Desaturase genes and uses thereof

The present invention relates to the identification and isolation of genes encoding enzymes (ie, Thraustochytrium aureum Δ5-desaturase, Saprolegnia diclina Δ5-desaturase, Saprolegnia diclina Δ6-desaturase and Isochrysis galbana Δ5-desaturase). In particular, Δ5-desaturase is, for example, from dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) and (n-3) -eicosatetraenoic acid (20: 4n-3) to eicosapentaenoic acid (20: 5n-). Catalyze the conversion to 3). Δ6-desaturase catalyzes the conversion of, for example, α-linolenic acid (ALA) to stearidonic acid (STA). This conversion product can then be used as a substrate in the production of other polyunsaturated fatty acids (PUFAs). This product or other polyunsaturated fatty acids can be added to pharmaceutical compositions, nutritional compositions, animal feeds and other products (eg cosmetics).

Desaturase is essential for the production of long-chain polyunsaturated fatty acids with a number of important functions. For example, polyunsaturated fatty acids (PUFAs) are a major component of cell membranes and are present in the phospholipid form. It also functions as a precursor for mammalian prostacyclins, eicosanoids, leukotrienes and pstaglandins. In addition, PUFAs are required for proper development and tissue formation and repair of the developing infant brain. In view of the biological significance of PUFAs, attempts have been made to efficiently produce PUFAs and their intermediates.

In addition to Δ5-desaturase and Δ6-desaturase, many enzymes are involved in PUFA biosynthesis. For example, elongase (elo) converts γ-linolenic acid (GLA) to dihomo-γ-linolenic acid (DGLA) and stearidonic acid (18: 4n-3) to (n-3) -eicosatetraenoic acid ( 20: 4 Catalyzes the conversion to n-3). Linolenic acid (LA, 18: 2-Δ9,12 or 18: 2n-6) is produced from oleic acid (18: 1-Δ9) by Δ12-desaturase. GLA (18: 3-Δ6,9,12) is produced from linolenic acid by Δ6-desaturase.

It should be noted that oleic acid cannot be converted to linolenic acid because animals cannot be unsaturated beyond position Δ9. Similarly, α-linolenic acid (ALA, 18: 3-Δ9,12,15) cannot be synthesized by mammals. On the other hand, α-linolenic acid is converted to stearidonic acid (STA, 18: 4-Δ6,9,12,15) by Δ6-desaturase in mammals and algae (PCT publication WO 96/13591 and The Faseb Journal, Abstracts, Part). I, Abstract 3093, page A532 (Experimental Biology 98, San Francisco, CA, April See 18-22,1988), also US Pat. No. 5,552,306), which can then be extended to (n-3) -eicosatetraenoic acid (20: 4-Δ8,11,14,17). This polyunsaturated fatty acid (ie 20: 4-Δ8,11,14,17) is then subjected to eicosapentaenoic acid (EPA, 20: 5-Δ5,8,11,14) by the enzyme-like Δ5-desaturase of the present invention. , 17) can be converted. Other eukaryotes, such as fungi and plants, have enzymes that are unsaturated with carbon-12 (see PCT Publication WO 94/11516 and US Pat. No. 5,443,974) and carbon 15 (see PCT Publication WO 93/11245). Thus, animal major polyunsaturated fatty acids are derived from food and / or linolenic acid or α-linolenic acid desaturation and elongation. In view of these issues, in microorganisms, plants or animal systems that can isolate genes involved in PUFA synthesis from naturally producing species of these fatty acids and modify them to produce one or more PUFAs in commercial quantities. Expressing these genes is very beneficial.

<p> One of the most important long-chain PUFAs mentioned above is arachidonic acid (AA). AA is contained in filamentous fungi and can be purified from mammalian tissues such as liver and adrenal gland. As mentioned above, AA production from dihomo-γ-linolenic acid is catalyzed by Δ5-desaturase. EPA is also an important long-chain PUFA. EPA is found in fungi and is also found in fish oil. As mentioned above, EPA is produced from (n-3) -eicosapentaenoic acid and catalyzed by Δ5-desaturase. In view of the above, there is a clear need for Δ5-desaturase and Δ6-desaturase enzymes, the genes encoding these enzymes, and recombinant production methods for these enzymes. Furthermore, oils containing PUFAs at a higher concentration than the natural concentration and oils containing a high concentration of new PUFAs are also required. Such oils can only be produced by isolation and expression of the Δ5-desaturase and Δ6-desaturase genes.</p><p> All US patents, priority documents and publications cited herein are incorporated herein by reference in their entirety.</p>

<p> The present invention relates to SEQ ID NO: 13 (FIG. 2), SEQ ID NO: 19 (FIG. 4), SEQ ID NO: 28 (FIG. 6), SEQ ID NO: 30 (FIG. 8), SEQ ID NO: 32 (FIG. 10) and SEQ ID NO: 34 (FIG. 14). With respect to an isolated nucleotide sequence or fragment comprising, or complementary to, at least about 50% of a nucleotide sequence comprising. In particular, isolated nucleotides can be represented by SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34. These sequences can encode functionally active desaturases that utilize polyunsaturated fatty acids as substrates.</p><p> Furthermore, the present invention has desaturase activity and has at least 50% concordance or at least 50% concordance with the amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35. With respect to an isolated nucleotide sequence or fragment comprising or complementary to a nucleotide sequence encoding a polypeptide having similarities.</p><p> The nucleotide sequence is obtained from, for example, fungi such as Saprolegnia diclina (SEQ ID NO: 13 and SEQ ID NO: 19) or Thraustochytrium aureum (SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32) or algae such as Isochrysis galbana (SEQ ID NO: 34).</p><p> The present invention is a purified protein or polypeptide encoded by the above nucleotide sequence (SEQ ID NO: 14 (FIG. 3), SEQ ID NO: 20 (FIG. 5), SEQ ID NO: 29 (FIG. 7), SEQ ID NO: 31 (FIG. 9), SEQ ID NO: It also relates to 33 (Fig. 11) and SEQ ID NO: 35 (Fig. 15)).</p><p> Furthermore, the present invention desaturates carbon 5 or carbon 6 polyunsaturated fatty acids and the amino acid sequences of the purified proteins (ie, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 and sequences. It also relates to purified polypeptides having at least about 50% amino acid concordance or similarity to number 35).</p><p> Furthermore, the present invention also relates to a method for producing a desaturase (ie, Δ5 or Δ6). The method comprises a) isolating a nucleotide sequence appropriately comprising SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 13, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, and b) i) isolated nucleotide sequence and this. The vector is introduced into the host cell at the stage of constructing a vector containing an ii) promoter or a predetermined type of regulatory sequence operably linked to ii) and c) at a time and condition sufficient for the expression of Δ5-desaturase or Δ6-desaturase as appropriate. Including the stage to do. The host cell can be, for example, a eukaryotic cell or a prokaryotic cell. In particular, the prokaryotic cells can be, for example, Escherichia coli, cyanobacteria or Bacillus subtilis. Eukaryotic cells can be, for example, mammalian cells, insect cells, plant cells or fungal cells (eg, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida species, Lipomyces starkey, Yarrowia lipolytica, Kluyveromyces species, Hansenula species, Trichoderma species or Pichia species). it can.</p><p> Furthermore, the present invention a) a nucleotide sequence represented by SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, and b) a promoter or regulatory operably linked thereto. It also relates to vectors containing sequences. The present invention also relates to host cells containing this vector. The host cell can be, for example, a eukaryotic cell or a prokaryotic cell. The eukaryotic and prokaryotic cells available are as described above.</p><p> Furthermore, the present invention is a plant cell, plant or plant tissue containing the above vector, and as a result of expression of the nucleotide sequence of the vector, at least one polyunsaturated fatty acid is produced by the plant cell, plant or plant tissue. It also relates to plant cells, plants or plant tissues. Polyunsaturated fatty acids can be selected from the group consisting of, for example, AA, EPA, GLA or STA, depending on whether the nucleotide sequence encodes, for example, Δ5- or Δ6-desaturase. The present invention also relates to one or more vegetable oils or acids expressed by the plant cells, plants or plant tissues.</p><p> Furthermore, the present invention also relates to a transgenic plant comprising the vector, wherein polyunsaturated fatty acids are produced in the seeds of the transgenic plant as a result of expression of the nucleotide sequence of the vector.</p><p> Furthermore, the present invention is a mammalian cell containing the above vector, and when the cell is grown in a medium containing a fatty acid selected from the group consisting of, for example, LA, ALA, DGLA and ETA, the nucleotide sequence of the vector is expressed. As a result, the mammalian cells whose production levels of AA, EPA, GLA and / or STA are changed are also involved.</p><p> It should also be noted that the present invention relates to transgenic non-human mammals whose genome contains a DNA sequence encoding a Δ5-desaturase or a Δ6-desaturase operably linked to a promoter or regulatory sequence. The DNA sequence can be represented by SEQ ID NO: 13 (Δ6), SEQ ID NO: 19 (Δ5), SEQ ID NO: 28 (Δ5), SEQ ID NO: 30 (Δ5), SEQ ID NO: 32 (Δ6) and SEQ ID NO: 34 (Δ5). Furthermore, the present invention also relates to liquids (eg, milk) produced by transgenic non-human mammals and optionally containing at a detectable concentration of at least Δ5-desaturase or at least Δ6-desaturase.</p><p> Furthermore, the present invention isolates the nucleotide sequence represented by a) eg, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 34 as a method for producing polyunsaturated fatty acids (ie, the "first" method). Steps, b) constructing a vector containing the isolated nucleotide sequence, c) introducing the vector into host cells under sufficient time and conditions for expression of the Δ5-desaturase enzyme, and d) expressing human Δ5. -It also relates to methods involving the step of exposing the desaturase enzyme to substrate polyunsaturated fatty acids and converting the substrate to product polyunsaturated fatty acids. Each substrate polyunsaturated fatty acid can be, for example, DGLA or 20: 4n-3, and the product polyunsaturated fatty acid can be, for example, AA or EPA. The method can further include exposing the product polyunsaturated fatty acid to an elongase or desaturase to convert the product polyunsaturated fatty acid to another polyunsaturated fatty acid (ie, the "second" method). In this method, which involves additional steps (ie, the "second" method), each product polyunsaturated fatty acid can be, for example, AA or EPA, and the "other" polyunsaturated fatty acid can be adrenoic acid or (n-3). )-Can be docosapentaenoic acid. Methods involving additional steps can further include the step of exposing another polyunsaturated fatty acid to an additional desaturase or elongase to convert another polyunsaturated fatty acid to the final polyunsaturated fatty acid (ie, "third"). Method). The final polyunsaturated fatty acid can be, for example, (n-6) -docosapentaenoic acid or docosahexaenoic acid (DHA) acid.</p><p> Furthermore, the present invention comprises, as a method for producing polyunsaturated fatty acids, a) a step of isolating the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 33, and b) a step of constructing a vector containing the isolated nucleotide sequence. , C) The step of introducing the vector into the host cell under sufficient time and conditions for the expression of the Δ6-desaturase enzyme, and d) the exposed Δ6-desaturase enzyme to the substrate polyunsaturated fatty acid to produce the substrate. It relates to a method including a step of converting to a saturated fatty acid. The substrate polyunsaturated fatty acids can be, for example, LA or ALA, and the product polyunsaturated fatty acids can be, for example, GLA or STA. The method can further include the step of exposing the product polyunsaturated fatty acid to an elongase (or desaturase) to convert the product polyunsaturated fatty acid to another polyunsaturated fatty acid. In this method involving additional steps, each product polyunsaturated fatty acid can be, for example, GLA or STA, and the "other" polyunsaturated fatty acid can be DGLA or eicosatetraenoic acid (ETA). .. Methods involving additional steps can further include the step of exposing another polyunsaturated fatty acid to an added desaturase (or elongase) to convert the other polyunsaturated fatty acid to the final polyunsaturated fatty acid. The final polyunsaturated fatty acid can be, for example, AA or EPA.</p><p> The present invention is at least one selected from the group consisting of the product polyunsaturated fatty acids produced by the method, another polyunsaturated fatty acid produced by the method and the final polyunsaturated fatty acid produced by the method. It also relates to nutritional compositions containing the species' polyunsaturated fatty acids. The product polyunsaturated fatty acids can be, for example, AA, EPA, GLA or STA, depending on whether the Δ5- or Δ6-desaturase nucleotide sequence is used. Another polyunsaturated fatty acid can also be, for example, adrenoic acid, (n-3) -docosapentaenoic acid, DGLA and EPA, depending on which of the Δ5- or Δ6-desaturase nucleotide sequences is also used. The final polyunsaturated fatty acid can also be, for example, (n-6) -docosapentaenoic acid, DHA, AA or EPA, depending on whether the Δ5- or Δ6-desaturase nucleotide sequence is used as well.</p><p> The present invention is 1) at least one selected from the group consisting of a "product" PUFA produced by the above method, a "different" PUFA produced by the above method and a "final" PUFA produced by the above method. Also related to pharmaceutical compositions containing polyunsaturated fatty acids (PUFAs) and 2) pharmaceutically acceptable carriers.</p><p> Furthermore, the present invention comprises an animal feed comprising at least one PUFA selected from the group consisting of the product PUFA produced by the method, another PUFA produced by the method and the final PUFA produced by the method. Or related to cosmetics. These PUFAs are as listed above and are illustrated in FIG.</p><p> Furthermore, the present invention also relates to a method of administering a sufficient amount of the above nutritional composition to a patient as a method of preventing or treating a condition caused by inadequate intake of polyunsaturated fatty acids.</p><p> It should also be noted that each nucleotide and amino acid sequence described herein is designated by a specific SEQ ID NO:. The (attached) sequence listing shows each of these sequences and their corresponding numbers.</p>

The present invention presents the nucleotide and translated amino acids of the Δ5-desaturase gene derived from Saprolegnia diclina, the Δ6-desaturase gene derived from Saprolegnia diclina, the two Δ5-desaturase genes derived from Thraustochytrium aureum, and the Δ6-desaturase gene derived from Isochrysis galbana. Regarding sequences. Furthermore, the present invention also relates to the use of these genes and the enzymes encoded by these genes. For example, the enzyme corresponding to the gene can be used in the production of polyunsaturated fatty acids (eg, arachidonic acid, eicosapentaenoic acid and / or adrenoic acid) that can be added to pharmaceutical compositions, nutritional compositions and other valuable products. it can.

Δ5-desaturase gene, Δ6-desaturase gene and enzymes encoded by them As mentioned above, the enzymes encoded by the Δ5-desaturase gene and the Δ6-desaturase gene of the present invention are essential for the production of highly unsaturated polyunsaturated fatty acids having lengths greater than 20 and 18, respectively. .. The nucleotide sequence of the isolated Thraustochytrium aureum Δ5-desaturase gene (ATCC34304) is shown in FIG. 6, and the amino acid sequence of the corresponding purified protein is shown in FIG. The nucleotide sequence of the isolated Saprolegnia diclina Δ5-desaturase gene is shown in FIG. 4, and the amino acid sequence of the corresponding purified protein is shown in FIG. The nucleotide sequence of the isolated Saprolegnia diclina Δ6-desaturase gene is shown in FIG. 2, and the amino acid sequence of the corresponding purified protein is shown in FIG. The nucleotide sequence of the isolated Thraustochytrium aureum (BICC7091) Δ5-desaturase gene is shown in FIG. 8, and the amino acid sequence of the corresponding purified protein is shown in FIG. Isolated Thraustochytrium The nucleotide sequence of the aureum Δ6-desaturase gene is shown in FIG. 10, and the amino acid sequence of the corresponding purified protein is shown in FIG. Finally, the nucleotide sequence of the isolated Isochrysis galbana Δ5-desaturase gene is shown in FIG. 14, and the amino acid sequence of the corresponding purified protein is shown in FIG.

As an example of the importance of the genes of the invention, the isolated Δ5-desaturase gene converts DGLA to AA or eicosapentaenoic acid to EPA. AA cannot be synthesized without, for example, the Δ5-desaturase gene and the enzyme encoded by it. The isolated Δ6-desaturase gene of the present invention, for example, converts linolenic acid (18: 2n-6) to γ-linolenic acid (GLA) and γ-linolenic acid (GLA) to stearidonic acid (STA).

The present invention relates to SEQ ID NO: 19 (ie, the nucleotide sequence of the Δ5-desaturase gene of Saprolegnia diclina), SEQ ID NO: 13 (ie, the nucleotide sequence of the Δ6-desaturase gene of Thraustochytrium aureum), SEQ ID NO: 28 (ie, Thraustochytrium aureum). (ATCC34304) Δ5-desaturase gene nucleotide sequence), SEQ ID NO: 30 (ie, Thraustochytrium aureum (BICC7091) Δ5-desaturase gene nucleotide sequence), SEQ ID NO: 32 (ie Thraustochytrium aureum Δ6-desaturase gene) and SEQ ID NO: 34 (That is, Isochrysis Contains at least about 50%, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, most preferably at least about 90% of the nucleotide sequence of the galbana Δ5-desaturase gene). It should be noted that this also relates to a complementary (ie, sequence-matched) nucleotide sequence (and the corresponding protein encoded by it). (All integers from 50% to 100% with respect to humanity percentage are also included in the scope of the invention.) Such sequences can be of human and other non-human origin (eg C. elegans or mouse).

Furthermore, the invention is derived from the nucleotide sequences of the invention (ie, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 34) and other origins, as described above for complementarity or correspondence. Also related to fragments and derivatives of sequences with. Functional equivalents of the above sequences (ie, sequences having Δ5-desaturase activity or Δ6-desaturase activity as appropriate) are also included in the present invention.

The present invention desaturates polyunsaturated fatty acids at the 5th or 6th carbon position, and the amino acid sequence of the protein encoded by the nucleotide sequence (ie, SEQ ID NO: 14 (FIG. 3), SEQ ID NO: 20 (FIG. 5), At least about 50%, preferably at least about 60%, more preferably at least about 50% of SEQ ID NO: 29 (FIG. 7), SEQ ID NO: 31 (FIG. 9), SEQ ID NO: 33 (FIG. 11) and SEQ ID NO: 35 (FIG. 15)). It also relates to purified polypeptides having at least about 70%, more preferably at least about 80%, and most preferably at least about 90% amino acid similarity or concordance. The similarity or agreement of all integers of 50 to 100% shall also be included in the scope of the present invention.

The term "match" refers to the degree of association between the nucleotides of two sequences across a particular comparison window or segment. That is, the degree of coincidence is defined as the same, correspondence, or equivalence between the same strands (sense or antisense) of two types of DNA segments. "Sequence match percentage" compares two optimally aligned sequences over a specific region, measures the number of positions where the bases match in both sequences to determine the number of matching positions, and compares the number of such positions. Calculated by dividing by the total number of positions in the segment and multiplying by 100. Optimal sequence alignment is the algorithm of Smith &amp; Waterman, Appl.Math.2: 482 (1981), the algorithm of Needleman &amp; Wunsch, J.Mol.Biol.48: 443 (1970), Pearson &amp; Lipman, Proc.Natl.Acad. Computer programs that implement the method of Sci (USA) 85: 2444 (1988) and related algorithms (eg Clustal Macaw) Pileup (http://cmgm.stanford.edu/biochem218/11Multiple.pdf; Higgins et al., CABIOS.5L151-153 (1989)), FASTDB (Interlligenetics), BLAST (National Center for Biomedical Information; Altschul et al., Nucleic Acids Research) 25: 3389-3402 (1997)), PILEUP (Genetics Computer Group, It can be implemented by Madison, WI) or TGAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, Madison, WI). (See US Pat. No. 5,912,120.) For the purposes of the present invention, "complementarity" is defined as the degree of association between two types of segments. This is determined by measuring the ability of the sense strand of one DNA segment to hybridize with the antisense strand of the other DNA segment under appropriate conditions to form a double helix. In the double helix, adenine appears on one strand and thymine appears on the other strand. Similarly, if guanine is present on one strand, then cytosine is present on the other. The greater the relevance between the nucleotide sequences of the two DNA segments, the greater the ability to form hybrid duplicates between the strands of the two DNA segments.

The "similarity" between two amino acid sequences is defined as the presence of a series of identical conserved amino acid residues in both sequences. The higher the similarity between the two amino acid sequences, the higher the correspondence, identity or equivalence of the two sequences. (The "match" between two amino acid sequences is defined as the presence of a series of strictly identical or invariant amino acid residues in both sequences.) Of "complementary", "match" and "similarity" The definition is well known to those skilled in the art.

By "encoded by" is meant a nucleic acid sequence encoding a polypeptide sequence, the polypeptide sequence or a portion thereof preferably containing at least three amino acids from the polypeptide encoded by the nucleic acid sequence. It contains an amino acid sequence containing at least 8, more preferably at least 15.

The present invention has PUFA desaturase activity and is SEQ ID NO: 13 (FIG. 2), SEQ ID NO: 19 (FIG. 4), SEQ ID NO: 28 (FIG. 6), SEQ ID NO: 30 (FIG. 8), SEQ ID NO: 32 (FIG. 10) or sequence. It also relates to an isolated nucleotide sequence that is capable of hybridizing under stringent conditions comparable to nucleic acids containing or having a nucleotide sequence complementary to the nucleotide sequence containing number 34 (FIG. 14). A nucleic acid molecule is "hybridizable" with another nucleic acid molecule when the single-stranded form of the nucleic acid molecule can be annealed with another nucleic acid molecule under suitable temperature and ionic strength conditions (Sambrook et al., "Molecular Cloning: A". Laboratory Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New See York). Temperature and ionic strength conditions determine the "stringency" of hybridization. "Hybridization" requires that the two nucleic acids contain complementary sequences. On the other hand, mismatches between bases may occur depending on the stringency of hybridization. The stringency suitable for hybridizing nucleic acids depends on the length and complementarity of the nucleic acids. Such variables are well known to those of skill in the art. More specifically, the higher the similarity or homology between the two nucleotide sequences, the higher the Tm value of the hybrid of nucleic acids having these sequences. For hybrids over 100 nucleotides in length, the formula for Tm has been derived (see Sambrook et al., Supra). The location of the mismatch is important in hybridization with short nucleic acids, and the length of the oligonucleotide determines its specificity (see Sambrook et al., Supra).

As used herein, an "isolated nucleic acid fragment or sequence" is a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, unnatural or modified nucleotide bases. An isolated nucleic acid fragment in the form of a DNA polymer can consist of one or more segments of cDNA, genomic DNA or synthetic DNA (a "fragment" of a particular nucleotide is at least about identical or complementary to a region of a particular nucleotide sequence. It means a polynucleotide sequence containing a contiguous sequence of 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10 nucleotides, even more preferably at least about 15 nucleotides, most preferably at least about 25 nucleotides. Nucleotides (which exist in the 5'-monophosphate form) are represented by their one-letter notation as follows. "A" is adenylic acid or deoxyadenylic acid (RNA or DNA, respectively), "C" is cytidine or deoxycytidilic acid, "G" is guanylic acid or deoxyguanylic acid, "U" is uridylic acid, "T" Is deoxythymidylate, "R" is purine (A or G), "Y" is pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "I" is inosin. , "N" represents any nucleotide.

The terms "functionally equivalent fragment or subfragment" and "functionally equivalent fragment or subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment that alters gene expression or maintains the ability to express a given phenotype regardless of whether the fragment or subfragment encodes an active enzyme. For example, fragments or subfragments can be used in the design of chimeric constructs to express a given phenotype in transformed plants. Chimeric constructs can be designed for co-suppression or antisense use by ligating nucleic acid fragments or subfragments in the appropriate direction to the plant promoter sequence, whether or not they encode the active enzyme.

Terms such as "homology", "homology", "substantially similar |" and "substantially corresponding" are used interchangeably herein. These terms mean that mutations in one or more nucleotide bases do not change the ability of nucleic acid fragments that mediate gene expression or express a given phenotype. These terms refer to mutations in the nucleic acid fragments of the invention (eg, 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 original unmodified fragment. Therefore, as will be apparent to those skilled in the art, the present invention relates to sequences other than the specific exemplary sequences.

"Gene" means a nucleic acid fragment that expresses a specific protein including a regulatory sequence (5'non-coding sequence) preceding the coding sequence and a regulatory sequence (3'non-coding sequence) following the coding sequence.

"Natural gene" means a gene that naturally exists with its own regulatory sequence. On the other hand, "chimeric construct" generally means a combination of nucleic acid fragments that do not coexist naturally. Thus, chimeric constructs can contain regulatory and coding sequences from different origins, or can contain regulatory and coding sequences that are derived from the same origin but are usually arranged in a different way than naturally occurring ( The term "isolated" means removing the sequence from its natural environment.) A "foreign" gene means a gene that is not normally present in the host organism but has been introduced into the host organism by gene transfer. Foreign genes can include natural genes inserted into non-natural organisms or chimeric constructs. A "transgene" is a gene introduced into the genome by a transformation method.

"Coding sequence" means a DNA sequence encoding a specific amino acid sequence. A "regulatory sequence" is a nucleotide that is located upstream (5'non-coding sequence), inside, or downstream (3'non-coding sequence) of a coding sequence and acts on transcription, RNA processing or stability or translation of the relevant coding sequence. Means an array. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, introns and polyadenylation recognition sequences.

"Promoter" means a coding sequence or a DNA sequence capable of controlling the expression of functional RNA. Promoters are composed of proximal and distal upstream elements, which are often referred to as enhancers. Thus, an "enhancer" can stimulate promoter activity and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. The promoter sequence may be located inside the transcriptional portion of the gene and / or downstream of the transcription sequence. The promoter may be entirely derived from a natural gene, may be composed of different elements derived from different naturally occurring promoters, or may contain a synthetic DNA segment. As will be appreciated by those skilled in the art, different promoters can induce gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental conditions. Promoters that express genes in most cell types in most cases are commonly referred to as "constitutive promoters." New promoters of various types useful in plant cells are constantly being discovered, and numerous examples are described in Okamuro and Goldbert, (1989) Biochemistry of Plants 15: 1-82. Furthermore, since the exact boundaries of the regulatory sequences have been completely determined in most cases, it is possible that the DNA fragments of a given variant may have the same promoter activity.

An "intron" is an intervening sequence of a gene that does not encode part of a protein sequence. Therefore, such sequences are transcribed into RNA but then excised and not translated. The term is also used for excised RNA sequences. The "exon" is part of the sequence of the transcribed gene and is present in the mature messenger RNA derived from the gene, but it does not necessarily have to be part of the sequence encoding the final gene product.

"Translation leader sequence" means a DNA sequence located between a gene promoter sequence and a coding sequence. The translation leader sequence resides in the fully processed mRNA upstream of the translation initiation sequence. Translation leader sequences can affect the processing of primary transcripts into mRNA, mRNA stability or translation efficiency. An example of a translation reader sequence can be found in the literature (Turner, R. and Foster, GD (1995) Molecular Biotechnology 3:225).

"3'Non-coding sequence" means a DNA sequence located downstream of the coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory sequences capable of acting on mRNA processing or gene expression. Be done. The polyadenylation signal is generally characterized by acting on the addition of the polyadenylate region to the 3'end of the mRNA precursor. The use of various 3'non-coding sequences is illustrated in Ingelbrecht et al., (1989) Plant Cell 1: 671-680.

"RNA transcript" means a product obtained by RNA polymerase-catalyzed transcription of a DNA sequence. When an RNA transcript is a perfect complementary copy of a DNA sequence, it is referred to as the primary transcript, but it may also be an RNA sequence induced by post-transcriptional processing of the primary product, in which case it is referred to as mature RNA. "Messenger RNA (mRNA)" is RNA that does not have an intron and means that it can be translated into protein by cells. "CDNA" is complementary to an mRNA template and means DNA synthesized from this template using reverse transcriptase. The cDNA may be single-stranded or may be converted to double-stranded using the Klenow fragment of DNA polymerase I. "Sense" RNA includes mRNA and is intracellular or in It means an RNA transcript that can be translated into a protein in vitro. "Antisense RNA" means an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and blocks the expression of the target gene (US Pat. No. 5,107,065). The antisense RNA may be complementary to any portion of the specific gene transcript, i.e., it may be any of a 5'non-coding sequence, a 3'non-coding sequence, an intron, or a coding sequence. "Functional RNA" means antisense RNA, ribozyme RNA or other RNA that cannot be translated but acts on cellular processes. The terms "complement" and "inverse complement" are used interchangeably herein with respect to mRNA transcripts to mean message antisense RNA.

The term "endogenous RNA" is an arbitrary nucleic acid sequence present in the genome of the host prior to transformation with the recombinant construct of the invention, whether introduced by natural or unnatural ie recombinant means, mutagenesis, etc. Means any RNA encoded by.

The term "non-natural" means man-made that does not usually match what is naturally occurring.

The term "operably linked" means binding a nucleic acid sequence with a single nucleic acid fragment such that the function of one nucleic acid sequence is regulated by the other. For example, a promoter is operably linked to a coding sequence when it can regulate the expression of the coding sequence (ie, when the coding sequence is under transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in either the sense or antisense direction. In another example, the complementary RNA region of the invention may be operably linked directly or indirectly into the target mRNA 5'or the target mRNA 3'or the target mRNA, or the first complementary region. May be operably linked to 5'of the target mRNA and its complement may be operably linked to 3'.

As used herein, the term "expression" refers to the production of a functional end product. Gene expression requires transcription of the gene and translation of mRNA into a precursor or mature protein. "Antisense inhibition" means the production of an antisense RNA transcript capable of suppressing the expression of a target protein. "Co-suppression" means the production of a sense RNA transcript capable of suppressing the expression of the same or substantially similar foreign or endogenous genes (US Pat. No. 5,231,020).

A "mature" protein means a post-translationally processed polypeptide, i.e., a polypeptide from which pre or propebutide present in the primary translation product has been removed. A "precursor" protein means a primary product of translation of mRNA, i.e., in which pre and propebutide are still present. Pre and prepebutide include, but are not limited to, intracellular localization signals.

"Stable transformation" means introducing a nucleic acid fragment into the genome of a host organism containing both a nuclear genome and an organelle genome to carry out genetically stable inheritance. In contrast, "transient transformation" means introducing a nucleic acid fragment into the nucleus of a host organism or a DNA-containing organelle for gene expression without integration or stable inheritance. A host organism containing a transformed nucleic acid fragment is referred to as a "transgenic" organism. Preferred cell transformation methods for rice, maize and other monocotyledonous plants are particle acceleration or "gene gun" transformation techniques (Klein et al., (1987) Nature (London) 327: 70-73; US Pat. No. 4,945,050). Alternatively, use the Agrobacterium method (Ishida Y. et al., 1996, Nature Biotech. 14: 745-750) using a suitable Ti plasmid containing the transgene. As used herein, the term "transformation" means both stable transformation and transient transformation.

The standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring. Described in detail by Harbor, 1989 (hereinafter referred to as "Sambrook").

The term "recombination" means an artificial combination of two isolated sequence segments, for example by manipulating isolated segments of nucleic acid by chemical synthesis or genetic engineering techniques.

"PCR" or "polymerase chain reaction" is a technique for synthesizing large amounts of specific DNA segments and consists of a series of iterative cycles (Perkin Elmer Cetus Instruments, Norwalk, CT). In general, double-stranded DNA is heat-denatured, and two primers complementary to the 3'boundary of the target segment are annealed at low temperature and then extended at medium temperature. A set of these three consecutive stages is called a cycle.

Polymerase chain reaction (PCR) is a powerful technique used to amplify DNA millions of times by repeated replication of the template in a short period of time. (Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51: 263-273 (1986); Erlich et al., European Patent Application No. 50,424; European Patent Application No. 84,796; European Patent Application No. 258,017; European Patent Application No. 237,362; Mullis, European Patent Application 201,184, Mullis et al., US Pat. No. 4,683,202; Erlich, US Pat. No. 4,582,788; and Saiki et al., US Pat. No. 4,683,194). This method initiates DNA synthesis using a combination of specific oligonucleotides synthesized in vitro. Primer design depends on the sequence of DNA you want to analyze. This method is performed by melting the template at high temperature, annealing the primers to complementary sequences in the template, and then performing multiple cycles (usually 20-50) of replicating the template with DNA polymerase.

The PCR reaction product is separated on an agarose gel and then analyzed by ethidium bromide staining and UV illumination. Alternatively, radioactive dNTPs may be added to the PCR to label the product. In this case, the PCR product is visualized by exposing the gel to an X-ray film. Radiolabeled PCR products also have the advantage of being able to quantify individual amplification product levels.

The terms "recombinant construct," "expression construct," and "recombinant expression construct" are used interchangeably herein. These terms refer to functional units of genetic material that can be inserted into the genome of a cell using standard methods well known to those of skill in the art. Such a structure may be used as it is, or may be incorporated into a vector and used. When using a vector, the choice of vector depends on the method used to transform the host plant, as is well known to those of skill in the art. For example, a plasmid vector can be used. Genetic elements that need to be placed on the vector for effective transformation, selection and proliferation of host cells containing any of the isolated nucleic acid fragments of the invention are well known to those of skill in the art. Expression of various levels and patterns occurs as a result of individual transformation events (Jones et al., (1985) EMBO J. 4: 2411-214; De Almeida et al., (1989) Mol. Gen. Genetics. Since 218: 78-86), it is also obvious to those skilled in the art that multiple events must be screened to obtain a sequence showing the desired expression level and pattern. Such screening can be performed by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

Production of Δ5-desaturase enzyme and Δ6-desaturase enzyme Once the gene encoding any one of the desaturase enzymes has been isolated, it can then be introduced into a prokaryotic or eukaryotic host cell by using a vector or construct. The vector (eg, bacteriophage, cosmid or plasmid) induces the expression of a nucleotide sequence encoding either a Δ5-desaturase enzyme or a Δ6-desaturase enzyme and a desaturase that is functional in the host cell and is encoded by the nucleotide sequence. It can include any regulatory sequence (eg, promoter) that is capable. Regulatory sequences are operably associated or operably linked to nucleotide sequences. (As mentioned above, a regulatory sequence is said to be "operably linked" to the coding sequence when it acts on the transcription or expression of the coding sequence.) Available promoters include, for example, alcohol dehydrogenase, glyco. Ceraldehyde-3-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglycerate kinase, acid phosphatase, T7, TPI, lactase, metallothioneine, cytomerogalovirus very early, whey acid protein, those derived from genes encoding glucoamylase , Promoters activated in the presence of galactose (eg, GAL1 and GAL10). In addition, nucleotide sequences encoding other proteins, oligosaccharides, lipids, etc. and other regulatory sequences such as polyadenylation signals (eg, SV-40T-antigen, ovalbumin or bovine growth hormone polyA signal) are also placed in the vector. You may. The choice of sequence to place in the construct depends on the desired expression product and the type of host cell.

As described above, once the vector has been constructed, it can then be introduced into selected host cells by methods known to those of skill in the art, such as transfection, transformation and electroporation (Molecular Cloning: A Laboratory Manual, No. 2). Edition, Vol.1-3, edited by Sambrook et al., Cold Spring Harbor Laboratory Press (1989)). The host cells are then cultured under appropriate conditions to allow gene expression to produce the desired PUFAs, which are then recovered and purified.

Examples of available prokaryotic host cells include, for example, bacteria such as Escherichia coli, Bacillus subtilis and cyanobacteria (eg Spirulina species, ie cyanobacteria). Examples of eukaryotic host cells available include, for example, mammalian cells, plant cells, yeast cells (eg Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Lipomyces starkey, Candida species (eg Yarrowia (Candida) lipolytica), Kluyveromyces species, Pichia species, Trichoderma). Species or Hansenula species), or fungal cells (eg, filamentous fungal cells, such as Aspergillus, Neurospora and Penicillium). It is preferable to utilize Saccharomyces cerevisiae (baker's yeast) cells.

Expression in host cells can be transient or stable. Transient expression can occur if the introduced construct contains a functional expression signal in the host cell but is poorly integrated into the host cell because it does not replicate or if the host cell is not proliferating. Transient expression can also be achieved by inducing the activity of a regulatory promoter operably linked to the gene of interest, but such induction systems often have low basal expression levels. Stable expression can be achieved by incorporating into the host genome or by introducing constructs that autonomously replicate in the host cell. After selecting stable expression of the gene of interest using a selectable marker that is placed on the expression construct or that transfects the expression construct, cells expressing the marker can be selected. If stable expression is the result of integration, the integration site of the construct may be random within the host genome, or the construct may contain a region homologous to the host genome sufficient to target recombination with the host gene site. May be targeted using. When targeting constructs to endogenous sites, all or part of the transcriptional and translational regulatory regions can be provided by the endogenous sites.

Transgenic mammals can also be used to express the enzyme of interest (ie, one or more of the Δ5-desaturase, one or more of the Δ6-desaturase, or a combination thereof) and ultimately the PUFA of interest. More specifically, once the above construct is created, it can be inserted into the pronucleus of the embryo. The embryo is then transplanted into the recipient female. Alternatively, a nuclear introduction method is available (Schnieke et al., Science). 278: 2130-2133 (1977)). Pregnancy and childbirth are then possible (see, eg, US Pat. No. 5,750,176 and US Pat. No. 5,700,671). Subsequent milk, tissue or other body fluid samples from offspring should contain PUFAs at concentrations different from those normally detected in non-transgenic animals. Monitor subsequent generations to see if altered or increased concentrations of PUFAs are being produced, and thus if the gene encoding the desired desaturase enzyme is integrated into its genome. Mammals to be used as hosts can be selected from the group consisting of, for example, mice, rats, rabbits, pigs, goats, sheep, horses and cows. However, any mammal can be used as long as it has the ability to integrate the DNA encoding the enzyme of interest into its genome.

To express a desaturase polypeptide, functional transcription and translation initiation and termination regions are operably linked to the DNA encoding the desaturase polypeptide. Transcription and translation initiation and termination regions vary from the DNA to be expressed, genes known or possible to be expressed in the desired system, expression vectors, chemical synthesis, or from endogenous sites in the host cell. Derived from non-exclusive origin. Expression in plant tissues and / or plant parts exhibits some efficiency, especially if the tissues or parts are those with early recovery of seeds, leaves, fruits, flowers, roots, etc. Expression can be targeted at that location in plants by utilizing specific regulatory sequences such as those described in U.S. Pat. Nos. 5,463,174, 4,943,674, 5,106,739, 5,175,095, 5,420,034, 5,188,958 and 5,589,379. .. Alternatively, the expressed protein may be an enzyme that produces a product that can be incorporated into the liquid fraction directly or after further modification from the host plant. Expression of the desaturase gene or antisense desaturase transcript can change the concentration of specific PUFAs or derivatives thereof contained in plant parts and / or plant tissues. The desaturase polypeptide coding region can also be expressed alone or with other genes to produce tissue and / or plant parts that contain higher concentrations of the desired PUFA or whose PUFA composition is closer to human milk (Prieto et al., PCT published WO 95/24494). The termination region can be derived from the 3'region of the gene from which the initiation region was obtained or another gene. Numerous termination regions are known and found to be satisfactory in a variety of hosts from the same and different genera and species. The termination region is generally selected for convenience rather than specific properties.

As mentioned above, plants (eg Glycine max (soybean) or Brassica Napus) or plant tissue may be used as hosts or host cells to express desaturase enzymes and used for the production of polyunsaturated fatty acids. More specifically, the desired PUFA can be expressed in seeds. A method for separating seed oil is known in the art. That is, in addition to providing a PUFA source, manipulating the seed oil component by expressing the desaturase gene and possibly other desaturase genes and elongase genes provides seed oil that can be added to nutritional compositions, pharmaceutical compositions, animal feeds and cosmetics. can do. Again, a vector containing the DNA sequence encoding the desaturase operably linked to the promoter is introduced into the plant tissue or plant under sufficient time and conditions for expression of the desaturase gene. The vector may also contain one or more genes encoding other enzymes (eg, Δ4-desaturase, elongase, Δ12-desaturase, Δ15-desaturase, Δ17-desaturase and / or Δ19-desaturase). The relevant substrate on which the enzyme acts (eg, DGLA (for Δ5-desaturase), ALA (for Δ6-desaturase), etc.) may be produced in a plant tissue or plant, or the enzyme that produces such a substrate is encoded. The vector to be used may be introduced into a plant tissue, plant cell or plant. In addition, the substrate may be sprayed onto plant tissue expressing the appropriate enzyme. These various techniques can also be used to produce PUFAs (eg, n-6 unsaturated fatty acids (eg, AA) or n-3 fatty acids (eg, EPA or STA)) by using plant cells, plant tissues or plants. it can. It should also be noted that the present invention also relates to transgenic plants containing the above vectors, for example plants that produce polyunsaturated fatty acids in the seeds of transgenic plants as a result of expression of the nucleotide sequence of the vector.

Regeneration, development and culture of plants from single plant protoplast transformants or various transformed explants is also well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, CA, (1988)). This regeneration and proliferation method generally includes the step of selecting transformed cells and the step of culturing the individualized cells in the normal embryogenesis-cutting seedling stage. Transgenic embryos and seeds are regenerated as well. The obtained transgenic cuttings are then planted in a suitable plant growth medium such as soil.

The development or regeneration of plants containing exogenous genes encoding the protein of interest is well known in the art. It is preferable to self-pollinate the regenerated plant to provide an isomorphic transgenic plant. Or. Pollen obtained from regenerated plants is crossed with seed-growing plants of an agronomically important line. Conversely, pollen from these important series of plants is used to pollinate regenerated plants. The transgenic plants of the invention containing the desired polypeptide are grown using methods well known to those of skill in the art.

There are various methods for regenerating plants from plant tissues. The specific regeneration method depends on the starting plant tissue and the specific plant species to be regenerated. The methods for transforming dicotyledonous plants mainly using Agrobacterium Tumefaciens to obtain transgenic plants are cotton (US patent 5,004,863, US patent 5,159,135, US patent 5,518,908), soybean (US patent 5,569,834). No., US Pat. No. 5,416,011, McCabe et al., Biol Technology 6: 923 (1988), Christou et al., Plant Physiol. 87: 671-674 (1988)), Brassica (US Pat. No. 5,463,174), Dicotyledon (Cheng et al., Plant) Cell Rep. 15: 653-657 (1996), McKently et al., Plant Cell Rep. 14: 699-703 (1995)), Papaya and Peas (Grant et al., Plant Cell Rep. 15: 254-258, (1995)) Has been reported.

Transformation of monocotyledonous plants using electroporation, particle impact and Agrobacterium has also been reported. Transformation and plant regeneration are asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84: 5354, (1987)), oats (Wan and Lemaux, Plant Physiol 104: 37 (1994)), maize ( Rhodes et al., Science 240: 204 (1988), Bordon-Kamm et al., Plant Cell 2: 603-618 (1990), Fromm et al., Biol Technology 8: 833 (1990), Koziel et al., Biol Technology 11; 194, (1993), Armstrong et al., Crop Science 35: 550-557 (1995)), Oats (Somers et al., Biol Technology 10: 1589 (1992)), Orchardgrass (Horn et al., Plant Cell) Rep.7: 469 (1986)), Rice (Toriyama et al., TheorAppl.Genet.205: 34, (1986); Part et al., Plant Mol.Biol.32: 1135-1148, (1996); Abedinia et al., Austin. J.Plant Physiol.24: 133-141 (1997); Zhang and Wu, Theor.Appl.Genet.76: 835 (1988); Zhang et al., Plant Cell Re.7: 379, (1988); Battraw and Hall, Plant Sci. 86: 191-202 (1992); Christou et al., Bio / Technology 9: 957 (1991)), rye (De la Pena et al., Nature 325: 274 (1987)), sugar cane (Bower and Birch, Plant J) .2: 409 (1992)), Hirohanousinokegusa (Wang et al., Biol Technologt 10: 691 (1992); US Pat. No. 5,631,152).

Gene expression assays based on transient expression of cloned nucleic acid constructs have been developed by introducing nucleic acid molecules into plant cells by polyethylene glycol treatment, electroporation or particle impact (Marcotte et al., Nature 335: 454-457). (1988); Marcotte et al., Plant Cell 1: 523-532 (1989); McCarty et al., Cell 66: 895-905 (1991); Hattori et al., Genes Dev. 6: 609-618 (1992); Goff et al., EMBO J.9: 2517-2522 (1990)).

Transient expression systems can be used to functionally analyze gene constructs (generally Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995)). Of course, any of the nucleic acid molecules of the invention can be permanently or transiently introduced into plant cells along with other genetic elements such as vectors, promoters, enhancers and the like.

In addition to the above methods, standard materials describing specific conditions and procedures for the construction, manipulation and isolation of macromolecules (eg, DNA molecules, plasmids, etc.), the preparation of recombinant organisms, and the screening and isolation of clones are well known to those skilled in the art. (Eg Sambrook et al., "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et al., Genome Analysis: Detecting Genes, 1 , Cold Spring Harbor, New York (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, New York (1998); Plant Molecular Biology: A Laboratory Manual, Clark ed., Springer, New York (1997)).

A substrate that can be produced by a host cell, either naturally or recombinantly, and an enzyme that can be encoded by a DNA sequence present in a vector that is later introduced into the host cell are shown in FIG.

In view of the above, the present invention comprises, as a method for producing a desaturase enzyme (that is, Δ5 or Δ6), 1) a step of isolating the nucleotide sequence of the gene encoding the desaturase enzyme, and 2) a step of constructing a vector containing the desaturase enzyme. And 3) a method comprising the step of introducing the vector into a host cell at a time and condition sufficient for the production of the desaturase enzyme.

The present invention also relates to a method for producing a polyunsaturated fatty acid, which comprises the step of exposing the acid to an enzyme such that desaturase converts the acid to a polyunsaturated fatty acid. For example, exposure of 20: 3n-6 to the Δ5-desaturase enzyme converts it to AA. The AA is then exposed to elongase and the AA is extended to adrenoic acid (ie 22: 4n-6). Alternatively, Δ5-desaturase may be used to convert 20: 4n-3 to 20: 5n-3 and then exposed to elongase to convert to (n-3) -docosapentaenoic acid. The (n-3) -docosapentaenoic acid can then be converted to DHA by using Δ4-desaturase. That is, Δ5-desaturase can be used in the production of polyunsaturated fatty acids that can be used for specific effective purposes.

For the role of Δ6-desaturase, linolenic acid can be exposed to the enzyme as it converts the acid to GLA. GLA can then be converted to DGLA using elongase. DGLA can then be converted to AA by exposing DGLA to Δ5-desaturase. Alternatively, ALA can be exposed to Δ6-desaturase to convert ALA to STA. The STA can then be converted to 20: 4n-3 by using elongase. It can then be converted to EPA by exposing 20: 4n-3 to Δ5-desaturase. That is, Δ6-desaturase can be used for the production of PUFA having advantageous properties and the production of other PUFAs.

Use of the Δ5-desaturase gene, the Δ6-desaturase gene, and the enzymes encoded by these genes As mentioned above, isolated desaturase genes and the desaturase enzymes encoded by these genes have many uses. For example, the enzyme corresponding to the gene can be used indirectly or directly in the production of polyunsaturated fatty acids, for example Δ5-desaturase can be used in the production of AA, adrenoic acid or EPA. Δ6-desaturase can be used indirectly or directly in the production of GLA, DGLA, STA or 20: 4n-3. ("Direct" means the situation where the enzyme directly converts an acid to another acid used in the composition (eg DGLA to AA conversion). "Indirect" means that the acid is desaturase to another acid (ie,). After conversion to an intermediate pathway (eg DGLA to AA), the latter acid is converted to the use of a non-desaturase enzyme (eg from AA to adrenoic acid by elongase) or another desaturase enzyme (eg from AA to EPA by Δ17-desaturase). Means the situation to be converted). These polyunsaturated fatty acids (ie, polyunsaturated fatty acids produced directly or indirectly by the activity of desaturase enzymes) can be added, for example, to nutritional compositions, pharmaceutical compositions, cosmetics and animal feeds, any of these. Is also included in the present invention. These uses are described in detail below.

Nutritional composition The present invention includes nutritional compositions. Such compositions, when taken into the body for the purposes of the present invention, (a) strengthen or energize tissues and / or (b) maintain, restore or assist adequate nutritional or metabolic function. Includes any food or preparation for human consumption (including gastric or intestinal consumption) that acts as such.

The nutritional composition of the present invention contains at least one oil or acid produced directly or indirectly by using the desaturase gene according to the present invention, and may be in solid form or liquid form. In addition, the composition may be supplemented with edible mass nutrients, vitamins and minerals in an amount required for a particular application. The amount of such ingredients ranges widely depending on whether the composition is used for healthy infants, toddlers or adults, or for those with special requirements such as those with a given metabolic state (eg, metabolic disorders).

Examples of high-volume nutrients that can 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, soybean oil and mono and diglycerides. Examples of such carbohydrates include, but are not limited to, sucrose, edible lactose and hydrolyzed starch. Furthermore, examples of proteins available in the nutritional compositions of the present invention include, but are not limited to, soy protein, electrodialysis whey, electrodialysis defatted milk, whey, or hydrolysates of these proteins.

As vitamins and minerals, calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C and E complexes are used as the nutritional composition of the present invention. Can be added to things.

The components used in the nutritional composition of the present invention are derived from semi-purified or purified products. Semi-refined or refined means a material produced by refining or synthesizing a natural material.

Examples of nutritional compositions of the present invention include, but are not limited to, infant formulas, dietary supplements, food substitutes and rehydration compositions. Particularly advantageous nutritional compositions include enteral and non-enteral infant formulas, special infant formulas, elderly supplements, and gastrointestinal disorders and / or malabsorption supplements. Not limited.

The nutritional composition of the present invention can be added to foods even when dietary supplementation is not required. For example, the composition can be added to any type of food, such as margarine, prepared butter, cheese, milk, yogurt, chocolate, candy, snacks, salad oil, cooking oil, cooking fat, meat, fish and beverages. These include, but are not limited to.

In a preferred embodiment of the present invention, the nutritional composition is an enteral nutritional product, more preferably an adult or pediatric enteral nutritional product. This composition can be administered to adults or children who are stressed by chronic or acute illness conditions or who require special treatment. In addition to the polyunsaturated fatty acids produced according to the present invention, the composition may contain a large amount of nutrients, vitamins and minerals as described above. The large amount of nutrients can be added in an amount equivalent to that contained in human milk or in terms of energy, that is, in terms of calories.

Methods for prescribing liquid or solid enteral and non-enteral nutritional preparations are well known in the art (see also Examples below).

Enteral preparations may be used, for example, in the Immediate Use (RTF) formula after sterilization, or may be stored as a concentrated liquid or powder. The powder can be produced by spray-drying the preparation prepared as described above and rehydrating and reconstitution of the concentrate. Adult and pediatric nutritional products are well known and commercially available in the art (eg Rose Products Division, Abbott Laboratories, Columbus, Ohio Products Similac®, Ensure®, Jevity® and Alimentum (eg. Registered trademark)). The oil or acid produced according to the present invention can be added to any of these formulations.

The energy density of the nutritional composition of the present invention can be about 0.6 Kcal to about 3 Kcal / ml in the liquid form. In the solid or powder form, about 1.2 ~&gt; 9 Kcal / g, preferably about 3-7 Kcal / g, can be added to the nutritional supplement. Generally, the osmotic pressure of a liquid product is less than 700 mOsm, more preferably less than 660 mOsm.

In addition to the PUFA produced according to the present invention, a large amount of nutrients, vitamins and minerals can be added to the nutritional preparation as described above. The presence of these additional ingredients helps the individual to consume a minimum daily requirement of these ingredients. In addition to providing PUFA, it may be desirable to add zinc, copper, folic acid and antioxidants to the composition. These substances strengthen the immune system under stress, which is even more beneficial to the individual to whom the composition is administered. These ingredients can also be added to pharmaceutical compositions.

In a more preferred embodiment, the nutritional composition comprises an antioxidant and at least one PUFA plus a carbohydrate source, with at least 5% by weight of the carbohydrate being edible oligosaccharides. In a more preferred embodiment, the nutritional composition further comprises protein, taurine and carnitine.

As described above, PUFAs or derivatives thereof produced by the present invention are added to substitute foods or supplements, especially infant formulas, and used for the prevention or treatment of malnourished patients and malnourished or other conditions or disease conditions. be able to. As a background, the fatty acid profile of human milk is DHA about 0.15% to about 0.36%, EPA about 0.03% to about 0.13%, AA about 0.30% to about 0.88%, DGLA about 0.22% to about 0.67%, and GLA about 0.27%. It should be noted that it is ~ about 1.04%. That is, fatty acids such as AA, EPA and / or docosahexaenoic acid (DHA) produced by the present invention may, for example, modify the composition of infant formula or non-human mammals so as to favorably increase the PUFA concentration of human milk. It can be used to change the PUFA concentration normally contained in breast milk. In particular, the composition used for pharmaceuticals or food supplements, especially breast milk substitutes or supplements, preferably contains one or more of AA, DGLA and GLA. The oil more preferably contains about 0.3-30% AA, about 0.2-30% DGLA and / or about 0.2-30% GLA.

The present invention relates to non-enteral nutritional compositions containing from about 2 to about 30% by weight of fatty acids when calculated as triglycerides. Suitable compositions contain from about 1 to about 25% by weight of the total PUFA composition as GLA (US Pat. No. 5,196,198). In some cases, other vitamins, especially fat-soluble vitamins (eg vitamins A, D, E) and L-carnitine may also be added. If desired, a preservative such as α-tocopherol may be added in an amount of about 0.1% by weight.

In addition, the ratio of AA, DGLA and GLA can be adjusted for specific intended end applications. When formulated as a breast milk supplement or substitute, compositions containing AA, DGLA and GLA are provided, respectively, in a ratio of about 1:19:30 to about 6: 1: 0.2. For example, animal milk can have an AA: DGLA: GLA ratio of 1: 19: 30 ~ 6: 1: 0.2, somewhere between about 1: 1: 1, 1: 2: 1, and 1: 1: 4. It is preferable to take a ratio. When simultaneously produced in host cells, it is preferable to strictly control the PUFA ratio by adjusting the conversion rate of precursor substrates such as GLA and DGLA to AA. For example, 5% to 10% can be used as the conversion rate from DGLA to AA to make the AA to DGLA ratio about 1:19, and the conversion rate to make the AA to DGLA ratio about 6: 1. About 75% to 80% can be used. Therefore, adjusting the PUFA concentration and ratio by regulating the timing, degree and specificity of desaturase expression and the expression of other desaturases and elongases, whether in cell culture or in the host animal. Can be done. The PUFAs / acids produced by the present invention (eg AA and EPA) may then be blended with other PUFAs / acids (eg GLA) in desired concentrations and ratios.

In addition, PUFAs produced according to the present invention or host cells containing them can be used as animal feed supplements to alter animal tissues or milk fatty acid compositions to those desired by human or animal consumption.

Pharmaceutical composition The present invention also relates to pharmaceutical compositions containing one or more of the acids and / or oils produced by the methods described herein using the desaturase genes described herein. More specifically, such pharmaceutical compositions include one or more acids and / or oils and pharmaceutically acceptable standard well-known non-toxic carriers, adjuvants or vehicles such as phosphate buffered salts solution, water, ethanol, etc. It can include polyols, vegetable oils, wetting agents or emulsions (eg water / oil emulsions). The composition may be in liquid form or solid form. For example, the composition can be in the form of tablets, capsules, drinking solutions or powders, injections, or topical ointments or creams. For example, in the case of a dispersant, the desired particle size can be maintained, and proper fluidity can be maintained by using a surfactant. For example, it may be desirable to add isotonic agents such as sugars and sodium chloride. In addition to such an inert diluent, adjuvants such as wetting agents, emulsifying suspensions, sweeteners, seasonings and flavors may be added to the composition.

In addition to the active compound, the suspension is a suspension of, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacant gum or a mixture of these substances. Can include agents.

Solid dosage forms such as tablets and capsules can be produced using techniques well known in the art. For example, PUFAs produced according to the present invention can be added to conventional tablet bases such as lactose, corn starch and corn starch with binders (eg arabic gum, corn starch or gelatin), disintegrants (eg potato starch or alginic acid) and lubricants (eg stearate or stearate). It can be tableted by adding magnesium acid). Capsules can be produced by encapsulating these excipients in gelatin capsules with antioxidants and the relevant PUFAs. Make sure that the antioxidant and PUFA components meet the above criteria.

For intravenous administration, PUFA produced by the present invention or a derivative thereof may be added to a commercially available preparation such as Intralipids (registered trademark). Typical healthy adult plasma fatty acid profiles are AA 6.64 to 9.46%, DGLA 1.45 to 3.11%, and GLA 0.02 to 0.08%. These PUFAs or their metabolic precursors can be administered alone or in combination with other PUFAs to reach the normal fatty acid profile in the patient. If desired, each component of the formulation may be provided individually in the form of a single or multiple-use kit. Typical doses of specific fatty acids are 0.1 mg to 20 g (up to 100 g) / day, preferably 10 mg to 1, 2, 5 or 10 g / day.

Possible routes of administration of the pharmaceutical composition of the present invention include, for example, enteral (eg, oral and rectal) and non-enteral. For example, the liquid preparation can be administered, for example, orally or rectally. In addition, the homogeneous mixture can be completely dispersed in water and mixed with physiologically acceptable diluents, preservatives, buffers or propellants under sterile conditions to form sprays or inhalants. The route of administration naturally depends on the desired effect. For example, when the composition is used for the treatment of rough skin, dryness or aging, the treatment of skin damage or burns, or the treatment of skin or hair damaged by a disease or disorder, it may be applied topically.

The patient dose of the composition can be determined by one of ordinary skill in the art and will vary depending on various factors such as the patient's weight, the patient's age, the patient's immune status and the like.

In dosage form, the composition can be, for example, a liquid, a dispersion, a suspension, an emulsion or a sterile powder to be reconstituted later.

The present invention also relates to the treatment of various diseases by the use of the pharmaceuticals and / or nutritional compositions described herein. In particular, the compositions of the present invention can be used in the treatment of post-angiogenic restenosis. In addition, symptoms of inflammation, rheumatoid arthritis, asthma and psoriasis can also be treated with the compositions of the present invention. Since PUFAs are also known to be involved in calcium metabolism, the compositions of the present invention may also be useful for the treatment or prevention of osteoporosis and renal or ureteral stones.

Furthermore, the compositions of the present invention can also be used in the treatment of cancer. Malignant cells have been shown to have altered fatty acid composition. It has been shown that the addition of fatty acids suppresses their growth, causes cell death and increases chemotherapeutic sensitivity. Furthermore, the compositions of the present invention appear to be useful in the treatment of cachexia associated with cancer.

The compositions of the present invention can also be used in the treatment of diabetes (see, eg, US Pat. No. 4,826,877 and Horrobin et al., Am.J.Clin.Nutr. Vol.57 (Suppl.) 732S-737S). Changes in fatty acid metabolism and composition have been shown in diabetic animals.

In addition, the compositions of the present invention containing PUFAs produced directly or indirectly by the use of desaturase enzymes can also be used to treat eczema, lower blood pressure and improve test values. Furthermore, the compositions of the present invention include platelet coagulation, induction of vasodilation, lowering of cholesterol levels, suppression of vascular smooth muscle and fibrous tissue growth (Brenner et al., Adv.Exp.Med.Biol.Vol.83, p.85-101). , 1976), Alleviation or prevention of gastrointestinal bleeding and other side effects of non-steroidal anti-inflammatory drugs (see US Pat. No. 4,666,701), Prevention or treatment of endometriosis and premenstrual syndrome (see US Pat. No. 4,758,592), It can also be used to treat muscle pain, encephalomyelitis and chronic fatigue (see US Pat. No. 5,116,871) after viral infection.

Other uses of the compositions of the present invention include the treatment of AIDS, multiple sclerosis and inflammatory skin diseases, and health maintenance.

Furthermore, the compositions of the present invention can also be used for cosmetic purposes. It may be added to an existing cosmetic composition to form a mixture, or it may be used as a single composition.

Veterinary use Since animals have many of the same requirements and conditions as humans, the pharmaceutical and nutritional compositions can be used in animals (ie, domestic or non-livestock) as well as in humans. For example, the oil or acid of the present invention can be used in animal or aquaculture feed additives, animal substitute feeds, animal vitamins or animal topical ointments.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.

Design of degenerate oligonucleotides for desaturase isolation from fungi and cDNA library construction Analysis of the fatty acid composition of Saprolegnia diclina (S. diclina) (ATCC 56851) reveals significant amounts of arachidonic acid (ARA, 20: 4 n-6) and eicosapentaenoic acid (EPA, 20: 5 n-3). It has been found. Therefore, this organism has active Δ6-desaturase and dihomo-γ-linolenic acid (DGLA,) capable of converting linolenic acid (LA, 18: 2n-6) to γ-linolenic acid (GLA, 18: 3n-6). It appeared to contain the active Δ5-desaturase that converts 20: 3 n-6) to arachidonic acid (ARA, 20: 4 n-6) (Fig. 1). In addition, S. diclina appeared to also contain Δ17-desaturase, which can desaturate ARA to EPA.

Analysis of the fatty acid composition of Thraustochytrium aureum (T.aureum) (ATCC34304) revealed that not only ARA and EPA but also adrenoic acid (ADA, 22: 4n-6) and ω6-docosapentaenoic acid (ω6-DPA, 22:) Long-chain PUFAs such as 5n-6), ω3-docosapentaenoic acid (ω3-DPA, 22: 5n-3) and docosahexaenoic acid (DHA, 22: 6n-3) were also detected. Thus, in addition to Δ6, Δ5 and Δ17-desaturase, T. aureum converts ADA to ω3-DPA or ω6-DPA to DHA Δ19-desaturase and / or ADA to ω6-DPA or ω3-DPA to DHA. It probably contained Δ4-desaturase. Therefore, we aimed to confirm the function by isolating these putative desaturase genes from S. diclina and T. aureum and finally expressing them in an alternative host.

A cDNA library was constructed for each organism to isolate the gene encoding the functional desaturase enzyme. Saprolegnia diclina (ATCC56851) was cultured in potato dextrose medium Dfco # 336 (Difco Laboratories, Detroit, Michigan) at room temperature for 4 days with constant stirring. Mycelium was recovered by filtering through several layers of cheesecloth and the culture was ground in liquid nitrogen using a mortar and pestle. Total RNA was purified using the Qiagen RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer's protocol.

T. aureum (ATCC34304) cells were cultured under light irradiation in BY + medium (Difco # 790) at room temperature for 4 days with constant stirring (250 rpm) to obtain maximum biomass. These cells were collected by centrifugation at 5000 rpm for 10 minutes and rinsed with RNase-free ice-cold water. These cells were then lysed with a French press at 10,000 psi and the lysed cells were collected directly in TE buffered phenol. After repeated extraction of phenol: chloroform (1: 1 v / v), protein was removed from the cytolytic solution by chloroform extraction. Nucleic acids were precipitated from the aqueous phase at -70 ° C for 30 minutes using 0.3 M (final concentration) sodium acetate (pH 5.6) and 1 volume of isopropanol. The precipitated nucleic acid is centrifuged at 15,000 rpm for 30 minutes at 4 ° C, collected, dried under reduced pressure for 5 minutes, and then 1 × DNase buffer (20 mM Tris-Cl, pH 8.0; 5 mM MgCl).₂) Medium DNase I (without RNase) was treated at room temperature for 15 minutes. The reaction was stopped with 5 mM EDTA (pH 8.0) and RNA was further purified using the Qiagen RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer's protocol.

MRNA was isolated from total RNA of each organism using oligo dT cellulose resin. Next, double-stranded cDNA was synthesized using the pBluescript II XR library construction kit (Stratagene, La Jolla, CA) and then directionally cloned into the pBluescript II SK (+) vector (5'EcoRI / 3'XhoI). ). S.diclina and T.aureum libraries are clones with an average insert size of about 700 bp each about 2.5 x 10⁶Included. Genomic DNA from PUFAs that produce S. diclina and T. aureum cultures by grinding the culture in liquid nitrogen is isolated and purified using the Qiagen Genomic DNA Extraction Kit (Qiagen, Valencia, CA). did.

The approach adopted was to design degenerate oligonucleotides (ie, primers) that exhibit amino acid motifs conserved in known desaturases. Using these primers in the PCR reaction, fragments containing conserved regions in the putative desaturase gene could be identified from the fungus. Since only the Δ5 and Δ6-desaturase genes (Genbank accession numbers AF067650 and AB020032, respectively) have been identified from Mortierella alpina as fungal desaturases, the desaturase sequences from animals as well as plants were considered when designing these degenerate primers. Known Δ5 and Δ6-desaturase sequences from the following organisms were used in the design of these degenerate primers. Mortierella alpina, Borago officinalis, Heliantus annuus, Brassica napus, Dictyostelium discoideum, Rattus norvegicus, Mus musculus, Homo sapien, Caenorhabditis elegans, Arabidopsis thaliana and Ricinus comunis. The degenerate primers used were as follows, and the CODEHOP Blockmaker program (http://blocks.fhcrc.org/codehop.html) was used.

<chemistry num="1"><img file="JP4347397B2_D0001.tif" /></chemistry>

In addition, two other primers were designed based on the second and third preserved "histidine boxes" present in the known Δ6-desaturase.

<chemistry num="2"><img file="JP4347397B2_D0002.tif" /></chemistry>

The degenerate code of the oligonucleotide sequence is B = C, G, T; H = A, C, T; S = C, G; R = A, G; Y = C, T; D = A + T + C; N = A, C, G, or T / U, unknown or other.

Isolation of the Δ6-desaturase nucleotide sequence from Saprolegnia diclina (ATCC56851) Total RNA was isolated from Saprolegnia diclina (ATCC 56851) using the lithium chloride method (Hoge et al., Exp. Mycology (1982) 6: 225-232). SuperScript Preamplification System (Life Technologies, Rockville, MD) and Oligo (dT) included in the kit_12-185 μg of total RNA was reverse transcribed using primers to prepare first-strand cDNA.

Various permutations and combinations of the degenerate oligonucleotides were used in the PCR reaction to isolate the Δ6-desaturase gene. Of the various primer combinations tested, RO834 / FO838 was the only primer that produced a clear band. First strand cDNA template 2 μl, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂PCR amplification was performed in 100 μl volumes containing 200 μM of each deoxyribonucleotide triphosphate and 2 pmol of each primer. Thermocycles were performed at two different annealing temperatures of 42 ° C and 45 ° C, these two PCR reactions were combined and degraded on a 1.0% agarose gel, QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA). ) Was used to gel-purify the ~ 100 bp band. T4 DNA polymerase (Life Technologies, Rockville, MD) was used to "fill" the protruding ends of these fragments as specified by the manufacturer, and these DNA fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, CA). .. The recombinant plasmid was transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, CA) and the clones were sequenced.

In this way, two clones showing sequence homology to the previously identified Δ6-desaturase were isolated. These clones are described as follows.

When clone # 20-2 was partially sequenced, the deduced amino acid sequence from 702 bp showed a 30.2% match with Δ6-desaturase from Mortierella alpina as the highest score in the TfastA search.

b. When clone # 30-1 was partially sequenced, the 687 bp estimated amino acid sequence showed a 48.5% match with Mortierella alpina's Δ6-desaturase as the highest score in the TfastA search. These two sequences overlap each other and appear to belong to a single putative Δ6-desaturase from S. diclina. Next, using this novel Δ6-desaturase sequence, primers were designed to search the 3'and 5'ends of the full-length Δ6-desaturase gene from the cDNA library prepared from S. diclina mRNA.

To isolate the 3'end, plasmid DNA and oligonucleotides RO923 (SEQ ID NO: 7) (5'-CGGTGCAGTGGTGGAAGAACAAGCACAAC-3') and RO899 (SEQ ID NO: 8) (5'-AGCGGATAACAATTTCACACAGGAAACAGC) purified from the cDNA library as templates PCR amplification was performed using -3'). Oligonucleotide RO923 was designed based on the # 20-2 fragment of this putative Δ6-desaturase, and oligonucleotide RO899 corresponds to the sequence from the pBluescript II SK (+) vector used to generate the cDNA library. Amplification was performed using 10 pmol of each primer and Taq PCR Master Mix (Qiagen, Valencia, CA). The sample was first denatured at 94 ° C for 3 minutes, followed by 30 cycles of 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 2 minutes. The reaction was terminated after a 10 minute final extension cycle at 72 ° C. The PCR fragment was degraded on a 0.8% agarose gel and Qiagen Gel Extraction Purified using Kit. T4 DNA polymerase (Life Technologies, Rockville, MD) was used to "fill" the ends of these fragments as specified by the manufacturer, and these DNA fragments were cloned into the PCR-Blunt vector (Invitrogen, Carlbad, CA). .. The recombinant plasmid was transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, CA) and the clones were sequenced. It was confirmed that clone sd2-2 contained a 958 bp insert, contained the 3'end of the putative Δ6-gene based on sequence homology with known Δ6-desaturase, and further contained a "TAA" stop codon and a Poly A tail. It was.

To isolate the 5'end of this Δ6-desaturase from Saprolegnia diclina, the oligonucleotide RTO939 (SEQ ID NO: 9) (5'-CGTAGTACTGCTCGAGGAGCTTGAGCGCCG-3') was used based on the sequence of the # 30-1 fragment identified earlier. Designed. This oligonucleotide was used in combination with RO898 (SEQ ID NO: 10) (5'-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3') (designed based on the sequence of the pBluescript SK (+) vector) and the 5'end of Δ6-desaturase from the cDNA library. Was PCR amplified. In this case, the Advantage-GC cDNA PCR kit (Clonetech, Palo) is used to solve the PCR amplification problem that a GC-rich region that is expected to exist at the 5'end of this Δ6-desaturase occurs. Alto, CA) was used. The PCR thermodynamic cycle conditions were as follows. First, the mold was denatured at 94 ° C for 1 minute, followed by 30 cycles [94 ° C for 30 seconds, 68 ° C for 3 minutes], and finally an extension cycle at 68 ° C for 5 minutes. The PCR product thus obtained was cloned into a PCR-Blunt vector (Invitrogen, Carlsbad, CA) according to the same protocol as above. Thus, a clone sd21-2 containing a 360 bp insert containing the "ATG" initiation site of the novel Δ6-desaturase was obtained. When the putative amino acid sequence of this fragment was aligned with the known Δ6-desaturase, it showed a degree of agreement of 37-45%.

The entire novel Δ6-desaturase gene was isolated by PCR amplification using the following oligonucleotides using the S. diclina cDNA library or S. diclina genomic DNA as a template.

<chemistry num="3"><img file="JP4347397B2_D0003.tif" /></chemistry>An oligonucleotide containing the sequence from the 5'end of clone sd21-2 and the EcoRI site (underlined) to facilitate cloning into a yeast expression vector.

<chemistry num="4"><img file="JP4347397B2_D0004.tif" /></chemistry>An oligonucleotide containing a sequence from the 3'end of sd2-2 containing a stop codon and a HindIII site (underlined) for cloning into a yeast expression vector.

cDNA library plasmid template 200 ng and each primer 10 pmol and Taq PCR Master Mix (Qiagen, Valencia, CA) or genomic DNA 200 ng and each primer 10 pmol and Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, CA) PCR amplification was performed using. The thermodynamic cycle conditions were as follows. First, the mold was denatured at 94 ° C for 1 minute, followed by 30 cycles [94 ° C for 30 seconds, 68 ° C for 3 minutes], and finally an extension cycle at 68 ° C for 5 minutes. The PCR product thus obtained is digested with EcoRI / HindIII, cloned into a yeast expression vector pYX242 (Invitrogen, Carlsbad, CA) to prepare clones pRSP1 (derived from genomic DNA) and pRSP2 (derived from library), and then sequenced. And used for the expression test.

The Δ6-desaturase full-length gene insert was 1362 bp long (SEQ ID NO: 13, FIG. 2) and contained an open reading frame encoding the 453 amino acids, starting with the first ATG. (The nucleotide sequence encoding Δ6-desaturase was deposited as plasmid pRSP1 in the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110 under the Budapest Treaty and was given accession number PTA-2829.) Full length. The amino acid sequence of the gene (SEQ ID NO: 14, Figure 3) contained a region homologous to the Δ6-desaturase from Mortierella alpina, Caenorhabiditis elegans and Borago officinalis. In addition, three preserved "histidine boxes" present in all known membrane-bound desaturases (Oduley et al., (1994) The Plant Cell 6: 147-158) was also included. These were located at amino acid positions 171-176, 208-212 and 391-395. Like other membrane-bound Δ6-desaturases, the third histidine box motif (HXXHH) in S. diclina Δ6-desaturase was found to be QXXHH. This sequence also contained a cytochrome b5 domain at the 5'end. This cytochrome b5 domain is present in many membrane-bound desaturase enzymes, and cytochrome b5 is thought to function as an electron donor in these enzymes. The presence of this domain appears to be advantageous when expressing desaturase in heterologous strains for PUFA production. The base composition of this gene appears to be important, as it has been proposed to be used in plants to reconstitute the PUFA biosynthetic pathway. (It is known that some recombinant genes are difficult to express because their base composition is different from that of the host. The total G + C content of this gene is 59%, and it has been successfully expressed in plants. Approximate to M. alpina desaturase.)

Isolation of the Δ5-desaturase nucleotide sequence from Saprolegnia diclina (ATCC56851) Saprolegnia diclina (ATCC 56851) produces both arachidonic acid (ARA, 20: 4 n-6) and eicosapentaenoic acid (EPA, 20: 5 n-3), so it is probably dihomo-γ-linolenic acid (DGLA, 20 :). It was thought to have a Δ5-desaturase capable of converting 3n-6) to arachidonic acid (ARA, 20: 4 n-6).

To isolate Δ5-desaturase from S. diclina, various combinations of degenerate primers were used in the PCR reaction using first-strand cDNA as a template, as in the case of Δ6-desaturase isolation. First-strand cDNA template 2 μl, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl in a final reaction volume of 50 μl₂A clear 588 bp band was obtained with primer vs. RO753 and RO754 using PCR conditions of 200 μM each deoxyribonucleotide triphosphate, 2 pmol of each primer and 1 U of cDNA polymerase (Clonetech, Palo Alto, CA). The thermal cycle was carried out as follows. After initial denaturation at 94 ° C for 3 minutes, denaturation was performed at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension at 72 ° C for 1 minute for 35 cycles. After this, a final extension was performed at 72 ° C for 7 minutes, and the reaction was completed at 4 ° C. When this fragment thus prepared was cloned (clone # 18-1), sequenced, and translated, it had an amino acid match of 43% with Mortierella alpina Δ5-desaturase (Genbank accession number AF067654) and Dictyostelium. The degree of agreement with discoideum Δ5-desaturase (Genbank accession number AB029311) was 38.7%. Secondary PCR fragments were identified using primers RO834 and RO838 in the reactions described in Example 2. When this fragment, about 1000 bp long, was cloned (clone # 20-8), the deduced amino acid sequence from 775 bp showed 42% concordance with Dictyostelium discoideum Δ5-desaturase (Genbank accession number AB029311). These two sequences # 18-1 and # 20-8 overlap each other and appear to belong to a single putative Δ5-desaturase from S. diclina. These sequences were then used to design primers to search for the 3'and 5'ends of the novel Δ5-desaturase from a cDNA library made from S. diclina mRNA.

To isolate the 3'end of this putative Δ5-desaturase, plasmid DNA purified from the cDNA library was used as a template and oligonucleotides RO851 (SEQ ID NO: 15) (5'-CCATCAAGACGTACCTTGCGATC-3') and RO899 ( PCR amplification was performed using SEQ ID NO: 8) (5'-AGCGGATAACAATTTCACACAGGAAACAGC-3').

Oligonucleotide RO851 is designed based on the # 18-1 fragment of this putative Δ5-desaturase, and oligonucleotide RO899 corresponds to a sequence from the pBluescript II SK (+) vector. Template plasmid DNA 200 ng, each primer 10 pmol and Taq PCR Master Amplification was performed using Mix (Qiagen, Valencia, CA). The sample was first denatured at 94 ° C for 3 minutes, followed by 35 cycles consisting of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 1 minute. The reaction was terminated after a final extension cycle of 7 minutes at 72 ° C. The PCR fragment was cloned into a PCR-Blunt vector (Invitrogen, Carlsbad, CA) according to the protocol described in Example 2. The recombinant plasmid was transformed into TOP10 supercompetent cells (Invitrogen, Carlsbad, CA) and the clones were sequenced. Clone sd12-11 contained the 3'end of the putative Δ5-gene based on sequence homology with known Δ5-desaturase, and also contained a 648 bp insert containing a "TAA" stop codon and a poly-A tail.

The 5'end of this Δ5-desaturase was isolated from Saprolegnia diclina using primers RO941 and RO898. This oligonucleotide RO941 (SEQ ID NO: 16) (5'-GCTGAACGGGTGGTACGAGTCGAACGTG-3') was designed based on the sequence of the # 20-8 fragment identified earlier. cDNA library plasmid DNA was used as a template and this oligonucleotide was used in combination with PCR amplification (designed based on the sequence of the pBluescript SK (+) vector) RO898 (SEQ ID NO: 10) (5'-CCCAGTCACGACGTTGTAAAACGACGGCCAG-3'). .. Here we have the Advantage-GC cDNA PCR Kit (Clonetech, Palo) Alto, CA) was used according to the manufacturer's protocol, and the thermal cycle conditions were as follows. After initial denaturation at 94 ° C for 1 minute, [denaturation at 94 ° C for 30 seconds, annealing and elongation at 68 ° C for 3 minutes] 30 cycles, and final extension cycle at 68 ° C for 5 minutes. did. These PCR products were purified in the same manner as above, cloned into a PCR-Blunt vector (Invitrogen, Carlsbad, CA), and sequenced. The clone sd24-1 was confirmed to contain a 295 bp insert containing a putative "ATG" initiation site for the novel Δ5-desaturase. Analysis of the deduced amino acid sequence of this fragment revealed a region highly homologous to the known Δ5-desaturase, and that a cytochrome b5 domain was also present.

The full-length Δ5-desaturase gene was isolated by PCR amplification using the following oligonucleotides using S. diclina genomic DNA as a template.

RO953 (SEQ ID NO: 17) (5'-ACGAGA) containing the sequence from the 5'end of clone sd24-1 and the EcoRI site (underlined) to facilitate cloning into a yeast expression vector.<u style="single">GAATTC</u>ATGGCCCCGCAGACGGAGCTCCGCCAGCGC-3'), and b. RO956 (SEQ ID NO: 18) (5'-AAAAGA) containing the sequence from the 3'end of sd12-11 containing a stop codon and the XhoI site (underlined) for cloning into a yeast expression vector.<u style="single">CTCGAG</u>TTAGCCCATGTGGATCGTGGCGGCGATGCCCTGC-3').

The PCR amplification conditions for the "full length" gene were the same as described for amplification of Δ6-desaturase from genomic DNA (Example 2). The PCR product thus obtained was digested with EcoRI / XhoI and cloned into the yeast expression vector pYX242 (Invitrogen, Carlsbad, CA). The cloned pRSP3 (derived from genomic DNA) was found to contain a 1413 bp insert and was used in the expression test.

The 1413 bp full-length gene of the estimated Δ5-desaturase from S. diclina (SEQ ID NO: 19, FIG. 4) contained an open reading frame encoding 471 amino acids (SEQ ID NO: 20, FIG. 5). (The nucleotide sequence encoding Δ5-desaturase was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110 on January 23, 2001 (as plasmid pRSP3) under the Budapest Treaty and has a accession number. PTA-2928 was given.) This translated protein has an amino acid match of 40.5% with Mortierella alpina Δ5-desaturase (Genbank accession number AF067654) and Dictyostelium. The degree of agreement with discoideum Δ5-desaturase (Genbank accession number AB029311) was 39.5%. It also contained three preserved "histidine boxes" at amino acid positions 186-190, 223-228, and 406-410. Similar to Δ6-desaturase, this sequence also contained a cytochrome b5 domain at the 5'end. The total G + C content of this gene was 61.5%.

Expression of S. diclina desaturase gene in baker's yeast A clone pRSP2 composed of full-length Δ6-desaturase cloned into pYX242 (Invitrogen, Carlsbad, CA) and a clone pRSP3 composed of full-length Δ5-desaturase cloned into pYX242 were transformed into competent Saccharomyces cerevisiae strain 334. Yeast transformation was performed using the Alkali-Cation Yeast Transformation Kit (BIO101, Vista, CA) according to the conditions specified by the manufacturer. Transformants were selected for leucine auxotrophy in leucine-deficient medium (DOB [-Leu]). To detect the specific desaturase activity of these clones, the transformants were grown in the presence of 50 μM of the specific fatty acid substrate below.

Stearic acid (18: 0) (conversion to oleic acid exhibits Δ9-desaturase activity).

b. Oleic acid (18: 1) (conversion to linolenic acid exhibits Δ12-desaturase activity).

c. Linolenic acid (18: 2 n-6) (conversion to α-linolenic acid exhibits Δ15-desaturase activity, conversion to γ-linolenic acid exhibits Δ6-desaturase activity).

d. α-linolenic acid (18: 3 n-3) (conversion to stearidonic acid exhibits Δ6-desaturase activity).

e. Dihomo γ-linolenic acid (20: 3 n-3) (conversion to arachidonic acid exhibits Δ5-desaturase activity).

Negative control strain contains S. cerevisiae containing the unmutated pYX242 vector It was set to 334 and propagated at the same time. The culture was vigorously stirred (250 rpm) and grown at 24 ° C. for 48 hours in the presence of various substrates at 50 μM (final concentration). Cells were pelleted, vortexed in methanol, and chloroform and tritridecanoine (as an internal standard) added. These mixtures were incubated overnight at room temperature for at least 1 hour or at 4 ° C. The chloroform layer was extracted and filtered through a Whatman filter using 1 g of anhydrous sodium sulfate to remove granules and residual water. The organic solvent was evaporated under a stream of nitrogen at 40 ° C. Next, the lipid extracted for gas chromatography analysis (GC) was derivatized to fatty acid methyl ester (FAME) by adding 2 ml of 0.5N potassium hydroxide in methanol to a closed tube. The sample was heated from 95 ° C to 100 ° C for 30 minutes and cooled to room temperature. About 2 ml of 14% boron trifluoride in methanol was added, and heating was repeated. After the extracted lipid mixture had cooled, 2 ml of water and 1 ml of hexane were added and FAME was extracted for GC analysis. The conversion was calculated by dividing the product by the sum of (product + added substrate) and multiplying by 100.

Table 1 shows the enzymatic activity of the isolated gene based on the conversion rate of the added substrate. A pRSP1 clone containing the Δ6-desaturase gene from S. diclina converts 28% of the 18: 2n-6 substrate to 18: 3n-3 and 37% of the 18: 3n-3 substrate to 18: 4n-3. Converted. This confirms that the gene encodes Δ6-desaturase. There was no background (non-specific conversion of substrate) in this case. (The tables cited herein are summarized at the end.) A pRSP3 clone containing the Δ5-desaturase gene from S. diclina can convert 27% of the added 20: 3n-6 substrate to 20: 4n-6, and the enzyme encoded by this gene is Δ5-desaturase. It shows that there is. Again, no background substrate conversion was detected. This data shows that desaturases with various substrate specificities can be expressed in heterologous systems and can be used to produce polyunsaturated fatty acids.

Table 2 shows the target fatty acid as a percentage of the total lipid extracted from S. cerevisiae 334 containing the designated plasmid. No glucose was added to the growth medium. Each lipid was separated using affinity gas chromatography. The product was identified using GC / MS. As is clear from this table, the substrate added from the outside is taken up by recombinant yeast when added in free form, and is taken up by the membrane. In yeast clones (pRSP1) containing the Δ6-desaturase gene, GLA (γ-18: 3) was identified as a novel PUFA when LA (18: 2) was added as a substrate, and DGLA (20: 3) was used as a substrate. When added, arachidonic acid was detected in yeast (pRSP3) containing the Δ5-desaturase gene.

Simultaneous expression of S. diclina desaturase and elongase The plasmids pRSP1 (6Δ) and pRSP3 (Δ5) were co-transformed with pRAE73-A3, which is a clone containing the human elongase gene (SEQ ID NO: 21), in the yeast expression vector pYES2, which is a yeast as described in Example 4. .. This erongase gene catalyzes part of the extension phase of the PUFA pathway. Co-transformants were selected in minimal medium lacking leucine and uracil (DOB [-Leu-Ura]).

Table 3 shows that when the substrate LA (18: 2 n-6) 50 μM is added, Δ6-desaturase converts this substrate to GLA (18: 3 n-6), and elongase adds two carbons to GLA and DGLA. It shows that (20: 3n-6) can be produced. Substrate-to-end product conversion by these co-transformants is 26.4% and no background is observed from the negative control. Similarly, co-transformants can act on ALA (18: 3n-3) to finally form (20: 4n-3) with a conversion rate of 34.39%. Therefore, S. diclina Δ6-desaturase was able to produce the product in a heterologous expression system, and was able to produce the expected PUFA by further utilizing this expression system with another heterologous enzyme from the PUFA biosynthetic pathway.

Table 4 shows the results of the pRSP3 (Δ5) / human elongase simultaneous transformation experiment. In this case, the substrate GLA (18: 3 n-6) is converted to DGLA (20: 3 n-6) by human elongase, which is further converted to ARA (20: 4 n-6) by the action of S. diclina Δ5-desaturase. ). Substrate-to-end product conversion by these co-transformants was 38.6%, with no background observed from the negative control.

The other substrate tested in this case was STA (18: 4n-3), which was finally converted to EPA (20: 5n-3) by the joint action of the two enzymes. Similar results were observed when pRSP1 and pRSP3 were co-transformed with the elongase gene (pRSP2) (SEQ ID NO: 22) derived from M. alpina, and both genes were found to be functional in the presence of each other. (See Tables 3 and 4).

Isolation of the Δ5-desaturase nucleotide sequence from Thraustochytrium aureum (ATCC34303) To isolate the putative desaturase, total RNA was isolated as described in Example 2. Approximately 5 μg of total RNA was reverse transcribed using a SuperScript Preamplification system (Life Technologies, Rockville, MD) as shown in Example 2 to prepare first-strand cDNA. First-strand cDNA template 2 μl, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl using degenerate primers RO834 (SEQ ID NO: 1) and 838 (SEQ ID NO: 4) designed by the block maker program in a 50 μl reaction.₂, 200 μM of each deoxyribonucleotide triphosphate, 2 pmol of each primer and cDNA polymerase (Clonetech, Palo Alto, CA) were used in combination. The thermal cycle was carried out as follows. After initial denaturation at 94 ° C for 3 minutes, denaturation was performed at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension at 72 ° C for 1 minute for 35 cycles. After this, a final extension was performed at 72 ° C for 7 minutes. Two weak bands of about 1000 bp were separated on a 1% agarose gel, excised and purified using the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA). T4 at the end as described in Example 2 It was packed with DNA polymerase and the blunt-ended fragment was cloned into a PCR-Blunt vector (Invitrogen, Carlbad, CA). When the obtained clone was sequenced, a 680 bp partial sequence from clones 30-9 was confirmed, and the translation of its 226 amino acids had an amino acid concordance of 31.5 with Δ6-desaturase (Genbank accession number AW281238) from adult zebrafish. %Met. Human Δ6-desaturase (Genbank accession number AF126799), Physcomitrella patens (moss) Δ6-desaturase (Genbank accession number AJ222980), Brassica napus (canola) Δ8-sphingolipid desaturase (Genbank accession number AJ224160) and human Δ5-desaturase (ATCC) A similar degree of amino acid homology (29.6% to 28.7%) was observed with No. 203557 and Genbank Accession No. AF199596). Since there was a considerable degree of amino acid homology with the known desaturase, the activity of the full-length gene encoding the putative desaturase during yeast expression was determined.

Primer RO936 (SEQ ID NO: 23) was used to isolate the 3'end of the gene by reacting with 100 ng of purified plasmid from the T. aureum cDNA library in a Taq PCR Master Mix (Qiagen, Valencia, CA) medium reaction volume of 100 μl. (5'-GTCGGGCAAGGCGGAAAAGTACCTCAAGAG-3') and RO899 (SEQ ID NO: 8) (5'-AGCGGATAACAATTTCACACAGGAAACAGC-3') 10 pmol were used in combination. The thermodynamic cycle conditions were carried out as follows. After initial melting at 94 ° C for 3 minutes, 30 cycles of 94 ° C for 1 minute, 60 ° C for 1 minute and 72 ° C for 3 minutes were performed. After this, a 10-minute extension step was performed at 72 ° C. Several bands containing the expected dimensions of 1.2 kb were separated and purified on a 1% agarose gel as described above. Similarly, as described above, the ends of the fragments were blunted, cloned into PCR-Blunt and sequenced. When fragment # 70-2 of about 1.2 kb was sequenced, it contained an open reading frame and a stop codon that overlapped fragment 30-9.

To isolate the 5'end of the gene, RO937 (SEQ ID NO: 24) (5'-AAACCTGTAGACAATGTGGAGGGGCGTGGG-3') and RO899 (SEQ ID NO: 8) were used in a 50 μl PCR reaction with 100 ng of purified plasmid DNA from the library. Using 10 pmol of each primer, the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, CA) was used according to the manufacturer's protocol. The thermodynamic cycle conditions were as follows. After initial denaturation at 94 ° C for 1 minute, [denaturation at 94 ° C for 30 seconds, annealing and elongation at 68 ° C for 3 minutes] 30 cycles, and final extension cycle at 68 ° C for 5 minutes. did. A band of about 500 bp in the expected dimensional range was gel purified as described above, blunt-ended and cloned into PCR-Blunt. Clone 95-2 contained an open reading frame and a starting codon. This fragment also overlapped with clones 30-9 and certainly appeared to be part of the same gene.

To isolate the full-length gene, the following primers with restriction sites 3'and 5'(underlined), respectively, cleaved at EcoRI and XhoI, ie 5'primers RO972 (SEQ ID NO: 25) (5'-ATACTT)<u style="single">GAATTC</u>ATGGGACGCGGCGGCGAAGGTCAGGTGAAC-3'), 3'Primer RO949 (SEQ ID NO: 26) (5'-CTTATA<u style="single">CTCGAG</u>CTAAGCGGCCTTGGCCGCCGCCTGGCC-3') and 3'primer RO950 (SEQ ID NO: 27) (5'-CTTATA<u style="single">CTCGAG</u>TAAATGGCTCGCGAGGCGAAGCGAGTGGC-3') was designed. Primer RO949 containing a stop codon has a GC content of 66%, while another primer RO950 outside the stop codon has a GC content of only 56%, so in early isolation attempts there were two primers at the 3'end of the gene. It was used. 50 μl with RO972 / RO949 and RO972 / 950 using the Advantage-GC cDNA PCR kit (Clonetech, Palo Alto, CA) under the same conditions as described in the previous section. A PCR reaction was performed. Only the RO972 / 950 primer pair produced a band of about 1.6 kb. Bands of similar size were obtained when genomic DNA was used as a template (under the same conditions as the target 100 ng). Fragments were separated on an agarose gel as described above, gel purified, blunt-ended and cloned into PCR-Blunt. Fragments are sequenced, numerous clones are cleaved with EcoRI / XhoI to excise full-length genes, and pre-existing with shrimp alkaline phosphatase (Roche, Indianopolis, IN) using a Rapid ligation kit (Roche, Indianopolis, IN). It was connected to the processed pYX242 EcoRI / XhoI. Clone 99-3 (referred to as pRTA4) contained a 1317 bp full-length gene (SEQ ID NO: 28, FIG. 6) and an open reading frame of 439aa (SEQ ID NO: 29, FIG. 7). (The nucleotide sequence encoding Δ5-desaturase is based on the Budapest Treaty, ATCC, 10801 University Boulevard, Manassas, VA It was deposited in 20110 on January 23, 2001 and was given the accession number PTA-2927. ) This gene contained three histidine boxes at amino acids 171-175, 208-212, and 376-380. Translation of the 5'end of this gene shows homology with cytochrome b5.

Expression of T. aureum desaturase gene in baker's yeast A clone pRTA4 containing the full-length gene was transformed into yeast host S. cerevisiae 334 as described in Example 4 and plated on selective medium. As shown in Table 5, 50 μM of exogenous free fatty acid was added as a substrate to the minimum medium lacking leucine, and the culture medium was grown at 24 ° C. for 48 hours. Substrate conversion was only from DGLA (20: 3n-6) to ARA (20: 4n-6). 23.7% of the added DGLA is converted, indicating that this gene encodes Δ5-desaturase.

Table 6 shows some of the fatty acids as a percentage of the lipids extracted from the yeast host. There was no background for Δ5-desaturase activity (detection of ARA observed in negative controls containing the yeast expression plasmid PYX242).

Simultaneous expression of T. aureum desaturase and elongase The yeast expression vector pYES2 as described in Example 4 was co-transformed with the plasmid pRTA4 with a further enzyme of the PUFA pathway, pRAE73-A3 containing the human erongase gene, in a minimal medium lacking leucine and uracil. Co-transformers were selected.

Table 7 shows that Δ5-desaturase can produce ALA and elongase can produce ADA by adding 2 carbons to ALA when the substrate DGLA 100 μM is added. The conversion rate of T. aureum Δ5-desaturase, which is composed of both ARA and ADA (product), is 16.7%, and no background is observed from the negative control.

As a result of the above, T. aureum Δ5-desaturase can produce a product in a heterologous expression system, and if this expression system is further utilized by another heterologous enzyme from the PUFA biosynthetic pathway, expected PUFA can be produced.

Isolation of the Δ5-desaturase nucleotide sequence from Thraustochytrium aureum BIC C7091 (T7091) Partial desaturase candidates were isolated using the degenerate primer pair RO834 / RO836 described in Example 1. Genomic DNA was prepared from Thraustochytrium aureum BICC7091 (Bicon India Ltd., Banglore, India) using the DNeasy plant maxi kit (Qiagen, Valencia, CA). Primers RO834 (5'-GTB TAY GAYGTB ACC GAR TGG GTB AAG CGY CAY CCB GGH GGH-3') (SEQ ID NO: 1) and RO838 (5'-CAT GGT VGG RAA SAG RTG RTG YTC RAT CTG RTA GTT-3') (SEQ ID NO: 36) was used to amplify T7091 gDNA. Isolated T7091 gDNA 5 μl, 0.2 m dNTP PCR amplification was performed in a 100 μl volume containing mix, 50 pM for each primer, 10 μl of 10 × buffer and 1.0 U of cDNA polymerase. The following thermodynamic cycle conditions were performed on the Perkin Elmer 9600. After 3 minutes at 94 ° C, 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 1 minute were performed. After PCR, a 7-minute addition-elongation step was performed at 72 ° C. The PCR amplification mixture was run on a 1% agarose gel and about 1.2Kb and 1.4Kb amplified fragments were gel purified using the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA). T4 DNA polymerase (Life Technologies, Rockville, MD) is used to "fill" the protruding ends of these fragments, the isolated fragments are cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, CA) and the recombinant plasmids are top 10 super It was transformed into competent cells (Invitrogen, Carlsbad, CA).

Twenty-four clones were prepared and sequenced using the ABI 373A DNA Sequencer (Applied Biosystems, Foster City, CA). The translated sequence was used as a query and the GenEmb1 database (Genetics Computer Group (GCG) (Madison, WI) was searched using the tFastA algorithm (Pearson and Lipman search for similarity between protein query sequence and arbitrary group of nucleotide sequences). The gene fragment T7091B2 was in agreement with a known database and was further investigated. T7091B2 had a 26.8% concordance with human Δ5-desaturase (GenBank Accession No. AF225273) and 362 amino acids.

New primers were designed based on the T7091B2 internal sequence to isolate the 3'and 5'ends. Approximately 2 x 10 clones with an average insert size of 1 Kb using new and vector primers⁷The T7091 cDNA library containing the cells was PCR amplified. Vector Primer RO898 (5'-CCC AGT CAC GAC GTT GTA AAA CGA CGG CCA G-3') (SEQ ID NO: 10) and RO899 (5'-AGC GGA TAA CAA TTT CAC ACA GGA AAC AGC-3') (SEQ ID NO: 10) Primers that produced more 5'and 3'sequences in clone T7091B2 when used in combination with 8) were RO1065 (5'-CGA CAA GAG GAA GAG TGT CCA AAT C-3') (SEQ ID NO: 37) and RO1064, respectively. It was (5'-CGCCTT CAA GAG TTT TTG TAC GGA ATT GGG-3') (SEQ ID NO: 38). Two new primers were designed based on the T7091B2 5'and 3'sequences.

RO1097 (5'-CTT GTA) with additional NcoI restriction site (underlined part) <u style="single">CCA</u><u style="single">TGG</u> GTC GCG GAG CAC AGG GAG-3') (SEQ ID NO: 39).

b.RO1098 (5'-TG<u style="single">A</u><u style="single">AGC</u><u style="single">TT</u>A CTC GCT CTT GGC AGC TTG GCC-3') (SEQ ID NO: 40).

This novel Δ5-desaturase was isolated entirely by PCR amplification using T. aureum 7091 gDNA. Isolated T7091 gDNA 1 μl, 0.2 μM dNTP mix, each primer 50 pM, 10 × buffer 5 μl, 50 mM DDL₄ PCR was performed in a 50 μl volume containing 1.5 μl and 0.5 U of Taq DNA polymerase. The following thermodynamic cycle conditions were performed on the Perkin Elmer 9600. After 3 minutes at 94 ° C, 30 cycles of 95 ° C for 45 seconds, 55 ° C for 30 seconds and 68 ° C for 2 minutes were performed. The PCR amplification mixture was run on a gel, an amplification fragment of about 1.3 Kb was gel purified, and the isolated fragment was cloned into a pYX242 (NcoI / EcoRV) vector. Two clones, pRAT-2a and 2c, were prepared and sequenced. (The plasmid pRAT-2c was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 as of January 2002 and given a accession number based on the Budapest Treaty.) The sequences differ by 4 amino acids. (Fig. 12), the translation sequence was 67.4% concordance with T. aureum (ATCC34303) Δ5-desaturase (see Example 6) and 436 amino acids.

Isolation of Δ6-desaturase nucleotide sequence from Thraustochytrium aureum BIC C7091 (T7091) 373 templates from the T7091 cDNA library were sequenced and the viability of the library was examined. The translated sequence was used as a query and the GenEmb1 database was searched using the tFastA algorithm. Clone 602187281R1 (or shortened to clone 281) was consistent with several known desaturases. The 5'and 3'ends of the EST clone were sequenced to design amplification primers. Primer RO1107 (5'-TTT AA) with additional NcoI restriction site (underlined part)<u style="single">C</u><u style="single">CAT</u><u style="single">GG</u>RO1108 (5'-GGG AAG) with G CCG CGG CGG CGA GAA AAG-3') (SEQ ID NO: 41) and additional HindIII restriction site (underlined) <u style="single">AAG</u><u style="single">CTT</u> T7091 gDNA was PCR amplified using TCT ACT GCG CCT TGG CTT TCT TTG-3') (SEQ ID NO: 42).

PCR was performed in a 50 μl volume containing Taq DNA polymerase as described above. The PCR amplification mixture was run on a gel, an amplification fragment of about 1.3 Kb was gel purified, and the isolated fragment was cloned into a pYX242 (NcoI / EcoRV) vector. Two clones, pRAT-1a and 1b, were prepared and sequenced. The sequences differed by one amino acid (Fig. 13), and the translated sequence was 25% concordance between human Δ5-desaturase and 430 amino acids . (The plasmid pRAT-1a was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 as of January 2002 and given the ATCC accession number --- based on the Budapest Treaty.)

Expression of the T. aureum 7091 desaturase gene in baker's yeast Clones pRAT-2a and pRAT-2c composed of full-length human Δ5-desaturase cloned to pYX242 (Invitrogen, Carlsbad, CA) and clones pRAT-1a and pRAT- consisting of full-length human Δ6-desaturase cloned to pYX242 1b was transformed into the competent Saccharomyces cerevisiae strain 334. Yeast transformation was performed using the Alkali-Cation Yeast Transformation Kit (BIO101, Vista, CA). Transformants were selected for leucine auxotrophy in leucine-deficient medium (DOB [-Leu]). Since the translation sequence of pRAT-1 cDNA does not have a strong agreement with any specific known desaturase, several fatty acid substrates were tested to examine the activity of the expressing enzyme. To detect the specific desaturase activity of pRAT-1a and pRAT-1b clones, transformants were grown in the presence of 100 μM of the following specific fatty acid substrate.

Linolenic acid (LA, 18: 2 n-6) (conversion to α-linolenic acid exhibits Δ15-desaturase activity, conversion to γ-linolenic acid exhibits Δ6-desaturase activity).

b. α-linolenic acid (ALA, 18: 3 n-3) (conversion to stearidonic acid exhibits Δ6-desaturase activity).

c. Conversion to ω6-esicosazienoic acid (EDA, 20: 2n-6) (dihomo-γ-linolenic acid) shows Δ8-desaturase activity).

d. Dihomo γ-linolenic acid (DGLA, 20: 3n-3) (conversion to arachidonic acid exhibits Δ5-desaturase activity).

The substrate for the pRAT-2 clone was 100 μM DGLA. S. cerevisiae 334 containing the unmodified pYX242 vector was used as a negative control. Both pRAT-1a and pRAT-2c are also present in S. cerevisiae 334 containing pRAE-73-A3 (ie, a vector containing a human erongase enzyme that converts 18C fatty acids to 20C fatty acids, SEQ ID NO: 21) (see Example 5). Was simultaneously transformed. The substrates of the pRAT-1a / pRAE-73-A3 clone were LA and ALA, and the substrates of the pRAT-2c / pRAI-73-A3 clone were LA, ALA and GLA. The culture was grown in selective medium at 24 ° C for 48 hours in the presence of a specific substrate. Fatty acid analysis was performed as summarized in Example 4.

Table 8 shows an example comparing AA production with strain 334 (pRAT-2c) with AA production with control strain 334 (pYX242). AA production was 22.98%, which was not detectable in the control strain. The 334 (pRAT-2a) strain also did not produce a detectable amount of AA (data not shown). The difference in 4 amino acids between the 2 clones (see Figure 12, underlined) rendered the enzyme expressed from the 334 (pRAT-2a) strain inactive. When co-expressed with pRAE-73-A3 in the presence of GLA, further Δ5-desaturase activity from clone pRAT-2c was detected.

Table 9 shows the results of fatty acid analysis of 334 (pRAT-1a) and 334 (pYX242) strains expressed in the presence of substrates LA, ALA, EDA and DGLA. The Δ6-unsaturation of LA produces GLA, the Δ8-unsaturation of EDA produces DGLA, and the Δ5-unsaturation of DGLA produces AA. All three types of activity were detected in both strains containing the T7091 gene compared to the control strain. According to the conversion rate, this enzyme is an active Δ6-desaturase that can function like a Δ5 or Δ8-desaturase if the substrate is appropriate. However, the conversion rate of each of these to unsaturated fatty acids is lower than that of the Δ6-unsaturated substrate. Single amino acid mutations between the two clones (see Figure 13, underlined) did not affect enzyme activity (data not shown). The conversion rate is shown in the lower column of Table 9. Furthermore, Δ6-desaturase activity was also detected when pRAT-1a was co-expressed with pRAE-73-A3 in the presence of LA or ALA. Since it is difficult to determine whether the production of new fatty acids is due to the activity of desaturase, the activity of elongase, or the combination of the two enzymes, the conversion rate was not calculated in the co-expression experiment.

Two novel gene isolation methods have been developed to identify the desaturase gene from T. aureum 7091. The T7091 Δ5-desaturase and Δ6-desaturase genes were isolated by these methods. Due to the presence of these genes and suitable substrates, S. cerevisiae produced significantly higher levels of unsaturated fatty acids than the control strain. Simultaneous light transformation of the construct containing the T7091B2 gene (pRAT-2c) and the human elongase gene (pRAE-73-A3) into yeast converted the substrate GLA to AA. As a result of this experiment, it was confirmed that the enzyme expressed from pRAT-2c unsaturateds DGLA (produced by elongase) to AA. Simultaneous light conversion of the construct containing the "281" gene (pRAT-1a) and the human elongase gene (pRAE-73-A3) to yeast resulted in the conversion of substrate LA to DGLA. As a result of this experiment, it was confirmed that the enzyme expressed from pRAT-1a unsaturateds LA to GLA.

Isolation of the Δ5-desaturase nucleotide sequence from Isochrys galbana 1323 Partial desaturase candidates were isolated using the degenerate primer pair RO834 / RO836 described in Example 1. Genomic DNA was prepared from Isochrys galbana CCMP1323 (Provasoli-Guillard National Center for the Culture of Marine Phytoplankton (CCMP), West Boothbay Harbor, MA) using the DNeasy plant maxi kit (Qiagen, Valencia, CA). Primers RO834 (5'-GTB TAY GAYGTB ACC GAR TGG GTB AAG CGY CAY CCB GGH GGH-3') (SEQ ID NO: 1) and RO838 (5'-CAT GGT VGG RAA SAG RTG RTG YTC RAT CTG RTA GTT-3') I.galbana using (SEQ ID NO: 10) The gDNA was amplified. Isolated I. galbana gDNA 1 μl, 0.2 μM dNTP mix, each primer 50 pM, 10 × buffer 5 μl, 50 mM DDL₄ PCR was performed in a 50 μl volume containing 1.5 μl and 0.5 U of Taq DNA polymerase. The following thermodynamic cycle conditions were performed on the Perkin Elmer 9600. After 3 minutes at 94 ° C, 30 cycles of 95 ° C for 45 seconds, 55 ° C for 30 seconds and 68 ° C for 2 minutes were performed. The PCR amplification mixture was run on a 1.0% agarose gel and an amplified fragment of approximately 1.1 Kb was gel-purified using the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA). T4 DNA polymerase (Life Technologies, Rockville, MD) is used to "fill" the protruding ends of the fragment, the isolated fragment is cloned into the PCR-Blunt vector (Invitrogen, Carlsbad, CA), and the recombinant plasmid is TOP10 supercompetent. Transformed into cells (Invitrogen, Carlsbad, CA).

Six clones were prepared and sequenced using the ABI 373A DNA Sequencer (Applied Biosystems, Foster City, CA). All sequences were identical. The translation sequence was 47.5% concordance with T.aureum (ATCC34303) Δ5-desaturase and 335 amino acids in clone pRTA4 (Example 6) and T.aureum BIC C7091Δ5-desaturase and 278 amino acids in clone pRAT-2c (Example 9). The degree of agreement was 45.3%.

To isolate the 5'and 3'ends, novel primers were designed based on the internal sequence of the isolated I.galbana fragment. RO1235 (5'-CGA AGT TGG TGA AGA TGT AGG TGC CG-3') (SEQ ID NO: 43) is used at the 5'end of the gene, and RO1232 (5'-GAG CGA CGC GTA) is used at the 3'end of the gene. CAA CAA CTT TCA CGT-3') (SEQ ID NO: 44) was used. Approximately 1.4 μg of total RNA from the manufacturer with rapid amplification of the cDNA ends or RACE included with the GeneRacer® kit (Invitrogen, Carlsbad, CA) and the Superscript II® enzyme (Invitrogen, Carlsbad, CA). It was used as directed and reverse transcribed to produce a cDNA target. Perkin Elmer for terminal initial amplification The following thermodynamic cycle protocol was performed on the 9600. After initial melting at 94 ° C for 2 minutes, 5 cycles of 94 ° C for 30 seconds and 72 ° C for 3 minutes, 94 ° C for 30 seconds, 70 ° C for 30 seconds and 72 ° C for 3 minutes for 10 cycles. After 20 cycles of 94 ° C for 30 seconds, 68 ° C for 30 seconds and 72 ° C for 3 minutes, extension was performed at 72 ° C for 10 minutes. This primary PCR reaction consists of 10 pmol RO1235 or RO1232 and GeneRacer® 5'primers (5'-CGA CTG GAG CAC GAG GAC ACT GA-3') (SEQ ID NO: 45) or GeneRacer® 3'primers, respectively. 5'-GCT GTC AAC GAT ACG CTA CGT AAC G-3') (SEQ ID NO: 46), 1 l of Thermozyme® (Invitrogen, Carlsbad, CA) and 1 μl of cDNA in a final volume of 50 μl according to the manufacturer's instructions. Performed using.

Initial reaction product 2 μl, nested primer RO1234 (5'-AGC TCC AGG TGA TTG TGC ACG CGC AG-3') (SEQ ID NO: 47) or RO1233 (5'-GAC TTT GAG AAG CTG CGC CTC GAG CTG-3') ( SEQ ID NO: 48) 10 pmol and GeneRacer® nested 5'primer (5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3') (SEQ ID NO: 49) and GeneRacer® nested 3'primer (5', respectively. '-CGC TAC GTA ACG GCA TGA CAG TG-3') (SEQ ID NO: 50) 30 pmol and DDL₄Nesting reactions were performed using Platinum Taq® PCRx (Clonetech, Palo Alto, CA) using Platinum Taq® according to the manufacturer's instructions. The following thermodynamic cycle parameters were performed on a Perkin Elmer 9600. After initial melting at 94 ° C for 2 minutes, 5 cycles for 30 seconds at 94 ° C and 2 minutes at 72 ° C, 5 cycles for 30 seconds at 94 ° C and 2 minutes at 70 ° C, and 30 seconds at 94 ° C. After 20 cycles of 30 seconds at 65 ° C and 2 minutes at 68 ° C, stretching was performed at 68 ° C for 10 minutes. When the PCR product was analyzed on an agarose gel, a band of about 800 bp was detected in the 5'reaction and a band of about 1.2 kb was detected in the 3'reaction. It was then cloned into PCR Blunt (Invitrogen, Carlsbad, CA), transformed into Top10 supercompetent cells (Invitrogen, Carlsbad, CA), sequenced, and an open reading frame with both start and stop codons was detected. Was done.

Primer RO1309 (5'-ATG ATG) with restriction sites for cloning (EcoRI and SalI, respectively) <u style="single">GAA</u><u style="single">TTC</u> ATG GTG GCA GGC AAA TCA GGC GC-3') (SEQ ID NO: 51) and RO1310 (5'-AAT AAT <u style="single">GTC</u><u style="single">GAC</u> The full-length gene was isolated using CTA GTG CGT GTG CTC GTG GTA GG-3') (SEQ ID NO: 52). As mentioned above, targeting 2 μl of cDNA and DDL₄Platinum Taq® PCRx (Clonetech, Palo Alto, CA) was used with 10 pmol primers RO1309 and 1310 according to the manufacturer's protocol. The thermodynamic cycle parameters were performed as follows. After initial melting at 94 ° C for 2 minutes, 5 cycles for 30 seconds at 94 ° C and 2 minutes at 72 ° C, 5 cycles for 30 seconds at 94 ° C and 2 minutes at 70 ° C, and 30 seconds at 94 ° C. After 20 cycles of 30 seconds at 65 ° C and 2 minutes at 68 ° C, stretching was performed at 68 ° C for 10 minutes. The single product of the reaction was gel purified using the QiaQuick gel purification kit (Qiagen, Valencia, CA), cleaved with EcoRI and SalI, and pYX242 using the Rapid ligation kit (Roche, Indianopolis, IN). It was ligated to EcoRI / XhoI linearized DNA and named pRIG-1. The clone pRIG-1 contained a 1329 bp full-length gene (SEQ ID NO: 34, FIG. 14) and an open reading frame of 442 amino acids (SEQ ID NO: 35, FIG. 15). (The plasmid pRIG-1 was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 as of January 2002 and given the ATCC accession number --- based on the Budapest Treaty.)

Expression of the I. galbana desaturase gene in baker's yeast A clone pRIG-1 containing a full-length gene was transformed into yeast host S. cerevisiae 334 as described in Example 4 and plated on selective medium. As shown in Table 10, 50 μM of foreign free fatty acid was added as a substrate to the minimum medium lacking leucine, and the culture medium was grown at 24 ° C. for 48 hours. Substrate conversions were from ETA (20: 4n-3) to EPA (20: 5n-3) and DGLA (20: 3n-6) to AA (20: 4n-6). The conversion rate to ARA is 45.4% and the conversion rate to EPA is 59.75%, indicating that this gene encodes Δ5-desaturase. Table 10 shows some of the fatty acids as a percentage of the lipids extracted from the yeast host. There was little or no background for Δ5-desaturase activity (detection of ARA or EPA observed in negative controls containing the yeast expression plasmid pYX242).

Co-expression of I.galbana desaturase gene and elongase The yeast expression vector pYES2 as described in Example 4 was co-transformed with the PUFA pathway addition enzyme pRAE73-A3 containing the human erongase gene, and the plasmid pRIG-1 was co-transformed with the minimum medium lacking leucine and uracil. I chose a body. When a substrate such as DGLA or ETA is added, Δ5-desaturase actively produces ARA or EPA, and elongase can add two carbons to produce ADA or 3-DPA. Thus, I. galbana Δ5-desaturase can produce a product in a heterologous expression system, and further utilization of this expression system by another heterologous enzyme from the PUFA biosynthetic pathway can produce the expected PUFA.

Nutritional composition The PUFAs described in detail are available in a variety of nutritional supplements, infant formulas, milk substitutes and other nutritional solutions.

I. Infant formula A. Isomil® Iron-blended soymilk: Uses: Beverages for infants, toddlers and adults with milk allergies or hypersensitivity. Nutrition for patients with diseases for which lactose must be avoided (lactase deficiency, lactose intolerance and galactosemia).

Features: -Use soy protein isolate to avoid symptoms of milk protein allergy or hypersensitivity. -A lactose-free formula was used to avoid lactose-related diarrhea. -Low osmotic pressure (240 mOs / kg water) to reduce the risk of osmotic diarrhea. -Uses complex carbohydrates (corn syrup and sucrose) to enhance carbohydrate absorption and reduce the risk of exceeding the absorption capacity of the damaged gastrointestinal tract. -Contains 1.8 mg of iron (as iron sulfate) per 100 calories to help prevent iron deficiency. -Contains recommended values of vitamins and minerals. -Contains vegetable oils to provide recommended values of essential fatty acids. -Milk white, milky consistency and good aroma.

Ingredients: (parbe) water 85%, corn syrup 4.9%, sugar (zinc sulfate) 2.6%, soybean oil 2.1%, soybean protein isolate 1.9%, palm oil 1.4%, calcium citrate 0.15%, 0 tertiary calcium phosphate .11%, potassium citrate, potassium monophosphate, potassium chloride, mono and diglyceride, soy lecithin, caraginan, ascorbic acid, L-methionine, magnesium chloride, potassium diphosphate, sodium chloride, choline chloride, taurine, Ferrous sulfate, m-inositol, α-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, riboflavin, pyridoxin hydrochloride, folic acid, manganese sulfate , Potassium iodide, Phylloquinone, Biotin, Sodium selenate, Vitamin D3 and Cyanocobalamine.

B. Isomil® DF Prepared Soymilk for Diarrhea: Uses: Short-term diet for food management of diarrhea in infants.

Features: -The first infant formula to which dietary fiber from soy fiber was added, especially for diarrhea control. -It has been clinically found to shorten the period of watery loose stools during periods of mild to severe diarrhea in babies. -Nutritionally complete to meet the nutritional requirements of the baby. Soy protein isolates supplemented with -L-methionine meet or exceed the requirements for all essential amino acids in infants. -A lactose-free formula was used to avoid lactose-related diarrhea. -Low osmotic pressure (240 mOs / kg water) to reduce the risk of osmotic diarrhea. -Uses complex carbohydrates (corn syrup and sucrose) to enhance carbohydrate absorption and reduce the risk of exceeding the absorption capacity of the damaged gastrointestinal tract. -Satisfy or exceed the vitamin and mineral levels recommended by the American Academy of Pediatrics Nutrition Commission and specified in Infant Formula. -Contains 1.8 mg of iron (as iron sulfate) per 100 calories to help prevent iron deficiency. -Contains vegetable oils to provide recommended values of essential fatty acids.

Ingredients: (parbe) water 86%, corn syrup 4.8%, sugar (zinc) 2.5%, soybean oil 2.1%, soybean protein isolate 2.0%, palm oil 1.4%, soybean fiber 0.77%, calcium chloride 0.12% , Tertiary calcium phosphate 0.11%, potassium citrate 0.10%, potassium chloride, potassium monophosphate, mono and diglyceride, soybean lecithin, caraginan, magnesium chloride, ascorbic acid, L-methionine, potassium dibasic phosphate, sodium chloride, Choline chloride, taurine, ferrous sulfate, m-inositol, α-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, riboflavin, pyridoxin hydrochloride , Folic acid, manganese sulfate, potassium iodide, phylloquinone, biotin, sodium selenate, vitamin D3 and cyanocobalamine.

C.Isomil® SF Iron-containing sucrose-free soymilk: Uses: Beverages for infants, toddlers and adults with milk protein allergies or hypersensitivity or sucrose intolerance. Nutrition for patients with diseases where lactose and sucrose must be avoided.

Features: -Use soy protein isolate to avoid symptoms of milk protein allergy or hypersensitivity. -Lactose-free formulation to avoid lactose-related diarrhea (carbohydrate source is Polycose® glucose polymer). -Sucrose-free for patients who are intolerant of sucrose. -Low osmotic pressure (180 mOs / kg water) to reduce the risk of osmotic diarrhea. -Contains 1.8 mg of iron (as iron sulfate) per 100 calories to help prevent iron deficiency. -Contains recommended values of vitamins and minerals. -Contains vegetable oils to provide recommended values of essential fatty acids. -Milk white, milky consistency and good aroma.

Ingredients: (parbe) water 75%, hydrolyzed corn starch 11.8%, soybean oil 4.1%, soy protein isolate 4.1%, coconut oil 2.8%, modified corn starch 1.0%, tricalcium phosphate 0.38%, potassium citrate 0.17% , Potassium chloride 0.13%, mono and diglyceride, soy lecithin, magnesium chloride, ascorbic acid, L-methionine, calcium carbonate, sodium chloride, choline chloride, caraginan, taurine, ferrous sulfate, m-inositol, α-tocopheryl acetate, Zinc sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, riboflavin, pyridoxin hydrochloride, folic acid, manganese sulfate, potassium iodide, phylloquinone, biotin, sodium selenate, Vitamin D3 and cyanocobalamin.

D. Isomil® 20 Immediate Iron Formulated Soymilk, 20 Cal / ounce: Uses: If you need soy intake.

Ingredients: (parbe) water 85%, corn syrup 4.9%, sugar (zinc sulfate) 2.6%, soybean oil 2.1%, soybean protein isolate 1.9%, palm oil 1.4%, calcium citrate 0.15%, calcium triphosphate 0.11 %, Potassium Citrate, Primary Potassium Phosphate, Potassium Chloride, Mono and Diglyceride, Soy Lecithin, Caraginan, Ascorbic Acid, L-Metionine, Magnesium Chloride, Potassium Secondary Phosphate, Sodium Chloride, Choline Chloride, Taurine, Zinc Sulfate Iron, m-inositol, α-tocopheryl acetate, zinc sulfate, L-carnitine, niacinamide, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, riboflavin, pyridoxin hydrochloride, folic acid, manganese sulfate, iodine Potassium chloride, phylloquinone, biotin, sodium selenate, vitamin D3 and cyanocobalamine.

E. Similac® Infant Formula: Uses: If you need infant formula: If you decide to stop breastfeeding under the age of 1, if you need supplementation of breastfeeding, or as a daily diet when breastfeeding is not adopted.

Features: -Used quality and quantity of protein suitable for good growth and reduced the risk of milk-related intestinal bleeding by heat denaturation. -(Double homogenization) Uses fats derived from vegetable oil blends to provide essential linolenic acid that is easy to absorb. -Contains carbohydrates as lactose in a proportion similar to human milk. -Low renal solute load to minimize stress on growing organs. -Powder, concentrate and ready-to-use forms.

Ingredients: (-D) water, defatted milk, lactose, soy oil, palm oil, mono and diglyceride, soy lecithin, ascorbic acid, caraginan, choline chloride, taurine, m-inositol, α-tocopheryl acetate, zinc sulfate, niacinamide , Ferrous sulfate, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, folic acid, manganese sulfate, phylloquinone, biotin, sodium selenate, vitamin D3 and cyanocobalamin.

F.Similac® NeoCare Iron-containing preterm infant formula: Uses: Meet special nutritional requirements for preterm infants after discharge. Similac NeoCare is a nutritionally complete formulation developed to catch up with growth delays and provide the additional calories, proteins, vitamins and minerals needed to support growth.

Features: -Reduce the amount of heat and the need for vitamin supplementation. Higher calorific value (22 Cal / ounce) than standard formulation (20 Cal / ounce). -A high-absorption fat blend with medium-chain triglyceride (MCToil) was used to help meet the special digestive requirements of preterm infants. -Compounded with higher levels of protein, vitamins and minerals per 100 calories to prolong nutritional supplements initiated during hospitalization. -Increased calcium and phosphorus concentrations to improve bone mineralization.

Ingredients: -D coconut syrup solids, defatted milk, lactose, concentrated whey protein, soybean oil, high oleic safflower oil, distillate palm oil (medium chain triglyceride), palm oil, potassium citrate, calcium triphosphate, carbonic acid Calcium, ascorbic acid, magnesium chloride, potassium chloride, sodium chloride, taurine, ferrous sulfate, m-inositol, choline chloride, ascorbic palmitate, L-carnitine, α-tocopheryl acetate, zinc sulfate, niacinamide, mixed tocopherol, Sodium citrate, calcium pantothenate, cupric sulfate, thiamine hydrochloride, vitamin A palmitate, β-carotene, riboflavin, pyridoxin hydrochloride, folic acid, manganese sulfate, phylloquinone, biotin, sodium selenate, vitamin D3 and cyanocobalamin.

G.Similac Natural Care Ready-to-use low-iron milk enhancer, 24 Cal / ounce: Uses: Mix with breast milk or give to infants who are underweight at birth as a substitute for breast milk.

Ingredients: -D water, defatted milk, hydrolyzed corn starch, lactose, distillate palm oil (medium chain triglyceride), concentrated whey protein, soybean oil, palm oil, calcium tertiary phosphate, potassium citrate, magnesium chloride, sodium citrate, Ascorbic acid, calcium carbonate, mono and diglyceride, soy lecithin, caraginan, choline chloride, m-inositol, taurine, niacinamide, L-carnitine, α-tocopheryl acetate, zinc sulfate, potassium chloride, calcium pantothenate, ferrous sulfate , Dercoside sulfate, riboflavin, vitamin A palmitate, thiamine hydrochloride, pyridoxin hydrochloride, biotin, folic acid, manganese sulfate, phylloquinone, vitamin D3, sodium selenate and cyanocobalamin.

The various PUFAs of the present invention can be substituted and / or added to the above infant formula and other infant formulas known in the art.

II. Nutritional products A.ENSURE® Uses: ENSURE is a low-residue liquid food whose main purpose is to be used as an oral nutritional supplement with or between meals, or in appropriate amounts as a substitute. ENSURE is a lactose-free, gluten-free diet and is suitable for use in adjusted diets, including low-cholesterol diets. It is mainly an oral supplement, but it can also be supplied by tube.

Patient status: -Patients with adjusted diet, -Elderly people with dangerous nutrition, -Patients with involuntary weight loss, -Patients recovering from illness or surgery, -Patients who need a low-residue diet.

Ingredients: -D water, sugar (sugar), maltodextrin (corn), calcium and sodium caseate, high oleic acid saflower oil, soybean protein isolate, soybean oil, canola oil, potassium citrate, tertiary phosphorus Potassium acid, sodium citrate, magnesium chloride, magnesium diphosphate, artificial flavor, sodium chloride, soy lecithin, choline chloride, ascorbic acid, caraginan, zinc sulfate, ferrous sulfate, α-tocopheryl acetate, gellan gum, niacinamide , Calcium pantothenate, manganese sulfate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, pyridoxin hydrochloride, riboflavin, folic acid, sodium molybdate, chromium chloride, biotin, potassium iodide, sodium selenate.

B.ENSURE® BARS: Uses: ENSURE BARS is a fully balanced supplement that is supplemented between meals or with meals. A delicious and nutritious substitute for other snacks. ENSURE BARS has a lactose content of &lt;1g per bottle, and the chocolate fudge brownie flavor is gluten-free. (Honey Graham Crunch flavor contains gluten.) Patient status: -Patients who need additional calories, protein, vitamins and minerals. -Especially useful for those who do not have enough heat and nutrition. -Those who have the ability to chew and swallow. -Cannot be used by people with peanut allergies or any type of nut allergy.

Ingredients: Honeygraham Crunch-High fructose corn syrup, soy protein isolate, brown sugar, honey, maltodextrin (corn), crisprice (rice flour, sugar [sucrose], salt [sodium chloride] and malt), oat wheat Wheat, partially hydrolyzed crushed oil and soybean oil, soybean polysaccharide, glycerin, concentrated whey protein, polydextrin, fructose, calcium caseate, cocoa powder, artificial flavor, canola oil, high oleic acid safflower oil, defatted milk powder, whey powder , Soy lecithin and corn oil. Manufactured in a nut processing facility.

Vitamin and Minerals: Calcium Tritrate Phosphate, Potassium Diphosphate, Magnesium Oxide, Salt (Sodium Chloride), Potassium Chloride, Ascorbic Acid, Ferrite Orthophosphate, α-tocopheryl Acetate, Niacinamide, Zinc Oxide, Calcium Pantothenate, Copper gluconate, manganese sulfate, riboflavin, β-carotene, pyridoxin hydrochloride, thiamine mononitrate, folic acid, biotin, chromium chloride, potassium iodide, sodium selenate, sodium molybdate, phylloquinone, vitamin D3 and cyanocobalamine.

Protein: Honey Graham Crunch-The protein source is a blend of soy protein isolate and milk protein. Soy protein isolate 74% Milk protein 26%.

Fat: Honey Graham Crunch-The fat source is a blend of partially hydrolyzed cottonseed oil, soybean oil, canola oil, high oleic safflower oil and soy lecithin. Partially hydrolyzed cottonseed oil and soybean oil 76% Canola oil 8% High oleic safflower oil 8% Corn oil 4% Soy lecithin 4%.

Carbohydrates: Honey Graham Crunch-Carbohydrate sources are a combination of high fructose corn syrup, brown sugar, maltodextrin, honey, crisprice, glycerin, soybean polysaccharide and oat bran. High fructose corn syrup 24% Brown sugar 21% Maltodextrin 12% Honey 11% Chris Price 9% Glycerin 9% Soy polysaccharide 7% Oat bran 7%.

C.ENSURE® HIGH PROTEIN: Uses: ENSURE HIGH PROTEIN is a concentrated high protein liquid food for those who need additional calories, protein, vitamins and minerals in their diet. It can be used as an oral nutritional supplement with or between meals, or in appropriate amounts as a substitute meal. ENSURE HIGH PROTEIN is lactose-free and gluten-free and is suitable for use by those recovering from general surgery or hip fractures and patients at risk of stress ulcers.

Patient status: -Patients who require additional calories, proteins, vitamins and minerals, such as those recovering from general surgery or hip fracture, patients at risk of stress ulcers and patients on a low cholesterol diet.

Features: -Low saturated fat, -Total fat 6g, cholesterol &lt;5mg, per serving -Rich and creamy texture, -Good protein, calcium and other essential vitamin and mineral sources, -Low cholesterol edible, -Lactose-free and easy to digest.

component: Vanilla Supreme: -D water, sugar (sugar), maltodextrin (corn), calcium and sodium caseate, high oleic acid saflower oil, soybean protein isolate, soybean oil, canola oil, potassium citrate, tertiary Potassium phosphate, sodium citrate, magnesium chloride, magnesium diphosphate, artificial flavors, sodium chloride, soy lecithin, choline chloride, ascorbic acid, caraginan, zinc sulfate, ferrous sulfate, α-tocopheryl acetate, gelangum, niacin Amido, calcium pantothenate, manganese sulfate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, pyridoxin hydrochloride, riboflavin, folic acid, sodium molybdate, chromium chloride, biotin, potassium iodide, sodium selenite, phylloquinone, vitamins D3 and cyanocobalamine.

Protein: The source of protein is a blend of casein and soybeans, two proteins of high biological value. Sodium caseinate and calcium 85% Soy protein isolate 15%.

Fat: The fat source is a blend of three oils: high oleic safflower oil, canola oil and soybean oil. High oleic safflower oil 40% Canola oil 30% 30% soybean oil.

The fat level of ENSURE HIGH PROTEIN meets the American Heart Association (AHA) guidelines. 6 g of fat in ENSURE HIGH PROTEIN corresponds to 24% of total calories, 2.6% of fat is derived from saturated fatty acids and 7.9% is derived from polyunsaturated fatty acids. These values are within the AHA guidelines of total fat-derived calorie &lt;30%, saturated fatty acid-derived calorie &lt;10%, and polyunsaturated fatty acid-derived total calorie &lt;10%.

Carbohydrates: ENSURE HIGH PROTEIN contains a combination of maltodextrin and sucrose. Mild sweetness and flavor variety (vanilla supreme, chocolate royal, wild berry and banana) with VARI-FLABORS® flavor packs of pecan, cherry, strawberry, lemon and orange to prevent flavor loss and patient compliance To encourage.

Vanilla and other non-chocolate flavors: Sucrose 60% Maltodextrin 40%.

chocolate: Sucrose 70% Maltodextrin 30%.

D.ENSURE® LIGHT Uses: ENSURE LIGHT is a low-fat liquid food intended to be used with or between meals as an oral nutritional supplement. ENSURE LIGHT is lactose-free and gluten-free and is suitable for use in adjusted diets, including low-cholesterol diets.

Patient status: -Normal or overweight patients who require additional nutrition with supplements that are 50% less fat and 20% less calorie than ENSURE. -Healthy adults who do not have a proper diet and need additional nutrition.

Features: -Low fat and low saturation fat. -Total fat 3g, cholesterol &lt;5mg per serving. -Rich and creamy texture. -A good source of calcium and other essential vitamins and minerals. -Low cholesterol edible. -Lactose-free and easy to digest.

component: French vanilla: -D water, maltodextrin (corn), sugar (sugar), calcium caseate, saflower oil with high oleate, canola oil, magnesium chloride, sodium citrate, potassium citrate, potassium diphosphate, Magnesium dibasic phosphate, natural and artificial flavors, calcium tertiary phosphate, cellulose gel, choline chloride, soy lecithin, caraginan, salt (sodium chloride), ascorbic acid, cellulose gum, ferrous sulfate, α-tocopheryl acetate, zinc sulfate , Niacinamide, manganese sulfate, calcium pantothenate, cupric sulfate, thiamine hydrochloride, vitamin A palmitate, pyridoxin hydrochloride, riboflavin, chromium chloride, folic acid, sodium molybdate, biotin, potassium iodide, sodium selenate, phylloquinone , Vitamin D3 and cyanocobalamine.

Protein: The protein source is calcium caseinate. 100% calcium caseinate.

Fat: The fat source is a blend of two oils, high oleic safflower oil and canola oil. High oleic safflower oil 70% Canola oil 30%.

ENSURE LIGHT fat levels meet American Heart Association (AHA) guidelines. 3g of fat in ENSURE LIGHT corresponds to 13.5% of total calories, 1.4% of fat is derived from saturated fatty acids and 2.6% is derived from polyunsaturated fatty acids. These values are within the AHA guidelines of total fat-derived calorie &lt;30%, saturated fatty acid-derived calorie &lt;10%, and polyunsaturated fatty acid-derived total calorie &lt;10%.

Carbohydrates: ENSURE LIGHT contains a combination of maltodextrin and sucrose. The chocolate flavor also contains corn syrup. Mild sweetness and flavor variety (French vanilla, chocolate supreme, strawberry swirl) with VARI-FLABORS® flavor packs of pecan, cherry, strawberry, lemon and orange to prevent flavor loss and promote patient compliance To do.

Vanilla and other non-chocolate flavors: Sucrose 51% Maltodextrin 49%.

chocolate: Sucrose 47.0% Corn syrup 26.5% Maltodextrin 26.5%.

Vitamins and Minerals: 8 ounces of ENSURE LIGHT per serving provides at least 25% of the RDI of 24 major vitamins and minerals.

Caffeine: Chocolate flavor contains 2.1 mg of caffeine per ounce of 8 fluids.

E.ENSURE PLUS® Uses: ENSURE PLUS is a high calorie, low residue liquid food usually used when protein concentration requires additional calorie and nutrition. Its main purpose is to use it as an oral nutritional supplement with or between meals, or as a substitute meal in appropriate amounts. ENSURE PLUS is lactose-free and gluten-free. It is mainly an oral nutritional supplement, but it can also be supplied by tube.

Patient status: -Patients who require additional calories and nutrition at normal protein concentrations while limiting the amount. -Patients who need to gain weight or maintain a healthy weight.

Features: -Rich and creamy texture, -A good source of essential vitamins and minerals.

component: Vanilla: -D water, corn syrup, maltodextrin (corn), corn oil, sodium caseteate and calcium, sugar (sugar), soybean protein isolate, magnesium chloride, potassium citrate, calcium tertiary phosphate, soybean lecithin, Natural and artificial flavors, sodium citrate, potassium chloride, choline chloride, ascorbic acid, caraginan, zinc sulfate, ferrous sulfate, α-tocopheryl acetate, niacinamide, calcium pantothenate, manganese sulfate, cupric sulfate, thiamine hydrochloride , Pyridoxin hydrochloride, riboflavin, vitamin A palmitate, folic acid, biotin, chromium chloride, sodium molybdate, potassium iodide, sodium selenate, phylloquinone, cyanocobalamine and vitamin D3.

Protein: The source of protein is a blend of casein and soybeans, two proteins of high biological value. Sodium caseinate and calcium 84% Soy protein isolate 16%.

Fat: The source of fat is corn oil. 100% corn oil.

Carbohydrates: ENSURE PLUS contains a combination of maltodextrin and sucrose. Add VARI-FLABORS® flavor packs of pecans, cherries, strawberries, lemons and oranges to a mild sweetness and flavor variety (vanilla, chocolate, strawberry, coffee, butter pecans and eggnog) to prevent flavor loss and prevent flavor loss. Facilitates patient compliance.

Vanilla, strawberry, butter pecan, and coffee flavors: Corn syrup 39% Maltodextrin 38% Sucrose 23%.

Chocolate and Eggnog Flavors: Corn syrup 36% Maltodextrin 34% 30% sucrose.

Vitamins and Minerals: 8 ounces of ENSURE PLUS per serving provides at least 15% of the RDI of 25 major vitamins and minerals.

Caffeine: Chocolate flavor contains 3.1 mg of caffeine per ounce of 8 fluids. Coffee flavors contain trace amounts of caffeine.

F.ENSURE PLUS® HN Uses: ENSURE PLUS HN is a nutritionally complete high calorie, high nitrogen liquid food for those with high calorie and protein requirements or low capacity capacity. It may be used for oral supplementation or for complete tube feeding. ENSURE PLUS HN is lactose-free and gluten-free.

Patient status: -Patients with high calorie and protein requirements, such as after surgery or injury. -Early satiety patients with low capacity.

Features: -Supplementary or complete nutrition, -Oral or tube supply, -1.5CaVmL, -High nitrogen, -High calorific value density.

component: Vanilla: -D water, maltodextrin (corn), sodium caseteate and calcium, corn oil, sugar (sucrose), soybean protein isolate, magnesium chloride, potassium citrate, calcium triphosphate, soybean lecithin, natural and artificial Flavor, sodium citrate, choline chloride, ascorbic acid, taurine, L-carnitine, zinc sulfate, ferrous sulfate, α-tocopheryl acetate, niacinamide, caraginan, calcium pantothenate, manganese sulfate, cupric sulfate, thiamine hydrochloride , Pyridoxin hydrochloride, riboflavin, vitamin A palmitate, folic acid, biotin, chromium chloride, sodium molybdate, potassium iodide, sodium selenate, phylloquinone, cyanocobalamine and vitamin D3.

G.ENSURE® POWDER Uses: ENSURE POWDER (reconstituted with water) is a low-residue liquid food intended primarily for use with or between meals as an oral nutritional supplement. ENSURE POWDER is a lactose-free, gluten-free diet and is suitable for use in adjusted diets, including low-cholesterol diets.

Patient status: -Patients with adjusted diet, -Elderly people with dangerous nutrition, -Patients recovering from illness / surgery, -Patients who need a low-residue diet.

Features: -Easy to mix, easy to mix -Low saturated fat, -Total fat 9g, cholesterol &lt;5mg, per serving -High vitamins and minerals, -Low cholesterol edible, -Lactose-free and easy to digest.

Ingredients: -D corn syrup, maltodextrin (corn), sugar (ascorbic acid), corn oil, sodium caseate and calcium, soybean protein isolate, artificial flavor, potassium citrate, magnesium chloride, sodium citrate, tertiary Calcium phosphate, potassium chloride, soybean lecithin, ascorbic acid, choline chloride, zinc sulfate, ferrous sulfate, α-tocopheryl acetate, niacinamide, calcium pantothenate, manganese sulfate, thiamine hydrochloride, cupric sulfate, pyridoxin hydrochloride, riboflavin, Vitamin A palmitate, folic acid, biotin, sodium molybdenate, chromium chloride, potassium iodide, sodium selenate, phylloquinone, vitamin D3 and cyanocobalamine.

Protein: The source of protein is a blend of casein and soybeans, two proteins of high biological value. Sodium caseinate and calcium 84% Soy protein isolate 16%.

Fat: The source of fat is corn oil. 100% corn oil.

Carbohydrates: ENSURE POWDER contains a combination of corn syrup, maltodextrin and sucrose. Add VARI-FLABORS® flavor packs of pecans, cherries, strawberries, lemons and oranges to the mild sweetness of ENSURE POWDER to prevent flavor loss and promote patient compliance.

vanilla: Corn syrup 35% Maltodextrin 35% 30% sucrose.

H.ENSURE® PUDDING Uses: ENSURE PUDDING is a concentrated nutritional supplement that provides balanced nutrition in a non-liquid form for use with or between meals. Suitable for consistency-adjusted diets (eg, soft diets, purees or whole solutions) and for people with swallowing disorders. ENSURE PUDDING is gluten-free.

Patient status: -Patients on a consistency-adjusted diet (eg, soft diet, puree or whole solution), -Patients with swallowing disorders.

Features: -Rich, creamy and good texture, -Good source of essential vitamins and minerals, -Convenient, no refrigeration required -No gluten.

Nutritional profile per 5 ounces: calorie 250, protein 10.9%, total fat 34.9%, carbohydrates 54.2%.

component: Vanilla: -D defatted milk, water, sugar (sugar), partially hydride soybean oil, modified dietary starch, magnesium sulfate, sodium stearoyl lactylate, sodium diphosphate, artificial flavor, ascorbic acid, zinc sulfate, sulfuric acid Ferrite, α-tocopheryl acetate, choline chloride, niacinamide, manganese sulfate, calcium pantothenate, FD &amp; C yellow # 5, potassium citrate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, pyridoxin hydrochloride, riboflavin, FD &amp; C Yellow # 56, folic acid, biotin, phylloquinone, vitamin D3 and cyanocobalamine.

Protein: The source of protein is skim milk. 100% skim milk.

Fat: The source of fat is hydrogenated soybean oil. 100% hydrogenated soybean oil.

Carbohydrates: ENSURE PUDDING contains a combination of sucrose and modified dietary starch. Mild sweetness and flavor variety (vanilla, chocolate, butterscotch and tapioca) to prevent flavor loss. The product contains 9.2g of lactose per serving.

Vanilla and other non-chocolate flavors: Sucrose 56% Lactose 27% Modified food starch 17%.

chocolate: Sucrose 58% Lactose 26% Modified food starch 16%.

I.ENSURE® WITH FIBER: Uses: ENSURE WITH FIBER is a nutritionally complete fiber-containing liquid food for those who have access to fortified fiber and nutrition. ENSURE WITH FIBER is suitable for those who do not need a low residue diet. It can be supplied orally or by tube, and can be used as a nutritional supplement for a normal diet or as a substitute diet in an appropriate amount. ENSURE WITH FIBER is lactose-free and gluten-free and is suitable for use in adjusted diets, including low-cholesterol diets.

Patient status: -Patients with access to fortified fiber and nutrition.

Features: -Newly improved formulations of low-saturated fats, high vitamins and minerals, -Total fat 6g, cholesterol &lt;5mg, per serving -Rich and creamy texture, -Good fiber source, -Good source of essential vitamins and minerals, -Low cholesterol edible, -Lactose-free and gluten-free.

component: Vanilla: -D water, maltodextrin (corn), sugar (zinc sulfate), sodium caseteate and calcium, oat wheat fiber, high oleic acid saflower oil, canola oil, soybean protein isolate, soybean oil, soybean fiber, Tricalcium Phosphate, Magnesium Chloride, Potassium Citrate, Cellulose Gel, Soy Lecithin, Potassium Derphate, Sodium Citrate, Natural and Artificial Flavors, Choline Chloride, Magnesium Phosphate, Ascorbic Acid, Cellulose Gum, Potassium Chloride, Caraginan, Ferrous sulfate, α-tocopheryl acetate, zinc sulfate, niacinamide, manganese sulfate, calcium pantothenate, cupric sulfate, vitamin A palmitate, thiamine hydrochloride, pyridoxin hydrochloride, riboflavin, folic acid, chromium chloride, biotin, molybdenum acid Sodium, potassium iodide, sodium citrate, phylloquinone, vitamin D3 and cyanocobalamine.

Protein: The source of protein is a blend of casein and soybeans, two proteins of high biological value. Sodium caseinate and calcium 80% Soy protein isolate 20%.

Fat: The fat source is a blend of three oils: high oleic safflower oil, canola oil and corn oil. High oleic safflower oil 40% Canola oil 40% 20% corn oil.

ENSURE WITH FIBER fat levels meet American Heart Association (AHA) guidelines. 6 g of fat in ENSURE WITH FIBER corresponds to 22% of total calories, 2.01% of fat is derived from saturated fatty acids and 6.7% is derived from polyunsaturated fatty acids. These values are within the AHA guidelines of total fat-derived calorie 30%, saturated fatty acid-derived calorie 10%, and polyunsaturated fatty acid-derived total calorie 10%.

Carbohydrates: ENSURE WITH FIBER contains a combination of maltodextrin and sucrose. Add VARI-FLABORS® flavor packs of pecans, cherries, strawberries, lemons and oranges to a mild sweetness and flavor variety (vanilla, chocolate and butter pecans) to prevent flavor loss and promote patient compliance.

Vanilla and other non-chocolate flavors: Maltodextrin 66% Sucrose 25% Oat fiber 7% 2% soy fiber.

chocolate: Maltodextrin 55% Sucrose 36% Oat fiber 7% 2% soy fiber.

Fiber: The fiber blend used in ENSURE WITH FIBER is composed of oat fiber and soy polysaccharide. The result of this blend is approximately 4 g of total dietary fiber per 8 ounce can. The ratio of insoluble fiber to soluble fiber is 95: 5.

The various nutritional supplements described above are known to those skilled in the art and can be substituted and / or supplemented with PUFAs produced according to the present invention.

J.Oxepa® nutritional product Oxepa is a low-carbohydrate, high-density calorie enteral nutritional preparation intended for dietary management of ARDS patients or patients at risk thereof. It has a special ingredient combination including a patented oil blend containing eicosapentaenoic acid (fish oil-derived EPA), γ-linolenic acid (rildisa oil-derived GLA) and a high concentration of antioxidants.

Calorie Distribution: Calorie density is high to minimize the volume required to meet energy requirements, 1.5 Cal / mL (355 Cal / 8 fl oz). Table A shows the calorific value distribution of Oxepa.

<tables num="1"><img file="JP4347397B2_D0005.tif" /></tables>

fat: -Oxepa has a fat content of 22.2 g (93.7 g / L) per 8 ounces of fluid. -The fat source is an oil blend consisting of 31.8% canola oil, 25% medium chain triglyceride (MCT), 20% volage oil, 20% fish oil, and 3.2% soy lecithin. A typical fatty acid profile of Oxepa is shown in Table B. -Oxepa provides balanced amounts of poly-saturated fatty acids, mono-saturated fatty acids and saturated fatty acids as shown in Table VI. -Medium chain triglyceride (MCT), which is equivalent to 25% of the fat blend, is absorbed into the intestinal tract without being emulsified by bile acids, making it easier to empty the stomach.

Various fatty acid components of Oxepa® nutritional products can be substituted and / or supplemented with PUFAs produced according to the present invention.

<tables num="2"><img file="JP4347397B2_D0006.tif" /></tables> Fatty acids make up about 95% of total fat.

Table C. Oxepa Fat Profile Total calorific value derived from fat% 55.2 Polyunsaturated fatty acids 31.44g / L Mono-unsaturated fatty acids 25.53g / L Saturated fatty acids 32.38g / L n-6 to n-3 ratio 1.75: 1 Cholesterol 9.49 mg / 8 fluid ounce 40.1 mg / L.

carbohydrate: -Carbohydrate content is 25.0 g (105.5 g / L) per 8 ounces of fluid. -Carbohydrate sources are maltodextrin (complex carbohydrate) 45% and sucrose (monosaccharide) 55%, both of which are easy to digest and absorb. -Oxepa is a high-fat, low-carbohydrate that minimizes carbon dioxide (CO2) production. High CO2 levels can make weaning difficult for ventilator-dependent patients. Low-carbohydrate levels may also be useful in patients with stress-induced hyperglycemia. -Oxepa is lactose-free.

The glycerol portion of dietary carbohydrates, protein-derived amino acids and fats can be converted to glucose in the body. Through this process, the carbohydrate requirements of glucose-dependent tissues (eg, central nervous system and red blood cells) are met. However, a carbohydrate-free diet can lead to ketosis, which is an over-catabolism of tissue proteins, and a decrease in water and electrolytes. These effects can be prevented by consuming 50-100 g of edible carbohydrates daily if caloric intake is adequate. Oxepa's carbohydrate levels are also sufficient to minimize gluconeogenesis if the energy requirements are met.

Protein: -Oxepa contains 14.8 g (62.5 g / L) of protein per 8 fluid ounces. -Total calorific value nitrogen ratio (150: 1) meets the requirements of stress patients. -Oxepa provides enough protein to help maintain anabolic and lean body mass without exacerbating respiratory problems. High protein intake is a problem in patients with respiratory failure. Protein has little effect on CO2 production, but a high-protein diet increases the desire for ventilation. -The protein sources of Oxepa are 86.8% sodium caseinate and 13.2% calcium caseinate. -Oxepa's protein-based amino acid profile meets or exceeds the high-quality protein standards set by the National Academy of Sciences.

* Oxepa is gluten-free.

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<figref num="1">The fatty acid biosynthesis pathway and the role of Δ5-desaturase and Δ6-desaturase in this pathway are shown.</figref><figref num="2">The nucleotide sequence encoding the Δ6-desaturase of Saprolegnia diclina (ATCC56851) (SEQ ID NO: 13) is shown.</figref><figref num="3">The amino acid sequence of Δ6-desaturase of Saprolegnia diclina (ATCC56851) (SEQ ID NO: 14) is shown.</figref><figref num="4">The nucleotide sequence encoding Δ5-desaturase of Saprolegnia diclina (ATCC56851) (SEQ ID NO: 19) is shown.</figref><figref num="5">The amino acid sequence of Δ5-desaturase of Saprolegnia diclina (ATCC56851) (SEQ ID NO: 20) is shown.</figref><figref num="6">The nucleotide sequence encoding the Δ5-desaturase of Thraustochytrium aureum (ATCC34304) (SEQ ID NO: 28) is shown.</figref><figref num="7">The amino acid sequence of Δ5-desaturase of Thraustochytrium aureum (ATCC34304) (SEQ ID NO: 29) is shown.</figref><figref num="8">The nucleotide sequence encoding Δ5-desaturase from Thraustochytrium aureum (BICC7091) (SEQ ID NO: 30) is shown.</figref><figref num="9">The translated amino acid sequence of Δ5-desaturase from Thraustochytrium aureum (BICC7091) (SEQ ID NO: 31) is shown.</figref><figref num="10">The nucleotide sequence encoding Δ6-desaturase from Thraustochytrium aureum (BICC7091) (SEQ ID NO: 32) is shown.</figref><figref num="11">The translated amino acid sequence of Δ6-desaturase from Thraustochytrium aureum (BICC7091) (SEQ ID NO: 33) is shown.</figref><figref num="12">The degree of Δ5-desaturase amino acid sequence agreement between the pRAT-2a and pRAT-2c clones is shown.</figref><figref num="13">The degree of Δ6-desaturase amino acid sequence agreement between the pRAT-1a and pRAT-1b clones is shown.</figref><figref num="14">The nucleotide sequence encoding the Δ5-desaturase gene from Isochrysis galbana CCMP1323 (SEQ ID NO: 34) is shown.</figref><figref num="15">The translated amino acid sequence from Δ5-desaturase from Isochrysis galbana CCMP1323 (SEQ ID NO: 35) is shown.</figref>