JP5004083B2 - Recombinant diacylglycerol acyltransferase with high enzymatic activity - Google Patents

Description

  The present invention relates to a method for producing a diacylglycerol acyltransferase, which is a synthase of triacylglycerol, which is a major component of storage lipids, with a highly active recombinant, and the resulting highly active recombinant diacylglycerol acyltransferase itself. Is.

A diacylglycerol acyltransferase is a synthase of triacylglycerol, which is a major component of storage lipids, and an enzyme having an important role in the synthesis of storage lipids. Therefore, it is a target for modifying lipid production by oily microorganisms and plants (Non-Patent Document 1), and on the contrary, to improve lipid accumulation related to metabolic syndrome such as obesity, hyperlipidemia, diabetes, etc. (Non-Patent Document 2).
The diacylglycerol acyltransferase (hereinafter also referred to as DGAT) is a gene sequence of DGAT1 (Non-patent Document 3) having homology to cholesterol acyltransferase and DGAT purified from Mortierella lamaniana bar angerspora based on the gene sequence. And DGAT2 (Non-patent Document 4). There is no homology between the two. Both have a homologous gene (ortholog) in microorganisms, plants and animals, but their roles in triacylglycerol synthesis are thought to be different.
In mice, the destruction of DGAT1 did not significantly reduce triacylglycerol and was resistant to diet-induced obesity (Non-Patent Document 5), whereas the destruction of DGAT2 showed a marked reduction in triacylglycerol. Died immediately after birth (Non-patent Document 6). In addition, even in budding yeast, the disruption of the DGAT2 type DGA1 gene resulted in a significant reduction in triacylglycerol synthesis than the disruption of the DGAT1 type ARE1, ARE2 gene (Non-patent Document 7). From the above results, it is considered that the DGAT2 type enzyme is responsible for the main reaction of triacylglycerol synthesis. Hereinafter, in the present invention, when simply referred to as diacylglycerol acyltransferase or DGAT, it refers to a DGAT2 type enzyme.

As described above, DGAT is an important enzyme in lipid production or lipid accumulation, and the search for a substance that activates or inhibits the enzyme activity is important for improvement of lipid production and disease associated with lipid accumulation. It is. However, DGAT is a membrane-bound unstable enzyme, and a natural source of stable enzyme is not known. On the other hand, a DGAT gene is obtained (Non-Patent Document 4, Non-Patent Document 8, Patent Document 1, Patent Document 2) and expressed in insect cells, Saccharomyces cerevisiae, etc. to obtain a gene product having enzyme activity. However, the enzyme activity obtained from the reports varies. When DGAT2 of castor seeds or drill seeds was expressed in yeast, the DGAT activity at the microsomal membrane was 20-50 pmol / min / mg protein (Non-patent Documents 9 and 10). On the other hand, when the DGAT2 of Mortierella, Lamaniana, Bar, and Agrispora, and the DGA1 gene of Saccharomyces cerevisiae are expressed in insect cells, the activity in the microsomal membrane is as high as 30-200 nmol / min / mg protein. As shown (Non-patent Document 4, FIG. 5), in another experiment of the same report, the numerical value of the picomolar unit, which is about 100 pmol / min / mg protein of enzyme activity under the same conditions (Non-patent Document 4, FIG. 7) In view of this, it is possible that the high activity value described above may be mislabeled, and at least stable high activity is not exhibited. In other reports, even when mouse DGAT2 is expressed in insect cells, the enzyme activity of the microsomal membrane is about 600 pmol / min / mg protein (the enzyme activity when not expressed is about 200 pmol / min / mg). protein) (Non-patent Document 8), which does not exceed the order of picomolar. In either case, the expression of DGAT was only confirmed by the enzyme activity on the membrane surface, and reports that these expressed DGAT were used stably were further isolated and had high specific activity. There is no report that a DGAT having the above has been obtained.
International Publication 2000/001713 (WO00 / 001713) International Publication 2000/004682 (WO 02/04682) JP2006-042738 Lung, S.-C. and Weselake, RJ Lipids, 41, 1073 (2006) Coleman, RA and Lee, DP Prog. Lipid Res. 43, 134 (2004) Cases, S. et al, Proc. Natl. Acad. Sci. USA 95, 13018 (1998) Lardizabal, K. et al, J. Biol. Chem. 276, 38862 (2001) Smith, SJ et al, Nature Genetics 25, 87 (2000) Stone, SJ et al, J. Biol. Chem. 279, 11767 (2004) Sandager, L. et al, J. Biol. Chem. 277, 6478 (2002) Cases S. et al, J. Biol. Chem. 42, 38870 (2001) Kroon, JTM et al. Phytochemistry 67, 2541 (2006) Shockey, JM et al. Plant Cell, 18, 2294 (2006) Nikawa, J. and Kawabata, M., Nucleic Acids Res. 26, 860 (1998)

  An object of the present invention is to provide a method for expressing a DGAT gene in a large amount in a state having a high enzyme activity, and a recombinant DGAT having a high enzyme activity, and also a DGAT or a group having a high activity at a high concentration. This is to establish a system for searching for a substance that activates or inhibits DGAT activity using a transformed host (survival state, crushed material or culture solution) containing a modified DGAT.

As a result of various studies to solve the above problems, the present inventors have used a mutant microorganism in which the SNF2 gene is disrupted as a host for expressing the DGAT gene, so that the enzyme activity is extremely high as a DGAT gene product to be expressed. We succeeded in expressing high DGAT in large quantities.
The present inventors previously identified the SNF2 gene of Saccharomyces cerevisiae by transposon insertion mutation as a gene that suppresses lipid synthesis, and by destroying this gene, the lipid productivity of this mutant strain is improved. (Patent Document 2), and obtained the knowledge that the lipid content can be increased to about 30% by overexpressing the DGA1 gene, which is a DGAT gene of Saccharomyces cerevisiae, in this SNF2 gene disrupted strain (Japanese Patent Application 2006-31705). From these findings, although it was predicted that DGAT activity was increased in the SNF2 gene-disrupted strain, even when the DGA1 gene was overexpressed in a normal wild strain of Saccharomyces cerevisiae used as a control, it was already 17% Although the DGAT activity is increased in the SNF2 gene-disrupted strain in view of the increase in the lipid content, the increase in the DGAT activity is several times to 10 times that in the wild strain. It was expected to be about double.
However, when the DGAT activity in the SNF2 gene disrupted strain was actually measured, a surprising increase in enzyme activity of several hundred times was observed. The present invention has been made based on the finding that the DGAT activity in the SNF2 gene-disrupted strain is much higher than expected.
Moreover, it was confirmed that even when a tag peptide was attached to the DGAT protein, the high enzymatic activity of the expressed DGAT gene product was not lost, and it was found that the recombinant DGAT could be easily purified as needed. Furthermore, since the enzyme activity of DGAT in the above-mentioned transformed microorganism is extremely high and stable, it has been found that a clear enzyme activity can be detected in a crushed solution without further concentration and purification of the enzyme protein. And the screening system of the activator or inhibitor of DGAT using the crushing liquid containing the same was established, and it came to complete this invention.

That is, the present invention is as shown in the following (1) to (7).
[1] A recombinant diacylglycerol acyltransferase obtained by culturing the obtained transformant by introducing a diacylglycerol acyltransferase gene into a host cell in which the SNF2 gene or its ortholog gene has been disrupted or reduced in function.
[2] The recombinant diacylglycerol acyltransferase according to [1], wherein the host cell is Saccharomyces cerevisiae in which the SNF2 gene is disrupted or has a reduced function.
[3] The recombinant diacylglycerol acyltransferase according to [1] or [2] above, wherein the diacylglycerol acyltransferase gene is a DGA1 gene of Saccharomyces cerevisiae.
[4] The above-mentioned [3], wherein the transformant in which the DGA1 gene is introduced into Saccharomyces cerevisiae in which the SNF2 gene is disrupted or reduced in function further has the LEU2 gene introduced therein. Recombinant diacylglycerol acyltransferase.
[5] The product of the diacylglycerol acyltransferase gene has been modified to have a tag peptide, and has been uniformly purified from the expression product obtained by culturing the transformant according to the properties of the introduced tag peptide. The recombinant diacylglycerol acyltransferase according to any one of [1] to [4] above, wherein
[6] A diacylglycerol acyltransferase gene is introduced into a host cell in which the SNF2 gene or its orthologue gene has been disrupted or reduced in function, the transformant is cultured, and the resulting expression product is combined. A method for producing a recombinant diacylglycerol acyltransferase, comprising obtaining a substituted diacylglycerol acyltransferase.
[7] The recombinant diacylglycerol acyl according to [6] above, wherein the SGA2 gene is transformed or introduced into a Saccharomyces cerevisiae in which the SNF2 gene is disrupted or reduced in function. A method for producing a transferase.
[8] The method for producing a recombinant diacylglycerol acyltransferase according to [7], wherein a LEU2 gene is also introduced when the DGA1 gene is introduced for transformation.
[9] The product of the diacylglycerol acyltransferase gene is modified so as to have a tag peptide, and is uniformly purified from the expression product obtained by culturing the transformant according to the properties of the introduced tag peptide. The method for producing a recombinant diacylglycerol acyltransferase according to any one of the above [6] to [8], comprising the step of:
[10] The method for producing a recombinant diacylglycerol acyltransferase according to any one of [6] to [9], wherein the transformant is cultured in a nitrogen source-deficient medium.
[11] Screening of a substance that activates or inhibits the enzyme activity of diacylglycerol acyltransferase, wherein the recombinant diacylglycerol acyltransferase according to any one of [1] to [5] is used. Method.

  According to the present invention, a method of expressing a DGAT gene in a large amount and in a state having high enzyme activity can be provided, and at the same time, a recombinant DGAT having high enzyme activity can be provided. In addition, since the transformed host before purification contains highly active recombinant DGAT, the activator or inhibitor of DGAT enzyme can be used as it is in the transformed host or its disrupted solution as in the case of recombinant DGAT. Thus, a simple screening method could be provided. In particular, since DGAT inhibitors are targets for drug discovery for diseases such as obesity and diabetes, the present invention leads to the provision of drug discovery for diseases such as obesity and diabetes.

In the present invention, a DGAT gene, which is an enzyme that synthesizes triacylglycerol, which is a major storage lipid, is expressed in a host cell in which the SNF2 gene or its ortholog is disrupted or reduced in function, thereby obtaining a DGAT protein having high enzyme activity. To do.
The SNF2 gene of Saccharomyces cerevisiae has a wide range of orthologs in prokaryotes and eukaryotes, and has the universal function of reconstructing chromatin that wraps gene DNA. Therefore, microorganisms other than Saccharomyces cerevisiae that have disrupted the ortholog of the SNF2 gene, or animal and plant cells can be used as hosts. Similarly, when a DGAT gene is introduced and expressed in these host cells, recombinant DGAT with high enzyme activity can be obtained.
In the present invention, the method for obtaining a mutant in which the SNF2 gene is disrupted can be performed by the methods already described (Non-patent Document 11, Patent Document 3) and the like.
For example, by creating a DNA in which the upstream and downstream sequences of the target gene to be disrupted are added to both ends of the marker gene, and transforming the host using the DNA, the introduced DNA will be disrupted. It is possible to cause homologous recombination in the upstream and downstream regions of the gene to destroy the target gene. Mutants in which the gene of interest is disrupted can be identified by phenotypic changes due to the expression of marker genes.
In the present invention, other techniques can be used as long as they can delete or reduce the function of the gene. These include a method of transforming using a fragment obtained by isolating the gene to be disrupted, cleaving with an appropriate restriction enzyme, and inserting the marker gene into the cleavage site, There are a method of amplifying and transforming by using a plumer containing a part of the above, a method of transforming, or a method of screening for a mutant of a target gene by causing a random mutation using a mutagen or the like.

  The DGAT gene to be expressed in the present invention is a gene belonging to the DGAT2 family having homology with the DGA1 gene of Saccharomyces cerevisiae, and can be derived from any organism as long as it can be expressed in the host organism. Even if it is available. The organism from which the DGAT gene to be expressed is derived may be the same as the organism from which the host cell is to be expressed, or may be another organism. The optimal DGAT gene may be selected and used by considering the ease of expression, the strength of the enzyme activity, the substrate specificity of the enzyme, and the like. The DGAT gene of the present invention is not only a natural DGAT gene but also a fragment, a nucleic acid obtained by modifying a part of the base sequence, or a primer or probe derived from the sequence of the natural DGAT gene. It is included when the nucleic acid can be obtained under conditions and the expression product is a protein having DGAT activity similar to that of natural DGAT.

The expression method of the DGAT gene in the present invention may be incorporated into a vector such as a plasmid that replicates extrachromosomally or may be incorporated into a vector that is incorporated into the chromosome. A method of directly introducing a DGAT gene such as a liposome method can also be used.
In addition, when a DGA1 gene is expressed by transforming Saccharomyces cerevisiae using a vector into which the DGA1 gene is inserted, it is known that the lipid content is higher when the LEU2 gene is expressed at the same time ( Japanese Patent Application No. 2006-31705) and LEU2 gene are preferably added at the same time for transformation.

Since the DGAT having high enzyme activity expressed in the present invention can be easily detected in the lysate itself (or cell culture solution) of the expressed organism, further cell fractionation is performed for activity measurement. It is not necessary to prepare a membrane fraction or to perform solubilization or purification with a surfactant. Therefore, in the screening of compounds that examine the influence of DGAT derived from various organisms on the enzyme activity, it is possible to establish the screening without trouble.
Recombinant DGAT with high enzyme activity expressed in the present invention retains high enzyme activity even though it can be easily purified with a tag peptide attached. Tag peptides include 6xHis, GST (glutathione S transferase), FLAG, maltose binding protein, and the like, and any of them may be used as long as it is not provided at a position that affects enzyme activity. Recombinant DGAT with a tag peptide can be easily and uniformly purified by an affinity column that specifically recognizes each tag peptide after solubilization with a surfactant. Therefore, by culturing the obtained transformant in large quantities, recombinant DGAT with high enzyme activity can be easily obtained in a state of being purified in large quantities. Such a DGAT sample is not a highly sensitive measurement method using a commonly used radioisotope, but can also be measured by a spectroscopic method, and since there are few impurities, it has a nonspecific effect. Since it can be eliminated to the minimum, it can be used for high-throughput screening of substances that activate or inhibit DGAT activity.

  The recombinant DGAT obtained in the present invention is known to be the same as natural yeast DGAT at the amino acid sequence level because it is a known expression product of the DGA1 gene, but has been conventionally recognized as DGAT activity. In view of the extremely high activity that cannot be predicted from the activity value, there is a high probability that it is a novel protein that has undergone post-expression modification. In other words, depending on the environment in the host cell called SNF2 gene disrupted yeast, it was subjected to some modification such as glycosylation or phosphate group addition or the modification was released, and a new and excellent enzyme protein could be provided. Conceivable. As described later, at least a novel recombinant DGAT that is distinguished from a protein recognized as a natural DGAT is provided, or at least a novel recombinant DGAT composition is provided. I can say that. Since it can be said that other microorganisms or animal and plant cells in which the ortholog gene of the SNF2 gene is disrupted can also provide a similar environment, the DGAT protein expressed in the transformant of the microorganism or animal and plant cells also has a high enzymatic activity. A variant is obtained.

In the present invention, a normal culture solution or medium for transformed cells can be used for culturing a transformant expressing the DGAT gene. For example, in the case of transformed yeast, an SD medium (medium in which glucose is used as a carbon source, yeast nitrogen base is used as a nitrogen source, and required nutrients are added) as described in Examples below can be used. However, with respect to the type of carbon source, nitrogen source, culture temperature, culture time, and the like of the medium, optimum conditions for obtaining a large amount of recombinant DGAT having high enzyme activity can be appropriately selected. As these conditions, the amount of nitrogen source in the medium is important. If a nitrogen source-deficient medium is used in the range where microorganisms can grow, the expression level of recombinant DGAT with high enzyme activity can be increased along with the increase in lipid content of microorganisms. Can be significantly increased.
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples.

[Example 1]
Obtaining recombinant DGAT with high enzymatic activity by expressing DGAT gene in Saccharomyces cerevisiae SNF2 gene disruption strain Saccharomyces cerevisiae SNF2 gene mutant strain (BY4741Δsnf2 strain) (Mat a leu2Δ0 his3Δ1 ura3Δ0 met15Δ0 SNF2: kanMX) (manufactured by Invitrogen) or wild strain (BY4741 strain) (Mat a leu2Δ0 his3Δ1 ura3Δ0 met15Δ0) (manufactured by Invitrogen) was used as a host. As the DGAT gene to be expressed, DGA1, which is Saccharomyces cerevisiae's own DGAT gene, was used. DGA1 was obtained by amplification by PCR using Saccharomyces cerevisiae genomic DNA as a template and primers for DGA1 in Table 1. This primer is designed to amplify the full length of the gene based on the already determined genomic DNA sequence, and contains a restriction enzyme SacI recognition site and an XbaI recognition site at its ends. PCR amplification conditions were as follows: 0.4 units KOD plus polymerase (Toyobo), 0.2 mM dNTP mixture, 0.5 μM primer, 10 ng Saccharomyces cerevisiae genomic DNA in the attached buffer containing 1 mM MgCl2. (20 μl of total reaction solution). Amplification was performed by reacting at 94 ° C. for 2 minutes and then repeating 25 times with 94 ° C. (15 seconds) / 55 ° C. (30 seconds) / 68 ° C. (90 seconds) as one cycle. The reaction solution was subjected to 0.7% agarose gel electrophoresis, and after ethidium bromide staining, it was irradiated with ultraviolet rays to confirm that a single band of the expected size was obtained. The amplified DGA1 was purified by PCR purification kit (Qiagen), excluding primers. FAA3 was similarly amplified by PCR and obtained. However, 1.2 mM MgCl 2 was used, and the amplification cycle was 94 ° C. (15 seconds) / 60 ° C. (30 seconds) / 68 ° C. (120 seconds).

The obtained DGA1 gene is subjected to restriction enzyme treatment with SacI and XbaI (manufactured by Nippon Gene Co., Ltd.) to create a sticky end by SacI cleavage and a sticky end by XbaI cleavage at both ends. As a vector, pL1091-5 (multi-copy vector with 2 μm as the replication initiation site, which has an ADH1 promoter highly expressed in glucose medium for expression of the insert gene, has the URA3 gene as a selection marker in yeast, and is selected in E. coli. These vectors were similarly cleaved with SacI and XbaI.
Both the insert and vector were removed by PCR purification kit to remove low-molecular oligonucleotides cleaved with restriction enzymes, and the insert gene was incorporated into the vector by ligation. Ligation was carried out by mixing at a vector: insert ratio of about 1/1 to 1/10 and reacting at 16 ° C. for 1 hour to 3 hours using Ligation high (manufactured by Toyobo).
Transformation of the ligated vector into E. coli was performed using E. coli JM109 strain competent cells (ECOS, Nippon Gene). Ligation reaction solution was added to competent cells, incubated for 5 minutes in ice, then heat shocked at 42 ° C for 45 seconds, spread on LB agar medium containing ampicillin and cultured at 37 ° C overnight for resistance to ampicillin resistance. The presence or absence of colonies was confirmed. A colony grown in a medium containing ampicillin is considered to be Escherichia coli transformed with a vector having an ampicillin resistance gene.
Among the obtained colonies, colony PCR was performed in order to detect a vector containing the insert gene. Primers for this PCR (pVT100L-5 ′ and pVT100L-3 ′) were designed from the sequences of the ADH1 promoter and terminator (Table 1), and the insertion of the insert gene into the multicloning site between the ADH1 promoter and terminator was performed. This can be confirmed by amplification of the band corresponding to the size. The PCR solution at this time was prepared by rinsing the tip of a bamboo skewer with a colony pierced in 1 unit Ex Taq (Takara), 0.2 mM dNTP mixture, and 0.5 μM primer suspended in the attached buffer. (20 μl of total reaction solution). Amplification was performed by reacting at 95 ° C. for 1 minute, and then repeating 25 times with 95 ° C. (30 seconds) / 48 ° C. (30 seconds) / 72 ° C. (2 minutes) as one cycle.
E. coli transformed with the vector containing the insert gene was transformed into a single colony and then cultured overnight at 37 ° C. in LB medium supplemented with ampicillin. The vector contained in the obtained Escherichia coli was purified using a plasmid extraction kit (QIAprep Spin Miniprep Kit, manufactured by Qiagen). The purified vector was treated with various restriction enzymes to confirm that the target insert gene was inserted in the correct direction. In addition, the correct integration of the DGA1 gene sequence into the obtained plasmid was confirmed by determining the DNA sequence of the insert region. For sequencing, fluorescent labeling was performed using a Thermo Sequenase Cy5.5 dye terminator cycle sequencing kit (manufactured by Veritas), and a nucleotide sequence was determined using a Long-Read Tower DNA sequencer (manufactured by Veritas).

The Saccharomyces cerevisiae BY4741 wild strain and Δsnf2 strain were transformed with the vector into which the DGA1 gene was inserted (pL1091-5 / DGA1). At that time, the LEU2 gene (Japanese Patent Application No. 2006-31705) having an action of increasing the lipid content was also added for transformation. Specifically, pL1177-2, a vector in which the URA3 gene, which is a selection marker for pL1091-5, was changed to the LEU2 gene was used. Similarly, the vector (pL1177-2 / FAA3) into which the FAA3 gene (one of the acyl CoA synthase genes) (Japanese Patent Application No. 2006-31705), which has been found to increase lipid content, has been inserted is also pL1177-2 Used for transformation instead of. As a control, a combination using pL1091-5 not containing the DGA1 gene was also used. Transformation was performed using a yeast transformation kit (Sc EasyComp Transformation Kit, manufactured by Invitrogen). SD agar medium (20 g / l glucose, 6.7 g / l yeast nitrogen base w / o amino acids, 20 mg / l histidine, 20 mg / l) without nutrients (uracil and leucine) that can be synthesized by the marker gene Colonies growing on a medium supplemented with methionine and 20 g / l agar) were obtained, and single colonies were used as transformants.
The obtained transformant was cultured in a liquid medium on a rotary shaker at 30 ° C. and 120 rpm. Medium is SD nitrogen source deficient medium (NLSD) (20 g / l glucose, 1.7 g / l yeast nitrogen base w / o amino acids and ammonium sulfate, 1 g / l ammonium sulfate, 20 mg / l histidine, 20 mg / l l Medium containing methionine) was used. The culture was performed for 4 days and 7 days.
After incubation, the cells are sedimented by centrifugation (3000 rpm, 5 minutes), and the cells are disrupted at 4 ° C. (10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 0.15 M KCl, 1 mM EDTA). ) And centrifugation, and then the cells were crushed. The cells were crushed by using an MSK homogenizer manufactured by Brown Co., Ltd., adding glass beads (diameter 0.45-0.5 mm) and cooling with carbon dioxide gas for 30 seconds. The solution after disrupting the cells was centrifuged (3000 rpm, 5 minutes), and the supernatant was obtained as a homogenate solution.

DGAT activity in the homogenate was performed based on the method already reported (Kamisaka, Y. et al. Lipids, 28, 583-587 (1993)). As the reaction solution, 10 mM phosphate buffer (pH 7.0), 0.15 M KCl , 3.4 μM (0.2 μCi / ml) [1- 14 C] oleoyl-CoA, 1 mM 1,2-diolein, 0.1% Triton X- An appropriate amount of homogenate was added to 100 to make the volume 100 μl, and the mixture was reacted at 30 ° C. for 5 minutes. After the reaction, 3 ml chloroform / methanol (1: 2) was added, and the mixture was left at room temperature for about 1 hour to extract lipids. Thereafter, 1 ml chloroform and 1 ml water were added to allow two-phase partitioning, and the lower chloroform layer was separated and concentrated with nitrogen gas. This lipid extract was applied to a silica gel 60 TLC plate (manufactured by Merck), and lipids were separated using hexane / diethyl ether / acetic acid (80: 40: 1) as a developing solvent. Scratch the spot of ¹⁴ C-triolein synthesized by transferring [1- ¹⁴ C] oleoyl-CoA to 1,2-dioleoin by DGAT activity, add a scintillation cocktail, and use a scintillation counter (Aroca) Radioactivity was measured and enzyme activity was calculated.
As a result, as shown in Table 2, by overexpressing the DGA1 gene in the Δsnf2 strain, an increase in the DGAT activity of about 500 to 700 times when the DGA1 gene was overexpressed in the wild strain was observed. This increase was much more pronounced than expected from the increase in lipid content by overexpressing the DGA1 gene in the Δsnf2 strain. This increase in DGAT activity was not significantly different between the homogenates on the 4th and 7th days of culture, and there was no significant difference when the vector used for transformation was pL1177-2 / FAA3 instead of pL1177-2. In addition, even when the Δsnf2 strain was transformed only with the control vector not containing the DGA1 gene, a slight increase in DGAT activity was observed compared to the case where no transformation was performed. An over 200-fold increase in activity was observed due to overexpression of the DGA1 gene. In addition, the detected DGAT activity does not significantly decrease the activity for at least 6 months by storing the homogenate at −80 ° C., and does not significantly decrease the activity by at least 3-4 freeze-thaw cycles. Because it was not, it was considered very stable.
From the above results, it was possible to obtain a strain disruption solution (homogenate) in which the DGAT activity as the gene product was remarkably increased by overexpressing the DGA1 gene in the Δsnf2 strain.

[Example 2]
Obtaining recombinant DGAT with high enzymatic activity expressed with tag peptide
It was examined whether or not a high DGAT activity was retained when a tag peptide was added to the DGA1 gene product and expressed in the SNF2 gene disrupted strain of Saccharomyces cerevisiae. If a tag peptide is attached to the protein, the tag peptide-attached protein can be easily purified using a column that specifically recognizes the tag peptide.
In this experiment, a general histidine tag (6xHis) is added as a tag peptide, and the 6xHis vector pYES2 / NTC (manufactured by Invitrogen) incorporates the DGA1 gene and 6xHis is present on the N-terminal side. A DNA construct expressing such a protein was cloned. The DGA1 gene in this case was also obtained by amplification from the genomic DNA of Saccharomyces cerevisiae by PCR. However, unlike the case of Example 1, it is necessary to construct a construct starting from the first methionine of the DGA1p ORF (start of DGA1 gene There is a stop codon immediately before the codon, and in the PCR product using the primer of Example 1, DGA1p is not translated following 6xHis.), Another combination of primers (DGA1-3, DGA1-2) was used ( Table 1). Each of these primers has an EcoRI recognition site and an XbaI recognition site, and since the construct amplified with this primer contains an EcoRI recognition site at the 3 ′ end from the DGA1 gene, both ends are treated with EcoRI. A sticky end was created by EcoRI cutting.
The vector pYES2 / NTC is also treated with EcoRI to create sticky ends by EcoRI cleavage at both ends, dephosphorylated with alkaline phosphatase (TSAP, Promega), and cleaved with restriction enzymes using PCR purification kit After removing the small oligonucleotide, the insert gene was incorporated into the vector by ligation. Ligation was carried out by mixing at a vector: insert ratio of about 1/1 to 1/10 and reacting at 16 ° C. for 1 hour to 3 hours using Ligation high (manufactured by Toyobo). After ligation, E. coli was transformed by the method described in Example 1, and a vector containing 6xHis-attached DGA1 was cloned. In this case, there are two ways of inserting DGA1, but the vector inserted so that the 5 ′ region of DGA1 follows 6 × His was confirmed by restriction enzyme treatment and obtained. In addition, the nucleotide sequence of this region was determined, and it was confirmed that the reading frame for translation into protein was as planned.
The obtained vector containing DGA1 with 6xHis is enzyme-treated with HindIII and NotI and with 6xHis.
The region of DGA1 was excised, ligated with pL1091-5 enzyme-treated with HindIII and NotI, E. coli was transformed, and a vector in which DGA1 with 6xHis was inserted into pL1091-5 was cloned. The purified vector was treated with various restriction enzymes to confirm that DGA1 with 6xHis was correctly inserted.
Transformation of Saccharomyces cerevisiae BY4741 wild strain and Δsnf2 strain using a vector in which DGA1 with 6xHis was inserted (pL1091-5 / DGA1 (6xHis)) The same was done. The obtained transformed strain was cultured, the cells were disrupted, and the DGAT activity of the homogenate was measured in the same manner as in Example 1. As a result, it was found that even in the case of overexpression of DGA1 with 6xHis, the DGAT activity in the wild strain was low and the DGAT activity in the Δsnf2 strain was remarkably high as in the case of overexpression of DGA1 without 6xHis (Table). 3). However, the homogenate activity on the 4th day of culture is about 30-40% higher than that without 6xHis, whereas the homogenate activity on the 7th day of culture is about 50-60% without 6xHis. Decreased. The addition of 6xHis seems to have slightly changed the metabolic rate in cells, but the details are unknown. However, although there was a slight change in activity, it was confirmed that a significantly high DGAT activity was detected when the DGA1 gene was expressed in the Δsnf2 strain, regardless of the application of 6xHis.

Next, the expression level as a protein when DGA1 with 6xHis was overexpressed was examined using an antibody against 6xHis. A homogenate of wild strain and Δsnf2 strain overexpressing DGA1 with 6xHis was applied to 12.5% SDS polyacrylamide gel electrophoresis (PAGE) and electrophoresed at 200 V for about 40 minutes. After electrophoresis, the gel was blotted on PVDF membrane (Hybond-P, manufactured by GE Healthcare). After blocking the membrane with 10% skin milk, mouse anti-6xHis antibody (GE Healthcare, diluted 1: 2000) was used. In addition, it was incubated for 1 hour. After incubation, the membrane was washed three times and incubated with peroxidase-labeled sheep anti-mouse antibody (GE Healthcare, 1: 2000 dilution) for 1 hour. After incubation, the membrane was washed 3 times, chemiluminescence detection reagent (ECL, manufactured by GE Healthcare) was added, and chemiluminescence generated in the band corresponding to 6xHis-added DGA1p was image analyzer (LAS1000plus, manufactured by Fuji Film). Detected with.
As a result, it was found that the amount of DGA1 protein expressed per cell total protein (band near 50 kDa) was basically the same when expressed in the wild strain and when expressed in the Δsnf2 strain. (FIG. 1). Actually, when each band was quantified, the amount of the homogenate on the 4th day of the strain obtained by transforming the Δsnf2 strain with pL1091-5 / DGA1 (6xHis) pL1177-2 was about 50%. Except that the Δsnf2 strain was transformed with pL1091-5 / DGA1 (6xHis) pL1177-2 / FAA3, the amount of homogenate on the 7th day was almost the same as that of the other, except that it was about twice the amount. That is, a difference of 100 times or more of the DGAT activity between the wild strain and the Δsnf2 strain cannot be explained by a difference of about twice the protein expression level. It is apparently based on the properties of the recombinant DGAT itself obtained, and it is highly likely that a novel recombinant DGAT having extremely high enzyme activity was provided. The amount of DGA1 protein per total protein in the cells is similar to that in the wild strain and Δsnf2 strain as described above, but the Δsnf2 strain has higher growth than the wild strain, and the amount of total protein contained is also large. The amount of DGA1 protein per culture medium was several times higher in the Δsnf2 strain expressing DGA1 than in the wild strain expressing DGA1.

On the other hand, some minor bands other than 50 kDa predicted from the molecular weight were observed, but their appearance patterns were different between the wild strain and the Δsnf2 strain. For example, a band around 20 kDa was commonly detected in the wild strain, but not in the Δsnf2 strain. Conversely, in the homogenate on day 4 of the Δsnf2 strain, multiple bands were detected around 25 kDa, but not in the wild strain. Since it is impossible to deny the possibility that the presence of such a minor band affects a remarkable difference in enzyme activity, the cause of the extremely high DGAT activity of the present application will be discussed below based on the above results.
First, the highest possibility is that the degree of post-translational modification of the DGAT protein expressed in the Δsnf2 strain as described above may have changed. It is also well known that enzyme activity changes due to modifications such as phosphorylation, sugar chain addition, and fatty acid addition, so protein molecular species that have undergone such specific post-translational modification (or the modification has been released) Therefore, it is considered that the enzyme activity was remarkably increased. In that case, the obtained expression product is different from the conventional DGAT protein as a chemical substance because it has the same amino acid sequence but a different group that modifies the chain of the amino acid sequence. This is a new substance that is a different substance and has extremely high enzyme activity.
Another possibility is that the expressed protein, including modifications, is the same in the wild strain and the Δsnf2 strain and is the same as the protein, but in the environment within the Δsnf2 strain in which the SNF2 gene is disrupted, the activator It is possible that the increase in the number of inhibitors or the decrease in inhibitory factors. According to FIG. 1 above, since the position of the minor band is slightly different between the case where it is expressed in the wild strain and the case where it is expressed in the SNF2-disrupted strain, these DGAT genes have an activation or inhibitory action. There is a possibility that it corresponds to a decomposition product of the product and its recombination product. In the latter case, the band that is present in the wild type in FIG. 1 and disappears from the DGAT protein of the present application is a strong inhibitor, whereas it was conventionally recognized as a DGAT protein including the inhibitor. In the present invention, as a result of suppressing the production of the inhibitor, it is possible that the activity of the original DGAT protein was exhibited. However, in view of the fact that the enzyme activity of natural DGAT, which has been confirmed on the membrane surface in the past, has been generally at a low level, or there is no example of stable and high activity, the same or different in the expression environment of natural DGAT. Since different inhibitory factors were present, if high-activity recombinant DGAT was obtained as a result of suppressing the production of these inhibitory factors in the present invention, eventually, including the conventional inhibitory factors, This means that a protein “DGAT” different from the low activity protein recognized as “DGAT” could be provided, and a new protein was provided.
As a final possibility, the possibility that the 25 kDa band newly appeared in the Δsnf2 strain in FIG. 1 was a strong activator of recombinant DGAT can be considered. However, according to FIG. 1, this 25 kDa band is strongly expressed in the 4-day culture and hardly exists in the 7-day culture. Nevertheless, the level of the enzyme activity of the recombinant DGAT is almost the same regardless of whether it is a 4-day culture strain or a 7-day culture strain. Then, it can be said that there is no possibility that the 25 kDa band is a strong activator.
Therefore, in any case, the present invention provides a novel recombinant DGAT having high enzyme activity, and at the same time, a culture solution containing recombinant DGAT as an expression product, a microbial cell homogenate, etc. are extremely high. The present invention also provides a novel protein composition having enzyme activity. And since it has stable and high activity even in the state of microbial cell homogenate or culture solution as well as the purified DGAT protein composition, it is an inhibitor or activator of direct DGAT activity. The screening method can be used.

[Example 3]
Screening of compounds that inhibit DGAT activity using recombinant DGAT with high enzyme activity Using the homogenate containing recombinant DGAT with high enzyme activity obtained in Example 1, a screening system for compounds that inhibit activity was constructed. The homogenate using those when cultured strains were transformed Δsnf2 shares pL1091-5 / DGA1 and PL1177-2 7 days, the [1- ¹⁴ C] oleoyl-CoA as described in Example 1 The activity of DGAT was measured by the measurement method used. As compounds, eugenol and piperine (Kimura et al J. Agric. Food Chem. 54, 3529 (2006), which were found in spices and found to inhibit lipid accumulation and lipid body formation in lipid-accumulating lipomyces yeasts. )) Was used. As a result, both eugenol and piperine were found to inhibit DGAT activity of homogenate (Table 4), and it was confirmed that a compound that inhibits DGAT activity can be screened using recombinant DGAT having high enzyme activity prepared in the present invention. It was done.

  DGAT is a major synthase of triacylglycerol, which is a major storage lipid, and considering that accumulation of storage lipid is one of the etiology of diseases such as obesity and diabetes, inhibitors of DGAT are It is promising as a target for drug discovery for various diseases. By using the DGAT protein with high enzyme activity that can be used according to the present invention, it has become possible to rapidly screen for inhibitors of DGAT that can be used for the discovery of these diseases using DGAT derived from various organisms. .

The expression level of recombinant DGAT in the homogenate of BY4741 transformant overexpressing the 6 × His added DGA1 gene. BY4741 wild strain and Δsnf2 strain were changed to pL1091-5 / DGA1 (6 × His) and pL1177-2 (+ DGA1 in the figure) or pL1091-5 / DGA1 (6 × His) and pL1177-2 / FAA3 (+ in the figure). The homogenate (2 μg protein) obtained by culturing the strain transformed with DGA1, FAA3) for 4 days and 7 days was electrophoresed, and 6 × His-added DGA1 protein was detected with anti-6 × His antibody. A band around 50 kDa corresponds to 6 × His-added DGA1 protein.

Claims (8)

Saccharomyces cerevisiae which SNF2 gene is decreased fracture or function, is introduced DGA1 gene derived from Saccharomyces cerevisiae, and culturing the transformant obtained, is obtained by purification of the resulting expression product set Modified diacylglycerol acyltransferase. 2. The recombinant diacyl according to claim 1 , wherein the transformant introduced with the DGA1 gene is further introduced with the LEU2 gene into Saccharomyces cerevisiae in which the SNF2 gene is disrupted or reduced in function. Glycerol acyltransferase. The product of the diacylglycerol acyltransferase gene is modified so as to have a tag peptide, and is uniformly purified from the expression product obtained by culturing the transformant according to the properties of the introduced tag peptide. The recombinant diacylglycerol acyltransferase according to claim 1 or 2 , characterized in that Saccharomyces cerevisiae which SNF2 gene is decreased fracture or function, transformed by introducing the DGA1 gene derived from Saccharomyces cerevisiae, by culturing the transformant, and purified the resulting expression products, assembled A method for producing a recombinant diacylglycerol acyltransferase, comprising obtaining a substituted diacylglycerol acyltransferase. The method for producing a recombinant diacylglycerol acyltransferase according to claim 4 , wherein when the DGA1 gene is introduced for transformation, the LEU2 gene is also introduced. The product of the diacylglycerol acyltransferase gene is modified so as to have a tag peptide, and a step of uniformly purifying from the expression product obtained by culturing the transformant according to the property of the introduced tag peptide. A method for producing a recombinant diacylglycerol acyltransferase according to claim 4, wherein the method comprises the steps of: The method for producing a recombinant diacylglycerol acyltransferase according to any one of claims 4 to 6 , wherein the transformant is cultured in a nitrogen source-deficient medium when cultured. A method for screening a substance that activates or inhibits the enzyme activity of diacylglycerol acyltransferase, wherein the recombinant diacylglycerol acyltransferase according to any one of claims 1 to 3 is used.