Glycerol-3-phosphatocyltransferase

FIELD: biotechnologies.

SUBSTANCE: invention also refers to an expression vector and a transformant containing polynucleotide, as well as a production method of lipid composition or composition based on fat acids using the transformant.

EFFECT: invention allows producing a new ferment with improved properties, which has glycerol-3-phosphatocyltransferase activity.

11 cl, 4 dwg, 14 tbl, 4 ex

 

The technical field to which the invention relates.

The present invention relates to polynucleotide, codereuse new glycerol-3-hospitalstrasse, and their application.

The level of technology

Fatty acids are important components of lipids, such as phospholipids, triacylglycerides etc. Fatty acids containing two or more unsaturated bonds, collectively referred to as polyunsaturated fatty acids (Pufas)are known to include, in particular, arachidonic acid, di-Homo-γ-linolenic acid, eykozapentaenovuyu acid and docosahexaenoic acid, etc. Some of these polyunsaturated fatty acids cannot be synthesized in the animal body. Therefore, it is necessary to use these polyunsaturated fatty acids are essential amino acids together with food.

In the body of the animal polyunsaturated fatty acids distributed in many organs and tissues. For example, arachidonic acid was isolated from the lipids extracted from the adrenal gland and liver of animals. This polyunsaturated fatty acid found in small quantities in the bodies of animals, and the extraction and isolation of polyunsaturated fatty acids from animal organs are not sufficient to ensure a large quantity of polyunsaturated fatty acids. Were therefore developed the Ana methods of obtaining polyunsaturated fatty acids by culturing various microorganisms. Among these microorganisms, the microorganismMortierellaknown as a microorganism capable of producing lipids containing polyunsaturated fatty acids such as arachidonic acid, etc. in Addition, there have also been attempts to produce polyunsaturated fatty acids in plants. Polyunsaturated fatty acids are spare lipids, such as triacylglyceride etc., and is known to accumulate in the cells of microorganisms or seeds of plants.

Triacylglyceride, which are spare lipids are producedin vivoin the following way. The acyl transfer to glycerol-3-phosphate with the formation of lysophosphatidic acid using glycerol-3-hospitalstrasse. Then, transferring acyl next is using lysophosphatidylserine with the formation of phosphatidic acid. This phosphatidic acid, in turn, dephosphorylates using hospitalfacility with the formation of diacylglycerol. Finally, the acyl transfer occurs through diacylglycerides with the formation of triacylglyceride.

In the above-described triacylglyceride biosynthesis pathway or phospholipid biosynthesis pathway is known that the reaction of acylation of glycerol-3-phosphate with the formation of lysophospholipids acid is mediated by glycerol-3-hospitalcenter the Oh (hereinafter sometimes "GPAT"; EC 2.3.1.15).

To date, the presence of GPAT genes was observed in several organisms. As GPAT genes mammals have been cloned two types of genes: microsomal (membrane-bound) and mitochondrial (membrane-bound) (non-patent literature 2). Similarly, as GPAT genes of plants were also cloned three types of genes: microsomal (membrane-bound), mitochondrial (membrane-bound) and chloroplast (free) (non-patent literature 3).

As GPAT genes derived from fungusSaccharomyces cerevisiaewere cloned two types of genes: microsomal (membrane-bound) GPT2/GAT1 (YKR067w) and SCT1/GAT2 (YBL011w); it is known that the simultaneous DeleteMovie both genes causes lethality (non-patent literature 4). For these fungal genes shown that GPT2 has the activity to use a wide range of fatty acids palmitic acid (16:0) to oleic acid (18:1) as substrate, whereas, whereas SCT1 has a strong selectivity, using a 16-carbon fatty acids such as palmitic acid (16:0) and palmitoleic acid (16:1)as the substrate (non-patent literature 4).

In addition, GPAT genes have also been cloned from many other kinds of organisms. First of all, about GPAT derived from microorganisms of the genusMortierellaable radio is activated lipids, reported the following.

For GPAT derived fromMortierella ramannianawas isolated microsomal GPAT, and shows that he is used as the acyl donor with selectivity, 5.4 times greater for oleic acid (18:1)than for palmitic acid (16:0) (non-patent literature 5). It is reported that GPAT derived fromMortierella alpina(hereinafter referred to sometimes as "M. alpina"), has a glycerol-3-hospitalstrasse activity in their microsomal fraction (non-patent literature 6).

It is shown that when GPAT present in microsomesM. alpina(in the membrane-bound state), reactsin vitrowith different acyl COA, GPAT uses as a substrate a wide range of polyunsaturated fatty acids, including oleic acid (18:1), linoleic acid (18:2), di-Homo-γ-linolenic acid (DGLA) (20:3) and arachidonic acid (20:4) (patent literature 1).

It is shown that when GPAT cloned fromM. alpina(ATCC #16266) (hereinafter referred to as MaGPAT1 (ATCC#16266)), expressed in transformantsYarrowia lipolyticadesigned to provide biosynthesis for the production of eicosapentaenoic acid (EPA) in the total amount of fatty acids, increased the composition of di-Homo-γ-linolenic acid (DGLA) (20:3) and the reduced composition of oleic acid (18:1). The results show that p is linecasino fatty acids with longer chain and a high degree of unsaturation are included selectively (patent literature 2).

In recent years it was reported that the homologue of GPAT, or MaGPAT2, was isolated fromM. alpina(1S-4), and showed substrate specificity different from MaGPAT1 (patent literature 3). That is, it is assumed that MaGPAT1 will demonstrate high specificity to palmitic acid and MaGPAT2 will demonstrate high specificity to oleic acid.

A RELATED AREA

The patent literature:

[Patent literature 1] instructions to WO 2004/087902

[Patent literature 2] USA 2006/0094091

[Patent literature 3] instruction to WO 2008/156026

Non-patent literature:

[Non-patent literature 1] Lipids, 39, 1147 (2004)

[Non-patent literature 2] Biochimica et Biophysica Acta, 1348, 17-26, 1997

[Non-patent literature 3] Biochimica et Biophysica Acta, 1348, 10-16, 1997

[Non-patent literature 4] The Journal of Biological Chemistry, 276 (45), 41710-41716, 2001

[Non-patent literature 5] The Biochemical Journal, 355, 315-322, 2001

[Non-patent literature 6] Biochemical Society Transactions, 28, 707-709, 2000

DISCLOSURE of INVENTIONS

Based on the above, it is desirable to develop a new homologue of GPAT, contributing to further strengthening and effective ways of fatty acid synthesis by effective products lysophosphatidic acid and triacylglycerol formed on the basis of them.

As a result of extensive studies, the authors present invention was able to clone the gene encoding the third homologue GPAT (aGPAT3) lipid-producing fungus M. alpinaand to improve the present invention. That is, the present invention relates to the following: polynucleotides, proteins, expression vectors, transformants, the method of obtaining food, etc. using the transformants, food, etc. obtained by the method, and so on.

Thus, the present invention relates to the following.

[1] Polynucleotide selected from the group consisting of the following (a)to(e):

(a) polynucleotide containing the nucleotide sequence with SEQ ID NO: 1 or 4;

(b) polynucleotide, codereuse protein consisting of the amino acid sequence of SEQ ID NO:2;

(c) polynucleotide, codereuse protein consisting of the amino acid sequence, where 1-100 amino acids deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity;

(d) polynucleotide, codereuse protein with amino acid sequence that is at least 85% homologous to the amino acid sequence SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity; and,

(e) polynucleotide that hybridizes with polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 or 4 in Estrich conditions, and which encodes a protein having glycerol-3-hospitalstrasse activity.

[2] Polynucleotide under item [1] above, as defined in any of paragraphs (f) or (g) below:

(f) polynucleotide, codereuse protein consisting of the amino acid sequence, where 1-10 amino acids deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity; and,

(g) polynucleotide, codereuse protein with amino acid sequence that is at least 90% homologous to amino acid sequence SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity.

[3] Polynucleotide under item [1] above, which contains the nucleotide sequence of SEQ ID NO:1, or 4.

[4] Polynucleotide under item [1] above, codereuse protein consisting of the amino acid sequence of SEQ ID NO:2.

[5] Polynucleotide on any of the PP [1]-[4] above, which is a DNA.

[6] the Protein encoded by polynucleotides on any of the PP [1]-[5] above.

[7] the Vector containing polynucleotide on any of the PP [1]-[5] above.

[8] Transformant non transformants of human rights, which entered polynucleotide on any of the PP [1]-[5] above.

[9] Transformant non transformants person, which introduced the vector at p. [7] above.

[10] T is informant by p. [8] or [9] above, where the transformant is a lipid-producing fungus.

[11] Transformant on p. [10] above, where the lipid-producing fungus is a fungusMortierella alpina.

[12] a Method for producing lipid composition or compositions based on fatty acids, which provides for the collection of the lipid composition and the composition based on fatty acids from the culture of transformant on any of the PP [8]-[11] above.

[13] a Food product, a pharmaceutical product, a cosmetic product or a soap containing lipid composition or a composition based on fatty acids collected using the method of production by p. [12] above.

Polynucleotide of the present invention can be used for transformation of lipid-producing fungus (for example,M. alpina), yeast, plants, etc, and transformants lipid-producing fungus, transformants of yeast transformants plants etc, thus obtained, can be used to obtain a composition based on fatty acids, food products, cosmetics, pharmaceuticals, Soaps, etc.

More specifically, the transformants of the present invention provide extremely high efficiency production of triglycerides, and most of the fatty acids increased in transformed, is due to the increase of fatty acids in triglycerides, Respectively, these transformants can be used effectively to obtain drugs and diet food products.

BRIEF DESCRIPTION of DRAWINGS

In Fig. 1-1 shows the alignment of genomic sequences and CDS sequences MaGPAT3e.

Fig. 1-2 is a continuation of Fig. 1-1.

Fig. 1-3 is a continuation of Fig. 1-2.

In Fig. 2-1 shows the CDS sequence and putative amino acid sequence MaGPAT3 (1S-4).

Fig. 2-2 is a continuation of Fig. 2-1.

In Fig. 3 shows the alignment of amino acid sequences MaGPAT1 ((1S-4) and (ACTT#16266)) and MaGPAT3 (1S-4).

In Fig. 4 shows the alignment of amino acid sequences of different homologous proteins GPAT (MaGPAT1, MaGPAT3, ScSCT1 and ScGPT2). Four domains, conservative for GPAT-homologues, and amino acid residues (asterisks), which are considered important for GPAT activity, and amino acid residues (crosses), which are considered important for the binding of glycerol-3-phosphate, in these domains were also stored in GPAT3.

The BEST mode for carrying out the INVENTION

Below the present invention is described in more detail. Following implementation options are intended only to describe the invention and not to limit it to only two versions of the implementation. The present invention can be implemented in different ways, the e beyond its essence.

All publications, published patent applications, patents and other patent literature cited in this application, incorporated herein by reference in full. This application hereby incorporates by reference the contents of the specification and drawings of Japanese patent application No. 2009-217646, filed September 18, 2009, from which priority was claimed.

The authors of the present invention was the first to clone the full-size cDNA gene (MaGPAT3)encoding a third homologue of glycerol-3-hospitalstrasse lipid-producing fungusM. alpineas will later be described in detail in the EXAMPLES. The authors present invention also determined the nucleotide sequence of the genomic DNA MaGPAT3M. alpineand the estimated amino acid sequence. The sequence of the ORF MaGPAT3 the putative amino acid sequence MaGPAT3, the sequence CDS MaGPAT3 and genomic sequence MaGPAT3 represent SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 4, respectively. Genomic sequence MaGPAT1M. alpinealso shown in SEQ ID NO:5. These polynucleotide and enzymes can be obtained using the methods described in the EXAMPLES below, the known methods of genetic engineering, known methods of synthesis, and so on.

1. Polynucleotide according to the invention

The first is x, the present invention relates to polynucleotide selected from the group consisting of the following (a)to(e):

(a) polynucleotide containing the nucleotide sequence of SEQ ID NO: 1 or 4;

(b) polynucleotide, codereuse protein consisting of the amino acid sequence of SEQ ID NO:2;

(c) polynucleotide, codereuse protein consisting of the amino acid sequence, where 1-100 amino acids deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity;

(d) polynucleotide, codereuse protein with amino acid sequence that is at least 85% homologous to the amino acid sequence SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity; and,

(e) polynucleotide that hybridizes with polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1 or 4 in harsh conditions, and which encodes a protein having glycerol-3-hospitalstrasse activity.

As used herein, the term "polynucleotide" means DNA or RNA.

As used herein, the term "polynucleotide that hybridizes in stringent conditions" refers to polynucleotide obtained when help is and how hybridization on the colonies, the method of hybridization on the plaque hybridization method according to the Southern or the like, using as a probe, for example, polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1, or 4, or the whole or part of polynucleotide consisting of a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2. As methods of hybridization used in a manner described for example in Sambrook &Russell, Molecular Cloning; A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001 and Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997", etc.

As used herein, the term "stringent conditions" can mean any of weakly-hard conditions, moderately stringent conditions or highly-stringent conditions. The term "low stringent conditions" represents, for example, 5x SSC, 5x denhardt's solution and 0.5% SDS, 50% formamide at 32ºC. The term "moderately stringent conditions" represents, for example, 5x SSC, 5x denhardt's solution and 0.5% SDS, 50% formamide at 42ºC. The term "very strict conditions" represents, for example, 5x SSC, 5x denhardt's solution and 0.5% SDS, 50% formamide at 50ºC. Under these conditions, DNA with higher homology, is expected to work effectively at higher temperatures, despite the fact that the stiffness of hybridization is determined by several factors, including those is the temperature, the concentration of probe, probe length, ionic strength, time, salt concentration and other, and a specialist in the area in question can properly find these factors to achieve the same stiffness.

When using commercially available kits for hybridization is possible to use, for example, Alkphos Direct Labeling and Detection System (GE Healthcare). In this case, in accordance with the attached Protocol, after incubation with the labeled probe during the night the membrane was washed with primary wash buffer containing 0.1% (weight/volume) SDS at 55°C, detektywa thereby hybridisable DNA.

In addition to the above polynucleotides, other polynucleotides that can be hybridized include DNA, with 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher 80% or higher, 81% or greater, 82% or higher, 83% or higher, 84% or higher, 85% or more, 86% or more, 87% or more, 88% or more, 89% or higher, 90 percent or more, 91% or more, 92% or more, 93% or more, 94% or higher 95% or greater, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1 per cent or higher, to 99.2% or higher of 99.3% or higher, 99.4 per cent or higher of 99.5% or higher, 99.6 percent or higher, of 99.7% or higher, 99.8% or higher or 99.9% or higher homology with DNA SEQ ID NO:1, or 4, or a DNA that encodes the amino acid sequence of SEQ ID NO:2, as calculated using the software on the search of homology, such as FASTA and BLAST, is using the default settings.

Homology between amino acid sequences or nucleotide sequences can be determined using the algorithm BLAST from Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Nail Acad. Sci. USA, 90: 5873, 1993). Programs called BLASTN and BLASTX have been developed based on the BLAST algorithm (Altschul S. F. et al., J. Mol. Biol. 215: 403, 1990). When the nucleotide sequence is analyzed using BLASTN, the parameters are, for example, score=100 and word length=12. When the nucleotide sequence is analyzed using BLASTX, parameters are, for example, score=50 and word length=3. When using the programs BLAST and Gapped BLAST for each of the programs use the default settings.

Polynucleotide of the present invention, described above, can be obtained by known methods of genetic engineering, by known methods of synthesis, and so on.

2. Proteins according to the invention

The present invention relates to proteins, is shown below.

(i) a Protein encoded by polynucleotides on any of the points (a)to(e) above.

(ii) a Protein comprising amino acid sequence SEQ ID NO:2.

(iii) a Protein containing the amino acid sequence where one or more amino acids deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO:2, and having a glycerol-3-hospitality sferazo activity.

(iv) a Protein with the amino acid sequence that is at least 85% homologous to the amino acid sequence SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity.

The proteins described above in (iii) or (iv), as a rule, are mutants of the natural protein SEQ ID NO:2 and include those proteins that can be artificially obtained using site-directed mutagenesis described, e.g., in Sambrook &Russell, Molecular Cloning: A Laboratory Manual, Vol. 3, Cold Spring Harbor Laboratory Press 2001", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997", "Nuc. Acids. Res., 10, 6487 (1982)", "Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)", "Gene, 34, 315 (1985)", "Nuc. Acids. Res., 13, 4431 (1985)", "Proc. Natl. Acad. Sci. USA, 82, 488 (1985)" etc.

As used herein, "protein containing the amino acid sequence where one or more amino acids deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity"includes proteins containing the amino acid sequence, where, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or one amino acid is deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO:2, and possessing glycero the-3-hospitalstrasse activity. Typically, the number of deletions, substitutions, insertions and/or additions is preferably smaller.

Such proteins include proteins with amino acid sequence homology of about 85% or more, 86% or more, 87% or more, 88% or more, 89% or higher, 90% or more, 91% or more, 92% or more, 93% or more, 94% or higher 95% or more, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1 per cent or higher, to 99.2% or more, of 99,3% or higher, and 99.4% or higher, 99.5% pure or higher, 99.6 percent or higher, of 99.7% or higher, 99.8% or higher or 99.9% or higher amino acid sequence of SEQ ID NO:2, and having a glycerol-3-hospitalstrasse activity. The larger the percentage of homology described above, the generally more preferred is a protein.

Glycerol-3-hospitalstrasse activity can be determined, for example, using the method described in J.B.C. 276 (45), 41710-41716(2001). To test GPAT activity, there is an experiment on complementarity with the use of yeast strains Δgpt2 and Δsct1. When polynucleotide encoding the enzyme is expressed in the strains Δgpt2 and Δsct1 (simultaneous DeleteMovie GPT2 and SCT1 leads to lethality) and the strains Δgpt2 and Δsct1 able to grow, protein and peptide encoded by polynucleotides, as it turns out, has GPAT activity.

The deletion, substitution, insertion and/or addition of one or several amino acid residues in the amino acid p is the sequence of the protein according to the invention means, that one or more amino acid residues deleted, substituted, built and/or added at one or more positions in the same sequence of amino acids. Two or more types of deletions, substitutions, insertions and additions can take place.

Examples of amino acid residues, which are used interchangeably below. Amino acid residues in the same group are interchangeable. Group a: leucine, isoleucine, norleucine, valine, Norvaline, alanine, 2-aminobutanoic acid, methionine, o-medicein, t-butylglycol, t-butylene and cyclohexylamine; Group B: aspartic acid, glutamic acid, itaporanga acid, isoglutamine acid, 2 - aminoadenosine acid and 2-aminopropane acid; Group C: asparagine and glutamine, Group D: lysine, arginine, ornithine, 2,4 - diaminobutane acid and 2,3-diaminopropionic acid; Group E: Proline, 3-hydroxyproline and 4-hydroxyproline; Group F: serine, threonine, homoserine; and Group G: phenylalanine and tyrosine.

The protein of the present invention can also be obtained using methods of chemical synthesis, such as the Fmoc method (fluorenylmethoxycarbonyloxy way), tBoc method (t-butyloxycarbonyl way), etc. in Addition, peptide synthesizers, available from Advanced Automation Peptide Protein Technologies, Perkin Elmer, Protein Technology Instrument, PerSeptive, Applied Biosstems, SHIMADZU Corp. etc. can also be used for chemical synthesis.

3. The vector according to the invention and the transformant introduced with the vector

In another embodiment, the present invention also relates to an expression vector containing polynucleotide according to the invention.

The vector according to the invention is usually manufactured so that it contained the expression cassette, containing:

(i) a promoter that can be transcribed in the cell-host;

(ii) any polynucleotide described in paragraphs (a) to(g) above, which is linked to a promoter; and,

(iii) signal, which acts in a cell-owner in respect of the termination of transcription and polyadenylation of the RNA molecule. Thus constructed vector is introduced into a cell of the host. Examples of host cells that can appropriately be used in the present invention include lipid-producing fungus, yeast and the like.

Lipid-producing fungus, which can be used are strains are described, for example, MYCOTAXON, Vol. XLIV, No. 2, str.257-265(1992). Specific examples include microorganisms belonging to the genusMortierellaincluding microorganisms belonging to the subgenusMortierellafor example,Mortierella elongataIFO8570,Mortierella exiguaIFO8571,Mortierella hygrophilaIFO5941,Mortierella alpinaIFO8568, ATCC16266, ATCC32221, ATCC42430, CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS58.72, CBS529.72, CBS608.70 and CBS754.68, etc. or microorganisms belonging to the subgenusMicromucorfor example,Mortierella isabellinaCBS194.28, IFO6336, IFO7824, IFO7873, IFO7874, IFO8286, IFO8308 and IFO7884,Mortierella nanaIFO8190,Mortierella ramannianaIFO5426, IFO8186, CBS112.08, CBS212.72, IFO7825, IFO8184, IFO8185 and IFO8287,Mortierella vinaceaCBS236.82, etc. Among other things,Mortierella alpinais preferred.

Examples of yeast areSaccharomyces cerevisiaeNBRC1951, NBRC1952, NBRC1953, NBRC1954 etc.

Introduction of the vector according to the invention in yeast and testing glycerol-3-hospitalstrasse activity of the protein GPAT3 encoded by the vector, no GPAT genes (Gpt2p and Sct1p) yeast is used as the host cell, provides a measure of the enzymatic activity only protein GPAT3. Accordingly, in the embodiment of the invention the yeast as a host cell, preferably, are deficient in the gene Gpt2p and gene Sct1p.

These cell host transformed by the vector according to the invention, produce more triglycerides than the cells of the host, which is not transformed with the vector according to the invention. A large part of the fatty acids are fatty acids that comprise triglycerides, increased by introducing a vector according to the invention.

The vectors used for the introduction of lipid-producing fungi include, as a non-limiting example, pDura5 (Appl. Environ. Biotechnol., 65, 419-425, (2004)).

As vectors that can be used for introduction into yeast, any suitable vector without particular restrictions, provided that the vector has the activity to expressionate insert into yeast cells, and includes, for example, pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228, 1995).

Promoters/terminators for regulation of gene expression in cells of the owners may be in any combination, provided that they operate in cells of the host. For example, you can use the promoter of the gene of histone H4.1, the promoter of glyceraldehyde-3-phosphate dehydrogenase, etc.

As a selective marker used for transformation, can be used auxotrophic markers (ura5, niaD), markers chemical resistance (hygromycin, zeocin), gene resistance to geneticin (G418r), the gene of resistance to copper (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, 81, 337 1984), resistance gene cerulenin (fas2m, PDR4) (Junji Inokoshi, et al., Biochemistry, 64, 660, 1992; Hussain et al., Gene, 101: 149, 1991, respectively).

For transformation of host cells, as a rule, it is possible to use known methods. For example, methods that can be used include, as non-limiting examples, the method of electroporation (Mackenxie D. A. et al., Appl. Environ. Environ., 66, 4655-4661, 2000), the method of delivery of the particles (the method described in JPA 2005-287403 "Method of Breeding Lipid-Producing Fungus"), the method spheroplasts (Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)), lidiasitaly method J. Bacteriology, 153 p163 (1983)) and the methods described in Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc.

In addition, can be made the reference to "Sambrook &Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001", "Methods in Yeast Genitics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)", etc. for General methods cloning.

4. The method of obtaining the lipid composition or compositions based on fatty acids according to the invention

In another embodiment, the present invention also relates to a method for producing a lipid or a composition based on fatty acids, which involves the use of transformant lipid-producing fungus or yeast described above.

As used herein, the term "lipid" is intended to denote a simple lipid, including a connection (e.g., glycerides), which consists of fatty acids and alcohols connected through ester bonds, or similar (for example, cholesterol ester), etc; a complex lipid, in which phosphoric acid, amino acid(s)saccharide(s) or the like is attached to part of the simple lipid; or a derivative of lipid, which is a hydrolyzate of the above lipid and insoluble in water.

As used herein, the term "oil and grease" is intended to denote SL is the author of the ester of glycerol and fatty acids (glycerides).

As used herein, the term "fatty acid" means an aliphatic monocarboxylic acid, a carboxylic acid having one carboxyl group and the carbon atoms connected to each other in a chain)represented by the General formula RCOOH (where R is an alkyl). Fatty acid include saturated fatty acid having no double bonds and unsaturated fatty acid containing a double bond in the hydrocarbon chain.

The lipid composition or the composition based on fatty acids of the present invention can be obtained as follows, from cells transformed in accordance with the present invention. After incubation, the transformed organism (e.g., a lipid-producing fungus and yeast) are processed in the usual way, including centrifugation, filtration, etc. to collect cell culture. The cells are thoroughly washed with water and preferably dried. The drying may be carried out by means of freeze drying, air drying, etc. Dried cells destroy the Dyno or by treatment with ultrasound, etc. if necessary, and then extracted with an organic solvent, preferably in a stream of nitrogen. The organic solvent which can be used include ether, formaldehyde, methanol, ethanol, chloroform, dichloromethane, petrol is hydrated ether and other In addition, good results can also be obtained by alternating extraction with methanol and petroleum ether, single-phase extraction chloroform-methanol-water. When removing the organic solvent by distillation under reduced pressure to obtain a lipid containing a fatty acid. Extracted fatty acids can also be atrificial way hydrochloric acid-methanol, etc.

In addition, separation of fatty acids from lipids containing fatty acids described above can be achieved through the concentration and separation in the usual way (for example, a method with the addition of urea, the method of separation with cooling, column chromatography, etc.) at saturation of mixed fatty acids or mixed esters of fatty acids.

The lipid composition or the composition based on fatty acids, obtained by the method of receiving according to the present invention, can be used to obtain, for example, food, pharmaceutical products, industrial materials (raw materials for cosmetic products, Soaps, etc.)containing oils and fats, and the like.

In yet another embodiment, the present invention relates to a method for producing food products, cosmetics, pharmaceuticals, Soaps, etc. using transformant lipid-producing fungus is whether the yeast of the present invention. The method involves the formation stage of lipids and fatty acids using transformant lipid-producing fungus or transformant yeast of the present invention. Food, cosmetics, pharmaceuticals, Soaps and other containing educated lipids or fatty acids, prepared in the traditional manner. Thus, foodstuffs, cosmetics, pharmaceuticals, Soaps and other prepared by the method according to the present invention, contain lipids and fatty acids formed using transformant lipid-producing fungus or transformant yeast of the present invention. The present invention also relates to prepared the way for food, cosmetics, drugs, pharmaceuticals, soap and so on.

The form of beauty (song) or pharmaceutical (composition) of the present invention is not particularly limited and may be in any form, including the state of solution, paste, gel, dry matter or powder. Also, a cosmetic composition or pharmaceutical composition of the present invention can be used as cosmetics or local agents for the skin, including oil, lotion, cream, emulsion, gel, shampoo, hair rinse, hair conditioner, enamel cream-based, lipstick, face powder, face mask, ointments, perfumes, powder, Cologne, toothpaste, soap, spray, cleansing foam, etc., means on care of the skin, slow down aging, anti-inflammatory agent for the skin, means for bathing, healing tonic essence to give beauty to the skin, sunscreen or protective and improving remedy for skin diseases caused by trauma, cracking or fracture of the skin, etc.

Cosmetic composition of the present invention can also be prepared accordingly with other oils and fats and/or coloring agents, flavoring agents, preservatives, surfactants, dyes, antioxidants, etc. if necessary. In addition, the pharmaceutical composition according to the present invention may also contain other pharmaceutically active ingredients (e.g., anti-inflammatory components or accessories (for example, the components of the lubricant or carrier). Examples of other components, which are widely used in cosmetic preparations or preparations for skin for external use include the remedy of acne, the means for prevention of dandruff and itching, desodorante and antiperspirant agent, remedy for burns, anti-mites and lice, means for softening keratin, agentpath dry skin, antiviral substance, a substance promoting percutaneous absorption, and the like.

The food product according to the present invention includes a dietary Supplement, health food, functional food, food for children, food for babies, modified milk for infants, modified milk for preterm infants, geriatric nutrition, etc. As used herein, under food refers to solid, liquid and liquid food, as well as their mixture, and collectively means the edible product.

The term "food additive" refers to food products enriched with specific food ingredients. The term "diet product" refers to foods that are beneficial to health, and includes dietary supplements, natural foods and diet foods. The term "functional food" refers to food to compensate for nutrients that help regulate the body functions. "Functional foods" are synonyms foods for specified health use. The term "food for young children" refers to a food product intended for children up to 6 years. The term "geriatric food product" refers to a food product, clicks BotName for in order to facilitate the digestion and assimilation compared to the raw food product. The term "modified milk for infants" refers to modified milk, intended for children up to one year. The term "modified milk for preterm infants" refers to modified milk intended for premature infants up to 6 months after birth.

These products include natural products (processed fats and oils), such as meat, fish and nuts; and foods with added fats and oils during cooking, for example, Chinese products, Chinese noodles, soups, etc.; foods prepared with the use of fats and oils as a heated environment, such as tempura or deep fried fish and vegetables, deep fried foods, fried tofu, Chinese fried rice, donuts, Japanese fried cookie dough or Corinto; products based on fat and oil or refined products with the addition of fats and oils during processing, for example, butter, margarine, mayonnaise, sauce, chocolate, pasta, fast food, candy, biscuit, cookies, cake, ice cream and foods with a plating or coating fats and oils after cooking is completed, for example, rice crackers, hard biscuits, bread sweet the bean paste, etc. However, food is not limited to products containing fats and oils, and other examples include agricultural products, such as bakery products, noodles, cooked rice, sweets (e.g., candy, chewing gum, gummy mass, tablet, Japanese sweets), soy cheese and processed products; fermented foods, such as Japanese rice wine or sake, medicinal liqueurs, sherry dessert cooking (mirin), vinegar, soy sauce and miso or bean paste, etc; food animal products, such as yogurt, ham, bacon, sausage, etc.; seafood such as minced and steamed fish cakes or kamaboko, deep fried fish cakes or Agean and ascended fish cakes or henpen etc. as well as fruit drinks, soft drinks, sports drinks, alcoholic drinks, tea, etc.

A food product of the present invention can also be in the form of pharmaceutical preparations such as capsules and the like, or in the form of a processed food product, such as a natural liquid food, food with a certain composition and basic food made with oil and fat according to the present invention together with proteins, sugars, trace elements, vitamins, emulsifiers, synthetic fragrances and etc., healing drinks, enteral nutrients, and the like.

As described above, fatty acids can effectively produce by gene expression GPAT3 of the present invention in the cell host.

In addition, the expression level of the gene can be used as an indicator to study the conditions of cultivation, control incubation, etc. for efficient production of fatty acids.

EXAMPLES

Further, the present invention is described in more detail with reference to EXAMPLES, but without intent to limit the invention to these EXAMPLES.

[EXAMPLE 1]

Analysis of the genomeM. alpina

The strain ofM. alpina1S-4 were seeded into 100 ml of medium GY2:1 (2% glucose, 1% yeast extract, pH 6,0), then were cultured with stirring at 28°C for 2 days. The cells of fungi collected by filtration, and genomic DNA was obtained using DNeasy (QIAGEN).

The nucleotide sequence described above, genomic DNA was determined using Roche 454 Genome Sequencer FLX Standard. This process included two rounds of determining the nucleotide sequence of the library fragments, and three rounds of determining the nucleotide sequence of the combined steam library. The obtained nucleotide sequences were collected in 300 superantigen.

Synthesis of cDNA strainM. alpinaThe strain 1S-4

The strain ofM. alpina1S-4 were seeded in 4ml of medium (2% glucose, 1% yeast extract, pH 6,0) and were cultured for 4 days at 28°C. Cells were collected by means of filtration, and RNA was extracted using the RNeasy Plant Kit (QIAGEN). cDNA was synthesized using SuperScript First-Strand System using RT-PCR (Invitrogen).

Search for homologues GPAT

Was a search for amino acid sequence MaGPAT1 (ATCC#16266) for comparison with the genomic nucleotide sequence of a strain ofM. alpina1S-4 using tblastn. As a result, we have determined supermontage containing the sequence shown in SEQ ID NO:4 and SEQ ID NO:5. It was believed that SEQ ID NO:5 represents the genomic sequence of GPAT1 obtained from a strain ofM. alpina1S-4 (hereinafter referred to as MaGPAT1). On the other hand, it was believed that SEQ ID NO:4 is a sequence encoding a new homologue of GPAT, based on the presence of the initiating codon or termination codon and comparison with MaGPAT1. In addition, it was decided that the remains of the first, third and 3278-3280 in SEQ ID NO:4 are initiating codon and the termination codon of this homologue, respectively. This gene was named MaGPAT3.

Cloning of the gene MaGPAT3

For cloning CDS gene MaGPAT3 were obtained with the following primers.

Eco-MaGPAT3-F: 5'-GAATTCATGGGTCTCCAGATCTATGACTTCGTCTC-3' (SEQ ID NO:6)

Sal-MaGPAT3-R: 5'-GTCGACTTATGCCTCCTTAGACTTGACTGCATCC-3' (SEQ ID NO:7)

Using the above cDNA as template, was performed by PCR amplification with KOD-Plus(TOYOBO) using primers Eco-MaGPAT3-F and Sal-MaGPAT3-R, and was amplified DNA fragment approximately 2.2 TPN Fragment was cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and the resulting plasmid was named pCR-MAGPAT3. The sequence of the insert of this plasmid, i.e, CDS-sequence of a gene MaGPAT3 shown in SEQ ID NO:3. The ORF sequence of the gene MaGPAT3 also presented in SEQ ID NO:1.

Analysis of the nucleotide sequence

Comparison of the genomic sequence (SEQ ID NO:4) and CDS sequences (SEQ ID NO:3) gene MaGPAT3 it is assumed that the genomic sequence of a gene must contain 7 exons and 6 introns and encode a protein consisting of 753 amino acid residues (Fig. 1 and 2). The sequence MaGPAT3 compared with the known sequence GPAT-homologM. alpina. CDS sequence MaGPAT3 showed homology 73,7% from GPAT1 (ATCC#16266) and 73.2 per cent from GPAT1 (1S-4). Amino acid sequence MaGPAT3 showed homology to 14.4% with GPAT2 (1S-4).

The putative amino acid sequence (SEQ ID NO:2) MaGPAT3 was presented for the analysis of homology with the amino acid sequence registered in GENEBANK nr, using BLASTp. As a result, amino acid sequence with the lowest E-value for the sequence, namely, amino acid sequence with high homology, were obtained fromCryptococcus neoformansvar.<> neoformansJEC21 GPAT-homolog (inventory number GENEBANK No. XP_569487), and homology of amino acid sequence, as it turned out, was at 40.3%. In addition, the putative amino acid sequence MaGPAT3 showed a 33.3% homology with the amino acid sequence Sct1p, which is derived from yeastS. cerevisiaeGPAT, and 31.5% with the amino acid sequence Gpt2p. Comparing amino acid sequences GPAT3 and GPAT1 derived fromM. alpina1S-4, and derived fromS. cerevisiaeGPAT Sct1 and Gpt2 (Fig. 4). Four domain conservative in GPAT-homologues, and amino acid residues (asterisks in Fig. 4), which is considered important for GPAT activity, and amino acid residues (crosses in Fig. 4)considered to be important for binding of glycerol-3-phosphate, in these areas, were also conservative in GPAT3.

[EXAMPLE 2]

Complementary experiment for yeastS. cerevisiae(Δsct1, Δgpt2)

In the yeastS. cerevisiae, SCT1 and GPT2 known as the genes responsible for GPAT activity; it is known that simultaneous deletion of this gene leads to lethality. In order to confirm whether the proteins encoded MaGPAT1 and MaGPAT3M. alpina,GPAT activity was conducted an additional experiment with Δsct1 and Δgpt2. The genotypes of the strains obtained in this experiment are summarized in table 1.

TABLE 1

Obtaining strain GP-1

Gene SCT1 Δgpt2-homozygous diploid yeast (catalog Number No. YSC1021-663938) from the Collection of yeast strains with knockouts of genes (Open Biosystems) was destroyed in the following way. First, DNA was extracted from cells of strainS. cerevisiaeS288C using Gen Toru-Kun (for yeast) (TAKARA BIO). Using DNA as the template, a partial sequence of a gene SCT1 was amplified using PCR with KOD-Plus (TOYOBO) using primer Xba1-Des-SCT1-F: 5'-TCTAGAATGCCTGCACCAAAACTCAC-3 (SEQ ID NO:8) and primer Xba1-Des-SCT1-R: 5'-TCTAGACCACAAGGTGATCAGGAAGA-3' (SEQ ID NO:9). Amplificatory the DNA fragment of about 1.3 TPN was cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and the resulting plasmid was named pCR-SCT1P. Then, plasmid YEp13 was digested with restriction enzymes SalI and XhoI. The resulting DNA fragment with a size of about 2.2 TPN, CDS containing LEU2 gene was Legerova with a DNA fragment of about 4.4 TPN obtained by splitting the plasmid pCR-SCT1P SalI, using Ligation High (TOYOBO)to construct a plasmid with the gene LEU2, embedded in the opposite direction with respect to gene SCT1, and this plasmid was named pCR-Δsct1:LEU2. pCR Δsct1: LEU2 was treated with restriction enzyme restr the functions XbaI, and above Δgpt2 homozygous diploid yeast transformed with the help of literaturnogo way. Transformants selectively the ability to grow on agar medium SD-Leu (2% agar) (6.7 g of yeast-based nitrogen without amino acids (DIFCO), 20 g glucose and 1.3 g of amino acids in the form of a powder (a mixture of 1.25 g of adrinaline, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11,25 g serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan and 0.6 g of uracil) 1l). From the cells of the obtained transformants was extracted DNA using the method described above. Using (1) a pair of primers SCT1outORF-F: 5'-AGTGTAGGAAGCCCGGAATT-3 (SEQ ID NO:10) and primer SCT1inORF-R: 5'-GCGTAGATCCAACAGACTAC-3' (SEQ ID NO:11) (0,5 TPN), and (2) a pair of primers SCT1outORF-F1 primer LEU2inORF-F: 5'-TTGCCTCTTCCAAGAGCACA-3' (SEQ ID NO:12) (1,2 TPN), performed PCR amplification to verify the genotype, i.e., SCT1/Δsct1: LEU2 and transformed yeast cell having this genotype was named strain GP-1.

Construction of strain MaGPAT1-D and strain MaGPAT3-D

In order to embed GPAT genes MaGPAT1 and MaGPAT3M. alpinain the yeast chromosome, plasmid pUC-URA3-MAGPAT1 and plasmid pUC-URA3-MAGPAT3 were constructed as follows.

Plasmid pUC-URA3-MAGPAT1

Multiple site for cloning plasmid pUC18 was modified to contain only the HindIII site. DNA is fragment size of about 1.2 TPN, which was obtained by splitting pURA34 (WO0131000) HindIII, was built into the HindIII site to construct plasmids pUC-URA3. After splitting pYE-MAGPAT1 (WO2008156026) restriction enzyme HindIII ends were small mistake using a Blunting Kit (TAKARA BIO). As a result, the DNA fragment of about 3.5 tpil built into the SmaI site of plasmid pUC-URA3, to construct a plasmid with the gene of GPAT1 and URA3 gene, integrated in the same direction. This plasmid was named pUC-URA3-MAGPAT1.

Plasmid pUC-URA3-MAGPAT3

Plasmid pCR-MAGPAT3 was treated with restriction enzymes EcoRI and SalI, and the resulting DNA fragment of about 2.3 TPN was built in sites EcoRI/SalI vector pYE22m (Biosci. Biotech. Biochem., 59, 1221-1228, 1995) for expression in yeast, to construct a plasmid pYE-MAGPAT3. Next, plasmid pYE-MAGPAT3 was treated with restriction enzyme HindIII, and then the ends were blunted using a Blunting Kit (TAKARA BIO). The resulting DNA fragment of about 3.6 TPN was built into the SmaI site of plasmid pUC-URA3, to construct a plasmid with the gene GPAT3 and URA3 gene integrated in the same direction. This plasmid was named pUC-URA3-MAGPAT3.

Strain GP-1 was transformed with the plasmid pUC-URA3-MAGPAT1 and plasmid pUC-URA3-MAGPAT3 with restriction enzyme HindIII, respectively, using literaturnogo way. Transformants selectively the ability to grow on agar medium SD-Ura (2% agar) (6.7 g of yeast-based nitrogen without amino acids (DIFCO), 20 g of glucose and 1.3 g of amino acids in the form of a powder (a mixture of 1.25 g of adrinaline, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine, 11,25 g serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan and 1.8 g of leucine) 1 liter). DNA was extracted from strains, optionally selected from the resulting transformants using Gen Toru-Kun (for yeast) (TAKARA BIO). In the transformant inserted into the URA3-MAGPAT1, genomic DNA is PCR amplified, using (3) a pair of primers GPAT1-f1: 5'-GTCAAGAAGGAATTCAAGGAGCTCAAG-3' (SEQ ID NO:13) and primer GPAT1-r2: 5'-CCTGGGATGATGGACAAGAACAATG-3' (SEQ ID NO:14) (1,1 TPN). In transformants with built-URA3-MAGPAT3 genomic DNA amplified using PCR, using (4) a pair of primers MaGPAT3-2F: 5'-TTTTGAACGCTTAAATGCTGGC-3' (SEQ ID NO:15) and primer 5'-GGTCTTTTGAAGCTCTGCACGCGAC-3' (SEQ ID NO:16) (1,1 TPN). Transformants in which the insertion of expression cassettes MaGPAT1 or expression cassettes MaGPAT3 in the genome could be confirmed, were called the strain MaGPAT1-D or strain MaGPAT3-D.

Sporulation and tetrad analysis

Strains MaGPAT1-D and MaGPAT3-D were sown on agar medium YPD, respectively, and incubated at 30°C for 2 days. Grown cells were seeded on agar medium for sporulation (0.5% potassium acetate, 2% agar) and incubated at 25°C for 4 days. The appropriate number spirulina cells were scraped and suspended in 100 m is l Zymolyase solution (0.125 mg/ml Zymolyase 100T, 1M sorbitol, 40 mm califofnia buffer (pH 6,8)). After incubation at room temperature for 30 minutes, the test tubes was added a solution of Zymolyase, and cells were transferred to ice. After the formation of ascospores was microscopically confirmed, four ascospores were isolated on agar medium YPD using a micromanipulator, and incubated at 30°C for 2 days to find all the colonies obtained from the relevant dispute. Obtained from spores clones were printed on agar medium SD-Ura agar and environment SD-Leu. Incubation was carried out at 30°C for 3 days to identify auxotrophy for uracil and auxotroph on Latino. The presence or absence of growth on each plate and the number of clones is given in table 2.

TABLE 2
Phenotypeobtained from MaGPAT1-Dobtained from MaGPAT3-D
SD-UraSD-Leu
OO1411
OX149
X O00
XX1611
Only4431

In spore clones from both strains, the ratio prototrophic for uracil and prototrophic on Latino strains: prototrophic for uracil and auxotrophic for Latino strains: auxotrophic for uracil and auxotrophic for Latino strains was approximately 1:1:1. The strain that is auxotrophic for uracil and prototrophic on Latino received was not. Further, in order to determine the genotype of the strain, procuring for uracil and procuring on Latino, and strain auxotrophic for uracil and auxotrophic for leucine, obtained from strain MaGPAT1-D and strain MaGPAT3-D, respectively, DNA was extracted from cells in the same manner as described above, and PCR was performed using (1) a pair of primers SCT1outORF-F and primer SCT1inORF-R and (2) a pair of primers SCT1outORF-F1 primer LEU2in ORF-F and the strain obtained from MaGPAT1-D, using (3) a pair of primers GPAT1-f1 primer GPAT1-r2, and strain, derived from MaGPAT3-D, (4) pair of primers MaGPAT3-2F and primer MaGPAT3-3R.

Strains prototroph for uracil and prototron on Latino, did not show any amplification by PCR with a pair of 1), however, showed amplification with a pair of (2). Therefore, it was demonstrated that these strains, as it turned out, are Δsct1:LEU2. In addition, since the pair (3) or (4) showed amplification, it was demonstrated that MaGPAT1 or MaGPAT3 inserted in these strains.

The above results showed that mutations Δgpt2 and Δsct1 in strains cause mortality atS. cerevisiaebut the strains become viable when the expression MaGPAT1 or MaGPAT3 obtained fromM. alpina. That is, MaGPAT1 or MaGPAT3 obtained fromM. alpina,was able to complementarity mutations Δgpt2 and Δsct1 in yeast strains. This suggests that the proteins encoded MaGPAT1 or MaGPAT3 obtained fromM. alpina,could have GPAT activity.

On the other hand, strains auxotrophic for uracil and auxotrophic for Latino, PCR amplification was observed with the pair (1), but was not observed with the pair (2), okazawa that these strains represented SCT1. The absence of amplification with the pair (3) or couple (4) showed that MaGPAT1 or MaGPAT3 obtained fromM. alpina,had not been built.

In addition, these strains were printed on agar medium SD-Met (2% agar) (6.7 g of yeast-based nitrogen without amino acids (DIFCO), 20 g glucose and 1.3 g of amino acids in the powder (a mixture of 1.25 g of adrinaline, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 1.5 g phenylalanine, 11,25 is serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, 1.8 g of leucine, 0.6 g of uracil) and agar medium SD-Lys (2% agar) (6.7 g of yeast-based nitrogen without amino acids (DIFCO), 20 g glucose and 1.3 g of amino acids in the powder (a mixture of 1.25 g of adrinaline, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.6 g of methionine, 1.5 g of phenylalanine, 11,25 g serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g of tryptophan, 1.8 g of leucine and 0.6 g of uracil) per 1 liter). Incubation was carried out at 30°C for 3 days to determine auxotrophic for methionine and auxotrophs of lysine. Based on the results, GP-11 (#SC-1), MaGPAT1-11 (#3b, #4a, #8a), MaGPAT3-11 (#2d, #19a, #32a), GP-21 (#SC-2), MaGPAT1-21 (#1a, #2d, #13a) and MaGPAT3-21 (#10a, #20c, #26b)selected from the strains for the respective genotypes are shown in TABLE 1, were presented for the following checks.

Compared the ability to produce fatty acids of strain only with SCT1 obtained from a yeast strain with only GPAT1 derived fromM. alpinathat strain only GPAT3 derived fromM. alpina,as GPAT gene. The above strains GP-11 and GP-21, containing only SCT1, derived from yeast, are as ura3 and leu2 and, thus, need to uracil and leucine. For complementaly auxotrophs to uracil and Latino, GP-11 and GP-21 were jointly transformed by the plasmid pESC-URA3 and the plasmid pESC-LEU2, respectively. The strains were selected to the to transformants with the ability to grow on agar medium SD-Ura,Leu (2% agar) (6.7 g of yeast-based nitrogen without amino acids (DIFCO), 20 g of glucose and 1.3 g of amino acids in the powder (a mixture of 1.25 g of adrinaline, 0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g phenylalanine, 11,25 g serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine and 1.2 g of tryptophan) 1 liter). Additionally, selected strains GP-12 (#1, 2, 3) and GP-22 (#1, #2, #3) were provided for subsequent checks.

[EXAMPLE 3]

Analysis of fatty acids yeast

Strains GP-12 (#1, 2, 3), MaGPAT1-11 (#3b, #4a, #8a), MaGPAT3-11 (#2d, #19a, #32a), GP-22 (#1, #2, #3), MaGPAT1-21 (#1a, #2d, #13a) and MaGPAT3-21 (#10a, #20c, #26b), obtained as described above were incubated as follows.

One platinum loop of each strain were seeded into 10 ml of liquid medium SD-Ura, Leu and cultured with stirring at 30°C for 1 day. Then 100 μl of the resulting culture was transferred into 10 ml liquid SD-Ura, Leu, and then were cultured with stirring at 30°C for 2 days. Yeast culture was centrifuged to collect cells. Cells were washed in 10 ml of sterile water and centrifuged again to collect cells. Cells liofilizirovanny. Fatty acids in the cells was converted into the methyl ester by the method of hydrochloric acid-methanol. The esters were extracted with hexane. After distillation of the hexane was analyzed by gas chromatography. The results of the analysis of the composition based on fatty acids are presented in tables 3-8.

Table 3 shows the em cell composition based on fatty acids (%) yeast met15, lys2. Numeric values are expressed using the average ± standard deviation (SD).

Table 3
StrainGP-12MaGPAT1-11MaGPAT3-11
16:020,54±0,1220,72±4,7729,32±0,80
16:137,35±0,1834,02±3,8127,87±0,64
18:06,54±0,10of 10.72±1,5513,42±0,45
18:125,62±0,1724,75±2,4420,97±0,53
18:1(n-7)2,99±0,062,43±1,320,59±0,51
Otherof 6.96±0,02of 7.36±1,277,83±0,26

Table 4 shows the density of yeast cells met15, lys2 after incubation. Numeric values are expressed using the average ±SD.

Table 4
StrainGP-12MaGPAT1-11MaGPAT3-11
The density of cells (g/l)0,84±0,030,90±0,030,92±0,03

Table 5 shows the level of production of fatty acids in yeast met15, lys2. Numeric values are expressed using the average ±SD.

Table 5
StrainGP-12MaGPAT1-11MaGPAT3-11
total fatty acids (mg/l)42,41±1,9165,79±24,3193,76±9,80

Table 6 shows the composition of cellular fatty acids (%) yeast MET15,LYS2. Numeric values are expressed using the average ±SD.

Table 6
StrainGP-22MaGPAT1-21MaGPAT3-21
16:07,73±0,694,47±0,23 5,88±0,53
16:151,71±0,6451,11±0,3650,63±0,96
18:03,54±0,082,60±0,02is 3.08±0,13
18:131,09±0,8734,11±0,6033,62±0,73
18:1(n-7)3,44±0,436,35±0,244,58±0,50
Other2,49±0,401,37±0,152,22±0,26

Table 7 shows the density of yeast cells MET15,LYS2 after incubation. Numeric values are expressed by the average±SD.

Table 7
StrainGP-22MaGPAT1-21MaGPAT3-21
The density of cells (g/l)4,88±0,224,93±0,214,84±0,22

Table 8 shows the level of production of fatty acids yeast MET15,LYS2. Numeric values you who awives using the average ±SD.

Table 8
StrainGP-22MaGPAT1-21MaGPAT3-21
total fatty acids (mg/l)109,09±14,99of 99.96±of 6.71121,85±13,92

In the respective cells, the density of cells depended on auxotrophs. Cell density yeast MET15,LYS2 was 5 times higher than the cell density of yeast met15,lys2, but any impact GPAT gene to its composition based on fatty acids was not observed (Tables 3 and 6). Further, in yeast MET15,LYS2, also not observed any significant difference in the ability to produce fatty acids, on the basis of differences in the gene GPAT.

On the other hand, in yeast met15,lys2 the level of production of fatty acids was higher by about 1.5 times in the strain MaGPAT1-11 and above about 2.2 times the strain MaGPAT3-11 obtained fromM. alpinegenome GPAT, compared with strain GP-12, with only SCT1 as GPAT gene. In addition, the level of production of fatty acids of strain MaGPAT3-11 was higher by about 1.4 times than that of strain MaGPAT1-11. Yeast met15,lys2 inhibited proliferation compared with yeast MET15,LYS2, and found that the expression obtained fromM. alpinegene is GPAT1 or gene GPAT3 significantly increased the level of production of fatty acids (tables 4 and 5). Comparison of compositions based on fatty acids showed an increase in the ratio of saturated fatty acid palmitic acid (16:0) or stearic acid (18:0) in the strain MaGPAT3-11, compared with strains G-12 and MaGPAT1-11 (table 3).

[EXAMPLE 4]

Analysis of the lipids of yeast

The level of each lipid and composition based on fatty acids tested for yeast met15,lys2 GP-12 (#1, 2, 3), MaGPAT1-11 (#3b, #4a, #8a) and MaGPAT3-11 (#2d, #19a and #32a)that showed differences in the level of production of fatty acids.

One platinum loop of each strain were seeded into 10 ml of liquid medium SD-Ura,Leu, and were cultured with stirring at 30ºC for 1 day. Then 1 ml of the obtained culture of each of the 2 strains were seeded into 10 ml of liquid medium SD-Ura,Leu, and then were cultured with stirring at 30ºC for 1 day. Yeast culture was centrifuged to collect cells. Cells were washed in 10 ml of sterile water and centrifuged again to collect cells. Cells liofilizirovanny. Fatty acids in the cells of one strain were converted into methyl esters by way of hydrochloric acid-methanol. The esters were extracted with hexane. After removal of the hexane by distillation analysis was performed using gas chromatography.

In addition, lipids were extracted from each of the strains as follows. Then there was added 1 ml of chloroform:methanol (2:1) and glass beads, and the cells were destroyed with the help of open the device for breaking beads and centrifuged, in order to select the supernatant. The remaining cells were additionally added 1 ml of chloroform:methanol (2:1), and the supernatant was collected, as described above, which is repeated in order to obtain the lipids in the end with 4 ml of chloroform:methanol (2:1). The solvent was removed by distillation, using the device speed-vac and the residue was dissolved in a small amount of chloroform. Thin-layer chromatography was performed under conditions of Silica Gel 60 Plate (Merck) and developing solvent hexane:diethyl ether:acetic acid = 70:30:1 in order to fractionate the lipids. Lipids were detected by spraying the solution primulina and irradiation of UV-rays. The fraction of triglycerides and the fraction of phospholipids were scraped, respectively, and collected in a test tube. Fatty acids were converted into methyl esters by way of hydrochloric acid-methanol. Analysis of fatty acids was performed using gas chromatography.

The results are presented in the following tables.

Table 9
The density of cells after incubation of yeast met15,lys2 (mean +SD)
StrainGP-12MaGPAT1-11MaGPAT3-11
(g/l)9,40±0,539,37±0,64 at 9.53±0,45

Table 10
The total level of fatty acids in yeast met15,lys2 (mean ±SD)
StrainGP-12MaGPAT1-11MaGPAT3-11
(mg/l)40,55±2,3460,74±4,9986,53±0,53

Table 11
The level of fatty acids in the phospholipid fraction of yeast met15,lys2 (mean ±SD)
StrainGP-12MaGPAT1-11MaGPAT3-11
(mg/l)of 10.73±4,9110,16±0,8411,22±3,01

Table 12
Composition based on fatty acids in the phospholipid fraction of yeast met15,lys2 (mean ±SD)
StrainGP-12MaGPAT1-11MaGPAT3-11
16:027,08±1,54 25,24±2,29as opposed to 28.18 per±1,52
16:131,33±4,7132,82±0,8231,97±1,69
18:08,63±of 4.38of 9.89±0,59of 9.56±was 2.76
18:127,44±6,0628,12±1,7826,97±3,24
Otherof 5.53±4,863,94±1,553,31±0,57

Table 13
The level of fatty acids in the triglyceride fraction of yeast met15,lys2 (mean ±SD)
StrainGP-12MaGPAT1-11MaGPAT3-11
(mg/l)17,18±0,1627,43±4,2335.32 per±3,97

Table 14
Composition based on fatty acids in the triglyceride fraction of yeast met15,lys2 (mean ± SD)
StrainGP-12 MaGPAT1-11MaGPAT3-11
16:0fall of 19.88±0,4620,72±4,1927,39±0,70
16:138,61±1,1533,90±3,1229,34±0,69
18:07,10±1,3211,16±1,05of 12.76±0,33
18:124,96±0,8824,53±3,1820,34±0,48
Other9,44±0,459,70±1,0610,17±0,25

At the cellular density GPAT genes did not influence. Levels of production of fatty acids in terms of the environment, strain MaGPAT1-11 GPAT gene derived fromMortierella alpinahas shown an increase of about 1.5 times, and the strain MaGPAT3-11 - an increase of about 2 times than the strain GP-12, with only SCT1 as GPAT gene.

The level of fatty acids and the composition based on fatty acids of phospholipids GPAT genes did not influence. On the other hand, the level of triglycerides was higher than 1.6 times the strain MaGPAT1-11 and 2 times higher in the strain MaGPAT3-11 GPAT gene derived fromMortierella alpinathan in the strain, GP-2, having only SCT1 as GPAT gene.

It was found that increased the total level of production of fatty acids in strain MaGPAT1-11 and the strain MaGPAT3-11, mainly due to an increase in triglycerides. Comparison of compositions based on fatty acids in the triglycerides revealed an increase in the ratio of saturated fatty acids in strain MaGPAT3-11, compared with the strain GP-12.

It was found that the ability to produce triglycerides, in particular, can be improved by gene expression MaGPAT3.

Industrial applicability

By the expression of polynucleotide of the present invention in the appropriate cell host can effectively produce triglycerides regardless of the rate of growth of the host cell. Furthermore, increased triglyceride levels also leads to an increase in fatty acids, which form triglycerides. In accordance with the present invention, triglycerides and fatty acids can be used for food, cosmetics, medicines, soap and so on.

1. Polynucleotide selected from the group consisting of the following (a)to(e), which encodes a protein with glycerol-3-hospitalstrasse activity:
(a) polynucleotide containing the nucleotide sequence of SEQ ID NO: 1 or 4;
(b) polynucleotide encoding a protein comprising consecutive amino acid the activity SEQ ID NO: 2;
(c) polynucleotide encoding a protein consisting of the amino acid sequence, where 1-10 amino acids deleted, replaced, built and/or added in the amino acid sequence of SEQ ID NO: 2, and having a glycerol-3-hospitalstrasse activity;
(d) polynucleotide, encoding a protein with amino acid sequence homology of at least 90% amino acid sequence of SEQ ID NO: 2, and having a glycerol-3-hospitalstrasse activity; and,
(e) polynucleotide that hybridizes with polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or 4 in harsh conditions, and which encodes a protein having glycerol-3-hospitalstrasse activity.

2. Polynucleotide according to claim 1, containing the nucleotide sequence of SEQ ID NO: 1, or 4.

3. Polynucleotide according to claim 1, encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.

4. Polynucleotide according to any one of claims 1 to 3, which is a DNA.

5. The protein encoded by polynucleotides according to any one of claims 1 to 4, which is used to obtain the lipid composition or the composition of fatty acids.

6. A vector containing polynucleotide according to any one of claims 1 to 4, which is used for the expression of glycerol-3-hospitalstrasse.

7. The transformant, not class is audica transformants person, which introduced polynucleotide according to any one of claims 1 to 4, which is used to obtain the lipid composition or the composition of fatty acids, where the transformant is a yeast or a lipid-producing fungus.

8. The transformant, which is not transformants person, which introduced the vector according to claim 6, which is used to obtain the lipid composition or the composition of fatty acids, where the transformant is a yeast or a lipid-producing fungus.

9. The transformant according to claim 7 or 8, wherein the transformant is a lipid-producing fungus.

10. The transformant according to claim 9, where the lipid-producing fungus is a Mortierella alpina.

11. The method of obtaining the lipid composition or compositions based on fatty acids, which provides for the collection of the lipid composition or compositions based on fatty acids from the culture of transformant according to any one of claims 7 to 10.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: genetic structure pAd-SM is produced, being built by homological recombination of the vector pAdEasy-1, containing the main part of genome of adenovirus and plasmid pAdTrack-CMV, where cDNA fragments of human genes SOX2 and C-MYC are placed, being connected with a nucleotide sequence that codes P2A-peptide. The plasmid pAd-SM produced as a result of homological recombination contains a fragment SOX2-P2A-C-MYC under control of a constitutive promotor CMV.

EFFECT: possibility to produce adenovirus particles for simultaneois delivery of genes and expression of proteins SOX2 and C-MYC in human cells, pAd-SM contains a gene that codes a fluorescent protein EGFP, under control of a CMV promotor, which makes it possible to track transduction of cells and elimination of virus DNA from a cell.

3 cl, 1 dwg

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.

EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.

12 cl, 15 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of general formula III and their pharmaceutically acceptable salts, A represents (C1-C6)alkyl-O-, phenyl-(C1-C6)alkyl-O-; aryl, selected from phenyl, naphthyl, and which is possibly substituted by 1-3 substituents, given in the invention formula; or heteroaryl, which has four or five carbon atoms and one heteroatom, selected from oxygen, nitrogen and sulphur, which is possibly substituted by 1-3 substituents, given in the invention formula; B represents phenyl, possibly substituted by 1-3 substituents, where substituents are selected from (C1-C6)alkyl, (C3-C7)cycloalkyl, (C1-C6)alkyl-O-, hydroxy, amino and halogeno; and R1 and R2 independently represent (C1-C6)alkyl, phenyl-(C1-C6)alkyl-, hydroxy-(C1-C6)alkyl, (C3-C7)cycloalkyl, (C2-C6)alkenyl or (C2-C6)alkynyl; on condition that R1 is different from R2; where absolute configuration of asymmetric R1 and R2 -carrying carbon atom is mainly R-configuration. Invention also relates to pharmaceutical composition, possessing ability to modulate gene expression, methods of modulation of gene expression in host cell, method of regulating expression of endogenous or heterologous gene in transgenic subject, method of regulating transgenic expression in transgenic subject, method of polypeptide production and to method of obtaining formula IV compound. Method includes stages: a) interaction of formula V compound with formula IV compound with obtaining formula VII compound; b) reduction of formula VII compound with obtaining formula VIII compound, b) interaction of formula VIII compound with formula B-CO-LG compound, where B has values, given above, and LG is leaving group representing -F, -Cl or -Br, with formation of formula IX compound, d) removal of group R7CO2- from formula IX compound with obtaining formula X compound, e) interaction of formula X compound with formula A-CO-LG compound, where A has values, given above, and LG is leaving group, representing -F, -Cl or -Br, with obtaining formula IV compound ( compounds of formulas V, VI, VII, VIII, IX, X are given in the invention formula).

EFFECT: obtaining formula III compounds, possessing ability to modulate gene expression.

19 cl, 4 ex, 2 tbl, 78 ex

FIELD: biotechnology.

SUBSTANCE: antibodies are used as pharmaceutical means for treatment or prevention of inflammatory diseases.

EFFECT: invention enables to obtain antibodies with effective neutralising activity against NR10.

7 cl, 33 dwg, 19 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: isolated recombinant adenosine deaminase is described, which comprises polypeptide SEQ ID NO: 1 or a version polypeptide SEQ ID NO: 1 of isolated recombinant adenosine deaminase, where the version polypeptide SEQ ID NO: 1 comprises one or more amino acid substitutions selected from the group consisting of: Gin instead Lysl98; Ala instead Thr245; and Arg instead of Gly351, and DNA encoding it. The conjugate of polyalkylene oxide with the said adenosine deaminase for treatment of adenosine deaminase-mediated diseases is presented where adenosine deaminase comprises from 11 to 17 chains of polyalkylene oxide with a molecular weight of 5 kDa for ADA protein. The methods of purification of the recombinant adenosine deaminase are proposed, including protein purification using ion exchange chromatography, or protein purification using hydrophobic interaction chromatography. Also the preparations of recombinant adenosine deaminase produced by these methods are provided.

EFFECT: invention enables to obtain recombinant adenosine deaminase, having increased stability.

14 cl, 1 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: method comprises culturing CHO cell in which the gene of taurine transporter (TauT) was artificially transferred, the DNA encoding the desired polypeptide and the DNA encoding dihydrofolate reductase (DHFR) was introduced in the presence of methotrexate concentration at which 90% or more cells, in which TauT was not introduced die within three weeks after subculturing. From the number of surviving CHO cells the CHO cell is selected which is capable to produce a desired polypeptide with a high yield. The cell prepared in this manner produces a desired polypeptide in higher yield than the CHO cell transfected with DNA encoding the desired polypeptide and DNA encoding DHFR, but not the genome of taurine transporter. The method of producing a desired polypeptide comprises culturing the above mentioned cell and isolating the desired peptide.

EFFECT: invention enables to produce the desired polypeptide with a high yield.

9 cl, 10 dwg, 6 ex

FIELD: biotechnology.

SUBSTANCE: proposed enzymatically inactive IgA1 protease with replacement Ser267Ala for use as a component of a polyvalent vaccine designed to protect people against meningococcal infection and other microorganisms, which pathogenicity is caused by IgA1 protease. The invention includes a polynucleotide encoding the said mutant form of IgA1 protease of Neisseria meningitidis of serogroup B, comprising the said polynucleotide, a recombinant plasmid DNA, the strain Escherichia coli - producer of a mutant form of IgA1 protease according to the invention, the method of preparing a recombinant form of the enzyme using a technology of recombinant DNA, and recombinant inactivated IgA1 protease.

EFFECT: increased level of immunogenicity.

6 cl, 1 tbl, 5 dwg, 7 ex

FIELD: biotechnology.

SUBSTANCE: versions of modified yeast cells are presented. Each of the versions produces hydrogen sulfide at a reduced level and includes exogenous polynucleotide encoding a polypeptide MET10 which catalyses the transformation reaction of sulfite to sulfide at a reduced level, where the amino acid at the position 662 of the polypeptide MET10 is Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Asn, Gin, Arg, Val, Trp or Tyr (SEQ ID NO: 5). The expression vector is described comprising the said exogenous polynucleotide. The culture of the said cells is described. A method of preparing the said yeast cell is proposed, including the replacement of endogenous polynucleotide encoding a sulfide-active polypeptide MET10 to polynucleotide encoding MET10 with the said replacement at the position 662 SEQ ID NO: 5, and the parent yeast cell produces hydrogen sulfide. The method of reduction of H2S level in fermentation medium is described, including the contact of the fermentation medium with the mentioned above yeast cell. The fermentation medium and the fermentation product are proposed containing the said cell or the cell culture.

EFFECT: invention enables to reduce the level of H2S in the fermentation medium in production of fermented beverages.

56 cl, 8 dwg, 11 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there are presented an expression system for high gene expression including a promoter, and at least one MAR sequence; an isolated and purified nucleic acid molecule representing a MAR sequence; a cell; a transgenic animal; a kit for high expression, as well as the use of said expression system for increased protein production.

EFFECT: invention may be used for producing a wide range of proteins including antibodies and interferons by transgenic animals and cell cultures.

7 cl, 15 dwg, 5 tbl, 1 ex

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex

FIELD: gene engineering, biotechnology, medicine.

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EFFECT: high specific diagnosis method with improved accuracy.

2 cl, 2 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: heterologous protein is obtained by cultivation of strain Corynebacterium glutamicus AJ 12036, which does not produce cell surface protein and contains gene expressing construct wherein nucleic acid sequence encoding signal peptide of cell surface protein of Corynebacterium glutamicus or C. ammoniagenes is bound in direct direction to promoter sequence, and nucleic acid sequence encoding heterologous protein is bound in direct direction to abovementioned nucleic acid sequence encoding signal peptide. Further heterologous protein secreted from cells is isolated.

EFFECT: high effective method for heterologous protein production.

6 cl, 8 tbl, 10 ex

FIELD: biotechnology, agriculture.

SUBSTANCE: one should carry out cell transformation of hard or soft wheat varieties with, at least, two product-coding genes and blocking the nutrition of Eurygaster integryceps Puton, where, at least, one of the genes mentioned is functionally connected with a promoter that provides constitutive expression of gene's product in a plant and, at least, the second of the mentioned genes is functionally connected with a promoter that provides tissue-specific expression of gene product in a plant. Wheat plant should be regenerated out of mentioned transformed cells. The innovation suggested enables to increase resistance of wheat varieties to harmful bugs (Eurygaster integryceps Puton) and, thus, obtain high-quality grain and keep the yields.

EFFECT: higher efficiency.

4 cl, 1 dwg, 7 ex, 3 tbl

FIELD: selection and biotechnology of plants, agriculture.

SUBSTANCE: invention relates to highly effective system of genetic transformation of sugar beet plants. Method involves Agrobacterium-mediated transformation of sugar beet plant cotyledonous unit meristematic cells cultured in vitro that did not enter in the stage of callus-formation or regeneration of sprouts. Then sprouts are regenerated from transformed cells. Invention provides enhancing effectiveness and frequency of transformation of sugar beet plants.

EFFECT: improved preparing method of plants.

5 cl, 1 dwg, 2 tbl, 6 ex

FIELD: biotechnology, medicine.

SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.

EFFECT: valuable medicinal properties of allergens.

33 cl, 62 dwg, 10 ex

FIELD: gene engineering, in particular production of transgenic potato with Colorado beetle resistance and biological and food safety.

SUBSTANCE: recombinant polynucleotide sequence of general formula X-A, wherein X is part of nucleotide sequence of introduced genetic construct representing in SEQ ID N 1; A is nucleotide sequence of flanking site of genomic DNA of genus Elizaveta potato, representing in SEQ ID N 2 is constructed; plant cell producing of cryIIIa protein and containing recombinant polynucleotide sequence, as well as potato transgenic plant and vegetative generation thereof with Colorado beetle resistance are obtained. Polynucleotide sequence is used in identification of plant cell producing of cryIIIa protein, plant and vegetative generation.

EFFECT: new potato genus with Colorado beetle resistance.

6 cl, 6 dwg, 7 tbl, 7 ex

FIELD: gene engineering, in particular production of transgenic potato with Colorado beetle resistance and biological and food safety.

SUBSTANCE: recombinant polynucleotide sequence of general formula X-A, wherein X is part of nucleotide sequence of introduced genetic construct representing in SEQ ID N 1; A is nucleotide sequence of flanking site of genomic DNA of genus Nevskiy potato, representing in SEQ ID N 2 is constructed; plant cell producing of cryIIIa protein and containing recombinant polynucleotide sequence, as well as potato transgenic plant and vegetative generation thereof with Colorado beetle resistance are obtained. Polynucleotide sequence is used in identification of plant cell producing of cryIIIa protein, plant and vegetative generation.

EFFECT: new potato genus with Colorado beetle resistance.

6 cl, 6 dwg, 7 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: DNA is constructed encoding protein, advancing fungus resistance to certain group of compounds. Alternatively DNA is constructed having defect in function and encoding protein, which advances lowering of GPI-anchored protein amount in fungus cell wall. Encoded protein is useful in production of antibody thereto which may by applied as active ingredient of antifungal agent.

EFFECT: new method for production of antifungal agent.

11 cl, 8 dwg, 1 tbl, 2 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for producing methanol. Method involves culturing recombinant microorganism E. coli at temperature 20-30°C. Prepared culture and cells isolated from this culture, or treated product of these cells are remained in contact with methane for producing methanol. Indicated recombinant microorganism ahs ability to convert methane to alcohol as result of transformation of DNA encoding enzyme methane oxygenase of soluble type. Invention provides expression of all components of methane oxygenase that allows realization of method for producing methanol under milder conditions.

EFFECT: improved producing method.

3 cl, 1 dwg, 1 tbl

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