB020032, respectively) , desaturase sequences from plants as well as animals were taken into consideration during the design of these degenerate primers.
- degenerate primers Mortierella alpina, Borago officinalis, Helianthus annuus, Brassica napus, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homo sapiens, Caenorhabditis elegans, Arabidopsis thaliana, and Ricinus communis.
- the degenerate primers used were as follows using the CODEHOPTM Blockmaker program (http: //blocks. fhcrc.org/codehop.html) : a.
- Protein motif 1 (SEQ ID NO: 9) : NH 3 - VYDVTEWVKRHPGG -COOH Primer RO834 (SEQ ID NO: 10) : 5 ' -GTBTAYGAYGTBACCGARTGGGTBAAGCGYCAYCCBGGHGGH-S ' b.
- Protein Motif 2 (SEQ ID NO: 11) : NH 3 - GASANWWKHQHNVHH -COOH Primer RO835 (Forward) SEQ ID NO:12) :
- RNA was first prepared.
- D. coronata ATCC 28565 cells were grown in BY+ Media (#790, Difco, Detroit, MI) at room temperature for 4 days, in the presence of light, and with constant agitation (250 rpm) to obtain the maximum biomass. These cells were harvested by centrifugation at 5000 rpm for 10 minutes and rinsed in ice-cold RNase-free water. These cells were then lysed in a French press at 10,000 psi, and the lysed cells were directly collected into TE buffered phenol.
- Proteins from the cell lysate were removed by repeated phenol :chloroform (1:1 v/v) extraction, followed by a chloroform extraction.
- the nucleic acids from the aqueous phase were precipitated at -70 0 C for 30 minutes using 0.3 M (final concentration) sodium acetate (pH 5.6) and one volume of isopropanol .
- the precipitated nucleic acids were collected by centrifligation at 15,000 rpm for 30 minutes at 4°C, vacuum- dried for 5 minutes and then treated with DNasel (RNase-free) in IX DNase buffer (20 mM Tris-Cl, pH 8.0; 5 mM MgCl 2 ) for 15 minutes at room temperature.
- the reaction was quenched with 5 mM EDTA (pH 8.0) and the RNA further purified using the Qiagen RNeasy Maxi kit (Qiagen, Valencia, CA) , as per the manufacturer's protocol.
- the first PCR reaction was performed using the Superscript Preamplification System for First Strand cDNA Synthesis Kit (Life Technologies, Rockville, MD), with 0.5 ⁇ g of oligo dT primer and 5 ⁇ l RNA (1 ⁇ g/1) , following the manufacturer's instructions .
- the second PCR reaction was performed using the degenerate primers RO834/RO838 (designed with the block maker program) and the first strand cDNA as target, in Perkin Elmer 9600.
- the PCR components were as follows: 2 ⁇ l of the first strand cDNA template, 1 ⁇ l 5OX dNTP mix, 0.2 pM final concentration of each primer, 5 ⁇ l 1OX KlenTaq PCR reaction buffer, and 1 ⁇ l of Advantage KlenTaq polymerase (Clonetech,
- Thermocycling was carried out as follows: an initial denaturation at 94 0 C for 3 minutes, followed by 35 cycles of denaturation at 94 0 C for 30 seconds; annealing at 60 0 C for 30 seconds; and extension at 72 0 C for 7 minute. This was followed by a final extension at 72 0 C for 7 minutes.
- the reaction was separated on a 1% agarose gel, and approximately 1.3 Kb DNA fragment was excised and purified with the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA) .
- RACE rapid amplification of cDNA ends
- RACE ready cDNA was used as a target for the reactions to isolate the full-length cDNA.
- approximately 5 ⁇ g of total RNA was used according to the manufacturer's direction with the GeneRacerTM kit (Invitrogen, Carlsbad, CA) and Superscript IITM enzyme (Invitrogen, Carlsbad, CA) for reverse transcription to produce cDNA target.
- thermocycling protocol for the initial amplification of the ends, the following thermocycling protocol was used in a Perkin Elmer 9600: initial melt at 94 °C for 2 minutes followed by 5 cycles of 94 0 C for 30 seconds and 72 0 C for 3 minutes, 10 cycles of 94 0 C 30 seconds, 70 °C for 30 seconds, and 72 °C for 3 minutes and 20 cycles of 94 °C for 30 seconds, 68 °C for 30 seconds and 72 °C for 3 minutes, followed by an extension of 72 °C for 10 minutes.
- the first PCR reactions were performed with 10 pMol of RO1526 (SEQ ID NO: 19) (5'-TGC CTC CGT ATT CTC CCT TAA CCA CAA C-3') or RO1528 (SEQ ID NO: 20) (5'-CTT CCA CAC TTT CCA CCC TGA TTC TTC CTG-3') and 30 pMol GeneRacerTM 3 prime primer (SEQ ID N0:21) (5'- GCT GTC AAC GAT ACG CTA CGT AAC G-3' ) ; or RO1524 (SEQ ID NO:22) (5' -TGA ATC CAA GTG GAG GGC ATG AAG ACA G-3') or RO1525 SEQ ID NO:23) (5' -CGG AGG GGA TGA TAC CAA ACC AAC TAG AGC-3' ) and GeneRacerTM 5 prime primer (SEQ ID NO:24) (5'- CGA CTG GAG CAC GAG GAC ACT GA-3')
- Each reaction contained 1 ul of cDNA in a final volume of 50 ul with Platimum TaqTM PCRx (Clonetech, Palo Alto, CA) using MgSO 4 according to the manufacturer's directions.
- a nested reaction was performed with 1 ⁇ l of the initial reaction, 10 pmol of nested primer RO1524 or RO1525 and 30 pmol of the GeneRacerTM nested 5 prime primer (SEQ ID NO:25) (5'- GGA CAC TGA CAT GGA CTG AAG GAG TA- 3'); or nested primer RO1526 or RO1528 and GeneRacerTM nested 3 prime primer (SEQ ID NO:26) (5'- CGC TAC GTA ACG GCA TGA CAG TG -3' ) using the same conditions as the first reaction. Agarose gel analysis of the PCR products showed bands from approximately 400 bp to 1.3 Kb for the four reactions.
- PCR was carried out using RACE cDNA using the following primers:
- PCR reactions were performed according to the manufacturer's directions, using Platinum Pfx Polymerase (Invitrogen, Carlsbad, CA) .
- the full-length PCR product was digested with Mzel and cloned into EcoRI-blunted (5') and Mzel (3') sites of the pYX242 vector. This converted the first Ala after the stop codon (SEQ ID NO:42) (*AIKEGAIL%) to a Met (SEQ ID NO:43) (*MIKEGAIL...) , and this clone was designated as pRDCIO ( Figure 5, Figure 8 & Figure 9) .
- genomic DNA was prepared using the DNeasy plant mini kit according to the manufacturer' s directions (Qiagen, Valencia, CA) .
- Primer RO1585 (SEQ ID NO: 31) (5'-AAA GGA TCC AAT ATG TTA ATA GGC GGC GTT AAG-3') was designed to convert the third isoleucine after the stop codon
- Primer RO1584 (SEQ ID NO: 32) (5'-ATC CTC GAG TTA AAT TTG GTC GTT GAT ATT GGT G-3' ) was designed for the 3' end of the full- length cDNA sequence.
- the PCR components were as follows: 3 ⁇ l of the gDNA, 1 ⁇ l 5OX dNTP mix, 0.2 pM final concentration of primers RO1583 and RO1584 or RO1585 and RO1584, 2 ⁇ l MgSO 4 , 5 ⁇ l 1OX PCR reaction buffer, and 0.2 ⁇ l of platinum TAQ HF polymerase (Clonetech, Palo Alto, CA) .
- thermocycling protocol was used in a Perkin Elmer 9600: initial melt at 94 0 C for 2 minutes followed by 5 cycles of 94 °C for 30 seconds and 72 0 C for 3 minutes, 10 cycles of 94 0 C 30 seconds, 70 0 C for 30 seconds, and 72 °C for 3 minutes and 20 cycles of 94 °C for 30 seconds, 68 0 C for 30 seconds and 72 °C for 3 minutes, followed by an extension of 72 0 C for 10 minutes.
- the reaction was separated on a 1% agarose gel, and approximately 1.4 Kb DNA fragment was excised and purified with the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA) .
- the full-length PCR product designated Del-D6 was digested with BamHI/XhoI and cloned into pESC-Ura vector. This construct was designated as pRDC-12.
- pRDC-12 Plasmid pRDC-12 was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110 on May 18, 2004 and was accorded accession number PTA-5975. Alignment of the amino acid sequence of this putative desaturase (Del-D6) encoded by pRDC-12, with the ⁇ 6-desaturase sequence from Mortierella alpina, revealed these proteins to share -51% sequence identity with each other ( Figure 12) .
- Del-D6 contains the three conserved 'histidine boxes' known to be required for the catalytic activity of these front end desaturase enzymes (Shanklin et al . , Biochemistry 33 (43) : 12787-94 (1994), Sayanova et al . , Plant Physiol. 121 (2) : 641-46 (1999) , and Periera et al. , Prostaglandins Leukot. Essent. Fatty Acids 68(2) :97-106 (2003)) ( Figure 13) .
- this sequence also contained a cytochrome b5 domain at the 5 ' -end (HPGG motif (SEQ ID NO:36)) ( Figure 13) implying that it uses cytochrome b5 as an electron donor during the desaturation reaction, as seen with other fungal and algal ⁇ 6-desaturases (Periera et al . , Prostaglandins Leukot . Essent . Fatty Acids 68(2) :97-106 (2003)) . The overall G+C content of this gene is 41.9%.
- Saccharomyces cerevisiae strain 334 Saccharomyces cerevisiae strain 334.
- Yeast transformation was carried out using the Alkali-Cation Yeast Transformation Kit (BIO 101, Vista, CA) according to conditions specified by the manufacturer.
- Transformants were selected for uracil auxotrophy on media lacking uracil (DOB [-Ura] ) .
- transformants were grown in the presence of 50 ⁇ M specific fatty acid substrates as listed below: a.
- Linoleic acid (LA, C18:2n- ⁇ ) (conversion to ⁇ - linolenic acid would indicate ⁇ l5-desaturase activity and conversion to ⁇ -linolenic acid would indicate ⁇ 6-desaturase activity) ; b. Alpha-linolenic acid (ALA, C18:3n-3) (conversion to stearidonic acid would indicate ⁇ 6-desaturase activity) ; c. (C20:2n-6) (conversion to dihomo-gamma-linolenic acid would indicate ⁇ 8-desaturase activity) ; d.
- the negative control strain was S. cerevisiae 334 containing the unaltered pYX242 vector, and these were grown simultaneously.
- the cultures were vigorously agitated (250 rpm) and grown for 48 hours a 24 0 C in the presence of 50 ⁇ M (final concentration) of the various substrates in 50 ml of media lacking uracil after inoculation with overnight growth of single colonies in yeast peptone dextrose broth (YPD) at 30 °C.
- the cells were pelleted, and the pellets vortexed in methanol; chloroform was added along with tridecanoin (as an internal standard) . These mixtures were incubated for at least an hour at room temperature or at 4 0 C overnight .
- the chloroform layer was extracted and filtered through a Whatman filter with 1 gm anhydrous sodium sulfate to remove particulates and residual water.
- the organic solvents were evaporated at 40 0 C under a stream of nitrogen.
- the extracted lipids were then derivitized to fatty acid methyl esters (FAME) for gas chromatography analysis (GC) by adding 2 ml of 0.5 N potassium hydroxide in methanol to a closed tube.
- the samples were heated to 95 0 C-IOO 0 C for 30 minutes and cooled to room temperature.
- Approximately 2 ml of 14% borontrifluoride in methanol was added and the heating repeated.
- 2 ml of water and 1 ml of hexane were added to extract the fatty acid methyl esters (FAME) for analysis by GC.
- the percent conversion was calculated by dividing the product produced by the sum of (the product produced + the substrate added) and then multiplying by 100.
- the plasmid pRDC-12 was co-transformed with pRSP-46, a clone that contains a M. alpina elongase gene from pRPBG-2 (see U.S. published patent application no. US2003/0177508A1 incorporated herein in its entirety by reference) .
- Table 2 shows that when 50 ⁇ M of the substrate LA (C18:2n-6) was added, the desaturase converted the LA to GLA, and the elongase was able to add two carbons to GLA to produce DGLA. No DGLA was produced by the control transformation 334 (pYX242/pESC-Ura) .
- D. coronata ⁇ 6-desaturase was able to produce a product in a heterologous expression system that was the substrate of another heterologous enzyme (the M. ⁇ lpina elongase) from the PUFA biosynthetic pathway to produce the expected PUFA.
- M. ⁇ lpina elongase another heterologous enzyme from the PUFA biosynthetic pathway to produce the expected PUFA.
- ⁇ 6-desaturase can indeed work with other heterologous enzymes in the PUFA pathway in a heterologous expression system such as yeast.
- ⁇ 6-desaturases are predicted to exist in a number of
- PUFA-producing fungi and algae based on the presence of GLA (18:3n-6) , ARA (20:4n-6) and/ or EPA (20:5n-3) in these organisms upon analysis of their total fatty acid profiles.
- RO834 SEQ ID NO: 8
- RO838 SEQ ID NO: 13
- Organisms that can be used to isolate ⁇ 6- desaturase genes would belong to the genera: Fungi such as Cunninghamella, Rhizopus, Gongronella, Allamyces, Synchytrium, Achlya, Phycomyces, Choanephora, Helicostylum, Entomopthora; Microalgae such as Chlorella, Dunaliella, Lauderia, Fucus, Sargassum, Layengaria, Colpomenia, Plocamium, Rhodomella,
Abstract
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EP05806616A EP1802747A2 (en) | 2004-09-01 | 2005-08-30 | Delta 6-desaturase genes and uses thereof |
AU2005282793A AU2005282793B2 (en) | 2004-09-01 | 2005-08-30 | Delta 6-desaturase genes and uses thereof |
CA002578049A CA2578049A1 (en) | 2004-09-01 | 2005-08-30 | Delta 6-desaturase genes and uses thereof |
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WO2007056823A1 (en) * | 2005-11-18 | 2007-05-24 | Commonwealth Scientific And Industrial Research Organisation | Feedstuffs for aquaculture comprising stearidonic acid feedstuffs for aquaculture |
US8778632B2 (en) | 2004-09-01 | 2014-07-15 | Abbott Laboratories | Δ6-desaturase genes and uses thereof |
JP2014168468A (en) * | 2007-01-03 | 2014-09-18 | Monsanto Technology Llc | Food composition incorporating further longer chain fatty acid |
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US7470532B2 (en) * | 2005-10-19 | 2008-12-30 | E.I. Du Pont De Nemours And Company | Mortierella alpina C16/18 fatty acid elongase |
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Cited By (4)
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US8778632B2 (en) | 2004-09-01 | 2014-07-15 | Abbott Laboratories | Δ6-desaturase genes and uses thereof |
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JP2014168468A (en) * | 2007-01-03 | 2014-09-18 | Monsanto Technology Llc | Food composition incorporating further longer chain fatty acid |
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BRPI0514783A (en) | 2008-06-24 |
WO2006028839A3 (en) | 2007-01-25 |
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CA2578049A1 (en) | 2006-03-16 |
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US20060048244A1 (en) | 2006-03-02 |
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