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The Benefits of Phosphatidic Acid Supplementation

The invention relates to the field of molecular biology, biochemistry and genetic engineering. Proposed a nucleic acid, characterized by a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ […]

The invention relates to the field of molecular biology, biochemistry and genetic engineering. Proposed a nucleic acid, characterized by a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 and has an activity of phosphatase phosphatidic acid corresponding protein a recombinant vector for protein expression cell and a method for producing a fatty acid composition. The invention can be used to produce polyunsaturated fatty acids in the food industry. 8 n. 1 ZP f-ly, Table 6., 7 ill., 8 pr.

Phosphatidic Acid

TECHNICAL FIELD

The present invention relates to a new phosphatidic acid phosphatase gene and use thereof.

BACKGROUND ART

Fatty acids containing two or more unsaturated bonds, collectively referred to as polyunsaturated fatty acids (PUFA), which are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. Some of these fatty acids are not synthesized in the body of an animal, and such fatty acids should be taken as a dietary essential fatty acids. Polyunsaturated fatty acids are widely distributed. For example, arachidonic acid is recovered from a lipid extracted from animal adrenal and liver. However, the data amount of polyunsaturated fatty acids contained in animal organs is small, and the amount of polyunsaturated fatty acids obtained and isolated in pure form only from animal organs is insufficient to provide them in bulk. Thus, by culturing various microorganisms bacterial methods for obtaining polyunsaturated fatty acids have been developed. In particular, it is known that microorganisms of the genus Mortierella produce lipids containing polyunsaturated fatty acids such as arachidonic acid.

Other attempts to produce polyunsaturated fatty acids were taken for plants. It is known that polyunsaturated fatty acids to form lipid storing such as triacylglycerols (also referred to as triglycerides, or TG), accumulated inside the microorganism cells or plant beans.

As lipid triacylglycerol for storing in the body is formed as follows: an acyl group is introduced into glycerol-3-phosphate using glycerol-3-phosphate acyltransferase to produce lysophosphatidic acid. The acyl group is introduced in the lysophosphatidic acid acyltransferase via lizofosfat for phosphatidic acid. Phosphatidic acid is then subjected to dephosphorylation using phosphatidic acid phosphatase to produce diacylglycerol. An acyl group is introduced via a diacylglycerol diacylglycerol acyltransferase to produce triacylglycerol.

In this pathway, phosphatidic acid (hereinafter also referred to as «PA» or 1,2-diacyl-sn-glycerol-3-phosphate) is a precursor of triacylglycerol and also a biosynthetic precursor of diacyl glycerophospholipids. In yeast cells, CDP diacylglycerol (CDP-DG) and the PA is synthesized from cytidine 5′-triphosphate (CTP) using tsitidiltransferazy phosphatidate and synthesized various phospholipids.

As described above, it is known that the biosynthesis reaction of diacylglycerol (hereinafter, also referred to as «DG») phosphatidic acid phosphatase catalyzed (EC 3.1.3.4, hereinafter also referred to as «PAP») through dephosphorylation of PA. It is known that the PAP is present in all organisms from bacteria to vertebrates.

Yeast ( Saccharomyces cerevisiae ), which are fungi possess two types of PAP (non-patent literature 1, 2 and 7). One of them is of Mg 2+ -dependent PAP (PAP1), and the other – is of Mg 2+ -independent PAP (PAP2). It is known that the gene encodes PAH1 PAP1 (Non-Patent Literature 3-5). Pah1Δ option also indicates PAP1 activity, which suggests the existence of other genes, testifying PAP1 activity. If you have a version pah1Δ, nuclear membrane and the ER membrane is abnormally expanded and expression of important genes for the biosynthesis of phospholipids abnormally enhanced (non-patent literature 6).

It is known that genes encoding PAP2, DPP1 LPP1 gene and characterize the gene most likely species PAP2 activity in yeast. The enzymes encoded by these genes have a broad substrate specificity and is also influenced by, for example, dephosphorylation of diacylglycerol pyrophosphate (DGPP), lysophosphatidic acid and isoprenoid sfingoosnovany phosphate phosphate.

It is known that the lipid producing fungi, Mortierella alpina , MaPAP1 contains a gene which is Mg 2+ -independent homologue PAP2 (Patent Literature 1).

The art knows of the existence of homologs of genes that is likely to encode a family of enzymes PAP1 or PAP2 family enzymes in other bacteria, but their function is not yet understood.

List of cited documents

patent literature

Patent Literature 1: International Publication № WO2009 / 008466

non-patent literature

Non-patent literature 1: Biochem. Biophys. Acta, 1348, 45-55, 1997

Non-patent literature 2: Trends Biochem. Sci., 31 (12), 694-699, 2006

Nonpatent literature 3: EMBO J., 24, 1931-1941, 2005

Nonpatent literature 4: J. Biol. Chem., 281 (14), 9210-9218, 2006

Nonpatent literature 5: J. Biol. Chem., 281 (45), 34537-34548, 2006

Non-patent literature 6: J. Biol. Chem., 282 (51), 37026-37035, 2007

Non-patent literature 7: J. Biol. Chem., 284 (5), 2593-2597, 2009

SUMMARY OF THE INVENTION

TECHNICAL PROBLEM

For most genes PAP, which had previously reported, however, no investigations were performed regarding the changes in the ratio of fatty acids in the composition of fatty acids produced by the host cells, the introduction of these genes into host cells and expression therein. There is a need to identify a new gene by introducing the gene into a host cell and expressing the gene product of which will be given fat composition with a fatty acid or an increased content of the specified fatty acids.

The object of the present invention is the provision of a protein or nucleic acid which allows host cells to produce a given fat composition with a fatty acid or with an increased content of fatty acids defined by protein expression in the host cells or the introduction of nucleic acid into host cells.

SOLUTION

The present inventors actively sought solution to the problems mentioned above. That is, the inventors analyzed the genome of producing lipids fungi Mortierella alpina and isolated from the genome sequences homologous to known genes of Mg 2+ -dependent phosphatidic acid phosphatase (PAP1). Moreover, for a continuous open reading frame (ORF) in the gene encoding PAP, full-length cDNA cloning was performed using cDNA library screening or PCR gene was introduced into host cells with high proliferative activity such as yeast. As a result, the inventors found that the protein encoded by the cloned cDNA has phosphatidic acid phosphatase activity, and the introduction of the cDNA into yeast reserve stocks increases lipid triacylglycerol in yeast. Thus, it has been successfully achieved cloning a gene related to a new phosphatidic acid phosphatase (PAP), and completed the present invention. Thus, the present invention is as described below.

(1) The nucleic acid according to any one of items (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatase phosphatidic acid;

(B) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein with the activity of phosphatase phosphatidic acid;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity;

(E) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and encodes a protein having phosphatidic acid phosphatase activity;

(F) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with phosphatidic acid phosphatase activity; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and comprising exon encoding a protein with the activity of phosphatase phosphatidic acid.

. (2) The nucleic acid according to (1), wherein the nucleic acid is from any one of (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatase activity phosphatidic acid;

(B) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C and encodes a protein having phosphatidic acid phosphatase activity;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity;

(E) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 in conditions of 2 × SSC at 50 ° C, and encodes a protein having phosphatidic acid phosphatase activity;

(F) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50 ° C and comprising exon encoding a protein having phosphatidic acid phosphatase activity; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and comprising exon encoding a protein with the activity of phosphatase phosphatidic acid.

(3) The nucleic acid according to any one of items (a) to (d), shown below:

(A) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 or a fragment thereof;

(B) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 or a fragment thereof;

(C) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9 or a fragment thereof; and

(D) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, or a fragment thereof.

(4) The nucleic acid according to any one of items (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having activity amplifying the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1;

(B) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having an activity of enhancing the production of DG and / or TG of PA in yeast strain deficient PAH1;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, and encodes a protein having an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(E) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and encodes a protein with an activity enhancing production DG and / or TG of PA in yeast strain deficient PAH1;

(F) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with an activity of enhancing production of DG and / or TG of PA in yeast strain deficient PAH1; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and comprising exon encoding a protein with the activity of enhancing the production of DG and / or TG of PA in yeast strain deficient PAH1.

(5) The nucleic acid according to paragraph (4), wherein the nucleic acid is any of (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having activity enhancing the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1;

(B) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C and encodes a protein with an activity enhancing production DG and / or TG of PA in yeast strain deficient PAH1;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(E) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 in conditions of 2 × SSC at 50 ° C, and encodes a protein having an activity of enhancing production of DG and / or TG of PA in yeast strain deficient PAH1;

(F) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50 ° C, and includes exon coding for a protein which has an activity of enhancing production of DG and / or TG of PA in yeast strain deficient PAH1; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein which has an activity enhancing production DG and / or TG of PA in yeast strain deficient PAH1.

(6) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase; and

(B) a protein which consists of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase.

(7) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase; and

(B) a protein which comprises the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase.

(8) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1; and

(B) a protein which consists of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing production of DG and / or TG of PA in yeast strain deficit PAH1.

(9) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing the production of diacylglycerol (DG) and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1; and

(B) a protein which comprises the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing production of DG and / or TG of PA in yeast strain deficit PAH1.

(10) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7.

(11) A recombinant vector comprising a nucleic acid according to any one of items (1) to (5).

(12) A transformant transformed with the recombinant vector according to (11).

(13) fatty acid composition comprising fatty acid or lipid obtained by culturing the transformant according to item (12).

(14) A method for producing a fatty acid composition characterized by obtaining a fatty acid from a culture or lipid obtained by culturing the transformant according to item (12).

(15) A food product comprising the fatty acid composition of the item (13).

USEFUL EFFECTS OF THE INVENTION

PAP of the present invention may enhance the ability of products of fatty acids and lipids in the reserve cells in which the introduced PAP, and preferably can intensify the production of polyunsaturated fatty acids in microorganisms and plants.

It is believed that the PAP of the present invention facilitates the production of fatty acids in the host cell, the fatty acid composition different from the composition of fatty acids produced by a host cell, which is not administered PAP. This may provide lipids with desired properties and effects, and thus is useful for use in the production of, for example, food products, cosmetics, pharmaceutical products, and soaps.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1-1 shows a comparison between the genomic sequence (SEQ ID NO: 5) and the ORF (SEQ ID NO: 1) MaPAH1.1, derived from a strain of M. alpina 1S-4.

Figure 1-2 is a continuation of figure 1-1.

Figure 1-3 is a continuation of figure 1-2.

Figure 1-4 is a continuation of figure 1-3.

Figure 2-1 shows a comparison between the genomic sequence (SEQ ID NO: 10) and ORF (SEQ ID NO: 6) MaPAH1.2, derived from a strain of M. alpina 1S-4.

Figure 2-2 is a continuation of figure 2-1.

Figure 2-3 is a continuation of figure 2-2.

Figure 2-4 is a continuation of figure 2-3.

Figure 3-1 shows the cDNA (SEQ ID NO: 4) MaPAH1.1, derived from a strain of M. alpina 1S-4 and produced on the basis of the amino acid sequence (SEQ ID NO: 2).

Figure 3-2 is a continuation of figure 3-1.

Figure 3-3 is a continuation of figure 3-2.

Figure 4-1 shows the cDNA (SEQ ID NO: 9) MaPAH1.2, derived from a strain of M. alpina 1S-4 and produced on the basis of the amino acid sequence (SEQ ID NO: 7).

Figure 4-2 is a continuation of figure 4-1.

Figure 5-1 shows a comparison of the amino acid sequence obtained (SEQ ID NO: 2) MaPAH1.1 and resulting amino acid sequence (SEQ ID NO: 7) MaPAH1.2, derived from a strain of M. alpina 1S-4, phosphatidic acid phosphatase family PAP1, ScPAH1 protein (SEQ ID NO: 19) derived from yeast, Saccharomyces cerevisiae , and Lipina amino acid sequence (SEQ ID NO: 20) derived from the mouse. The acid phosphatase phosphatidic family PAP1 N-terminal region is quite conservative and marked as Liping, conservative N-terminal region (pfam04571). In MaPAH1.1 and MaPAH1.2 N-terminal region it is also quite conservative. In this sequence, the presence of a glycine residue which are marked * (corresponding to the 80th amino acid of SEQ ID NO: 2, and the 80th amino acid of SEQ ID NO: 7), is necessary for PAP activity. Sequence, marked by double underline (corresponding to from 819th to 823 th amino acids of SEQ ID NO: 2 and from the 737 th to 741 th amino acid from SEQ ID NO: 7) represents DXDX motif (T / V), present in galogenokislot dehalogenase (HAD) -like domain. This motif is also conserved in MaPAH1.1 and MaPAH1.2. Sequences above and below the relatively motif is also conserved.

Figure 5-2 is a continuation of figure 5-1.

Figure 6-1 shows a comparison of the sequence of CDS (SEQ ID NO: 3) MaPAH1.1 sequence and CDS (SEQ ID NO: 8) MaPAH1.2, derived from a strain of M. alpina 1S-4.

Figure 6-2 is a continuation of figure 6-1.

Figure 6-3 is a continuation of 6-2 pieces.

Figure 7 shows a comparison of the amino acid sequence obtained (SEQ ID NO: 2) MaPAH1.1 derived from the amino acid sequence (SEQ ID NO: 7) MaPAH1.2, derived from a strain of M. alpina 1S-4.

DESCRIPTION OF EMBODIMENTS

The present invention relates to a new phosphatidic acid phosphatase gene derived from the genus Mortierella, where phosphatidic acid phosphatase dephosphorylates phosphatidic acid to form diacylglycerol.

According to the present invention, phosphatidic acid phosphatase is an enzyme catalyzing the reaction for the formation of diacylglycerol in dephosphorylation of phosphatidic acid. PAP substrate of the present invention is generally phosphatidic acid, but it is not limited.

Nucleic acid encoding phosphatidic acid phosphatase of the present invention

Phosphatidic acid phosphatase (PAP) in the present invention and includes MaPAH1.1 MaPAH1.2. Correspondence between cDNA, CDS and ORF, encoding and MaPAH1.1 MaPAH1.2, and between the amino acid sequences obtained are summarized in Table 1.

Table 1
MaPAH1.1 MaPAH1.2
SEQ ID NO The corresponding region in SEQ ID NO: 4 SEQ ID NO The corresponding region in SEQ ID NO: 9
cDNA SEQ ID NO: 4 ***** SEQ ID NO: 9 *****
CDS SEQ ID NO: 3 Regulations from 1 to 3985 SEQ ID NO: 8 Regulations 72 to 3791
ORF SEQ ID NO: 1 Regulations from 1 to 3982 SEQ ID NO: 6 Regulations 72 to 3788
The amino acid sequence SEQ ID NO: 2 ***** SEQ ID NO: 7 *****

Sequences MaPAH1.1 relating to the present invention include SEQ ID NO: 2, which is the amino acid sequence MaPAH1.1; SEQ ID NO: 1, which refers to a sequence region ORF MaPAH1.1; SEQ ID NO: 3, which refers to a sequence region CDS MaPAH1.1; and SEQ ID NO: 4, which is the nucleotide sequence for the cDNA MaPAH1.1. Among them, SEQ ID NO: 3 corresponds to nucleotides 1 to 3985 of SEQ ID NO: 4, whereas SEQ ID NO: 1 corresponds to nucleotides 1 to 3982 of SEQ ID NO: 4 and nucleotides 1 to 3982 of SEQ ID NO: 3. SEQ ID NO: 5 is the genomic nucleotide sequence encoding MaPAH1.1 the present invention. The genomic sequence of SEQ ID NO: 5 consists of eleven ten exons and introns. In SEQ ID NO: 5 exons region corresponds to nucleotides 1 to 182, from 370 to 584, from 690 to 1435, from 1536 to 1856, from 1946 to 2192, from 2292 to 2403, from 2490 to 2763, from 2847 to 3077, from 3166 to 3555, from 3648 to 3862 and from 3981 to 5034.

Sequences MaPAH1.2 relating to the present invention include SEQ ID NO: 7, which is an amino acid sequence MaPAH1.2; SEQ ID NO: 6, which is a sequence of the ORF MaPAH1.2; SEQ ID NO: 8, which is a sequence of the CDS MaPAH1.2, and SEQ ID NO: 9, which is the nucleotide sequence of the cDNA MaPAH1.2. Among them, SEQ ID NO: 8 corresponds to nucleotides 72-3791 SEQ ID NO: 9, whereas SEQ ID NO: 6 correspond to nucleotides 72 to 3788 of SEQ ID NO: 9 and between nucleotides 1 to 3717 of SEQ ID NO: 8. SEQ ID NO: 10 is a genomic nucleotide sequence encoding MaPAH1.2 the present invention. The genomic sequence of SEQ ID NO: 10 consists of eight exons and seven introns. In SEQ ID NO: 10 region with exons corresponds to nucleotides 1 to 454, from 674 to 1006, from 1145 to 1390, from 1479 to 1583, from 1662 to 1804, from 1905 to 2143, from 2243 to 3409 and from 3520 to 4552 .

Nucleic acids of the invention include single and double stranded DNA and RNA complementary to them, which can be either natural or artificially prepared. Examples of the DNA include, but are not limited to, genomic DNAs, cDNAs corresponding to genomic DNA, chemically synthesized DNA, PCR amplified DNA, combinations thereof and DNA / RNA hybrids.

Preferred embodiments for the nucleic acids of the invention comprise (a) nucleic acids comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, (b) nucleic acids comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, (c) nucleic acids having a nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9, and (d) nucleic acid comprising the nucleotide sequence recited in SEQ ID NO: 5 or SEQ ID NO: 10.

For these nucleotide sequences can be used on the nucleotide sequence data for EST or genomic DNA from organisms PAP activity to search a nucleotide sequence encoding a protein that is very similar to known proteins having activity PAP. Preferred organisms having activity PAP, lipids are producing fungi including, but not limited to, M. alpina .

To analyze EST cDNA library is first prepared. The cDNA library may be prepared, guided «Molecular Cloning, A Laboratory Manual 3rd ed.» (Cold Spring Harbor Press ( 2001)). Alternatively, a commercially available kit may be used to obtain a cDNA library. Examples of the method for preparing cDNA libraries suitable for the present invention are shown below. That is, a suitable strain of M. alpina , fungi producing lipids plated on appropriate medium and pre-cultured for an appropriate period of time. Culture conditions suitable for said preculture are, for example, medium composition of 1.8% glucose, 1% yeast extract and pH 6.0, the cultivation time is 3 to 4 days, and the cultivation temperature 28 ° C. Products preculture is then cultured under suitable basic conditions. Medium composition suitable for main culture includes, for example, 1.8% glucose, 1% soybean powder, 0.1% olive oil, 0.01% Adekanol, 0.3% KH 2 PO 4 , 0,1% Na 2 SO 4 , 0,05% CaCl 2 · 2H 2 O and 0,05% MgCl 2 · 6H 2 O and pH 6,0. Culture conditions suitable for main culture are, for example, shaking culture and aeration at 300 rev / min, 1 vvm and 26 ° C for 8 days. The appropriate amount of glucose may be added during culture. After culturing, the product is taken up in suitable times during main culture, from cells harvested for total RNA. Total RNA can be prepared by any known method such as the method using guanidine hydrochloride / CsCl. From the resultant total RNA, poly (A) + RNA can be isolated using a commercially available kit, and a cDNA library can be prepared using a commercially available kit. The nucleotide sequence of all clones obtained from the cDNA library was determined using primers designed on the basis of the vector to determine the nucleotide sequence of the insert. As a result, one can obtain EST. For example, when using a set ZAP-cDNA GigapackIII Gold Cloning Kit ( Stratagene Inc.) to obtain a cDNA library may direct cloning.

In the analysis of genomic DNA of an organism is cultured cells, having an activity PAP, and genomic DNA was prepared from the cells. Determine the nucleotide sequence of the obtained genomic DNA, and the assembly is subjected to the nucleotide sequence determined. The result obtained in the search performed superkontiga sequence sequence encoding an amino acid sequence having high homology with the amino acid sequence of a known protein PAP activity. From the sequence superkontiga being found in the search as a sequence encoding an amino acid sequence prepared primers. PCR was performed using the cDNA library as a template, and the resulting DNA fragment was introduced into plasmid cloning. For sample PCR using the cloned plasmid as a template and the above primers. Using these samples, cDNA library screening was performed.

Homology search between amino acid sequences derived from and MaPAH1.1 MaPAH1.2 the present invention was performed using BLASTp program concerning the amino acid sequences reported in GenBank. Data derived from the amino acid sequence MaPAH1.1 MaPAH1.2 and have the advantage of an alleged nuclear protein elongation and deformation (AAW42851), derived from the Cryptococcus neoformans var. neoformans JEC21, with the highest levels, and identity was 25.9% and 26.6%, respectively. MaPAH1.1 obtained and the amino acid sequences of the present invention MaPAH1.2 22.7% identical and 22.5%, respectively, with the amino acid sequence PAH1, derived from S. cerevisiae (to throughout the specification, also referred to as PAH1 yeast or ScPAH1 ), which was functionally analyzed among homologues PAP1 fungi.

The present invention also includes nucleic acids that are functionally equivalent to the nucleic acid including the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6 (hereinafter also referred to as “the nucleotide sequence of the present invention”) or nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 (hereinafter also referred to as “amino acid sequence of the present invention”). The term “functionally equivalent” means that the protein encoded by the nucleotide sequence of the present invention and a protein consisting of the amino acid sequence of the present invention have an activity of phosphatidic acid phosphatase (PAP). Furthermore, the term “functionally equivalent” means an activity which enhances the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a strain of yeast PAH1 deficient in expression of the protein encoded by the nucleotide sequence of the present invention, or protein consisting of the amino acid sequence of the present invention. Activity of the protein of the present invention against PAP and activity, contributing to enhancing production DG and / or TG of PA in yeast strain deficient PAH1, may be Mg 2+ -dependent or Mg 2+ -independent. Activity of the protein of the present invention is preferably Mg 2+ -dependent.

Certain nucleic acids that are functionally equivalent in relation to the nucleic acids of the invention include nucleic acids comprising nucleotide sequences set forth below in any one of (a) to (g). It should be noted that in the descriptions of the nucleotide sequences listed below, the term “activity of the present invention” refers to “PAP activity and / or activity, which enhances the production of DG and / or TG of PA in yeast strain deficient PAH1».

(A) A nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of the present invention.

The nucleotide sequence contained in a nucleic acid of the present invention relates to nucleotide sequences encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO 7, and has an activity of the present invention.

In particular, the nucleotide sequence contained in a nucleic acid of the present invention is a nucleotide sequence encoding a protein having the above activity of the present invention and consisting of:

(I) an amino acid sequence with deletion of one or more (preferably one to several (e.g., 1 to 400, 1 to 200, 1 to 130, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(Ii) an amino acid sequence with substitution of one or more (preferably one to several (e.g., 1 to 400, 1 to 200, 1 to 130, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(Iii) an amino acid sequence with addition of one or more (preferably one to several (e.g., 1 to 400, 1 to 200, 1 to 130, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; or

(Iv) the amino acid sequence of any combination of (i) to (iii), described above.

Among the above, it is preferably conservative substitutions, which means the replacement of specific amino acid residue by another residue having similar physical and chemical properties. It can be any substitution that does not substantially change the structural properties of the original sequences. For example, any substitution is possible provided that substitutable amino acids disrupt helical structure is not the original sequence or do not disrupt any other type of secondary structure characterizing the original sequence.

Conservative substitution is generally introduced by synthesis using biological systems or chemical peptide synthesis, preferably by chemical peptide synthesis. In such cases, the substituent group may include an artificial amino acid residue, peptide mimetic or reversed or inverted form, where the region without changing the amino acid sequence or inverted faces.

Non-limiting examples of interchangeable amino acid residues arranged and are listed below:

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-metilserin, t-butylglycine, t-butyl alanine, and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, izoasparaginovaya acid izoglutaminovaya acid, 2-aminoadipic acid and 2-aminosuberinovaya acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline and 4-hydroxyproline;

Group F: serine, threonine and homoserine; and

Group G: phenylalanine and tyrosine.

When non-conservative substitutions possible substitution of one element of the above class member from another class. In this case, in order to maintain the biological function of the protein of the present invention preferably consider indices of hydrophobicity of amino acids (amino acid hydrophobicity index) (Kyte, et al, J. Mol Biol, 157:… 105-131 (1982)).

In the case of a non-conservative substitution, amino acid substitutions can be performed on the basis of hydrophilicity.

It should be noted that as in a conservative substitution, and at a non-conservative substitution, amino acid residue corresponding to the 80th amino acid in SEQ ID NO: 2 or SEQ ID NO: 7, preferably glycine, and an area corresponding to amino acids 819 to 823 of SEQ ID NO: 2 or from amino acids 737 to 741 of SEQ ID NO: 7, preferably DXDX (T / V) (X is any amino acid).

Throughout the description and figures nucleotides, amino acids and their abbreviations are in accordance Commission on Biochemical Nomenclature IUPAC-IUB, or any other standard nomenclature used in the art, such as described in Immunology – A Synthesis (second edition, edited by ES Golub and DR Gren, Sinauer Associates, Sunderland, Massachusetts (1991)). Also, it assumes that amino acids that may have optical isomers are represented as L-isomers unless otherwise indicated.

Stereoisomers such as D-amino acids are amino acids mentioned above, synthetic amino acids such as α, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be members constituting the proteins of the present invention.

It should be noted that in the protein notation throughout the description, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard practice, and the symbol in the art.

Similarly, in the main, if not stated otherwise, left-hand end of single-stranded polynucleotide sequences is the 5′-end and left-hand direction of double- stranded polynucleotide sequences is referred to as the 5′-direction.

Those skilled in the art competent in the field of design and preparation of suitable mutant proteins as described herein, using methods known in the art. For example, the region in the protein molecule that is suitable to change the structure of the protein without altering the biological activity of the present invention, can be identified by detecting an area that appears to be less important for the biological activity of the protein. You can also identify residues or regions that are conserved between similar proteins. Furthermore it is also possible to introduce conservative amino acid substitution in a region, which appears to be important for biological activity or structure of the protein of the present invention, the protein without altering the biological activity and without adversely affecting the polypeptide structure of the protein.

In particular, the amino acid sequences and MaPAH1.1 MaPAH1.2, the amino acid sequences of approximately 100 amino acids in the N-terminal region, referred to as lipin, N-terminal conserved region: pfam04571) against enzyme family Mg 2+ -dependent phosphatase phosphatidic acid (PAP1), is quite conservative. In addition, each of the amino acid sequences and MaPAH1.1 MaPAH1.2 has “motive DXDX catalytic site (T / V)», which is a conservative motif galogenokislot dehalogenase (HAD) -like protein superfamily of enzymes. Figure 5 correspond to data based DIDGT sequence (corresponding to residues from 819 to 823 of SEQ ID NO: 2 and residues from 737 to 741 of SEQ ID NO: 7), marked by double underline. As a mutant of the present invention may be any mutant, which retains the conserved motif and maintains the activity described above. Reported that changes in site conserved motif in the yeast PAP1 lead to loss of PAP activity (J. Biol Chem, 282 (51):.. 37026-37035, (2007)).

Those skilled in the art could implement the so-called structural and functional studies, which allow to identify residues of the peptide, which are important for biological activity or structure of the protein of the present invention and the residues of the peptide similar to the residues in a protein will allow for a comparison of the amino acid residues between the two these peptides and thus predict which the residue in the protein similar to the protein of the present invention, an amino acid residue corresponding to amino acid residue important for biological activity or structure. Furthermore, by selecting an amino acid substitution is chemically similar to the predicted amino acid residue of the mutant can be selected which maintains the biological activity of the protein of the present invention. Similarly, those skilled in the art could also analyze the three-dimensional structure and amino acid sequence of the mutant protein. Thus, the analysis results can then be used to predict the alignment of amino acid residues included in the spatial structure of the protein. Because amino acid residues, which was predicted by the presence on the protein surface may be involved in important interactions with other molecules, those skilled in the art could obtain a mutant in which there is no replacement of the data of amino acid residues, which was predicted by the presence on the protein surface on the basis of analysis results as mentioned above. Moreover, those skilled in the art could obtain mutants with single amino acid replacement at any of amino acid residues that form the protein of the present invention. These mutants can be selected by any of the known assays for collecting information about the individual mutants, which in turn allows to assess the practical value of individual amino acid residues that form the protein of the present invention by comparing the case where the mutant with the replacement of specific amino acid residue exhibited a lower bioactivity than the biological activity of the protein of the present invention, the case where the mutant shows no biological activity, or where the mutant exhibits an unacceptable activity which leads to inhibition of the biological activity of the protein of the present invention. Furthermore, on the basis of the information received after the specific routine experimentation or in combination with other mutations, those skilled in the art may readily analyze amino acid substitutions, which are undesirable for mutants of the protein of the present invention.

As described above, a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 can be prepared by methods such as site-directed mutagenesis as described for example in «. Molecular Cloning, a Laboratory Manual 3rd ed» (Cold Spring Harbor Press ( 2001)); «Current Protocols in Molecular Biology» ( John Wiley & Sons (1987-1997); Kunkel, ( 1985), Proc Natl Acad Sci USA, 82: 488-92; and Kunkel, (1988), Method Enzymol…. . ., 85: 2763-6 Preparation of the mutant with a mutation comprising a deletion, substitution or addition of amino acids can be carried out, for example, using known methods such as Kunkel method or by a method with the introduction of gaps in the DNA double chain, a method using a set of to introduce mutations by site-directed mutagenesis, such as set QuikChange TM site-directed mutagenesis Kit (Stratagene production) systems for site-directed mutagenesis GeneTailor TM (Invitrogen production) or system for site-directed mutagenesis TaKaRa (e.g., Mutan-K, Mutan-Super Express Km; Takara Bio Inc. production).

Methods allowing to carry out deletion, substitution or addition of one or more amino acids in the amino acid sequence of the protein while maintaining its activity include, in addition to the above site-directed mutagenesis, a method of gene mutation treatment and a method for the selective cleavage of the gene and deletion, substitution or addition of selected nucleotide and then ligating gene.

The nucleotide sequence contained in the nucleic acid of the present invention is preferably a nucleotide sequence which encodes a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and having PAP activity.

The nucleotide sequence contained in the nucleic acid of the present invention preferably comprises a nucleotide sequence which encodes a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO : 7, and having an activity of the present invention.

The number of sites with mutations leading to amino acid replacement or modifications in the protein of the present invention are not limited provided that maintain the activity of PAP activity or enhancing production of DG and / or TG of PA in yeast strain deficient PAH1.

PAP activity or the activity-enhancing production DG and / or TG of PA in yeast strain deficient PAH1, can be evaluated by known methods, e.g., see J. Biol. Chem., 273, 14331-14338 (1998).

For example, “Activity PAP» of the present invention can be measured as described below: The source of the enzyme solution was prepared by destruction of the transformed cells expressing the PAP of the present invention, centrifuging and collecting the lysate supernatant. The resulting original enzyme solution can be subjected to further purification of PAP of the present invention. The source of the enzyme solution containing the present izobreteniyuili PAP PAP purified according to the present invention is added to the reaction solution containing 0.5 mM phosphatidic acid, 10 mM 2-mercaptoethanol and 50 mM Tris-HCl (pH 7,5), followed by carrying out the reaction at from 25 ° C to 28 ° C for a suitable time. The reaction was stopped by adding a mixture of chloroform and methanol, and isolated lipids. The resulting lipids are fractionated by thin layer chromatography to measure the amount of diacylglycerol obtained.

“Activity-enhancing production DG and / or TG of PA in yeast strain deficient PAH1» may be measured, for example as described below: a yeast strain deficient PAH1 obtained by destroying the gene of yeast ( S. cerevisiae ) ScPAH1. PAH1 strain deficient yeast as a host cell transformed with a vector comprising a nucleic acid encoding the PAP of the present invention and culturing the transformed strain is performed. Culture solution was centrifuged to collect cells. Cells are washed with water and lyophilized. To the dried cells were added chloroform and methanol, and destroy the cells using glass beads to isolate lipids. The isolated lipids are fractionated by thin layer chromatography and the amount of generated DG and / or TG. Yeast strain deficient PAH1, transformed with a vector not containing the nucleic acid encoding the PAP of the present invention is used as a control for comparison. If the amount of generated DG and / or TG increases in yeast strain deficient PAH1, transformed with a vector containing nucleic acid encoding the PAP of the present invention, the PAP is defined as PAP with the “activity-enhancing production DG and / or TG of PA in yeast strain deficit PAH1 ».

(B) nucleic acid which comprises a nucleotide sequence capable of hybridizing with the nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions, and encodes a protein having an activity of of the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to a nucleotide sequence that can hybridize with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions, and encodes a protein having activity according to the present invention.

This nucleotide sequence can be prepared, for example, using cDNA or genomic library by known hybridization methods such as colony hybridization, plaque hybridization or Southern blotting using samples obtained from an appropriate fragment in a manner known to those skilled in the art.

Detailed hybridization method mentioned in «Molecular Cloning, A Laboratory Manual 3rd ed.» (Cold Spring Harbor Press (2001), particularly Chapters 6 and 7), «Current Protocols in Molecular Biology» (John Wiley & Sons (1987-1997 ), in particular chapters 6.3 and 6.4) and «DNA Cloning 1:. Core Techniques, a Practical Approach 2nd ed» (Oxford University (1995), particularly chapter 2.10 for hybridization conditions).

The stringency of hybridization is determined mainly based on the hybridization conditions, more preferably under conditions of hybridization and washing conditions. The term “stringent conditions” as used throughout the specification is intended to include moderately or highly stringent conditions.

Specifically, examples of moderately stringent conditions include hybridization conditions of 1 × SSC to 6 × SSC at 42 ° C to 55 ° C, more preferably 1 × SSC to 3 × SSC at 45 ° C to 50 ° C, and most preferably 2 × SSC at 50 ° C. In the case of using a hybridization solution containing, for example, approximately 50% formamide, hybridization temperature used of from 5 ° C to 15 ° C lower than the temperature indicated above. washing conditions, for example, 0.5 × SSC to 6 × SSC at 40 ° C to 60 ° C. To a solution hybridization and washing solution to be added is usually from 0.05% to 0,2% SDS, preferably about 0,1% SDS.

Highly stringent (stringent conditions) include hybridization and / or washing at high temperature and / or lower salt concentration as compared to the moderately stringent conditions. Examples of the hybridization conditions comprise 0.1 × SSC to 2 × SSC at 55 ° C to 65 ° C, more preferably from 0.1 × SSC to 1 × SSC at 60 ° C to 65 ° C and most preferably 0, 2 × SSC at 63 ° C. wash conditions include, for example, 0.2 × SSC to 2 × SSC at 50 ° C to 68 ° C, and more preferably 0.2 × SSC at 60 ° C to 65 ° C.

Examples of hybridization conditions, in particular, used in the present invention include, but are not limited to, pre-hybridization in 5 × SSC, 1% SDS, 50 mM Tris-HCl (pH 7,5) and 50% formamide, incubated at 42 ° C , incubated overnight at 42 ° C in the presence of a probe to form hybrids, and washing three times in 0,2 × SSC, 0,1% SDS at 65 ° C for 20 minutes.

It is also possible to use a commercially available hybridization kit in which the radioactive compound is not used as a probe. In particular, it is used for hybridization, for example, a set of DIG nucleic acid detection kit (Roche Diagnostics) or ECL direct labeling & detection system (production Amersham).

Preferable examples of the nucleotide sequences included in the present invention include nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C, and encoding a protein having PAP activity.

(C) A nucleic acid comprising a nucleotide sequence which consists of the nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having the activity of the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to nucleotide sequences that consist of a nucleotide sequence of at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, and encode a protein having the activity of the present invention.

Preferably, such that the nucleic acid comprises a nucleotide sequence identical to at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98% or 99% or more) of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having activity according to the present invention.

The percentage similarity between two nucleotide sequences can be determined by visual evaluation and the mathematical calculation, but preferably determined by comparing sequence information of two nucleic acids, using a computer program. As computer programs for sequence comparison can be used, for example, the BLASTN program (Altschul et al, (1990), J. Mol Biol, 215:… 403-10), version 2.2.7, available through the National Library of Medicine, the web website: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html, or algorithm WU-BLAST 2.0. Standard default settings for WU-BLAST 2.0 are described at the specified website: http://blast.wustl.edu.

(D) nucleic acid comprising a nucleotide sequence that encodes an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and encodes a protein having activity according to the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to nucleotide sequences encoding the amino acid sequence 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and encodes a protein having an activity of the present invention. As the protein encoded by the nucleic acid of the present invention may be a protein having the amino acid sequence similarity with the amino acid sequences or MaPAH1.1 MaPAH1.2 provided that the protein is functionally equivalent protein having activity according to the present invention.

Specific protein examples include amino acid sequences with 75% or more, preferably 80% or more, more preferably 85% or more, and most preferably 90% or more (e.g., 95% or more, furthermore 98% or more) identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7.

The nucleotide sequence contained in the nucleic acid of the present invention is preferably a nucleotide sequence encoding the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and encoding a protein having activity present invention. More preferably, the nucleotide sequence encodes an amino acid sequence 95% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and encodes a protein having activity according to the present invention.

The percentage similarity between two amino acid sequences can be determined by visual evaluation and mathematical calculation, or can be determined using a computer program. Examples of such a computer program include BLAST, FASTA (Altschul et al, J. Mol Biol, 215:… 403-410 (1990)) and ClustalW. In particular, various conditions (parameters) for the similarity search using the BLAST program described by Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available via the website of the National Center for Biotechnology Information (NCBI) in the US or Japan-based DNA database (DDBJ) (Guide to BLAST, Altschul et al ., NCB / NLM / NIH Bethesda, MD 20894; Altschul et al).. Also you can use a program such as software GENETYX Ver. 7 (Genetyx Corporation), DINASIS Pro (Hitachisoft) or Vector NTI (Infomax) for the processing of genetic information to determine percent identity.

Specific alignment algorithm to align multiple amino acid sequences can also display the matching sequences in specific areas of short and thus can detect a region with very high sequence similarity to these short areas, even if between the full length sequences have significant similarity. Additionally, BLAST algorithm may use a matrix substitutions BLOSUM62 amino acids, and following separation parameters can be used: (A) inclusion filter to mask segments of the analyzed sequence having low compositional complexity (as defined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993) ; also see Wootton and Federhen, 1996, «Analysis of compositionally biased regions in sequence databases», Methods Enzymol, 266: 554-71) or for masking segments, consisting of internal repetitions with low recurrence (as defined by the program XNU Claverie and. States (Computers and Chemistry, 1993), and (B) the threshold of statistical significance for identifying correspondences regarding the sequences from the database, or expected compliance probability, found only at random, according to the statistical model E-score (Karlin and Altschul, 1990); if statistical significance is attributed to more than a predetermined threshold for the E-score, the match is not observed.

(E) nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, with stringent conditions, and encodes a protein having activity according to the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention covers nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under stringent conditions, and encoding a protein having activity according to the present invention.

A protein consisting of the amino acid sequence presented in SEQ ID NO: 2 or SEQ ID NO: 7, and the hybridization conditions described above. Examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under stringent conditions, and encoding a protein having activity according to the present invention.

(F) A nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions, and includes exon coding a protein having activity according to the present invention.

The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10 is the genomic DNA sequences encoding MaPAH1.1 MaPAH1.2 and the present invention respectively.

The nucleotide sequence contained in the nucleic acid of the present invention covers nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions, and includes exon coding for a protein having the activity of the present invention.

This nucleotide sequence can be prepared by methods known to those skilled in the art, e.g., from genomic libraries by known hybridization method such as colony hybridization, plaque hybridization or Southern blotting using probes derived from a suitable fragment. Hybridization conditions are described above.

(G) nucleic acid comprising a nucleotide sequence which consists of the nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein having the activity of the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to nucleotide sequences that consist of a nucleotide sequence identity of at least 70% with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and It encodes a protein having activity according to the present invention. Preferred examples of nucleotide sequences include sequences having a similarity of at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98 % or 99% or more) with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 and containing an exon encoding a protein having the activity of the present invention. The percentage similarity between two nucleotide sequences can be determined as described above.

The sequence of the genomic DNA of SEQ ID NO: 5 consists of eleven ten exons and introns. In SEQ ID NO: 5 regions with exons correspond to nucleotides 1 to 182, from 370 to 584, from 690 to 1435, from 1536 to 1856, from 1946 to 2192, from 2292 to 2403, from 2490 to 2763, from 2847 to 3077 from 3166 to 3555, from 3648 to 3862 and from 3981 to 5034. The sequence of the genomic DNA of SEQ ID NO: 10 consists of eight exons and seven introns. In SEQ ID NO: 10 region with exons correspond to nucleotides 1 to 454, from 674 to 1006, from 1145 to 1390, from 1479 to 1583, from 1662 to 1804, from 1905 to 2143, from 2243 to 3409 and from 3520 to 4552 .

In another embodiment, examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences containing regions with introns from the nucleotide sequence are 100% identical to the genomic DNA sequence represented in SEQ ID NO: 5 or SEQ ID NO: 10, and regions with exons have a nucleotide sequence identical to at least 70% or more, more preferably 75% or more and more preferably 80% or more (e.g., 85% or more, more preferably 90% or and most preferably more than 95%, 98% or 99% or more) of the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, where exon encodes a protein having activity according to the present invention.

In another embodiment, examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences containing regions with exons from the nucleotide sequence are 100% identical to the genomic DNA sequence represented in SEQ ID NO: 5 or SEQ ID NO: 10, and regions with introns from the nucleotide sequence identical to at least 70% or more, more preferably 75% or more and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more and most preferably 95%, 98% or 99% or more) of the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, wherein the area with the introns can be cut by the splicing, and thus the area with exons ligated, encoding a protein having activity according to the present invention.

In another embodiment, examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences containing regions with introns from the nucleotide sequence identical to at least 70% or more, more preferably 75% or more, and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98% or 99% or more) of genomic DNA sequences presented in SEQ ID NO: 5 or SEQ ID NO: 10 and a region to exon with the nucleotide sequence identical to at least 70% or more, more preferably 75% or more and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95% or more, 98% or more or 99% or more) of the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, wherein the area with the introns can be cut by splicing, and thus the area with ligated exons, encoding a protein having activity according to the present invention.

The percentage similarity between two nucleotide sequences can be determined in the manner described above.

A nucleic acid of the present invention relates to nucleic acids, each of which consists of the nucleotide sequence with deletion, substitution or addition of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having activity of the present invention. More specifically, the nucleic acid for use include any one of the following nucleotide sequences:

(I) a nucleotide sequence with deletion of one or more (preferably one to several (e.g., 1 to 1200, 1 to 1000, 1 to 750, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(Ii) a nucleotide sequence with the replacement of one or more (preferably one to several (e.g., 1 to 1200, 1 to 1000, 1 to 750, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(Iii) a nucleotide sequence with addition of one or more (preferably one to several (e.g., 1 to 1200, 1 to 1000, 1 to 750, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6; or

(Iv) a nucleotide sequence with any combination of from (i) to (iii) above, wherein the nucleotide sequence encodes a protein having activity according to the present invention.

A preferred embodiment of the nucleic acid of the present invention also includes nucleic acid fragment comprising a nucleotide sequence shown in any one of (a) to (d), shown below:

(A) the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(B) a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(C) the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9; and

(D) the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

(A) The nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, (b) a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and ( c) the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9 are provided as shown in table 1. The nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, as described above. Fragments of these sequences are ORF, CDS, a biologically active region, a region for use as a primer, as described below, and the region that can serve as a probe contained in the data of the nucleotide sequences, and may be of natural origin or artificially obtained.

A nucleic acid of the present invention relates to nucleic acids listed below.

(1) The nucleic acids represented by any one of (a) to (g) below:

(A) nucleic acids comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(B) nucleic acids which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions;

(C) nucleic acid comprising a nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(D) nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(E) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under stringent conditions;

(F) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions; and

(G) nucleic acid comprising a nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

(2) The nucleic acids described in (1) above, represented by any of (a) to (g) below:

(A) nucleic acids comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(B) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C;

(C) nucleic acid comprising a nucleotide sequence 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(D) nucleic acid comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(E) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under conditions of 2 × SSC at 50 ° C;

(F) nucleic acid to be hybridized with a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50 ° C; and

(G) nucleic acid comprising a nucleotide sequence 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

Phosphatidic acid phosphatase of the present invention

The protein of the present invention includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and proteins functionally equivalent to this protein. These proteins may be naturally occurring or artificially prepared. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, described above. The term “functionally equivalent proteins” refers to proteins having “the activity of the present invention” described in the above “nucleic acid encoding phosphatidic acid phosphatase of the present invention”.

In the present invention, examples of proteins which are functionally equivalent to a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, include, proteins presented below in (a) and (b):

(A) protein consisting of the amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of the present invention; and

(B) proteins comprising the amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of the present invention.

In the above, the amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 or an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2, described above in “nucleic acid encoding phosphatidic acid phosphatase of the present invention”. “A protein having the activity of the present invention” includes mutant proteins encoded by nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6; proteins with mutations caused by different types of modifications such as deletion, replacement and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; these modified proteins have, for example, modified amino acid side chains; and these proteins fused to other proteins, where the proteins have PAP activity and / or activity which enhances the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1.

The protein of the present invention may be prepared synthetically. In this case, the protein can be produced by chemical synthesis method such as Fmoc (method a fluorenilmetiloksikarbonilom) method or tBoc (tert-method butyloxycarbonyl). Furthermore, chemical synthesis can be used peptide synthesizers available from Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation or other manufacturers.

The protein of the present invention further includes the following proteins:

(1) (a) protein consisting of the amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(B) proteins comprising the amino acid sequence with 80% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; and

(2) the protein according to any one of paragraphs (a) and (b) below:

(A) protein consisting of the amino acid sequence with deletion, substitution or addition of 1 to 200 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; and

(B) protein consisting of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7.

Cloning of nucleic acids of the invention

PAP nucleic acid of the present invention can be cloned, for example using screening cDNA libraries using appropriate probes. Cloning can be performed by amplification by PCR using appropriate primers followed by ligation into a suitable vector. The cloned nucleic acid may further be subcloned into another vector.

It is possible to use commercially available plasmid vectors such as Script-pBlue TM SK (+) (Stratagene), pGEM-T (Promega), pAmp (TM: Gibco-BRL), p-Direct (Clontech) and pCR2.1-TOPO ( Invitrogen). When amplification by PCR, as primers can be any part of, for example, of the nucleotide sequence shown in SEQ ID NO: 4. For example, as the forward primer may be used NotI-PAH1-1-F: 5′- GCGGCCGCATGCAGTCCGTGGGAAG- 3 ‘(SEQ ID NO: 15) and as the reverse primer can be used MaPAH1-1-10R: 5′-TTCTTGAGTAGCTGCTGTTGTTCG-3’ (SEQ ID NO: 16). PCR was then performed using cDNA obtained from cells M. alpina , with the above primers, DNA polymerase, and any other compounds. While those skilled in the art can easily carry out the method according to, for example, «Molecular Cloning, A Laboratory Manual 3rd ed.» (Cold Spring Harbor Press (2001)), PCR conditions in the present invention may be, for example, described below.

Denaturation temperature from 90 ° C to 95 ° C.

annealing temperature: 40 ° C to 60 ° C.

Elongation temperature: from 60 ° C to 75 ° C.

Number of cycles: 10 or more cycles.

The resulting PCR products can be purified by known methods, for example using a set such as a set of GENECLEAN (Funakoshi Co., Ltd.), QIAquick PCR purification (QIAGEN) or ExoSAP-IT (GE Healthcare Bio-Sciences)); filter of cellulose or DEAE-tubes for dialysis. In the case of using an agarose gel after PCR product was subjected to agarose gel electrophoresis and nucleotide sequence fragments are excised from the agarose gel and purified, for example, using a set of GENECLEAN (Funakoshi Co., Ltd.), or set QIAquick Gel extraction (QIAGEN) or using the freeze-squeeze method.

The nucleotide sequence of the cloned nucleic acids may be determined by sequencing nucleotides.

Construction of expression vector for PAP and obtain transformants

The present invention also relates to a recombinant vector comprising a nucleic acid encoding the PAP of the present invention. The present invention further relates to a transformant transformed using this recombinant vector.

Recombinant vector and transformant can be obtained as follows: a plasmid with the nucleic acid encoding the PAP of the present invention, digested with a restriction enzyme. Examples of restriction enzymes include, but are not limited to, EcoRI, KpnI, BamHI and SalI. The end may be blunt using polymerase T4. The digested DNA fragment was isolated by agarose gel electrophoresis. This DNA fragment was inserted in known manner into an expression vector for the expression vector for PAP. This expression vector is introduced into a host cell to obtain a transformant for expression of the desired protein.

In this case, the kind of the expression vector and the host can be any type that allows to express the desired protein. Examples of a host include fungi, bacteria, plants, animals and their cells. Examples of fungi include filamentous fungi such as lipid-producing M. alpina , and yeast strains such as Saccharomyces cerevisiae . Examples of bacteria include Escherichia coli and Bacillus subtilis . Additional examples of plants include oil plants such as rapeseed, soybean, cotton, safflower and flax.

It is possible to use microorganisms that produce lipids, for example strains described in MYCOTAXON, Vol. XLIV, NO. 2, pp. 257-265 (1992), and specific examples thereof include microorganisms belonging to the genus Mortierella, such as microorganisms belonging to the subgenus Mortierella, such as Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina IFO8568, ATCC16266, ATCC32221, ATCC42430, CBS 219.35, CBS224.37, CBS250.53, CBS343.66 , CBS527.72, CBS528.72, CBS529.72, CBS608.70 and CBS754.68; and microorganisms belonging to the subgenus Micromucor , such as Mortierella spp isabellina CBS194.28, IFO6336, IFO7824, IFO7873, IFO7874, IFO8286, IFO8308, IFO7884, Mortierella spp nana IFO8190, Mortierella spp ramanniana IFO5426, IFO8186, CBS112.08, CBS212.72, IFO7825, IFO8184 , IFO8185, IFO8287 and Mortierella spp vinacea CBS236.82. In particular, it is preferred Mortierella spp exchange alpina .

When used as a host fungus, the nucleic acid of the present invention is preferably autonomously replicable in the host cell, or preferably has the structure suitable for inclusion in the fungal chromosome. Preferably, the nucleic acid also comprises a promoter and terminator. When used as the master M. alpina as an expression vector can be used, for example, pD4, pDuraSC or pDura5. Can be any promoter allowing expression in a host cell, and examples thereof include promoters derived from M. alpina , such as histonH4,1 gene promoter, GAPDH promoter gene (glyceraldehyde 3-phosphate dehydrogenase) gene promoter and TEF (factor translation and elongation).

Examples of the administration method of the recombinant vector into filamentous fungi such as M. alpina , include electroporation, spheroplast method with, particle delivery method, and direct microinjection of DNA into the nucleus. In the case of using an auxotrophic host strain transformed strain can be obtained by selecting a strain which grows on selective medium without a specific nutrient substance (s). Alternatively, when using a transformation marker gene for resistance to a drug, a colony of cells which are resistant to a drug, obtainable by culturing the host cells in a selective medium with drug.

When using yeast as a host, an expression vector can be used, for example, pYE22m. Alternatively, a commercially available yeast expression vectors such as pYES (Invitrogen) and pESC (STRATAGENE). Examples of hosts useful in the present invention include, but are not limited to, a strain of Saccharomyces cerevisiae EH13-15 (trp1, MATα). Promoter which may be used is, for example, a promoter derived from yeast, such as a promoter, GAPDH, gal1 promoter or promoter gal10.

Examples of the administration method of the recombinant vector in yeast includes the lithium acetate method, electroporation, spheroplast method with, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, encapsulation of polynucleotide (s) in liposomes, and direct microinjection of the DNA into the nucleus.

When used as a bacterial host such as E. coli , as an expression vector can be used, for example, pGEX and pUC18, available from Pharmacia. Promoters which may be used include promoters derived from, for example, E. coli or phage, such as trp promoter, lac, PL promoter and the promoter PR. Examples of the administration method of the recombinant vector into bacteria include electroporation and calcium chloride method.

A method for producing a fatty acid composition of the present invention

The present invention relates to a method for producing a fatty acid composition of the transformant described above, i.e. a method for producing a fatty acid composition of the culture product obtained by culturing the transformant. fatty acid composition contains a set of one or more fatty acids. As fatty acids may be free fatty acids or they may be presented in the form of lipids containing fatty acids, such as a triglyceride or a phospholipid. In particular, the fatty acid composition of the present invention can be prepared in the following manner. Alternatively, the fatty acid composition can also be obtained by any other known method.

As a medium for use in culturing the organism expressing the PAP, can be any culture solution (medium) having appropriate pH and osmotic pressure and containing a biological substance, such as nutrients, micronutrients, serum and antibiotics required for growth of each host. For example, in the case of the transformed yeast expressing PAP medium nonlimiting examples include SC-Trp medium, YPD medium and YPD5 environment. Composition particular environment, for example, a medium SC-Trp, as indicated below: One liter of the medium comprises 6.7 g of agar bases for yeast nitrogen without amino acids (DIFCO), 20 g glucose and 1.3 g amino acid powder with (a mixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 g tyrosine, 4.5 g valine, 6 g threonine and 0.6 g uracil).

Culture can be any conditions that are suitable for host growth and are suitable for maintaining stability of the enzyme produced. In particular, the degree of specific conditions include anaerobic conditions, cultivation time, temperature, humidity and static culturing or cultivation conditions using mixing, which can be varied. Cultivation can be carried out under the same conditions (one-step culture) or by so-called two-step or three-step culture using two or more different culture conditions. For the massive cultivation it is preferable to two or more staging cultivation due to the high efficiency of cultivation.

When a two-stage culturing using yeast as a host of the fatty acid composition of the present invention can be prepared as follows: In a preliminary culturing a transformant colony is transferred, for example, SC-Trp medium and cultured under shaking at 30 ° C for two days. Next, 500 l of the solution after preculture to main culture as added to 10 ml YPD5 medium (2% yeast extract, 1% polypeptone, 5% glucose), followed by culturing under shaking at 30 ° C for two days.

fatty acid composition of the present invention

The present invention also relates to a fatty acid composition as an aggregate of one or more fatty acids in cells expressing the PAP of the present invention, preferably a fatty acid composition obtained by culturing a transformant expressing the PAP of the present invention. Fatty acids can be in the form of free fatty acids or may be present in the form of lipids containing fatty acids, such as a triglyceride or a phospholipid.

Fatty acids contained in the fatty acid composition of the present invention are linear or branched monocarboxylic acids carbohydrate with a long chain, such examples include, but are not limited to, myristic acid (tetradecanoic acid) (14: 0), myristoleic acid (tetradecanoic acid) (14: 1), palmitic acid (hexadecanoic acid) (16: 0), palmitoleic acid (9-hexadecanoic acid) (16: 1), stearic acid (octadecanoic acid) (18: 0), oleic acid (cis-9 -oktadekanovaya acid) (18: 1 (9)), vaccenic acid (11-octadecanoic acid) (18: 1 (11)), linoleic acid (cis, cis-9,12 octadecadienoic acid) (18: 2 (9, 12)), α-linolenic acid (9,12,15-octadecatrienoic acid) (18: 3 (9,12,15)), γ-linolenic acid (6,9,12-octadecatrienoic acid) (18: 3 ( 6,9,12)), stearidonic acid (6,9,12,15-octadecatetraenoic acid) (18: 4 (6,9,12,15)), arachidic acid (eicosanoic acid) (20: 0), ( 8,11-eykozadienovaya acid) (20: 2 (8,11)), Mead acid (5,8,11-eykozatrienovaya acid) (20: 3 (5,8,11)), dihomo-γ-linolenic acid ( 8,11,14-eykozatrienovaya acid) (20: 3 (8,11,14)), arachidonic acid (5,8,11,14-eicosatetraenoic acid) (20: 4 (5,8,11,14)) , eicosatetraenoic acid (8,11,14,17-eicosatetraenoic acid) (20: 4 (8,11,14,17)), eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid) (20: 5 (5,8,11,14,17)), behenic acid (docosanoic acid) (22: 0), (7,10,13,16-docosatetraenoic acid) (22: 4 (7,10,13,16) ), (7,10,13,16,19-docosapentaenoic acid) (22: 5 (7,10,13,16,19)), (4,7,10,13,16-docosapentaenoic acid) (22: 5 (4,7,10,13,16)), (4,7,10,13,16,19-docosahexaenoic acid) (22: 6 (4,7,10,13,16,19)), lignoceric acid (tetrakozanovaya acid) (24: 0), nervonic acid (cis-15-tetradokozanovaya acid) (24: 1) and cerotic acid (geksakozanovaya acid) (26: 0). It should be noted that the names of the compounds are presented as generic names according Biochemical Nomenclature IUPAC, and their systematic names with the numeric values ​​that reflect the number of carbon molecules and position of double bonds are listed in parentheses.

fatty acid composition of the present invention may consist of any number and type of fatty acid, with the proviso that it is a combination of one or more fatty acids selected from fatty acids listed above.

Food or other products comprising fatty acid compositions of the present invention

The present invention also relates to a food product comprising the fatty acid composition as described above. The composition of fatty acids of the present invention can be used to produce food products containing fats and oils and receiving industrial raw materials (for example, raw materials for cosmetics, pharmaceuticals (e.g., for external skin application) and washing) by standard methods. Cosmetics (cosmetic compositions) or pharmaceuticals (pharmaceutical compositions) may be formulated in any dosage form including, but not limited to, solutions, pastes, gels, solids, and powders. Examples of food forms include pharmaceutical preparations such as capsules; liquid food naturally occurring-partially digested food and elemental liquid food, wherein the fatty acid composition of the present invention is mixed with proteins, sugars, fats, trace elements, vitamins, emulsifiers and flavorings; and it produced in the form of shapes such as drinkable preparations or as enteral nutrient.

In addition, examples of the food product of the present invention include, but are not limited to, nutritional supplements, dietetic foods, healthy foods, food for children, children’s modified milk, modified milk for premature infants and nutrition for the elderly. Throughout the specification, the term “food product” is used as a collective term in respect of edible substances in solid form in the fluid form, liquid form, or mixtures thereof.

The term “nutritional supplement” refers to food products enriched with specific nutritional ingredients. The term “dietetic foods” refers to food products, which is healthy or beneficial to health, and nutritional supplements include, natural foods and diet foods. The term “health foods” refers to food products to maintain the nutrients that support the functioning of the body, and is synonymous with the product for the special applications for health. The term “children’s foods” refers to foods that are given to children up to about the age of 6 years. The term “foods for the elderly” refers to food products for easy digestion and absorption when compared to untreated products. The term “modified milk for children” refers to modified milk, which is given to children aged up to about one year. The term “modified milk for premature infants” refers to modified milk, which give premature babies until about 6 months after birth.

Examples of these food products include natural foods (treated with fats and oils) such as meat, fish and nuts; food supplements with added fats and oils in the preparation, such as Chinese food, Chinese noodles and soups; foods prepared using fats and oils as a medium for heat treatment, such as tempura (deep fried fish and vegetables), deep fried foods, fried tofu, fried Chinese rice, donuts and Japanese fried cookies from shortcrust pastry ( Carinthia); Food products based on fats and oils or processed foods supplemented with fats and oils during preparation such as butter, margarine, mayonnaise, relish, chocolate, instant noodles, caramel, cookies, muffins, cakes and ice-cream; and food, covered or coated with fats and oils when cooking completion, such as rice crackers, hard biscuits and sweet bread from the beans. However, the food products of the present invention is not limited to foods containing fats and oils, and other examples of food products include agricultural foods such as bakery products, noodles, cooked rice, sweets (e.g., candies, chewing gum, toffees, tablets, Japanese sweets), tofu, and products derived from them; fermented foods, such as refined sake, medicinal liquor, spicy liqueur (mirin), vinegar, soy sauce and miso; Food products derived from farm animals, such as yogurt, ham, bacon and sausages; seafood such as fish paste (kamaboko), deep fried fish paste (ageten) and fish pie (hanpen); and fruit drinks, soft drinks, sports, alcoholic beverages and tea.

A method of analysis and selection of strains with nucleic acid encoding the PAP, or PAP protein of the present invention

The present invention also relates to a method of analysis and selection of lipid producing fungi with a nucleic acid encoding the PAP, or PAP protein of the present invention. Details are given below.

(1) Method of analysis

One embodiment of the present invention is a method of lipid producing fungus analysis using nucleic acid encoding the PAP, or PAP protein of the present invention. The assay method of the present invention, for example, analyze lipid producing fungi strain as an experimental strain for activity evaluations of the present invention, using primers or probes designed based on the nucleotide sequence of the present invention. This analysis can be carried out by known methods such as those described in WO № WO01 / 040514 and JP-A-8-205900. The analysis method is briefly described below.

The first step is to obtain a genome test strain. Gene may be prepared by any known method such as Hereford method or potassium acetate method (see, e.g., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, p. 130 (1990)).

Primers or probes are designed based on the nucleotide sequence of the present invention, preferably the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6. The data as primers or probes may be any region of the nucleotide sequence of the present invention, and they can be constructed in any method. The number of nucleotides in a polynucleotide used as a primer is generally 10 or more preferably from 15 to 25. The number of nucleotides, primers suitable for franking, usually ranges from 300 to 2000.

Obtained as described above, the primers or probes are used to assess the presence of analyte in the genome sequence of strain, specific for the nucleotide sequence of the present invention. The sequence, which is specific for the nucleotide sequences of the invention can be identified by known techniques. For example, a polynucleotide comprising a part or the entire sequence specific to the nucleotide sequence of the present invention, or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence used as a primer, and a polynucleotide comprising a part or the entire sequence located above or below this sequence or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence used as the other primer, and nucleic acid amplified from the test strain by PCR or other methods. Further, for example, it is possible to determine the presence or absence of the amplification product and the molecular weight of the amplification product.

PCR conditions suitable for the method of the present invention is not particularly limited and, for example, may be as specified below.

Denaturation temperature from 90 ° C to 95 ° C.

annealing temperature: 40 ° C to 60 ° C.

Elongation temperature: from 60 ° C to 75 ° C.

Number of cycles: 10 or more cycles.

The resulting reaction products may be separated by agarose gel electrophoresis or any other method to determine the molecular mass of the amplification product. The activity of the present invention for the test strain can predict or estimate by confirming that the molecular weight of the amplification product is sufficient to cover a nucleic acid molecule corresponding to a region specific for a nucleotide sequence of the present invention. Furthermore, the activity of the present invention can predict or estimate with higher accuracy by analyzing the nucleotide sequence of the amplification product by a method described above. Activity evaluation method of the present invention described above.

Alternatively, the analysis of the present invention the activity of the present invention with respect to test strain can be assayed by culturing the test strain and measuring the expression level of PAP, encoded by the nucleotide sequence of the present invention, for example the sequence shown in SEQ ID NO: 1 or SEQ ID NO : 6. PAP expression level can be measured by culturing the test strain under certain conditions and determining the amount of mRNA or protein PAP. The amount of mRNA or protein can be determined using known methods. For example, the amount of mRNA can be determined by Northern hybridization or quantitative PCR in real time, and the amount of protein can be determined by Western blotting (Current Protocols in Molecular Biology, John Wiley & Sons, 1994-2003).

(2) A method for breeding

Another embodiment of the present invention is a method of lipid producing fungi selection using nucleic acid encoding the PAP, or PAP protein of the present invention. When selection of the present invention, a strain having a desired activity can be selected by culturing the test strain, measuring the expression level of PAP, encoded by the nucleotide sequence of the present invention, for example the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, and selecting strains with the desired level of expression. Alternatively, the desired strain may be chosen when using standard strain, culturing the strain and the standard test strain separately, measuring the expression level of each of the strains and strain comparisons of standard expression level with the expression level of the test strain. Specifically, for example, a standard strain and test strains are cultured under appropriate conditions and measuring the expression level of each strain. The strain exhibiting the desired activity can be selected by selecting a test strain with higher or lower expression level compared to the standard strain. The desired activity may be determined, for example, by measuring the expression level of PAP and the fatty acid composition produced by PAP, as described above.

When selection of the present invention studied strain having the desired activity can be selected by culturing the test strains and selecting the strains with high or low activity of the present invention. The desired activity may be determined, for example, by measuring the expression level of PAP and the fatty acid composition produced by PAP, as described above.

Examples of the standard test strain and strain include, but are not limited to, strains transformed with a vector of the present invention is modified strains against suppression of expression of a nucleic acid of the present invention, the strains subjected to mutagenesis, and strains from natural mutations. The activity of the present invention can be measured, for example, the method described in the specification, “nucleic acid encoding phosphatidic acid phosphatase of the present invention”. Examples mutational processes include, but are not limited to, physical methods such as ultraviolet light or radiation; Chemical methods and exposure to chemicals such as EMS (etilmetan sulfonate) or N-methyl-N-nitrosoguanidine (see, e.g., Yasuji Oshima ed., Biochemistry Experiments vol. 39, Experimental Protocols for Yeast Molecular Genetics, pp. 67-75 , Japan Scientific Societies Press).

Examples of strains for use as a standard strain of the present invention or the test strains include, but are not limited to, fungi that produce lipids, and the yeast described above. In particular, a standard strain and the strain can be analyzed with any combination of strains belonging to different genera or species, can be analyzed simultaneously, and one or more of the test strains.

The present invention is hereinafter described in detail with the following examples which are not intended to limit the scope of the invention.

EXAMPLES

Example 1: Analysis of the genome of M. alpina

Strain M. alpina 1S-4 was inoculated into 100 ml medium GY2: 1 (2% glucose, 1% yeast extract, pH 6,0) and cultured with shaking at 28 ° C for 2 days. Cells were collected by filtration to obtain genomic DNA using DNeasy (QIAGEN).

The nucleotide sequence of the genomic DNA was determined by 454 Roche GS FLX Standard. In this case the nucleotide sequence fragments from the library were sequenced in two parts, and the nucleotide sequence of the library “partners» (mate pair) sequenced in three passes. The resulting nucleotide sequences were aligned to obtain 300 superkontigov.

Example 2: cDNA Synthesis and Construction of cDNA Library

Strain M. alpina 1S-4 was inoculated into 100 ml medium (1.8% glucose, 1% yeast extract, pH 6,0) and cultured with shaking at 28 ° C for 4 days. Cells were collected by filtration and total RNA was prepared by the method guanidine hydrochloride / CsCl.

From the total RNA by reverse transcription cDNA was synthesized using SuperScript II RT (Invitrogen), using random hexamer. Moreover, total RNA was isolated from poly (A) + RNA using a set of Oligotex-dT30 <Super> mRNA Purification Kit (Takara Bio Inc.). CDNA library was constructed using a set of ZAP-cDNA GigapackIII Gold Cloning Kit ( STRATAGENE).

Example 3: Search for homologs PAH1, derived from S. cerevisiae

The amino acid sequence of PAP gene activity from Saccharomyces cerevisiae , PAH1 (YMR165C) (herein also may be designated as ScPAH1), it was subjected to analysis tblastn compared with the genomic nucleotide sequences of the strain M. alpina 1S-4. As a result, the advantage possessed superkontigi comprising the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10. The gene related to SEQ ID NO: 5 was named MaPAH1.1, a gene related to SEQ ID NO: 10, It was named MaPAH1.2.

Example 4: Cloning and MaPAH1.1 MaPAH1.2

(1) Preparation of probe

For cDNA cloning and gene MaPAH1.1 MaPAH1.2 gene obtained nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10, and corresponding primers were selected based on the analysis results using BLAST program described above.

MaPAH1-1-3F: 5′-CGCCAATACATTGACGTTTTCAG-3 ‘(SEQ ID NO: 11)

MaPAH1-1-5R: 5’-AGTTCCAGTCATTGAACTCGGGTGC-3 ‘(SEQ ID NO: 12)

MaPAH1-2-3F: 5’-GAGCCCAGTTGACCTTTGAGGCATTC-3 ‘(SEQ ID NO: 13)

MaPAH1-2-5R: 5’-CACTGAGAACGAGACCGTGTTGGCG-3 ‘(SEQ ID NO: 14)

PCR was conducted using ExTaq (Takara Bio Inc.), using as a template the cDNA library constructed in Example 2, and a combination of primer and MaPAH1-1-3F MaPAH1-1-5R primer or primer combination and MaPAH1-2-3F prymera MaPAH1 -2-5R at 94 ° C for 2 min, then 30 cycles (94 ° C for 30 sec, 55 ° C for 30 sec and 72 ° C for 2 min). A DNA fragment of about 0.6 kb was obtained for each combination, using a set of cloned TOPO-TA cloning Kit (Invitrogen) and the nucleotide sequence of the insert in the resulting plasmid. The plasmid obtained by using the above-mentioned combination of primers, with the sequence corresponding to nucleotides 2352 to 3010 of SEQ ID NO: 4, identified as pCR-MaPAH1.1-P; a plasmid obtained by using the last of these primer combinations, with the corresponding sequence from nucleotides 1615 to 2201 of SEQ ID NO: 9, identified as pCR-MaPAH1.2-P.

Then samples were obtained by PCR using the plasmid as a data matrix, and the primers described above. In the reaction used ExTaq (Takara Bio Inc., Japan), except that instead of dNTP mixture attached to label the amplified DNA solution was used to label the PCR (Roche Diagnostics) with digoxigenin (DIG) for the probe and probe MaPAH1.1 MaPAH1.2 . Then, using these probes, screening of a cDNA library were carried out.

The hybridization conditions were chosen as described below.

Buffer: 5 × SSC, 1% SDS, 50 mM Tris-HCl (pH 7,5), 50% formamide.

Temperature: 42 ° C (overnight).

wash conditions: in 0,2 ×× SSC, 0,1% SDS solution (65 ° C) for 20 min (three times).

For the detection kit was used DIG nucleic acid detection kit (Roche Diagnostics ). For each plasmid DNA was excised using plasmid excision in vivo of the phage clones obtained by screening. Plasmid inserts with the greatest length among the plasmids obtained by screening using MaPAH1.1 probe having a sequence ranging from position 1307 m and more from the sequence shown in SEQ ID NO: 4, and was named plasmid pB-MaPAH1.1p. The results of comparison of the amino acid sequence shown ScPAH1 that the plasmid pB-MaPAH1.1p contains the region encoding the N-terminal region PAH1.1. Comparison of the genomic sequence (SEQ ID NO: 5), which is expected by the results of BLAST analysis using contains MaPAH1.1 gene with N-terminal region of the amino acid sequence shown ScPAH1 that ATG in position 1 to 3 in the sequence presented in SEQ ID NO: 5 is the initiation codon. Each frame of the plasmid pB-MaPAH1.1p was translated into the amino acid sequence. The amino acid sequence was compared with the amino acid sequence ScPAH1, obtained from S. cerevisiae . As a result, it was shown that the TGA at positions 3985 to 3987 in the sequence shown in SEQ ID NO: 4 is the stop codon. Thus, for cloning of full-length cDNA, the following primers were designed.

NotI-PAH1-1-F: 5′-GCGGCCGCATGCAGTCCGTGGGAAG-3 ‘(SEQ ID NO: 15)

MaPAH1-1-10R: 5’-TTCTTGAGTAGCTGCTGTTGTTCG-3 ‘(SEQ ID NO: 16)

PCR was conducted using ExTaq (Takara Bio Inc.), using the above cDNA as a template and a primer combination NotI-PAH1-1-F primer and MaPAH1-1-10R at 94 ° C for 2 min followed by 30 cycles of (94 ° C for 30 sec, 55 ° C for 30 sec and 72 ° C for 2 min). The resulting DNA fragment of approximately 1.5 kbp was cloned using a set of TOPO-TA cloning Kit (Invitrogen) and the nucleotide sequence of the insert. The plasmid into which the cloned DNA fragment comprising a sequence of nucleotides from 1 to 1500 of SEQ ID NO: 4, was named pCR-MaPAH1.1-Np. The DNA fragment of about 1.4 kb obtained by cutting the plasmid pCR-MaPAH1.1-Np with restriction enzymes NotI and XhoI, the DNA fragment of about 3.7 kb obtained by pB-MaPAH1.1p cutting the plasmid with restriction enzymes NotI and BamHI, and a DNA fragment of about 2.1 kb obtained by cutting the plasmid pB-MaPAH1.1p using restriction enzymes XhoI and BamHI, was connected via ligation (TOYOBO) to obtain cDNA plasmid pB-MaPAH1.1, which is believed to contain the full length cDNA MaPAH1.1. The cDNA sequence comprising the full length ORF of MaPAH1.1, shown in SEQ ID NO: 4.

Separately, the plasmid with the greatest length among the inserts of plasmids obtained by screening using MaPAH1.2 probe has a nucleotide sequence shown in SEQ ID NO: 9. The results of this sequence comparison ScPAH1 plasmid with sequence derived from S. cerevisiae , shown that includes the full-length cDNA plasmid ORF of MaPAH1.2. This plasmid was named pB-MaPAH1.2 cDNA.

(2) Sequence analysis

The cDNA sequence (SEQ ID NO: 4) of gene MaPAH1.1 includes CDS (SEQ ID NO: 3) consisting of a sequence of nucleotides from 1 to 3987 and ORF (SEQ ID NO: 1) comprising the sequence of nucleotides from 1 to 3984. Installed amino acid sequence encoded by the gene MaPAH1.1, presented in SEQ ID NO: 2. The genomic sequence MaPAH1.1 gene ORF was compared with the sequence (figure 1). As a result, it was shown that the genomic gene sequence MaPAH1.1 consists of eleven ten exons and introns.

The cDNA sequence (SEQ ID NO: 9) gene MaPAH1.2 includes CDS (SEQ ID NO: 8) consisting of a sequence of nucleotides from 72 to 3791, and ORF (SEQ ID NO: 6) consisting of a sequence of nucleotides from 72 to 3788 . Installed amino acid sequence encoding gene MaPAH1.2, presented in SEQ ID NO: 7. Genomic sequence MaPAH1.2 gene ORF was compared with the sequence (figure 2). The genomic sequence of the gene MaPAH1.2 consists of eight exons and seven introns.

cDNA sequence and MaPAH1.1 MaPAH1.2 and amino acid sequences respectively set shown in Figure 3 and Figure 4.

With the program carried BLASTp search of homology between amino acid sequences set MaPAH1.1 and MaPAH1.2 and amino acid sequences from GenBank. Both amino acid sequence homology to reach the highest level since 1 nuclear protein elongation and the alleged distortion protein (AAW42851), derived from Cryptococcus neoformans var. neoformans JEC21, but with low identity, i.e. 25.9% and 26.6% respectively.

Among fungal homologues PAP1 and amino acid sequences MaPAH1.1 MaPAH1.2, derived from M. alpina of the present invention are identical to 22.7% and 22.5% respectively, with the amino acid sequence ScPAH1, which was subjected to functional analysis. The amino acid sequence and MaPAH1.1 MaPAH1.2, derived from M. alpina in the present invention were compared with known amino acid sequences and ScPAH1 Lipina obtained from mouse (Figure 5). In PAP1 family of enzymes, the amino acid sequence of the N-terminal region is highly conserved and is called Liping, N-terminal conserved region (pfam04571). Also MaPAH1.1 and MaPAH1.2, derived from M. alpina of the present invention, a well-known enzyme and N-terminal region are quite conservative. Moreover, the sequence DIDGT, marked by double underline in Figure 5, is a protein superfamily of enzymes such galogenokislot dehalogenase (HAD), and corresponds to the catalytic site motif conserved DXDX (T / V).

Sequences of CDS MaPAH1.1 MaPAH1.2 and compared with each other in order to demonstrate the identity of 54.7% (Figure 6), while the identity between amino acid sequences set was 35.6% (Figure 7).

Example 5: Expression and MaPAH1.1 MaPAH1.2 yeast

Construction of the expression vector and MaPAH1.1 MaPAH1.2:

MaPAH1.1 For expression in yeast expression vectors were constructed as follows.

Yeast expression vector pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228, 1995) was cut with the restriction enzyme EcoRI, and blunt-ended using a set Blunting Kit (TaKaRa Bio Inc.). The resulting fragment and linker pNotI, phosphorylated (8-mer) (TaKaRa Bio Inc.), attached to each other by means of ligation (TOYOBO) to construct pYE22mN vector. PYE22mN vector was cut with restriction enzymes NotI and KpnI, the resulting fragment was fused to the DNA fragment of approximately 4.2 kbp obtained plasmid pB-MaPAH1.1 when cutting the cDNA with restriction enzymes KpnI and NotI to ensure plasmid pYE-MaPAH1.1. Separately, pYE22mN vector was cut with restriction enzymes NotI and KpnI, the resulting fragment was fused to a DNA fragment about 3.8 kb in length, resulting in cutting of the cDNA plasmid pB-MaPAH1.2 with restriction enzymes KpnI and NotI to ensure plasmid pYE-MaPAH1.2.

Getting strain ΔScpah1: URA3 S. cerevisiae

For cloning ScPAH1 gene obtained from strain S288C S. cerevisiae , the following primers were prepared:

Primer KpnI-PAH1-F: 5′-GGTACCATGCAGTACGTAGGCAGAGCTC-3 ‘(SEQ ID NO: 17) and

Primer XhoI-PAH1-R: 5’-CTCGAGTTAATCTTCGAATTCATCTTCG-3 ‘(SEQ ID NO: 18).

Strain S288C S. cerevisiae was cultured in a liquid medium YPD (2% yeast extract, 1% polypeptone, 2% glucose) at 30 ° C overnight. DNA was isolated from cells using the Dr. GenTLE (yeast) (TaKaRa Bio Inc.), and ScPAH1 gene was amplified by PCR with ExTaq, using as template DNA and primers derived KpnI-PAH1-F and XhoI-PAH1-R. The resulting DNA fragment of approximately 2.5 kbp was cloned using a set of TOPO TA cloning Kit, a clone with the correct nucleotide sequence was determined as pCR-ScPAH1. DNA fragment of about 0.4 kb obtained by cutting the pCR-ScPAH1 with restriction enzymes EcoRI and EcoRV, and a DNA fragment of about 2.1 kb obtained by cutting with pCR-ScPAH1 the restriction enzymes EcoRV and XhoI, were ligated into the vector pBluescriptIISK +, cut with restriction enzymes EcoRI and XhoI, to obtain plasmid pBScPAH1. PBScPAH1 plasmid was cut with restriction enzymes HincII and EcoRV and ligated with a fragment of DNA of approximately 1.2 kb obtained by cutting pURA34 plasmid (Japanese Unexamined Patent Application № 2001-120276) with the restriction enzyme HindIII, and then blunt-ended. The resulting product is a URA3 gene in the same direction as the gene ScPAH1, pBΔpah1 plasmid identified as: URA3. Further strain YPH499 S. cerevisiae (lys2-801amber ade2-101ochre ura3-52 trp1-Δ63 his3-Δ200 leu2-Δ1 a) (STARATAGENE) as a host transformed with a DNA fragment prepared by cutting plasmid pBΔpah1: URA3 with a restriction enzyme EcoRI. Transformed strain selected for the ability to grow on agar medium SC-Ura (one liter of the medium comprises 6.7 g of agar bases for yeast nitrogen without amino acids (DIFCO), 20 g glucose, 1.3 g amino acid powder (a mixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine, 11 25 g serine, 0.9 g tyrosine, 4.5 g valine, 6 g threonine and 1.2 g tryptophan) and agar medium (2% agar)). A strain whose PCR confirmed that Δpah1 structure: URA3 and introduced into a strain that is damaged ScPAH1 gene was determined as a strain ΔScpah1: URA3.

Preparation of transformed strain:

Strain ΔScpah1: URA3 was used as the host and transformed with a plasmid pYE22m, pYE-MaPAH1.1 or pYE-MaPAH1.2. The transformed strains were selected by ability to grow on agar medium SC-Ura, Trp (one liter of the medium comprises 6.7 g of agar bases for yeast nitrogen without amino acids (DIFCO), 20 g glucose, 1.3 g amino acid powder (a mixture of 1, 25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine , 11.25 g serine, 0.9 g tyrosine, 4.5 g valine and 6 g threonine) and agar medium (2% agar)). Two arbitrary shtmamma from corresponding strains transformed with each plasmid (control strains transformed with the plasmid pYE22m: C1 and C2, strains transformed with plasmid pYE-MaPAH1.1: MaPAH1.1-1 and MaPAH1.1-2, and strains transformed with plasmid pYE -MaPAH1.2: MaPAH1.2-1 and MaPAH1.2-2), was used in subsequent experiments.

Example 6: Measurement of the activity of Mg 2+ -dependent phosphatidic acid phosphatase (PAP1 activity)

Each yeast transformant strain was inoculated into 100 ml liquid medium SC-Ura, Trp and cultured with shaking at 30 ° C for one day. Initial enzyme solution prepared from the culture solution prepared as follows. In particular, the process carried out at 4 ° C or on ice. Cells were harvested from the culture solution by centrifugation and washed with water. Subsequently, cells were suspended in 5 ml of buffer A (50 mM Tris-HCl (pH 7,5), 0,3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). The cells were disrupted during treatment with French press (Thermo Fisher Scientific), Mini-Cell, three times at 16 psi. The cell lysate was centrifuged at 1500 × g for 10 minutes and the supernatant was collected as the source of enzyme solution. Protein concentration contained in the initial solution of the enzyme was measured using Protein Assay CBB Solution (5 ×) (Nacalai Tesque).

PAP1 activity was measured using a modified method according to Gil-Soo et al. (J. Biol. Chem., 282 (51), 37026-37035, (2007)) as described below. Because S. cerevisiae can not synthesize linoleic acid as substrate for PAP used dilinoleoil 1,2-glycero-sn-3-phosphate (18: 2-PA). We used five hundred microliters of the reaction solution. As the composition of the reaction solution was 100 .mu.l original enzyme solution, 50 mM Tris-HCl (pH 7,5), 100 ug / ml of 1,2-dilinoleoil-sn-glycero-3-phosphate, monosodium salt (Avanti Polar Lipids, Inc .), 1 mM MgCl 2 and 10 mM 2-mercaptoethanol. The reaction solution was maintained at 25 ° C for 30 minutes and then the reaction was terminated by the addition of chloroform: methanol (1: 2). Lipids were extracted by the method of Bligh-Dyer. By TLC (TLC) lipids fractionated on a silica gel 60 plate (Merck), using as eluent hexane: dietilovyyefir: acetic acid = 70: 30: 1. Lipids were visualized by spraying primulina solution (0.015% primulin in 80% aqueous acetone), and then irradiated with ultraviolet light. Fraction diacylglycerol (DG) was scraped from the plate and converted to fatty acid methyl ester by the method with hydrochloric acid / methanol. Next, the methyl ester fatty acids were extracted with hexane and the hexane was distilled off, followed by analysis by gas chromatography.

Table 2 shows the amounts of linoleic acid, transformed into DG fraction on the amount of protein in the initial enzyme solution.

table 2
transformed strain 18: 2 (pg / mg protein)
C1 15.43
C2 17.53
MaPAH1.1-1 56.03
MaPAH1.1-2 44.34
MaPAH1.2-1 19.45
MaPAH1.2-2 20,90

As indicated in Table 2, compared with C1 and C2, transformed using pYE22m, conversion activity 18: 2 to dilinoleina-PA (18: 2-DG) was about 3-fold and against MaPAH1.1-1 MaPAH1.1 -2 expressing MaPAH1.1, and approximately 1.2-fold and against MaPAH1.2-1 MaPAH1.2-2, expressing MaPAH1.2. This indicates that MaPAH1.1 MaPAH1.2 and have PAP activity.

PAP activity dependence of Mg 2+ was investigated as follows: five hundred microliters using reaction solution. Reaction and analysis were carried out under the same conditions as above, except that as the composition of the reaction solution was 100 .mu.l original enzyme solution, 50 mM Tris-HCl (pH 7,5), 100 ug / ml of 1,2-dilinoleoil -sn-glycero-3-phosphate, monosodium salt (Avanti Polar Lipids, Inc.), 2 mM EDTA and 10 mM 2-mercaptoethanol. In Table 3 shows amounts of linoleic acid in DG transferred fraction to the amount of protein in the initial enzyme solution.

Table 3
transformed strain 18: 2 (pg / mg protein)
C1 11.17
C2 10.77
MaPAH1.1-1 13.06
MaPAH1.1-2 11.39
MaPAH1.2-1 12.52
MaPAH1.2-2 10.93

As indicated in Table 3 for the conversion activity of each strain 18: 2 to dilinoleina-PA (18: 2-DG) was approximately the same.

This indicates that the activity of the PAP and MaPAH1.1 MaPAH1.2 depends of Mg 2+ and that MaPAH1.1 and MaPAH1.2 have PAP1 activity.

Example 7: Number of produced triacylglycerol

Triacylglycerol (throughout the specification called triglyceride or TG), which is a backup lipid, the lipid is obtained by further acylation of diacylglycerol product which is PAP protein. Measures the amount of the TG, produced by yeast transformants in which high espressirovany MaPAH1.1 or MaPAH1.2.

The cells of the transformants, yeast host strain deficient ScPAH1, seeded in 10 ml of liquid medium SC-Ura, Trp and cultured under static conditions at 30 ° C for 3 days. One milliliter of the culture solution was inoculated into 10 ml of YPDA liquid medium (2% w of yeast extract, 1% polypeptone, 2% glucose, 0.008% sulfate adenine), followed by culturing with shaking at 30 ° C for one day (n = 3) . Cells were harvested by centrifuging the culture solution, washed with water and lyophilized. To the dried cells were added chloroform and methanol (2: 1). Cells were again disrupted by glass beads and lipids were extracted using a total of 8 ml solvent. The extracted lipids were fractionated by TLC, as described above, and was scraped and analyzed by TG fraction. The results are shown in Table 4.

Table 4
Number * TG, produced in each medium
transformed strain mg / l
C1 11,01 ± 1,27
C2 11,54 ± 0,54
MaPAH1.1-1 16,01 ± 2,45
MaPAH1.1-2 17,09 ± 1,41
MaPAH1.2-1 14,29 ± 0,87
MaPAH1.2-2 13,32 ± 0,78
* In terms of fatty acids

As shown in Table 4, the amount of TG was approximately 1.5-fold in strain intensive expression MaPAH1.1, was approximately 1.2-fold in strain intensive expression MaPAH1.2, as compared with that in the control.

Example 8: Substrate specificity and MaPAH1.1 MaPAH1.2

Strain ΔScpah1: URA3, as a host, transformed with the plasmid pYE22m, pYE-MaPAH1.1 or pYE-MaPAH1.2. Four each transformant strain was used in subsequent experiments. Strains transformed with plasmid pYE22m, used as a control.

Each yeast transformant was inoculated into 10 ml liquid medium SC-Ura, Trp and cultured under static conditions at 27,5 ° C overnight. Each of the resulting culture solution was inoculated into 40 ml of liquid medium SC-Ura, Trp in an amount of 1.10 to two parallel and cultured under static conditions at 27,5 ° C for two days. From the obtained culture solutions were prepared stock solutions of the enzyme, as in Example 6, and protein concentration was measured.

PAP1 activity was measured as in Example 6 except that as the substrate was used for PAP dilinoleoil 1,2-glycero-sn-3-phosphate (18: 2-PA) and 1,2-dioleolil-sn-glycero-3 -phosphate (18: 1-PA).

Table 5 and Table 6, respectively indicated amounts of linoleic acid (18: 2) and oleic acid (18: 1) fraction was transferred into diacylglycerol (DG), on the amount of protein in the initial enzyme solution.

Table 5:
18: 2 DG on the amount of protein (micrograms / mg · protein)
The name of the sample Control MaPAH1.1 MaPAH1.2
average value CO average value CO average value CO
13.72 2.74 25.50 6.75 18,19 1.43
Table 6:
18: 1 to DG protein (pg / mg · protein)
The name of the sample Control MaPAH1.1 MaPAH1.2
average value CO average value CO average value CO
15.14 0.88 29,16 7.04 16.69 1.05

When used as a substrate 18: 2-PA, the activity types and MaPAH1.1 MaPAH1.2, derived from Mortierella, were 1.9-fold and 1.3-fold, respectively, compared to those in control.

When used as the substrate 18: 1-PA, and species MaPAH1.1 MaPAH1.2 activity were 1.9-fold and 1.1-fold, respectively, compared to those in control. 18: 1 – is a fatty acid that naturally produce yeast, and thus, it is initially present in the composition DG enzyme in the original solution. However, no differences were observed regarding the number of 18: 1 in the composition DG enzyme in the original solution, if the substrate is not added. Accordingly, it can be assumed that the difference in activity MaPAH1.1 MaPAH1.2 and the activity in the control as shown in Table 6, based on the effect in respect of 18: 1-PA, added as a substrate.

When comparing the activity of the same species of the enzyme for different substrates, MaPAH1.1 increased as the amount of 18: 1 and 18: 2 1.9-fold compared with the control, whereas the increased amount MaPAH1.2 18: 1 1.1-fold and the amount of 18 2 to 1.3 times compared with the control. This indicates that MaPAH1.1 exhibits the same activity in respect of both 18: 1-PA, and 18: 2-PA, but in the case MaPAH1.2 activity on 18: 2-PA higher than that against 18 1-PA.

These results indicate that possess MaPAH1.2 MaPAH1.1 and PAP activity. Furthermore, MaPAH1.2 exhibit higher activity towards 18: 2-PA, than for 18: 1-PA, indicating that MaPAH1.2 exhibits a higher activity for phosphatidic acids having a fatty acid composition with a higher degree of unsaturation

1. A nucleic acid, as described below:
(a) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(b) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions, and encodes a protein with the activity of phosphatase phosphatidic acid;
(c) a nucleic acid characterized by a nucleotide sequence which consists of a nucleotide sequence that is 95% or more identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;
(d) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(e) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 under stringent conditions, and encodes a protein having phosphatidic acid phosphatase activity;
(f) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions, and includes an exon coding for a protein with phosphatase activity phosphatidic acid; and
(g) a nucleic acid characterized by the nucleotide sequence which consists of a nucleotide sequence that is 95% or more identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with the activity of phosphatase phosphatidic acid.

2. Nucleic acid according to claim 1, wherein the nucleic acid is any nucleic acid below:
(a) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(b) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C, and encodes a protein with phosphatidic acid phosphatase activity;
(c) a nucleic acid characterized by a nucleotide sequence which consists of a nucleotide sequence that is 98% or more identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;
(d) a nucleic acid characterized by the nucleotide sequence encoding a protein which cons

The invention relates to the field of molecular biology, biochemistry and genetic engineering. Proposed a nucleic acid, characterized by a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 and has an activity of phosphatase phosphatidic acid corresponding protein a recombinant vector for protein expression cell and a method for producing a fatty acid composition. The invention can be used to produce polyunsaturated fatty acids in the food industry. 8 n. 1 ZP f-ly, Table 6., 7 ill., 8 pr.

 

TECHNICAL FIELD

The present invention relates to a new phosphatidic acid phosphatase gene and use thereof.

BACKGROUND ART

Fatty acids containing two or more unsaturated bonds, collectively referred to as polyunsaturated fatty acids (PUFA), which are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. Some of these fatty acids are not synthesized in the body of an animal, and such fatty acids should be taken as a dietary essential fatty acids. Polyunsaturated fatty acids are widely distributed. For example, arachidonic acid is recovered from a lipid extracted from animal adrenal and liver. However, the data amount of polyunsaturated fatty acids contained in animal organs is small, and the amount of polyunsaturated fatty acids obtained and isolated in pure form only from animal organs is insufficient to provide them in bulk. Thus, by culturing various microorganisms bacterial methods for obtaining polyunsaturated fatty acids have been developed. In particular, it is known that microorganisms of the genus Mortierella produce lipids containing polyunsaturated fatty acids such as arachidonic acid.

Other attempts to produce polyunsaturated fatty acids were taken for plants. It is known that polyunsaturated fatty acids to form lipid storing such as triacylglycerols (also referred to as triglycerides, or TG), accumulated inside the microorganism cells or plant beans.

As lipid triacylglycerol for storing in the body is formed as follows: an acyl group is introduced into glycerol-3-phosphate using glycerol-3-phosphate acyltransferase to produce lysophosphatidic acid. The acyl group is introduced in the lysophosphatidic acid acyltransferase via lizofosfat for phosphatidic acid. Phosphatidic acid is then subjected to dephosphorylation using phosphatidic acid phosphatase to produce diacylglycerol. An acyl group is introduced via a diacylglycerol diacylglycerol acyltransferase to produce triacylglycerol.

In this pathway, phosphatidic acid (hereinafter also referred to as «PA» or 1,2-diacyl-sn-glycerol-3-phosphate) is a precursor of triacylglycerol and also a biosynthetic precursor of diacyl glycerophospholipids. In yeast cells, CDP diacylglycerol (CDP-DG) and the PA is synthesized from cytidine 5′-triphosphate (CTP) using tsitidiltransferazy phosphatidate and synthesized various phospholipids.

As described above, it is known that the biosynthesis reaction of diacylglycerol (hereinafter, also referred to as «DG») phosphatidic acid phosphatase catalyzed (EC 3.1.3.4, hereinafter also referred to as «PAP») through dephosphorylation of PA. It is known that the PAP is present in all organisms from bacteria to vertebrates.

Yeast ( Saccharomyces cerevisiae ), which are fungi possess two types of PAP (non-patent literature 1, 2 and 7). One of them is of Mg 2+ -dependent PAP (PAP1), and the other – is of Mg 2+ -independent PAP (PAP2). It is known that the gene encodes PAH1 PAP1 (Non-Patent Literature 3-5). Pah1Δ option also indicates PAP1 activity, which suggests the existence of other genes, testifying PAP1 activity. If you have a version pah1Δ, nuclear membrane and the ER membrane is abnormally expanded and expression of important genes for the biosynthesis of phospholipids abnormally enhanced (non-patent literature 6).

It is known that genes encoding PAP2, DPP1 LPP1 gene and characterize the gene most likely species PAP2 activity in yeast. The enzymes encoded by these genes have a broad substrate specificity and is also influenced by, for example, dephosphorylation of diacylglycerol pyrophosphate (DGPP), lysophosphatidic acid and isoprenoid sfingoosnovany phosphate phosphate.

It is known that the lipid producing fungi, Mortierella alpina , MaPAP1 contains a gene which is Mg 2+ -independent homologue PAP2 (Patent Literature 1).

The art knows of the existence of homologs of genes that is likely to encode a family of enzymes PAP1 or PAP2 family enzymes in other bacteria, but their function is not yet understood.

List of cited documents

patent literature

Patent Literature 1: International Publication № WO2009 / 008466

non-patent literature

Non-patent literature 1: Biochem. Biophys. Acta, 1348, 45-55, 1997

Non-patent literature 2: Trends Biochem. Sci., 31 (12), 694-699, 2006

Nonpatent literature 3: EMBO J., 24, 1931-1941, 2005

Nonpatent literature 4: J. Biol. Chem., 281 (14), 9210-9218, 2006

Nonpatent literature 5: J. Biol. Chem., 281 (45), 34537-34548, 2006

Non-patent literature 6: J. Biol. Chem., 282 (51), 37026-37035, 2007

Non-patent literature 7: J. Biol. Chem., 284 (5), 2593-2597, 2009

SUMMARY OF THE INVENTION

TECHNICAL PROBLEM

For most genes PAP, which had previously reported, however, no investigations were performed regarding the changes in the ratio of fatty acids in the composition of fatty acids produced by the host cells, the introduction of these genes into host cells and expression therein. There is a need to identify a new gene by introducing the gene into a host cell and expressing the gene product of which will be given fat composition with a fatty acid or an increased content of the specified fatty acids.

The object of the present invention is the provision of a protein or nucleic acid which allows host cells to produce a given fat composition with a fatty acid or with an increased content of fatty acids defined by protein expression in the host cells or the introduction of nucleic acid into host cells.

SOLUTION

The present inventors actively sought solution to the problems mentioned above. That is, the inventors analyzed the genome of producing lipids fungi Mortierella alpina and isolated from the genome sequences homologous to known genes of Mg 2+ -dependent phosphatidic acid phosphatase (PAP1). Moreover, for a continuous open reading frame (ORF) in the gene encoding PAP, full-length cDNA cloning was performed using cDNA library screening or PCR gene was introduced into host cells with high proliferative activity such as yeast. As a result, the inventors found that the protein encoded by the cloned cDNA has phosphatidic acid phosphatase activity, and the introduction of the cDNA into yeast reserve stocks increases lipid triacylglycerol in yeast. Thus, it has been successfully achieved cloning a gene related to a new phosphatidic acid phosphatase (PAP), and completed the present invention. Thus, the present invention is as described below.

(1) The nucleic acid according to any one of items (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatase phosphatidic acid;

(B) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein with the activity of phosphatase phosphatidic acid;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity;

(E) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and encodes a protein having phosphatidic acid phosphatase activity;

(F) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with phosphatidic acid phosphatase activity; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and comprising exon encoding a protein with the activity of phosphatase phosphatidic acid.

. (2) The nucleic acid according to (1), wherein the nucleic acid is from any one of (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatase activity phosphatidic acid;

(B) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C and encodes a protein having phosphatidic acid phosphatase activity;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity;

(E) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 in conditions of 2 × SSC at 50 ° C, and encodes a protein having phosphatidic acid phosphatase activity;

(F) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50 ° C and comprising exon encoding a protein having phosphatidic acid phosphatase activity; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and comprising exon encoding a protein with the activity of phosphatase phosphatidic acid.

(3) The nucleic acid according to any one of items (a) to (d), shown below:

(A) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 or a fragment thereof;

(B) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 or a fragment thereof;

(C) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9 or a fragment thereof; and

(D) a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, or a fragment thereof.

(4) The nucleic acid according to any one of items (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having activity amplifying the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1;

(B) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having an activity of enhancing the production of DG and / or TG of PA in yeast strain deficient PAH1;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, and encodes a protein having an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(E) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and encodes a protein with an activity enhancing production DG and / or TG of PA in yeast strain deficient PAH1;

(F) a nucleic acid comprising a nucleotide sequence which under stringent conditions can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with an activity of enhancing production of DG and / or TG of PA in yeast strain deficient PAH1; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and comprising exon encoding a protein with the activity of enhancing the production of DG and / or TG of PA in yeast strain deficient PAH1.

(5) The nucleic acid according to paragraph (4), wherein the nucleic acid is any of (a) to (g), shown below:

(A) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having activity enhancing the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1;

(B) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C and encodes a protein with an activity enhancing production DG and / or TG of PA in yeast strain deficient PAH1;

(C) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(D) a nucleic acid comprising a nucleotide sequence encoding a protein which consists of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing production of DG and / or TG of the PA in the yeast strain deficient PAH1;

(E) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 in conditions of 2 × SSC at 50 ° C, and encodes a protein having an activity of enhancing production of DG and / or TG of PA in yeast strain deficient PAH1;

(F) a nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50 ° C, and includes exon coding for a protein which has an activity of enhancing production of DG and / or TG of PA in yeast strain deficient PAH1; and

(G) a nucleic acid comprising a nucleotide sequence consisting of the nucleotide sequence of 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein which has an activity enhancing production DG and / or TG of PA in yeast strain deficient PAH1.

(6) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase; and

(B) a protein which consists of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase.

(7) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase; and

(B) a protein which comprises the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase.

(8) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1; and

(B) a protein which consists of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing production of DG and / or TG of PA in yeast strain deficit PAH1.

(9) The protein of step (a) or (b), shown below:

(A) a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing the production of diacylglycerol (DG) and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1; and

(B) a protein which comprises the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of enhancing production of DG and / or TG of PA in yeast strain deficit PAH1.

(10) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7.

(11) A recombinant vector comprising a nucleic acid according to any one of items (1) to (5).

(12) A transformant transformed with the recombinant vector according to (11).

(13) fatty acid composition comprising fatty acid or lipid obtained by culturing the transformant according to item (12).

(14) A method for producing a fatty acid composition characterized by obtaining a fatty acid from a culture or lipid obtained by culturing the transformant according to item (12).

(15) A food product comprising the fatty acid composition of the item (13).

USEFUL EFFECTS OF THE INVENTION

PAP of the present invention may enhance the ability of products of fatty acids and lipids in the reserve cells in which the introduced PAP, and preferably can intensify the production of polyunsaturated fatty acids in microorganisms and plants.

It is believed that the PAP of the present invention facilitates the production of fatty acids in the host cell, the fatty acid composition different from the composition of fatty acids produced by a host cell, which is not administered PAP. This may provide lipids with desired properties and effects, and thus is useful for use in the production of, for example, food products, cosmetics, pharmaceutical products, and soaps.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1-1 shows a comparison between the genomic sequence (SEQ ID NO: 5) and the ORF (SEQ ID NO: 1) MaPAH1.1, derived from a strain of M. alpina 1S-4.

Figure 1-2 is a continuation of figure 1-1.

Figure 1-3 is a continuation of figure 1-2.

Figure 1-4 is a continuation of figure 1-3.

Figure 2-1 shows a comparison between the genomic sequence (SEQ ID NO: 10) and ORF (SEQ ID NO: 6) MaPAH1.2, derived from a strain of M. alpina 1S-4.

Figure 2-2 is a continuation of figure 2-1.

Figure 2-3 is a continuation of figure 2-2.

Figure 2-4 is a continuation of figure 2-3.

Figure 3-1 shows the cDNA (SEQ ID NO: 4) MaPAH1.1, derived from a strain of M. alpina 1S-4 and produced on the basis of the amino acid sequence (SEQ ID NO: 2).

Figure 3-2 is a continuation of figure 3-1.

Figure 3-3 is a continuation of figure 3-2.

Figure 4-1 shows the cDNA (SEQ ID NO: 9) MaPAH1.2, derived from a strain of M. alpina 1S-4 and produced on the basis of the amino acid sequence (SEQ ID NO: 7).

Figure 4-2 is a continuation of figure 4-1.

Figure 5-1 shows a comparison of the amino acid sequence obtained (SEQ ID NO: 2) MaPAH1.1 and resulting amino acid sequence (SEQ ID NO: 7) MaPAH1.2, derived from a strain of M. alpina 1S-4, phosphatidic acid phosphatase family PAP1, ScPAH1 protein (SEQ ID NO: 19) derived from yeast, Saccharomyces cerevisiae , and Lipina amino acid sequence (SEQ ID NO: 20) derived from the mouse. The acid phosphatase phosphatidic family PAP1 N-terminal region is quite conservative and marked as Liping, conservative N-terminal region (pfam04571). In MaPAH1.1 and MaPAH1.2 N-terminal region it is also quite conservative. In this sequence, the presence of a glycine residue which are marked * (corresponding to the 80th amino acid of SEQ ID NO: 2, and the 80th amino acid of SEQ ID NO: 7), is necessary for PAP activity. Sequence, marked by double underline (corresponding to from 819th to 823 th amino acids of SEQ ID NO: 2 and from the 737 th to 741 th amino acid from SEQ ID NO: 7) represents DXDX motif (T / V), present in galogenokislot dehalogenase (HAD) -like domain. This motif is also conserved in MaPAH1.1 and MaPAH1.2. Sequences above and below the relatively motif is also conserved.

Figure 5-2 is a continuation of figure 5-1.

Figure 6-1 shows a comparison of the sequence of CDS (SEQ ID NO: 3) MaPAH1.1 sequence and CDS (SEQ ID NO: 8) MaPAH1.2, derived from a strain of M. alpina 1S-4.

Figure 6-2 is a continuation of figure 6-1.

Figure 6-3 is a continuation of 6-2 pieces.

Figure 7 shows a comparison of the amino acid sequence obtained (SEQ ID NO: 2) MaPAH1.1 derived from the amino acid sequence (SEQ ID NO: 7) MaPAH1.2, derived from a strain of M. alpina 1S-4.

DESCRIPTION OF EMBODIMENTS

The present invention relates to a new phosphatidic acid phosphatase gene derived from the genus Mortierella, where phosphatidic acid phosphatase dephosphorylates phosphatidic acid to form diacylglycerol.

According to the present invention, phosphatidic acid phosphatase is an enzyme catalyzing the reaction for the formation of diacylglycerol in dephosphorylation of phosphatidic acid. PAP substrate of the present invention is generally phosphatidic acid, but it is not limited.

Nucleic acid encoding phosphatidic acid phosphatase of the present invention

Phosphatidic acid phosphatase (PAP) in the present invention and includes MaPAH1.1 MaPAH1.2. Correspondence between cDNA, CDS and ORF, encoding and MaPAH1.1 MaPAH1.2, and between the amino acid sequences obtained are summarized in Table 1.

Table 1
MaPAH1.1 MaPAH1.2
SEQ ID NO The corresponding region in SEQ ID NO: 4 SEQ ID NO The corresponding region in SEQ ID NO: 9
cDNA SEQ ID NO: 4 ***** SEQ ID NO: 9 *****
CDS SEQ ID NO: 3 Regulations from 1 to 3985 SEQ ID NO: 8 Regulations 72 to 3791
ORF SEQ ID NO: 1 Regulations from 1 to 3982 SEQ ID NO: 6 Regulations 72 to 3788
The amino acid sequence SEQ ID NO: 2 ***** SEQ ID NO: 7 *****

Sequences MaPAH1.1 relating to the present invention include SEQ ID NO: 2, which is the amino acid sequence MaPAH1.1; SEQ ID NO: 1, which refers to a sequence region ORF MaPAH1.1; SEQ ID NO: 3, which refers to a sequence region CDS MaPAH1.1; and SEQ ID NO: 4, which is the nucleotide sequence for the cDNA MaPAH1.1. Among them, SEQ ID NO: 3 corresponds to nucleotides 1 to 3985 of SEQ ID NO: 4, whereas SEQ ID NO: 1 corresponds to nucleotides 1 to 3982 of SEQ ID NO: 4 and nucleotides 1 to 3982 of SEQ ID NO: 3. SEQ ID NO: 5 is the genomic nucleotide sequence encoding MaPAH1.1 the present invention. The genomic sequence of SEQ ID NO: 5 consists of eleven ten exons and introns. In SEQ ID NO: 5 exons region corresponds to nucleotides 1 to 182, from 370 to 584, from 690 to 1435, from 1536 to 1856, from 1946 to 2192, from 2292 to 2403, from 2490 to 2763, from 2847 to 3077, from 3166 to 3555, from 3648 to 3862 and from 3981 to 5034.

Sequences MaPAH1.2 relating to the present invention include SEQ ID NO: 7, which is an amino acid sequence MaPAH1.2; SEQ ID NO: 6, which is a sequence of the ORF MaPAH1.2; SEQ ID NO: 8, which is a sequence of the CDS MaPAH1.2, and SEQ ID NO: 9, which is the nucleotide sequence of the cDNA MaPAH1.2. Among them, SEQ ID NO: 8 corresponds to nucleotides 72-3791 SEQ ID NO: 9, whereas SEQ ID NO: 6 correspond to nucleotides 72 to 3788 of SEQ ID NO: 9 and between nucleotides 1 to 3717 of SEQ ID NO: 8. SEQ ID NO: 10 is a genomic nucleotide sequence encoding MaPAH1.2 the present invention. The genomic sequence of SEQ ID NO: 10 consists of eight exons and seven introns. In SEQ ID NO: 10 region with exons corresponds to nucleotides 1 to 454, from 674 to 1006, from 1145 to 1390, from 1479 to 1583, from 1662 to 1804, from 1905 to 2143, from 2243 to 3409 and from 3520 to 4552 .

Nucleic acids of the invention include single and double stranded DNA and RNA complementary to them, which can be either natural or artificially prepared. Examples of the DNA include, but are not limited to, genomic DNAs, cDNAs corresponding to genomic DNA, chemically synthesized DNA, PCR amplified DNA, combinations thereof and DNA / RNA hybrids.

Preferred embodiments for the nucleic acids of the invention comprise (a) nucleic acids comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, (b) nucleic acids comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, (c) nucleic acids having a nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9, and (d) nucleic acid comprising the nucleotide sequence recited in SEQ ID NO: 5 or SEQ ID NO: 10.

For these nucleotide sequences can be used on the nucleotide sequence data for EST or genomic DNA from organisms PAP activity to search a nucleotide sequence encoding a protein that is very similar to known proteins having activity PAP. Preferred organisms having activity PAP, lipids are producing fungi including, but not limited to, M. alpina .

To analyze EST cDNA library is first prepared. The cDNA library may be prepared, guided «Molecular Cloning, A Laboratory Manual 3rd ed.» (Cold Spring Harbor Press ( 2001)). Alternatively, a commercially available kit may be used to obtain a cDNA library. Examples of the method for preparing cDNA libraries suitable for the present invention are shown below. That is, a suitable strain of M. alpina , fungi producing lipids plated on appropriate medium and pre-cultured for an appropriate period of time. Culture conditions suitable for said preculture are, for example, medium composition of 1.8% glucose, 1% yeast extract and pH 6.0, the cultivation time is 3 to 4 days, and the cultivation temperature 28 ° C. Products preculture is then cultured under suitable basic conditions. Medium composition suitable for main culture includes, for example, 1.8% glucose, 1% soybean powder, 0.1% olive oil, 0.01% Adekanol, 0.3% KH 2 PO 4 , 0,1% Na 2 SO 4 , 0,05% CaCl 2 · 2H 2 O and 0,05% MgCl 2 · 6H 2 O and pH 6,0. Culture conditions suitable for main culture are, for example, shaking culture and aeration at 300 rev / min, 1 vvm and 26 ° C for 8 days. The appropriate amount of glucose may be added during culture. After culturing, the product is taken up in suitable times during main culture, from cells harvested for total RNA. Total RNA can be prepared by any known method such as the method using guanidine hydrochloride / CsCl. From the resultant total RNA, poly (A) + RNA can be isolated using a commercially available kit, and a cDNA library can be prepared using a commercially available kit. The nucleotide sequence of all clones obtained from the cDNA library was determined using primers designed on the basis of the vector to determine the nucleotide sequence of the insert. As a result, one can obtain EST. For example, when using a set ZAP-cDNA GigapackIII Gold Cloning Kit ( Stratagene Inc.) to obtain a cDNA library may direct cloning.

In the analysis of genomic DNA of an organism is cultured cells, having an activity PAP, and genomic DNA was prepared from the cells. Determine the nucleotide sequence of the obtained genomic DNA, and the assembly is subjected to the nucleotide sequence determined. The result obtained in the search performed superkontiga sequence sequence encoding an amino acid sequence having high homology with the amino acid sequence of a known protein PAP activity. From the sequence superkontiga being found in the search as a sequence encoding an amino acid sequence prepared primers. PCR was performed using the cDNA library as a template, and the resulting DNA fragment was introduced into plasmid cloning. For sample PCR using the cloned plasmid as a template and the above primers. Using these samples, cDNA library screening was performed.

Homology search between amino acid sequences derived from and MaPAH1.1 MaPAH1.2 the present invention was performed using BLASTp program concerning the amino acid sequences reported in GenBank. Data derived from the amino acid sequence MaPAH1.1 MaPAH1.2 and have the advantage of an alleged nuclear protein elongation and deformation (AAW42851), derived from the Cryptococcus neoformans var. neoformans JEC21, with the highest levels, and identity was 25.9% and 26.6%, respectively. MaPAH1.1 obtained and the amino acid sequences of the present invention MaPAH1.2 22.7% identical and 22.5%, respectively, with the amino acid sequence PAH1, derived from S. cerevisiae (to throughout the specification, also referred to as PAH1 yeast or ScPAH1 ), which was functionally analyzed among homologues PAP1 fungi.

The present invention also includes nucleic acids that are functionally equivalent to the nucleic acid including the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 6 (hereinafter also referred to as “the nucleotide sequence of the present invention”) or nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 (hereinafter also referred to as “amino acid sequence of the present invention”). The term “functionally equivalent” means that the protein encoded by the nucleotide sequence of the present invention and a protein consisting of the amino acid sequence of the present invention have an activity of phosphatidic acid phosphatase (PAP). Furthermore, the term “functionally equivalent” means an activity which enhances the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a strain of yeast PAH1 deficient in expression of the protein encoded by the nucleotide sequence of the present invention, or protein consisting of the amino acid sequence of the present invention. Activity of the protein of the present invention against PAP and activity, contributing to enhancing production DG and / or TG of PA in yeast strain deficient PAH1, may be Mg 2+ -dependent or Mg 2+ -independent. Activity of the protein of the present invention is preferably Mg 2+ -dependent.

Certain nucleic acids that are functionally equivalent in relation to the nucleic acids of the invention include nucleic acids comprising nucleotide sequences set forth below in any one of (a) to (g). It should be noted that in the descriptions of the nucleotide sequences listed below, the term “activity of the present invention” refers to “PAP activity and / or activity, which enhances the production of DG and / or TG of PA in yeast strain deficient PAH1».

(A) A nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of the present invention.

The nucleotide sequence contained in a nucleic acid of the present invention relates to nucleotide sequences encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO 7, and has an activity of the present invention.

In particular, the nucleotide sequence contained in a nucleic acid of the present invention is a nucleotide sequence encoding a protein having the above activity of the present invention and consisting of:

(I) an amino acid sequence with deletion of one or more (preferably one to several (e.g., 1 to 400, 1 to 200, 1 to 130, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(Ii) an amino acid sequence with substitution of one or more (preferably one to several (e.g., 1 to 400, 1 to 200, 1 to 130, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(Iii) an amino acid sequence with addition of one or more (preferably one to several (e.g., 1 to 400, 1 to 200, 1 to 130, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; or

(Iv) the amino acid sequence of any combination of (i) to (iii), described above.

Among the above, it is preferably conservative substitutions, which means the replacement of specific amino acid residue by another residue having similar physical and chemical properties. It can be any substitution that does not substantially change the structural properties of the original sequences. For example, any substitution is possible provided that substitutable amino acids disrupt helical structure is not the original sequence or do not disrupt any other type of secondary structure characterizing the original sequence.

Conservative substitution is generally introduced by synthesis using biological systems or chemical peptide synthesis, preferably by chemical peptide synthesis. In such cases, the substituent group may include an artificial amino acid residue, peptide mimetic or reversed or inverted form, where the region without changing the amino acid sequence or inverted faces.

Non-limiting examples of interchangeable amino acid residues arranged and are listed below:

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, O-metilserin, t-butylglycine, t-butyl alanine, and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, izoasparaginovaya acid izoglutaminovaya acid, 2-aminoadipic acid and 2-aminosuberinovaya acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, and 2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline and 4-hydroxyproline;

Group F: serine, threonine and homoserine; and

Group G: phenylalanine and tyrosine.

When non-conservative substitutions possible substitution of one element of the above class member from another class. In this case, in order to maintain the biological function of the protein of the present invention preferably consider indices of hydrophobicity of amino acids (amino acid hydrophobicity index) (Kyte, et al, J. Mol Biol, 157:… 105-131 (1982)).

In the case of a non-conservative substitution, amino acid substitutions can be performed on the basis of hydrophilicity.

It should be noted that as in a conservative substitution, and at a non-conservative substitution, amino acid residue corresponding to the 80th amino acid in SEQ ID NO: 2 or SEQ ID NO: 7, preferably glycine, and an area corresponding to amino acids 819 to 823 of SEQ ID NO: 2 or from amino acids 737 to 741 of SEQ ID NO: 7, preferably DXDX (T / V) (X is any amino acid).

Throughout the description and figures nucleotides, amino acids and their abbreviations are in accordance Commission on Biochemical Nomenclature IUPAC-IUB, or any other standard nomenclature used in the art, such as described in Immunology – A Synthesis (second edition, edited by ES Golub and DR Gren, Sinauer Associates, Sunderland, Massachusetts (1991)). Also, it assumes that amino acids that may have optical isomers are represented as L-isomers unless otherwise indicated.

Stereoisomers such as D-amino acids are amino acids mentioned above, synthetic amino acids such as α, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other unconventional amino acids may also be members constituting the proteins of the present invention.

It should be noted that in the protein notation throughout the description, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard practice, and the symbol in the art.

Similarly, in the main, if not stated otherwise, left-hand end of single-stranded polynucleotide sequences is the 5′-end and left-hand direction of double- stranded polynucleotide sequences is referred to as the 5′-direction.

Those skilled in the art competent in the field of design and preparation of suitable mutant proteins as described herein, using methods known in the art. For example, the region in the protein molecule that is suitable to change the structure of the protein without altering the biological activity of the present invention, can be identified by detecting an area that appears to be less important for the biological activity of the protein. You can also identify residues or regions that are conserved between similar proteins. Furthermore it is also possible to introduce conservative amino acid substitution in a region, which appears to be important for biological activity or structure of the protein of the present invention, the protein without altering the biological activity and without adversely affecting the polypeptide structure of the protein.

In particular, the amino acid sequences and MaPAH1.1 MaPAH1.2, the amino acid sequences of approximately 100 amino acids in the N-terminal region, referred to as lipin, N-terminal conserved region: pfam04571) against enzyme family Mg 2+ -dependent phosphatase phosphatidic acid (PAP1), is quite conservative. In addition, each of the amino acid sequences and MaPAH1.1 MaPAH1.2 has “motive DXDX catalytic site (T / V)», which is a conservative motif galogenokislot dehalogenase (HAD) -like protein superfamily of enzymes. Figure 5 correspond to data based DIDGT sequence (corresponding to residues from 819 to 823 of SEQ ID NO: 2 and residues from 737 to 741 of SEQ ID NO: 7), marked by double underline. As a mutant of the present invention may be any mutant, which retains the conserved motif and maintains the activity described above. Reported that changes in site conserved motif in the yeast PAP1 lead to loss of PAP activity (J. Biol Chem, 282 (51):.. 37026-37035, (2007)).

Those skilled in the art could implement the so-called structural and functional studies, which allow to identify residues of the peptide, which are important for biological activity or structure of the protein of the present invention and the residues of the peptide similar to the residues in a protein will allow for a comparison of the amino acid residues between the two these peptides and thus predict which the residue in the protein similar to the protein of the present invention, an amino acid residue corresponding to amino acid residue important for biological activity or structure. Furthermore, by selecting an amino acid substitution is chemically similar to the predicted amino acid residue of the mutant can be selected which maintains the biological activity of the protein of the present invention. Similarly, those skilled in the art could also analyze the three-dimensional structure and amino acid sequence of the mutant protein. Thus, the analysis results can then be used to predict the alignment of amino acid residues included in the spatial structure of the protein. Because amino acid residues, which was predicted by the presence on the protein surface may be involved in important interactions with other molecules, those skilled in the art could obtain a mutant in which there is no replacement of the data of amino acid residues, which was predicted by the presence on the protein surface on the basis of analysis results as mentioned above. Moreover, those skilled in the art could obtain mutants with single amino acid replacement at any of amino acid residues that form the protein of the present invention. These mutants can be selected by any of the known assays for collecting information about the individual mutants, which in turn allows to assess the practical value of individual amino acid residues that form the protein of the present invention by comparing the case where the mutant with the replacement of specific amino acid residue exhibited a lower bioactivity than the biological activity of the protein of the present invention, the case where the mutant shows no biological activity, or where the mutant exhibits an unacceptable activity which leads to inhibition of the biological activity of the protein of the present invention. Furthermore, on the basis of the information received after the specific routine experimentation or in combination with other mutations, those skilled in the art may readily analyze amino acid substitutions, which are undesirable for mutants of the protein of the present invention.

As described above, a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 can be prepared by methods such as site-directed mutagenesis as described for example in «. Molecular Cloning, a Laboratory Manual 3rd ed» (Cold Spring Harbor Press ( 2001)); «Current Protocols in Molecular Biology» ( John Wiley & Sons (1987-1997); Kunkel, ( 1985), Proc Natl Acad Sci USA, 82: 488-92; and Kunkel, (1988), Method Enzymol…. . ., 85: 2763-6 Preparation of the mutant with a mutation comprising a deletion, substitution or addition of amino acids can be carried out, for example, using known methods such as Kunkel method or by a method with the introduction of gaps in the DNA double chain, a method using a set of to introduce mutations by site-directed mutagenesis, such as set QuikChange TM site-directed mutagenesis Kit (Stratagene production) systems for site-directed mutagenesis GeneTailor TM (Invitrogen production) or system for site-directed mutagenesis TaKaRa (e.g., Mutan-K, Mutan-Super Express Km; Takara Bio Inc. production).

Methods allowing to carry out deletion, substitution or addition of one or more amino acids in the amino acid sequence of the protein while maintaining its activity include, in addition to the above site-directed mutagenesis, a method of gene mutation treatment and a method for the selective cleavage of the gene and deletion, substitution or addition of selected nucleotide and then ligating gene.

The nucleotide sequence contained in the nucleic acid of the present invention is preferably a nucleotide sequence which encodes a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and having PAP activity.

The nucleotide sequence contained in the nucleic acid of the present invention preferably comprises a nucleotide sequence which encodes a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO : 7, and having an activity of the present invention.

The number of sites with mutations leading to amino acid replacement or modifications in the protein of the present invention are not limited provided that maintain the activity of PAP activity or enhancing production of DG and / or TG of PA in yeast strain deficient PAH1.

PAP activity or the activity-enhancing production DG and / or TG of PA in yeast strain deficient PAH1, can be evaluated by known methods, e.g., see J. Biol. Chem., 273, 14331-14338 (1998).

For example, “Activity PAP» of the present invention can be measured as described below: The source of the enzyme solution was prepared by destruction of the transformed cells expressing the PAP of the present invention, centrifuging and collecting the lysate supernatant. The resulting original enzyme solution can be subjected to further purification of PAP of the present invention. The source of the enzyme solution containing the present izobreteniyuili PAP PAP purified according to the present invention is added to the reaction solution containing 0.5 mM phosphatidic acid, 10 mM 2-mercaptoethanol and 50 mM Tris-HCl (pH 7,5), followed by carrying out the reaction at from 25 ° C to 28 ° C for a suitable time. The reaction was stopped by adding a mixture of chloroform and methanol, and isolated lipids. The resulting lipids are fractionated by thin layer chromatography to measure the amount of diacylglycerol obtained.

“Activity-enhancing production DG and / or TG of PA in yeast strain deficient PAH1» may be measured, for example as described below: a yeast strain deficient PAH1 obtained by destroying the gene of yeast ( S. cerevisiae ) ScPAH1. PAH1 strain deficient yeast as a host cell transformed with a vector comprising a nucleic acid encoding the PAP of the present invention and culturing the transformed strain is performed. Culture solution was centrifuged to collect cells. Cells are washed with water and lyophilized. To the dried cells were added chloroform and methanol, and destroy the cells using glass beads to isolate lipids. The isolated lipids are fractionated by thin layer chromatography and the amount of generated DG and / or TG. Yeast strain deficient PAH1, transformed with a vector not containing the nucleic acid encoding the PAP of the present invention is used as a control for comparison. If the amount of generated DG and / or TG increases in yeast strain deficient PAH1, transformed with a vector containing nucleic acid encoding the PAP of the present invention, the PAP is defined as PAP with the “activity-enhancing production DG and / or TG of PA in yeast strain deficit PAH1 ».

(B) nucleic acid which comprises a nucleotide sequence capable of hybridizing with the nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions, and encodes a protein having an activity of of the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to a nucleotide sequence that can hybridize with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions, and encodes a protein having activity according to the present invention.

This nucleotide sequence can be prepared, for example, using cDNA or genomic library by known hybridization methods such as colony hybridization, plaque hybridization or Southern blotting using samples obtained from an appropriate fragment in a manner known to those skilled in the art.

Detailed hybridization method mentioned in «Molecular Cloning, A Laboratory Manual 3rd ed.» (Cold Spring Harbor Press (2001), particularly Chapters 6 and 7), «Current Protocols in Molecular Biology» (John Wiley & Sons (1987-1997 ), in particular chapters 6.3 and 6.4) and «DNA Cloning 1:. Core Techniques, a Practical Approach 2nd ed» (Oxford University (1995), particularly chapter 2.10 for hybridization conditions).

The stringency of hybridization is determined mainly based on the hybridization conditions, more preferably under conditions of hybridization and washing conditions. The term “stringent conditions” as used throughout the specification is intended to include moderately or highly stringent conditions.

Specifically, examples of moderately stringent conditions include hybridization conditions of 1 × SSC to 6 × SSC at 42 ° C to 55 ° C, more preferably 1 × SSC to 3 × SSC at 45 ° C to 50 ° C, and most preferably 2 × SSC at 50 ° C. In the case of using a hybridization solution containing, for example, approximately 50% formamide, hybridization temperature used of from 5 ° C to 15 ° C lower than the temperature indicated above. washing conditions, for example, 0.5 × SSC to 6 × SSC at 40 ° C to 60 ° C. To a solution hybridization and washing solution to be added is usually from 0.05% to 0,2% SDS, preferably about 0,1% SDS.

Highly stringent (stringent conditions) include hybridization and / or washing at high temperature and / or lower salt concentration as compared to the moderately stringent conditions. Examples of the hybridization conditions comprise 0.1 × SSC to 2 × SSC at 55 ° C to 65 ° C, more preferably from 0.1 × SSC to 1 × SSC at 60 ° C to 65 ° C and most preferably 0, 2 × SSC at 63 ° C. wash conditions include, for example, 0.2 × SSC to 2 × SSC at 50 ° C to 68 ° C, and more preferably 0.2 × SSC at 60 ° C to 65 ° C.

Examples of hybridization conditions, in particular, used in the present invention include, but are not limited to, pre-hybridization in 5 × SSC, 1% SDS, 50 mM Tris-HCl (pH 7,5) and 50% formamide, incubated at 42 ° C , incubated overnight at 42 ° C in the presence of a probe to form hybrids, and washing three times in 0,2 × SSC, 0,1% SDS at 65 ° C for 20 minutes.

It is also possible to use a commercially available hybridization kit in which the radioactive compound is not used as a probe. In particular, it is used for hybridization, for example, a set of DIG nucleic acid detection kit (Roche Diagnostics) or ECL direct labeling & detection system (production Amersham).

Preferable examples of the nucleotide sequences included in the present invention include nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C, and encoding a protein having PAP activity.

(C) A nucleic acid comprising a nucleotide sequence which consists of the nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having the activity of the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to nucleotide sequences that consist of a nucleotide sequence of at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, and encode a protein having the activity of the present invention.

Preferably, such that the nucleic acid comprises a nucleotide sequence identical to at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98% or 99% or more) of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having activity according to the present invention.

The percentage similarity between two nucleotide sequences can be determined by visual evaluation and the mathematical calculation, but preferably determined by comparing sequence information of two nucleic acids, using a computer program. As computer programs for sequence comparison can be used, for example, the BLASTN program (Altschul et al, (1990), J. Mol Biol, 215:… 403-10), version 2.2.7, available through the National Library of Medicine, the web website: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html, or algorithm WU-BLAST 2.0. Standard default settings for WU-BLAST 2.0 are described at the specified website: http://blast.wustl.edu.

(D) nucleic acid comprising a nucleotide sequence that encodes an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and encodes a protein having activity according to the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to nucleotide sequences encoding the amino acid sequence 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and encodes a protein having an activity of the present invention. As the protein encoded by the nucleic acid of the present invention may be a protein having the amino acid sequence similarity with the amino acid sequences or MaPAH1.1 MaPAH1.2 provided that the protein is functionally equivalent protein having activity according to the present invention.

Specific protein examples include amino acid sequences with 75% or more, preferably 80% or more, more preferably 85% or more, and most preferably 90% or more (e.g., 95% or more, furthermore 98% or more) identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7.

The nucleotide sequence contained in the nucleic acid of the present invention is preferably a nucleotide sequence encoding the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and encoding a protein having activity present invention. More preferably, the nucleotide sequence encodes an amino acid sequence 95% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 and encodes a protein having activity according to the present invention.

The percentage similarity between two amino acid sequences can be determined by visual evaluation and mathematical calculation, or can be determined using a computer program. Examples of such a computer program include BLAST, FASTA (Altschul et al, J. Mol Biol, 215:… 403-410 (1990)) and ClustalW. In particular, various conditions (parameters) for the similarity search using the BLAST program described by Altschul et al. (Nucl. Acids. Res., 25, pp. 3389-3402, 1997) and are publicly available via the website of the National Center for Biotechnology Information (NCBI) in the US or Japan-based DNA database (DDBJ) (Guide to BLAST, Altschul et al ., NCB / NLM / NIH Bethesda, MD 20894; Altschul et al).. Also you can use a program such as software GENETYX Ver. 7 (Genetyx Corporation), DINASIS Pro (Hitachisoft) or Vector NTI (Infomax) for the processing of genetic information to determine percent identity.

Specific alignment algorithm to align multiple amino acid sequences can also display the matching sequences in specific areas of short and thus can detect a region with very high sequence similarity to these short areas, even if between the full length sequences have significant similarity. Additionally, BLAST algorithm may use a matrix substitutions BLOSUM62 amino acids, and following separation parameters can be used: (A) inclusion filter to mask segments of the analyzed sequence having low compositional complexity (as defined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993) ; also see Wootton and Federhen, 1996, «Analysis of compositionally biased regions in sequence databases», Methods Enzymol, 266: 554-71) or for masking segments, consisting of internal repetitions with low recurrence (as defined by the program XNU Claverie and. States (Computers and Chemistry, 1993), and (B) the threshold of statistical significance for identifying correspondences regarding the sequences from the database, or expected compliance probability, found only at random, according to the statistical model E-score (Karlin and Altschul, 1990); if statistical significance is attributed to more than a predetermined threshold for the E-score, the match is not observed.

(E) nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, with stringent conditions, and encodes a protein having activity according to the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention covers nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under stringent conditions, and encoding a protein having activity according to the present invention.

A protein consisting of the amino acid sequence presented in SEQ ID NO: 2 or SEQ ID NO: 7, and the hybridization conditions described above. Examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under stringent conditions, and encoding a protein having activity according to the present invention.

(F) A nucleic acid comprising a nucleotide sequence that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions, and includes exon coding a protein having activity according to the present invention.

The nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10 is the genomic DNA sequences encoding MaPAH1.1 MaPAH1.2 and the present invention respectively.

The nucleotide sequence contained in the nucleic acid of the present invention covers nucleotide sequences that can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions, and includes exon coding for a protein having the activity of the present invention.

This nucleotide sequence can be prepared by methods known to those skilled in the art, e.g., from genomic libraries by known hybridization method such as colony hybridization, plaque hybridization or Southern blotting using probes derived from a suitable fragment. Hybridization conditions are described above.

(G) nucleic acid comprising a nucleotide sequence which consists of the nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein having the activity of the present invention.

The nucleotide sequence contained in the nucleic acid of the present invention relates to nucleotide sequences that consist of a nucleotide sequence identity of at least 70% with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, and It encodes a protein having activity according to the present invention. Preferred examples of nucleotide sequences include sequences having a similarity of at least 75%, more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98 % or 99% or more) with the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 and containing an exon encoding a protein having the activity of the present invention. The percentage similarity between two nucleotide sequences can be determined as described above.

The sequence of the genomic DNA of SEQ ID NO: 5 consists of eleven ten exons and introns. In SEQ ID NO: 5 regions with exons correspond to nucleotides 1 to 182, from 370 to 584, from 690 to 1435, from 1536 to 1856, from 1946 to 2192, from 2292 to 2403, from 2490 to 2763, from 2847 to 3077 from 3166 to 3555, from 3648 to 3862 and from 3981 to 5034. The sequence of the genomic DNA of SEQ ID NO: 10 consists of eight exons and seven introns. In SEQ ID NO: 10 region with exons correspond to nucleotides 1 to 454, from 674 to 1006, from 1145 to 1390, from 1479 to 1583, from 1662 to 1804, from 1905 to 2143, from 2243 to 3409 and from 3520 to 4552 .

In another embodiment, examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences containing regions with introns from the nucleotide sequence are 100% identical to the genomic DNA sequence represented in SEQ ID NO: 5 or SEQ ID NO: 10, and regions with exons have a nucleotide sequence identical to at least 70% or more, more preferably 75% or more and more preferably 80% or more (e.g., 85% or more, more preferably 90% or and most preferably more than 95%, 98% or 99% or more) of the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, where exon encodes a protein having activity according to the present invention.

In another embodiment, examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences containing regions with exons from the nucleotide sequence are 100% identical to the genomic DNA sequence represented in SEQ ID NO: 5 or SEQ ID NO: 10, and regions with introns from the nucleotide sequence identical to at least 70% or more, more preferably 75% or more and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more and most preferably 95%, 98% or 99% or more) of the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, wherein the area with the introns can be cut by the splicing, and thus the area with exons ligated, encoding a protein having activity according to the present invention.

In another embodiment, examples of the nucleotide sequence contained in the nucleic acid of the present invention include nucleotide sequences containing regions with introns from the nucleotide sequence identical to at least 70% or more, more preferably 75% or more, and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95%, 98% or 99% or more) of genomic DNA sequences presented in SEQ ID NO: 5 or SEQ ID NO: 10 and a region to exon with the nucleotide sequence identical to at least 70% or more, more preferably 75% or more and more preferably 80% or more (e.g., 85% or more, more preferably 90% or more, and most preferably 95% or more, 98% or more or 99% or more) of the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, wherein the area with the introns can be cut by splicing, and thus the area with ligated exons, encoding a protein having activity according to the present invention.

The percentage similarity between two nucleotide sequences can be determined in the manner described above.

A nucleic acid of the present invention relates to nucleic acids, each of which consists of the nucleotide sequence with deletion, substitution or addition of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having activity of the present invention. More specifically, the nucleic acid for use include any one of the following nucleotide sequences:

(I) a nucleotide sequence with deletion of one or more (preferably one to several (e.g., 1 to 1200, 1 to 1000, 1 to 750, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(Ii) a nucleotide sequence with the replacement of one or more (preferably one to several (e.g., 1 to 1200, 1 to 1000, 1 to 750, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(Iii) a nucleotide sequence with addition of one or more (preferably one to several (e.g., 1 to 1200, 1 to 1000, 1 to 750, 1 to 500, 1 to 400, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 30, 1 to 25, 1 to 20 or 1 to 15, more preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)) nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6; or

(Iv) a nucleotide sequence with any combination of from (i) to (iii) above, wherein the nucleotide sequence encodes a protein having activity according to the present invention.

A preferred embodiment of the nucleic acid of the present invention also includes nucleic acid fragment comprising a nucleotide sequence shown in any one of (a) to (d), shown below:

(A) the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(B) a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(C) the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9; and

(D) the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

(A) The nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, (b) a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and ( c) the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9 are provided as shown in table 1. The nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10, as described above. Fragments of these sequences are ORF, CDS, a biologically active region, a region for use as a primer, as described below, and the region that can serve as a probe contained in the data of the nucleotide sequences, and may be of natural origin or artificially obtained.

A nucleic acid of the present invention relates to nucleic acids listed below.

(1) The nucleic acids represented by any one of (a) to (g) below:

(A) nucleic acids comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(B) nucleic acids which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions;

(C) nucleic acid comprising a nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(D) nucleic acid comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(E) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under stringent conditions;

(F) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions; and

(G) nucleic acid comprising a nucleotide sequence with 70% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

(2) The nucleic acids described in (1) above, represented by any of (a) to (g) below:

(A) nucleic acids comprising a nucleotide sequence encoding a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(B) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C;

(C) nucleic acid comprising a nucleotide sequence 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6;

(D) nucleic acid comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(E) nucleic acid to be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, under conditions of 2 × SSC at 50 ° C;

(F) nucleic acid to be hybridized with a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50 ° C; and

(G) nucleic acid comprising a nucleotide sequence 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

Phosphatidic acid phosphatase of the present invention

The protein of the present invention includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and proteins functionally equivalent to this protein. These proteins may be naturally occurring or artificially prepared. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, described above. The term “functionally equivalent proteins” refers to proteins having “the activity of the present invention” described in the above “nucleic acid encoding phosphatidic acid phosphatase of the present invention”.

In the present invention, examples of proteins which are functionally equivalent to a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, include, proteins presented below in (a) and (b):

(A) protein consisting of the amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of the present invention; and

(B) proteins comprising the amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having an activity of the present invention.

In the above, the amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7 or an amino acid sequence with 70% or more identical to the amino acid sequence shown in SEQ ID NO: 2, described above in “nucleic acid encoding phosphatidic acid phosphatase of the present invention”. “A protein having the activity of the present invention” includes mutant proteins encoded by nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6; proteins with mutations caused by different types of modifications such as deletion, replacement and addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; these modified proteins have, for example, modified amino acid side chains; and these proteins fused to other proteins, where the proteins have PAP activity and / or activity which enhances the production of diacylglycerol (DG), and / or triglyceride (TG) of phosphatidic acid (PA) in a yeast strain deficient PAH1.

The protein of the present invention may be prepared synthetically. In this case, the protein can be produced by chemical synthesis method such as Fmoc (method a fluorenilmetiloksikarbonilom) method or tBoc (tert-method butyloxycarbonyl). Furthermore, chemical synthesis can be used peptide synthesizers available from Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation or other manufacturers.

The protein of the present invention further includes the following proteins:

(1) (a) protein consisting of the amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7;

(B) proteins comprising the amino acid sequence with 80% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; and

(2) the protein according to any one of paragraphs (a) and (b) below:

(A) protein consisting of the amino acid sequence with deletion, substitution or addition of 1 to 200 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7; and

(B) protein consisting of the amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7.

Cloning of nucleic acids of the invention

PAP nucleic acid of the present invention can be cloned, for example using screening cDNA libraries using appropriate probes. Cloning can be performed by amplification by PCR using appropriate primers followed by ligation into a suitable vector. The cloned nucleic acid may further be subcloned into another vector.

It is possible to use commercially available plasmid vectors such as Script-pBlue TM SK (+) (Stratagene), pGEM-T (Promega), pAmp (TM: Gibco-BRL), p-Direct (Clontech) and pCR2.1-TOPO ( Invitrogen). When amplification by PCR, as primers can be any part of, for example, of the nucleotide sequence shown in SEQ ID NO: 4. For example, as the forward primer may be used NotI-PAH1-1-F: 5′- GCGGCCGCATGCAGTCCGTGGGAAG- 3 ‘(SEQ ID NO: 15) and as the reverse primer can be used MaPAH1-1-10R: 5′-TTCTTGAGTAGCTGCTGTTGTTCG-3’ (SEQ ID NO: 16). PCR was then performed using cDNA obtained from cells M. alpina , with the above primers, DNA polymerase, and any other compounds. While those skilled in the art can easily carry out the method according to, for example, «Molecular Cloning, A Laboratory Manual 3rd ed.» (Cold Spring Harbor Press (2001)), PCR conditions in the present invention may be, for example, described below.

Denaturation temperature from 90 ° C to 95 ° C.

annealing temperature: 40 ° C to 60 ° C.

Elongation temperature: from 60 ° C to 75 ° C.

Number of cycles: 10 or more cycles.

The resulting PCR products can be purified by known methods, for example using a set such as a set of GENECLEAN (Funakoshi Co., Ltd.), QIAquick PCR purification (QIAGEN) or ExoSAP-IT (GE Healthcare Bio-Sciences)); filter of cellulose or DEAE-tubes for dialysis. In the case of using an agarose gel after PCR product was subjected to agarose gel electrophoresis and nucleotide sequence fragments are excised from the agarose gel and purified, for example, using a set of GENECLEAN (Funakoshi Co., Ltd.), or set QIAquick Gel extraction (QIAGEN) or using the freeze-squeeze method.

The nucleotide sequence of the cloned nucleic acids may be determined by sequencing nucleotides.

Construction of expression vector for PAP and obtain transformants

The present invention also relates to a recombinant vector comprising a nucleic acid encoding the PAP of the present invention. The present invention further relates to a transformant transformed using this recombinant vector.

Recombinant vector and transformant can be obtained as follows: a plasmid with the nucleic acid encoding the PAP of the present invention, digested with a restriction enzyme. Examples of restriction enzymes include, but are not limited to, EcoRI, KpnI, BamHI and SalI. The end may be blunt using polymerase T4. The digested DNA fragment was isolated by agarose gel electrophoresis. This DNA fragment was inserted in known manner into an expression vector for the expression vector for PAP. This expression vector is introduced into a host cell to obtain a transformant for expression of the desired protein.

In this case, the kind of the expression vector and the host can be any type that allows to express the desired protein. Examples of a host include fungi, bacteria, plants, animals and their cells. Examples of fungi include filamentous fungi such as lipid-producing M. alpina , and yeast strains such as Saccharomyces cerevisiae . Examples of bacteria include Escherichia coli and Bacillus subtilis . Additional examples of plants include oil plants such as rapeseed, soybean, cotton, safflower and flax.

It is possible to use microorganisms that produce lipids, for example strains described in MYCOTAXON, Vol. XLIV, NO. 2, pp. 257-265 (1992), and specific examples thereof include microorganisms belonging to the genus Mortierella, such as microorganisms belonging to the subgenus Mortierella, such as Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina IFO8568, ATCC16266, ATCC32221, ATCC42430, CBS 219.35, CBS224.37, CBS250.53, CBS343.66 , CBS527.72, CBS528.72, CBS529.72, CBS608.70 and CBS754.68; and microorganisms belonging to the subgenus Micromucor , such as Mortierella spp isabellina CBS194.28, IFO6336, IFO7824, IFO7873, IFO7874, IFO8286, IFO8308, IFO7884, Mortierella spp nana IFO8190, Mortierella spp ramanniana IFO5426, IFO8186, CBS112.08, CBS212.72, IFO7825, IFO8184 , IFO8185, IFO8287 and Mortierella spp vinacea CBS236.82. In particular, it is preferred Mortierella spp exchange alpina .

When used as a host fungus, the nucleic acid of the present invention is preferably autonomously replicable in the host cell, or preferably has the structure suitable for inclusion in the fungal chromosome. Preferably, the nucleic acid also comprises a promoter and terminator. When used as the master M. alpina as an expression vector can be used, for example, pD4, pDuraSC or pDura5. Can be any promoter allowing expression in a host cell, and examples thereof include promoters derived from M. alpina , such as histonH4,1 gene promoter, GAPDH promoter gene (glyceraldehyde 3-phosphate dehydrogenase) gene promoter and TEF (factor translation and elongation).

Examples of the administration method of the recombinant vector into filamentous fungi such as M. alpina , include electroporation, spheroplast method with, particle delivery method, and direct microinjection of DNA into the nucleus. In the case of using an auxotrophic host strain transformed strain can be obtained by selecting a strain which grows on selective medium without a specific nutrient substance (s). Alternatively, when using a transformation marker gene for resistance to a drug, a colony of cells which are resistant to a drug, obtainable by culturing the host cells in a selective medium with drug.

When using yeast as a host, an expression vector can be used, for example, pYE22m. Alternatively, a commercially available yeast expression vectors such as pYES (Invitrogen) and pESC (STRATAGENE). Examples of hosts useful in the present invention include, but are not limited to, a strain of Saccharomyces cerevisiae EH13-15 (trp1, MATα). Promoter which may be used is, for example, a promoter derived from yeast, such as a promoter, GAPDH, gal1 promoter or promoter gal10.

Examples of the administration method of the recombinant vector in yeast includes the lithium acetate method, electroporation, spheroplast method with, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, encapsulation of polynucleotide (s) in liposomes, and direct microinjection of the DNA into the nucleus.

When used as a bacterial host such as E. coli , as an expression vector can be used, for example, pGEX and pUC18, available from Pharmacia. Promoters which may be used include promoters derived from, for example, E. coli or phage, such as trp promoter, lac, PL promoter and the promoter PR. Examples of the administration method of the recombinant vector into bacteria include electroporation and calcium chloride method.

A method for producing a fatty acid composition of the present invention

The present invention relates to a method for producing a fatty acid composition of the transformant described above, i.e. a method for producing a fatty acid composition of the culture product obtained by culturing the transformant. fatty acid composition contains a set of one or more fatty acids. As fatty acids may be free fatty acids or they may be presented in the form of lipids containing fatty acids, such as a triglyceride or a phospholipid. In particular, the fatty acid composition of the present invention can be prepared in the following manner. Alternatively, the fatty acid composition can also be obtained by any other known method.

As a medium for use in culturing the organism expressing the PAP, can be any culture solution (medium) having appropriate pH and osmotic pressure and containing a biological substance, such as nutrients, micronutrients, serum and antibiotics required for growth of each host. For example, in the case of the transformed yeast expressing PAP medium nonlimiting examples include SC-Trp medium, YPD medium and YPD5 environment. Composition particular environment, for example, a medium SC-Trp, as indicated below: One liter of the medium comprises 6.7 g of agar bases for yeast nitrogen without amino acids (DIFCO), 20 g glucose and 1.3 g amino acid powder with (a mixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine, 11.25 g serine, 0.9 g tyrosine, 4.5 g valine, 6 g threonine and 0.6 g uracil).

Culture can be any conditions that are suitable for host growth and are suitable for maintaining stability of the enzyme produced. In particular, the degree of specific conditions include anaerobic conditions, cultivation time, temperature, humidity and static culturing or cultivation conditions using mixing, which can be varied. Cultivation can be carried out under the same conditions (one-step culture) or by so-called two-step or three-step culture using two or more different culture conditions. For the massive cultivation it is preferable to two or more staging cultivation due to the high efficiency of cultivation.

When a two-stage culturing using yeast as a host of the fatty acid composition of the present invention can be prepared as follows: In a preliminary culturing a transformant colony is transferred, for example, SC-Trp medium and cultured under shaking at 30 ° C for two days. Next, 500 l of the solution after preculture to main culture as added to 10 ml YPD5 medium (2% yeast extract, 1% polypeptone, 5% glucose), followed by culturing under shaking at 30 ° C for two days.

fatty acid composition of the present invention

The present invention also relates to a fatty acid composition as an aggregate of one or more fatty acids in cells expressing the PAP of the present invention, preferably a fatty acid composition obtained by culturing a transformant expressing the PAP of the present invention. Fatty acids can be in the form of free fatty acids or may be present in the form of lipids containing fatty acids, such as a triglyceride or a phospholipid.

Fatty acids contained in the fatty acid composition of the present invention are linear or branched monocarboxylic acids carbohydrate with a long chain, such examples include, but are not limited to, myristic acid (tetradecanoic acid) (14: 0), myristoleic acid (tetradecanoic acid) (14: 1), palmitic acid (hexadecanoic acid) (16: 0), palmitoleic acid (9-hexadecanoic acid) (16: 1), stearic acid (octadecanoic acid) (18: 0), oleic acid (cis-9 -oktadekanovaya acid) (18: 1 (9)), vaccenic acid (11-octadecanoic acid) (18: 1 (11)), linoleic acid (cis, cis-9,12 octadecadienoic acid) (18: 2 (9, 12)), α-linolenic acid (9,12,15-octadecatrienoic acid) (18: 3 (9,12,15)), γ-linolenic acid (6,9,12-octadecatrienoic acid) (18: 3 ( 6,9,12)), stearidonic acid (6,9,12,15-octadecatetraenoic acid) (18: 4 (6,9,12,15)), arachidic acid (eicosanoic acid) (20: 0), ( 8,11-eykozadienovaya acid) (20: 2 (8,11)), Mead acid (5,8,11-eykozatrienovaya acid) (20: 3 (5,8,11)), dihomo-γ-linolenic acid ( 8,11,14-eykozatrienovaya acid) (20: 3 (8,11,14)), arachidonic acid (5,8,11,14-eicosatetraenoic acid) (20: 4 (5,8,11,14)) , eicosatetraenoic acid (8,11,14,17-eicosatetraenoic acid) (20: 4 (8,11,14,17)), eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid) (20: 5 (5,8,11,14,17)), behenic acid (docosanoic acid) (22: 0), (7,10,13,16-docosatetraenoic acid) (22: 4 (7,10,13,16) ), (7,10,13,16,19-docosapentaenoic acid) (22: 5 (7,10,13,16,19)), (4,7,10,13,16-docosapentaenoic acid) (22: 5 (4,7,10,13,16)), (4,7,10,13,16,19-docosahexaenoic acid) (22: 6 (4,7,10,13,16,19)), lignoceric acid (tetrakozanovaya acid) (24: 0), nervonic acid (cis-15-tetradokozanovaya acid) (24: 1) and cerotic acid (geksakozanovaya acid) (26: 0). It should be noted that the names of the compounds are presented as generic names according Biochemical Nomenclature IUPAC, and their systematic names with the numeric values ​​that reflect the number of carbon molecules and position of double bonds are listed in parentheses.

fatty acid composition of the present invention may consist of any number and type of fatty acid, with the proviso that it is a combination of one or more fatty acids selected from fatty acids listed above.

Food or other products comprising fatty acid compositions of the present invention

The present invention also relates to a food product comprising the fatty acid composition as described above. The composition of fatty acids of the present invention can be used to produce food products containing fats and oils and receiving industrial raw materials (for example, raw materials for cosmetics, pharmaceuticals (e.g., for external skin application) and washing) by standard methods. Cosmetics (cosmetic compositions) or pharmaceuticals (pharmaceutical compositions) may be formulated in any dosage form including, but not limited to, solutions, pastes, gels, solids, and powders. Examples of food forms include pharmaceutical preparations such as capsules; liquid food naturally occurring-partially digested food and elemental liquid food, wherein the fatty acid composition of the present invention is mixed with proteins, sugars, fats, trace elements, vitamins, emulsifiers and flavorings; and it produced in the form of shapes such as drinkable preparations or as enteral nutrient.

In addition, examples of the food product of the present invention include, but are not limited to, nutritional supplements, dietetic foods, healthy foods, food for children, children’s modified milk, modified milk for premature infants and nutrition for the elderly. Throughout the specification, the term “food product” is used as a collective term in respect of edible substances in solid form in the fluid form, liquid form, or mixtures thereof.

The term “nutritional supplement” refers to food products enriched with specific nutritional ingredients. The term “dietetic foods” refers to food products, which is healthy or beneficial to health, and nutritional supplements include, natural foods and diet foods. The term “health foods” refers to food products to maintain the nutrients that support the functioning of the body, and is synonymous with the product for the special applications for health. The term “children’s foods” refers to foods that are given to children up to about the age of 6 years. The term “foods for the elderly” refers to food products for easy digestion and absorption when compared to untreated products. The term “modified milk for children” refers to modified milk, which is given to children aged up to about one year. The term “modified milk for premature infants” refers to modified milk, which give premature babies until about 6 months after birth.

Examples of these food products include natural foods (treated with fats and oils) such as meat, fish and nuts; food supplements with added fats and oils in the preparation, such as Chinese food, Chinese noodles and soups; foods prepared using fats and oils as a medium for heat treatment, such as tempura (deep fried fish and vegetables), deep fried foods, fried tofu, fried Chinese rice, donuts and Japanese fried cookies from shortcrust pastry ( Carinthia); Food products based on fats and oils or processed foods supplemented with fats and oils during preparation such as butter, margarine, mayonnaise, relish, chocolate, instant noodles, caramel, cookies, muffins, cakes and ice-cream; and food, covered or coated with fats and oils when cooking completion, such as rice crackers, hard biscuits and sweet bread from the beans. However, the food products of the present invention is not limited to foods containing fats and oils, and other examples of food products include agricultural foods such as bakery products, noodles, cooked rice, sweets (e.g., candies, chewing gum, toffees, tablets, Japanese sweets), tofu, and products derived from them; fermented foods, such as refined sake, medicinal liquor, spicy liqueur (mirin), vinegar, soy sauce and miso; Food products derived from farm animals, such as yogurt, ham, bacon and sausages; seafood such as fish paste (kamaboko), deep fried fish paste (ageten) and fish pie (hanpen); and fruit drinks, soft drinks, sports, alcoholic beverages and tea.

A method of analysis and selection of strains with nucleic acid encoding the PAP, or PAP protein of the present invention

The present invention also relates to a method of analysis and selection of lipid producing fungi with a nucleic acid encoding the PAP, or PAP protein of the present invention. Details are given below.

(1) Method of analysis

One embodiment of the present invention is a method of lipid producing fungus analysis using nucleic acid encoding the PAP, or PAP protein of the present invention. The assay method of the present invention, for example, analyze lipid producing fungi strain as an experimental strain for activity evaluations of the present invention, using primers or probes designed based on the nucleotide sequence of the present invention. This analysis can be carried out by known methods such as those described in WO № WO01 / 040514 and JP-A-8-205900. The analysis method is briefly described below.

The first step is to obtain a genome test strain. Gene may be prepared by any known method such as Hereford method or potassium acetate method (see, e.g., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, p. 130 (1990)).

Primers or probes are designed based on the nucleotide sequence of the present invention, preferably the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6. The data as primers or probes may be any region of the nucleotide sequence of the present invention, and they can be constructed in any method. The number of nucleotides in a polynucleotide used as a primer is generally 10 or more preferably from 15 to 25. The number of nucleotides, primers suitable for franking, usually ranges from 300 to 2000.

Obtained as described above, the primers or probes are used to assess the presence of analyte in the genome sequence of strain, specific for the nucleotide sequence of the present invention. The sequence, which is specific for the nucleotide sequences of the invention can be identified by known techniques. For example, a polynucleotide comprising a part or the entire sequence specific to the nucleotide sequence of the present invention, or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence used as a primer, and a polynucleotide comprising a part or the entire sequence located above or below this sequence or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence used as the other primer, and nucleic acid amplified from the test strain by PCR or other methods. Further, for example, it is possible to determine the presence or absence of the amplification product and the molecular weight of the amplification product.

PCR conditions suitable for the method of the present invention is not particularly limited and, for example, may be as specified below.

Denaturation temperature from 90 ° C to 95 ° C.

annealing temperature: 40 ° C to 60 ° C.

Elongation temperature: from 60 ° C to 75 ° C.

Number of cycles: 10 or more cycles.

The resulting reaction products may be separated by agarose gel electrophoresis or any other method to determine the molecular mass of the amplification product. The activity of the present invention for the test strain can predict or estimate by confirming that the molecular weight of the amplification product is sufficient to cover a nucleic acid molecule corresponding to a region specific for a nucleotide sequence of the present invention. Furthermore, the activity of the present invention can predict or estimate with higher accuracy by analyzing the nucleotide sequence of the amplification product by a method described above. Activity evaluation method of the present invention described above.

Alternatively, the analysis of the present invention the activity of the present invention with respect to test strain can be assayed by culturing the test strain and measuring the expression level of PAP, encoded by the nucleotide sequence of the present invention, for example the sequence shown in SEQ ID NO: 1 or SEQ ID NO : 6. PAP expression level can be measured by culturing the test strain under certain conditions and determining the amount of mRNA or protein PAP. The amount of mRNA or protein can be determined using known methods. For example, the amount of mRNA can be determined by Northern hybridization or quantitative PCR in real time, and the amount of protein can be determined by Western blotting (Current Protocols in Molecular Biology, John Wiley & Sons, 1994-2003).

(2) A method for breeding

Another embodiment of the present invention is a method of lipid producing fungi selection using nucleic acid encoding the PAP, or PAP protein of the present invention. When selection of the present invention, a strain having a desired activity can be selected by culturing the test strain, measuring the expression level of PAP, encoded by the nucleotide sequence of the present invention, for example the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 6, and selecting strains with the desired level of expression. Alternatively, the desired strain may be chosen when using standard strain, culturing the strain and the standard test strain separately, measuring the expression level of each of the strains and strain comparisons of standard expression level with the expression level of the test strain. Specifically, for example, a standard strain and test strains are cultured under appropriate conditions and measuring the expression level of each strain. The strain exhibiting the desired activity can be selected by selecting a test strain with higher or lower expression level compared to the standard strain. The desired activity may be determined, for example, by measuring the expression level of PAP and the fatty acid composition produced by PAP, as described above.

When selection of the present invention studied strain having the desired activity can be selected by culturing the test strains and selecting the strains with high or low activity of the present invention. The desired activity may be determined, for example, by measuring the expression level of PAP and the fatty acid composition produced by PAP, as described above.

Examples of the standard test strain and strain include, but are not limited to, strains transformed with a vector of the present invention is modified strains against suppression of expression of a nucleic acid of the present invention, the strains subjected to mutagenesis, and strains from natural mutations. The activity of the present invention can be measured, for example, the method described in the specification, “nucleic acid encoding phosphatidic acid phosphatase of the present invention”. Examples mutational processes include, but are not limited to, physical methods such as ultraviolet light or radiation; Chemical methods and exposure to chemicals such as EMS (etilmetan sulfonate) or N-methyl-N-nitrosoguanidine (see, e.g., Yasuji Oshima ed., Biochemistry Experiments vol. 39, Experimental Protocols for Yeast Molecular Genetics, pp. 67-75 , Japan Scientific Societies Press).

Examples of strains for use as a standard strain of the present invention or the test strains include, but are not limited to, fungi that produce lipids, and the yeast described above. In particular, a standard strain and the strain can be analyzed with any combination of strains belonging to different genera or species, can be analyzed simultaneously, and one or more of the test strains.

The present invention is hereinafter described in detail with the following examples which are not intended to limit the scope of the invention.

EXAMPLES

Example 1: Analysis of the genome of M. alpina

Strain M. alpina 1S-4 was inoculated into 100 ml medium GY2: 1 (2% glucose, 1% yeast extract, pH 6,0) and cultured with shaking at 28 ° C for 2 days. Cells were collected by filtration to obtain genomic DNA using DNeasy (QIAGEN).

The nucleotide sequence of the genomic DNA was determined by 454 Roche GS FLX Standard. In this case the nucleotide sequence fragments from the library were sequenced in two parts, and the nucleotide sequence of the library “partners» (mate pair) sequenced in three passes. The resulting nucleotide sequences were aligned to obtain 300 superkontigov.

Example 2: cDNA Synthesis and Construction of cDNA Library

Strain M. alpina 1S-4 was inoculated into 100 ml medium (1.8% glucose, 1% yeast extract, pH 6,0) and cultured with shaking at 28 ° C for 4 days. Cells were collected by filtration and total RNA was prepared by the method guanidine hydrochloride / CsCl.

From the total RNA by reverse transcription cDNA was synthesized using SuperScript II RT (Invitrogen), using random hexamer. Moreover, total RNA was isolated from poly (A) + RNA using a set of Oligotex-dT30 <Super> mRNA Purification Kit (Takara Bio Inc.). CDNA library was constructed using a set of ZAP-cDNA GigapackIII Gold Cloning Kit ( STRATAGENE).

Example 3: Search for homologs PAH1, derived from S. cerevisiae

The amino acid sequence of PAP gene activity from Saccharomyces cerevisiae , PAH1 (YMR165C) (herein also may be designated as ScPAH1), it was subjected to analysis tblastn compared with the genomic nucleotide sequences of the strain M. alpina 1S-4. As a result, the advantage possessed superkontigi comprising the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10. The gene related to SEQ ID NO: 5 was named MaPAH1.1, a gene related to SEQ ID NO: 10, It was named MaPAH1.2.

Example 4: Cloning and MaPAH1.1 MaPAH1.2

(1) Preparation of probe

For cDNA cloning and gene MaPAH1.1 MaPAH1.2 gene obtained nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10, and corresponding primers were selected based on the analysis results using BLAST program described above.

MaPAH1-1-3F: 5′-CGCCAATACATTGACGTTTTCAG-3 ‘(SEQ ID NO: 11)

MaPAH1-1-5R: 5’-AGTTCCAGTCATTGAACTCGGGTGC-3 ‘(SEQ ID NO: 12)

MaPAH1-2-3F: 5’-GAGCCCAGTTGACCTTTGAGGCATTC-3 ‘(SEQ ID NO: 13)

MaPAH1-2-5R: 5’-CACTGAGAACGAGACCGTGTTGGCG-3 ‘(SEQ ID NO: 14)

PCR was conducted using ExTaq (Takara Bio Inc.), using as a template the cDNA library constructed in Example 2, and a combination of primer and MaPAH1-1-3F MaPAH1-1-5R primer or primer combination and MaPAH1-2-3F prymera MaPAH1 -2-5R at 94 ° C for 2 min, then 30 cycles (94 ° C for 30 sec, 55 ° C for 30 sec and 72 ° C for 2 min). A DNA fragment of about 0.6 kb was obtained for each combination, using a set of cloned TOPO-TA cloning Kit (Invitrogen) and the nucleotide sequence of the insert in the resulting plasmid. The plasmid obtained by using the above-mentioned combination of primers, with the sequence corresponding to nucleotides 2352 to 3010 of SEQ ID NO: 4, identified as pCR-MaPAH1.1-P; a plasmid obtained by using the last of these primer combinations, with the corresponding sequence from nucleotides 1615 to 2201 of SEQ ID NO: 9, identified as pCR-MaPAH1.2-P.

Then samples were obtained by PCR using the plasmid as a data matrix, and the primers described above. In the reaction used ExTaq (Takara Bio Inc., Japan), except that instead of dNTP mixture attached to label the amplified DNA solution was used to label the PCR (Roche Diagnostics) with digoxigenin (DIG) for the probe and probe MaPAH1.1 MaPAH1.2 . Then, using these probes, screening of a cDNA library were carried out.

The hybridization conditions were chosen as described below.

Buffer: 5 × SSC, 1% SDS, 50 mM Tris-HCl (pH 7,5), 50% formamide.

Temperature: 42 ° C (overnight).

wash conditions: in 0,2 ×× SSC, 0,1% SDS solution (65 ° C) for 20 min (three times).

For the detection kit was used DIG nucleic acid detection kit (Roche Diagnostics ). For each plasmid DNA was excised using plasmid excision in vivo of the phage clones obtained by screening. Plasmid inserts with the greatest length among the plasmids obtained by screening using MaPAH1.1 probe having a sequence ranging from position 1307 m and more from the sequence shown in SEQ ID NO: 4, and was named plasmid pB-MaPAH1.1p. The results of comparison of the amino acid sequence shown ScPAH1 that the plasmid pB-MaPAH1.1p contains the region encoding the N-terminal region PAH1.1. Comparison of the genomic sequence (SEQ ID NO: 5), which is expected by the results of BLAST analysis using contains MaPAH1.1 gene with N-terminal region of the amino acid sequence shown ScPAH1 that ATG in position 1 to 3 in the sequence presented in SEQ ID NO: 5 is the initiation codon. Each frame of the plasmid pB-MaPAH1.1p was translated into the amino acid sequence. The amino acid sequence was compared with the amino acid sequence ScPAH1, obtained from S. cerevisiae . As a result, it was shown that the TGA at positions 3985 to 3987 in the sequence shown in SEQ ID NO: 4 is the stop codon. Thus, for cloning of full-length cDNA, the following primers were designed.

NotI-PAH1-1-F: 5′-GCGGCCGCATGCAGTCCGTGGGAAG-3 ‘(SEQ ID NO: 15)

MaPAH1-1-10R: 5’-TTCTTGAGTAGCTGCTGTTGTTCG-3 ‘(SEQ ID NO: 16)

PCR was conducted using ExTaq (Takara Bio Inc.), using the above cDNA as a template and a primer combination NotI-PAH1-1-F primer and MaPAH1-1-10R at 94 ° C for 2 min followed by 30 cycles of (94 ° C for 30 sec, 55 ° C for 30 sec and 72 ° C for 2 min). The resulting DNA fragment of approximately 1.5 kbp was cloned using a set of TOPO-TA cloning Kit (Invitrogen) and the nucleotide sequence of the insert. The plasmid into which the cloned DNA fragment comprising a sequence of nucleotides from 1 to 1500 of SEQ ID NO: 4, was named pCR-MaPAH1.1-Np. The DNA fragment of about 1.4 kb obtained by cutting the plasmid pCR-MaPAH1.1-Np with restriction enzymes NotI and XhoI, the DNA fragment of about 3.7 kb obtained by pB-MaPAH1.1p cutting the plasmid with restriction enzymes NotI and BamHI, and a DNA fragment of about 2.1 kb obtained by cutting the plasmid pB-MaPAH1.1p using restriction enzymes XhoI and BamHI, was connected via ligation (TOYOBO) to obtain cDNA plasmid pB-MaPAH1.1, which is believed to contain the full length cDNA MaPAH1.1. The cDNA sequence comprising the full length ORF of MaPAH1.1, shown in SEQ ID NO: 4.

Separately, the plasmid with the greatest length among the inserts of plasmids obtained by screening using MaPAH1.2 probe has a nucleotide sequence shown in SEQ ID NO: 9. The results of this sequence comparison ScPAH1 plasmid with sequence derived from S. cerevisiae , shown that includes the full-length cDNA plasmid ORF of MaPAH1.2. This plasmid was named pB-MaPAH1.2 cDNA.

(2) Sequence analysis

The cDNA sequence (SEQ ID NO: 4) of gene MaPAH1.1 includes CDS (SEQ ID NO: 3) consisting of a sequence of nucleotides from 1 to 3987 and ORF (SEQ ID NO: 1) comprising the sequence of nucleotides from 1 to 3984. Installed amino acid sequence encoded by the gene MaPAH1.1, presented in SEQ ID NO: 2. The genomic sequence MaPAH1.1 gene ORF was compared with the sequence (figure 1). As a result, it was shown that the genomic gene sequence MaPAH1.1 consists of eleven ten exons and introns.

The cDNA sequence (SEQ ID NO: 9) gene MaPAH1.2 includes CDS (SEQ ID NO: 8) consisting of a sequence of nucleotides from 72 to 3791, and ORF (SEQ ID NO: 6) consisting of a sequence of nucleotides from 72 to 3788 . Installed amino acid sequence encoding gene MaPAH1.2, presented in SEQ ID NO: 7. Genomic sequence MaPAH1.2 gene ORF was compared with the sequence (figure 2). The genomic sequence of the gene MaPAH1.2 consists of eight exons and seven introns.

cDNA sequence and MaPAH1.1 MaPAH1.2 and amino acid sequences respectively set shown in Figure 3 and Figure 4.

With the program carried BLASTp search of homology between amino acid sequences set MaPAH1.1 and MaPAH1.2 and amino acid sequences from GenBank. Both amino acid sequence homology to reach the highest level since 1 nuclear protein elongation and the alleged distortion protein (AAW42851), derived from Cryptococcus neoformans var. neoformans JEC21, but with low identity, i.e. 25.9% and 26.6% respectively.

Among fungal homologues PAP1 and amino acid sequences MaPAH1.1 MaPAH1.2, derived from M. alpina of the present invention are identical to 22.7% and 22.5% respectively, with the amino acid sequence ScPAH1, which was subjected to functional analysis. The amino acid sequence and MaPAH1.1 MaPAH1.2, derived from M. alpina in the present invention were compared with known amino acid sequences and ScPAH1 Lipina obtained from mouse (Figure 5). In PAP1 family of enzymes, the amino acid sequence of the N-terminal region is highly conserved and is called Liping, N-terminal conserved region (pfam04571). Also MaPAH1.1 and MaPAH1.2, derived from M. alpina of the present invention, a well-known enzyme and N-terminal region are quite conservative. Moreover, the sequence DIDGT, marked by double underline in Figure 5, is a protein superfamily of enzymes such galogenokislot dehalogenase (HAD), and corresponds to the catalytic site motif conserved DXDX (T / V).

Sequences of CDS MaPAH1.1 MaPAH1.2 and compared with each other in order to demonstrate the identity of 54.7% (Figure 6), while the identity between amino acid sequences set was 35.6% (Figure 7).

Example 5: Expression and MaPAH1.1 MaPAH1.2 yeast

Construction of the expression vector and MaPAH1.1 MaPAH1.2:

MaPAH1.1 For expression in yeast expression vectors were constructed as follows.

Yeast expression vector pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228, 1995) was cut with the restriction enzyme EcoRI, and blunt-ended using a set Blunting Kit (TaKaRa Bio Inc.). The resulting fragment and linker pNotI, phosphorylated (8-mer) (TaKaRa Bio Inc.), attached to each other by means of ligation (TOYOBO) to construct pYE22mN vector. PYE22mN vector was cut with restriction enzymes NotI and KpnI, the resulting fragment was fused to the DNA fragment of approximately 4.2 kbp obtained plasmid pB-MaPAH1.1 when cutting the cDNA with restriction enzymes KpnI and NotI to ensure plasmid pYE-MaPAH1.1. Separately, pYE22mN vector was cut with restriction enzymes NotI and KpnI, the resulting fragment was fused to a DNA fragment about 3.8 kb in length, resulting in cutting of the cDNA plasmid pB-MaPAH1.2 with restriction enzymes KpnI and NotI to ensure plasmid pYE-MaPAH1.2.

Getting strain ΔScpah1: URA3 S. cerevisiae

For cloning ScPAH1 gene obtained from strain S288C S. cerevisiae , the following primers were prepared:

Primer KpnI-PAH1-F: 5′-GGTACCATGCAGTACGTAGGCAGAGCTC-3 ‘(SEQ ID NO: 17) and

Primer XhoI-PAH1-R: 5’-CTCGAGTTAATCTTCGAATTCATCTTCG-3 ‘(SEQ ID NO: 18).

Strain S288C S. cerevisiae was cultured in a liquid medium YPD (2% yeast extract, 1% polypeptone, 2% glucose) at 30 ° C overnight. DNA was isolated from cells using the Dr. GenTLE (yeast) (TaKaRa Bio Inc.), and ScPAH1 gene was amplified by PCR with ExTaq, using as template DNA and primers derived KpnI-PAH1-F and XhoI-PAH1-R. The resulting DNA fragment of approximately 2.5 kbp was cloned using a set of TOPO TA cloning Kit, a clone with the correct nucleotide sequence was determined as pCR-ScPAH1. DNA fragment of about 0.4 kb obtained by cutting the pCR-ScPAH1 with restriction enzymes EcoRI and EcoRV, and a DNA fragment of about 2.1 kb obtained by cutting with pCR-ScPAH1 the restriction enzymes EcoRV and XhoI, were ligated into the vector pBluescriptIISK +, cut with restriction enzymes EcoRI and XhoI, to obtain plasmid pBScPAH1. PBScPAH1 plasmid was cut with restriction enzymes HincII and EcoRV and ligated with a fragment of DNA of approximately 1.2 kb obtained by cutting pURA34 plasmid (Japanese Unexamined Patent Application № 2001-120276) with the restriction enzyme HindIII, and then blunt-ended. The resulting product is a URA3 gene in the same direction as the gene ScPAH1, pBΔpah1 plasmid identified as: URA3. Further strain YPH499 S. cerevisiae (lys2-801amber ade2-101ochre ura3-52 trp1-Δ63 his3-Δ200 leu2-Δ1 a) (STARATAGENE) as a host transformed with a DNA fragment prepared by cutting plasmid pBΔpah1: URA3 with a restriction enzyme EcoRI. Transformed strain selected for the ability to grow on agar medium SC-Ura (one liter of the medium comprises 6.7 g of agar bases for yeast nitrogen without amino acids (DIFCO), 20 g glucose, 1.3 g amino acid powder (a mixture of 1.25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine, 11 25 g serine, 0.9 g tyrosine, 4.5 g valine, 6 g threonine and 1.2 g tryptophan) and agar medium (2% agar)). A strain whose PCR confirmed that Δpah1 structure: URA3 and introduced into a strain that is damaged ScPAH1 gene was determined as a strain ΔScpah1: URA3.

Preparation of transformed strain:

Strain ΔScpah1: URA3 was used as the host and transformed with a plasmid pYE22m, pYE-MaPAH1.1 or pYE-MaPAH1.2. The transformed strains were selected by ability to grow on agar medium SC-Ura, Trp (one liter of the medium comprises 6.7 g of agar bases for yeast nitrogen without amino acids (DIFCO), 20 g glucose, 1.3 g amino acid powder (a mixture of 1, 25 g adenine sulfate, 0.6 g arginine, 3 g aspartic acid, 3 g glutamic acid, 0.6 g histidine, 1.8 g leucine, 0.9 g lysine, 0.6 g methionine, 1.5 g phenylalanine , 11.25 g serine, 0.9 g tyrosine, 4.5 g valine and 6 g threonine) and agar medium (2% agar)). Two arbitrary shtmamma from corresponding strains transformed with each plasmid (control strains transformed with the plasmid pYE22m: C1 and C2, strains transformed with plasmid pYE-MaPAH1.1: MaPAH1.1-1 and MaPAH1.1-2, and strains transformed with plasmid pYE -MaPAH1.2: MaPAH1.2-1 and MaPAH1.2-2), was used in subsequent experiments.

Example 6: Measurement of the activity of Mg 2+ -dependent phosphatidic acid phosphatase (PAP1 activity)

Each yeast transformant strain was inoculated into 100 ml liquid medium SC-Ura, Trp and cultured with shaking at 30 ° C for one day. Initial enzyme solution prepared from the culture solution prepared as follows. In particular, the process carried out at 4 ° C or on ice. Cells were harvested from the culture solution by centrifugation and washed with water. Subsequently, cells were suspended in 5 ml of buffer A (50 mM Tris-HCl (pH 7,5), 0,3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). The cells were disrupted during treatment with French press (Thermo Fisher Scientific), Mini-Cell, three times at 16 psi. The cell lysate was centrifuged at 1500 × g for 10 minutes and the supernatant was collected as the source of enzyme solution. Protein concentration contained in the initial solution of the enzyme was measured using Protein Assay CBB Solution (5 ×) (Nacalai Tesque).

PAP1 activity was measured using a modified method according to Gil-Soo et al. (J. Biol. Chem., 282 (51), 37026-37035, (2007)) as described below. Because S. cerevisiae can not synthesize linoleic acid as substrate for PAP used dilinoleoil 1,2-glycero-sn-3-phosphate (18: 2-PA). We used five hundred microliters of the reaction solution. As the composition of the reaction solution was 100 .mu.l original enzyme solution, 50 mM Tris-HCl (pH 7,5), 100 ug / ml of 1,2-dilinoleoil-sn-glycero-3-phosphate, monosodium salt (Avanti Polar Lipids, Inc .), 1 mM MgCl 2 and 10 mM 2-mercaptoethanol. The reaction solution was maintained at 25 ° C for 30 minutes and then the reaction was terminated by the addition of chloroform: methanol (1: 2). Lipids were extracted by the method of Bligh-Dyer. By TLC (TLC) lipids fractionated on a silica gel 60 plate (Merck), using as eluent hexane: dietilovyyefir: acetic acid = 70: 30: 1. Lipids were visualized by spraying primulina solution (0.015% primulin in 80% aqueous acetone), and then irradiated with ultraviolet light. Fraction diacylglycerol (DG) was scraped from the plate and converted to fatty acid methyl ester by the method with hydrochloric acid / methanol. Next, the methyl ester fatty acids were extracted with hexane and the hexane was distilled off, followed by analysis by gas chromatography.

Table 2 shows the amounts of linoleic acid, transformed into DG fraction on the amount of protein in the initial enzyme solution.

table 2
transformed strain 18: 2 (pg / mg protein)
C1 15.43
C2 17.53
MaPAH1.1-1 56.03
MaPAH1.1-2 44.34
MaPAH1.2-1 19.45
MaPAH1.2-2 20,90

As indicated in Table 2, compared with C1 and C2, transformed using pYE22m, conversion activity 18: 2 to dilinoleina-PA (18: 2-DG) was about 3-fold and against MaPAH1.1-1 MaPAH1.1 -2 expressing MaPAH1.1, and approximately 1.2-fold and against MaPAH1.2-1 MaPAH1.2-2, expressing MaPAH1.2. This indicates that MaPAH1.1 MaPAH1.2 and have PAP activity.

PAP activity dependence of Mg 2+ was investigated as follows: five hundred microliters using reaction solution. Reaction and analysis were carried out under the same conditions as above, except that as the composition of the reaction solution was 100 .mu.l original enzyme solution, 50 mM Tris-HCl (pH 7,5), 100 ug / ml of 1,2-dilinoleoil -sn-glycero-3-phosphate, monosodium salt (Avanti Polar Lipids, Inc.), 2 mM EDTA and 10 mM 2-mercaptoethanol. In Table 3 shows amounts of linoleic acid in DG transferred fraction to the amount of protein in the initial enzyme solution.

Table 3
transformed strain 18: 2 (pg / mg protein)
C1 11.17
C2 10.77
MaPAH1.1-1 13.06
MaPAH1.1-2 11.39
MaPAH1.2-1 12.52
MaPAH1.2-2 10.93

As indicated in Table 3 for the conversion activity of each strain 18: 2 to dilinoleina-PA (18: 2-DG) was approximately the same.

This indicates that the activity of the PAP and MaPAH1.1 MaPAH1.2 depends of Mg 2+ and that MaPAH1.1 and MaPAH1.2 have PAP1 activity.

Example 7: Number of produced triacylglycerol

Triacylglycerol (throughout the specification called triglyceride or TG), which is a backup lipid, the lipid is obtained by further acylation of diacylglycerol product which is PAP protein. Measures the amount of the TG, produced by yeast transformants in which high espressirovany MaPAH1.1 or MaPAH1.2.

The cells of the transformants, yeast host strain deficient ScPAH1, seeded in 10 ml of liquid medium SC-Ura, Trp and cultured under static conditions at 30 ° C for 3 days. One milliliter of the culture solution was inoculated into 10 ml of YPDA liquid medium (2% w of yeast extract, 1% polypeptone, 2% glucose, 0.008% sulfate adenine), followed by culturing with shaking at 30 ° C for one day (n = 3) . Cells were harvested by centrifuging the culture solution, washed with water and lyophilized. To the dried cells were added chloroform and methanol (2: 1). Cells were again disrupted by glass beads and lipids were extracted using a total of 8 ml solvent. The extracted lipids were fractionated by TLC, as described above, and was scraped and analyzed by TG fraction. The results are shown in Table 4.

Table 4
Number * TG, produced in each medium
transformed strain mg / l
C1 11,01 ± 1,27
C2 11,54 ± 0,54
MaPAH1.1-1 16,01 ± 2,45
MaPAH1.1-2 17,09 ± 1,41
MaPAH1.2-1 14,29 ± 0,87
MaPAH1.2-2 13,32 ± 0,78
* In terms of fatty acids

As shown in Table 4, the amount of TG was approximately 1.5-fold in strain intensive expression MaPAH1.1, was approximately 1.2-fold in strain intensive expression MaPAH1.2, as compared with that in the control.

Example 8: Substrate specificity and MaPAH1.1 MaPAH1.2

Strain ΔScpah1: URA3, as a host, transformed with the plasmid pYE22m, pYE-MaPAH1.1 or pYE-MaPAH1.2. Four each transformant strain was used in subsequent experiments. Strains transformed with plasmid pYE22m, used as a control.

Each yeast transformant was inoculated into 10 ml liquid medium SC-Ura, Trp and cultured under static conditions at 27,5 ° C overnight. Each of the resulting culture solution was inoculated into 40 ml of liquid medium SC-Ura, Trp in an amount of 1.10 to two parallel and cultured under static conditions at 27,5 ° C for two days. From the obtained culture solutions were prepared stock solutions of the enzyme, as in Example 6, and protein concentration was measured.

PAP1 activity was measured as in Example 6 except that as the substrate was used for PAP dilinoleoil 1,2-glycero-sn-3-phosphate (18: 2-PA) and 1,2-dioleolil-sn-glycero-3 -phosphate (18: 1-PA).

Table 5 and Table 6, respectively indicated amounts of linoleic acid (18: 2) and oleic acid (18: 1) fraction was transferred into diacylglycerol (DG), on the amount of protein in the initial enzyme solution.

Table 5:
18: 2 DG on the amount of protein (micrograms / mg · protein)
The name of the sample Control MaPAH1.1 MaPAH1.2
average value CO average value CO average value CO
13.72 2.74 25.50 6.75 18,19 1.43
Table 6:
18: 1 to DG protein (pg / mg · protein)
The name of the sample Control MaPAH1.1 MaPAH1.2
average value CO average value CO average value CO
15.14 0.88 29,16 7.04 16.69 1.05

When used as a substrate 18: 2-PA, the activity types and MaPAH1.1 MaPAH1.2, derived from Mortierella, were 1.9-fold and 1.3-fold, respectively, compared to those in control.

When used as the substrate 18: 1-PA, and species MaPAH1.1 MaPAH1.2 activity were 1.9-fold and 1.1-fold, respectively, compared to those in control. 18: 1 – is a fatty acid that naturally produce yeast, and thus, it is initially present in the composition DG enzyme in the original solution. However, no differences were observed regarding the number of 18: 1 in the composition DG enzyme in the original solution, if the substrate is not added. Accordingly, it can be assumed that the difference in activity MaPAH1.1 MaPAH1.2 and the activity in the control as shown in Table 6, based on the effect in respect of 18: 1-PA, added as a substrate.

When comparing the activity of the same species of the enzyme for different substrates, MaPAH1.1 increased as the amount of 18: 1 and 18: 2 1.9-fold compared with the control, whereas the increased amount MaPAH1.2 18: 1 1.1-fold and the amount of 18 2 to 1.3 times compared with the control. This indicates that MaPAH1.1 exhibits the same activity in respect of both 18: 1-PA, and 18: 2-PA, but in the case MaPAH1.2 activity on 18: 2-PA higher than that against 18 1-PA.

These results indicate that possess MaPAH1.2 MaPAH1.1 and PAP activity. Furthermore, MaPAH1.2 exhibit higher activity towards 18: 2-PA, than for 18: 1-PA, indicating that MaPAH1.2 exhibits a higher activity for phosphatidic acids having a fatty acid composition with a higher degree of unsaturation

1. A nucleic acid, as described below:
(a) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(b) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 under stringent conditions, and encodes a protein with the activity of phosphatase phosphatidic acid;
(c) a nucleic acid characterized by a nucleotide sequence which consists of a nucleotide sequence that is 95% or more identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;
(d) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(e) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 under stringent conditions, and encodes a protein having phosphatidic acid phosphatase activity;
(f) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10 under stringent conditions, and includes an exon coding for a protein with phosphatase activity phosphatidic acid; and
(g) a nucleic acid characterized by the nucleotide sequence which consists of a nucleotide sequence that is 95% or more identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with the activity of phosphatase phosphatidic acid.

2. Nucleic acid according to claim 1, wherein the nucleic acid is any nucleic acid below:
(a) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(b) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 under conditions of 2 × SSC at 50 ° C, and encodes a protein with phosphatidic acid phosphatase activity;
(c) a nucleic acid characterized by a nucleotide sequence which consists of a nucleotide sequence that is 98% or more identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 and encodes a protein having phosphatidic acid phosphatase activity;
(d) a nucleic acid characterized by the nucleotide sequence encoding a protein which consists of an amino acid sequence that is 98% or more identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and has an activity of phosphatidic acid phosphatase;
(e) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to a nucleotide sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, under conditions of 2 × SSC at 50 ° C, and encodes a protein having phosphatidic acid phosphatase activity;
(f) a nucleic acid characterized by the nucleotide sequence which can be hybridized with a nucleic acid consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10 under conditions of 2 × SSC at 50ºC, and includes exon coding a protein having phosphatidic acid phosphatase activity; and
(g) a nucleic acid characterized by the nucleotide sequence which consists of a nucleotide sequence that is 98% or more identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10, and includes an exon coding for a protein with the activity of phosphatase phosphatidic acid.

3. A nucleic acid, as described below:
(a) a nucleic acid characterized by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity;
(b) a nucleic acid characterized by the nucleotide sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity;
(c) a nucleic acid characterized by the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 9 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity; and
(d) a nucleic acid characterized by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity.

4. A protein as indicated below:
(a) a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity; and
(b) a protein consisting of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity.

5. A protein as indicated below:
(a) a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatase activity phosphatidic acid; and
(b) a protein consisting of the amino acid sequence 98% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity.

6. A protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity.

7. A recombinant vector comprising a nucleic acid according to any one of claims 1-3, for protein expression on pp.5-6.

8. A cell transformed with the recombinant vector according to claim 7, for protein expression on pp.5-6.

9. A process for preparing a fatty acid composition characterized by collecting a fatty acid from a culture or lipid obtained by culturing the transformant of claim 8.

3. A nucleic acid, as described below:
(a) a nucleic acid characterized by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity;
(b) a nucleic acid characterized by the nucleotide sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity;
(c) a nucleic acid characterized by the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 9 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity; and
(d) a nucleic acid characterized by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10 or a fragment thereof, and encodes a protein having phosphatidic acid phosphatase activity.

4. A protein as indicated below:
(a) a protein consisting of an amino acid sequence with deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity; and
(b) a protein consisting of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity.

5. A protein as indicated below:
(a) a protein consisting of an amino acid sequence with deletion, substitution or addition of 1 to 130 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatase activity phosphatidic acid; and
(b) a protein consisting of the amino acid sequence 98% or more identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity.

6. A protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and having phosphatidic acid phosphatase activity.

7. A recombinant vector comprising a nucleic acid according to any one of claims 1-3, for protein expression on pp.5-6.

8. A cell transformed with the recombinant vector according to claim 7, for protein expression on pp.5-6.

9. A process for preparing a fatty acid composition characterized by collecting a fatty acid from a culture or lipid obtained by culturing the transformant of claim 8.