TWI323281B - Saccharomyces cerevisiae yeast strain with functional expression of a glut transporter - Google Patents

Description

1323281 A7 B7 V. INSTRUCTION DESCRIPTION OF THE INVENTION (1) The present invention relates to a Saccharomyces cerevisiae strain in which a six-carbon sugar transporter is no longer synthesized due to deletion of a gene sequence, and as a result, it cannot be on a substrate having a six-carbon sugar as a carbon source. Growth, only after the expression of the GLUT4 gene, its ability to grow on the substrate 5 with hexose carbon as the sole carbon source is restored. Most of the heterologous cell lines transport glucose to the interior of the cell via a special transporter. Among them, different mechanisms for the transfer of glucose transport have been developed, namely, proton-to-co-transport, _, Na+glucose co-transporter, dependent-binding protein system, dish acid transferase system, and diffusion-promoting system 10. In the case of eukaryotic cells In the case of mammals, the glucose transporter family, which is encoded by the gene in Saccharomyces cerevisiae, transmits glucose uptake by promoting diffusion. These transporters belong to the larger sugar transport superfamily, which is characterized by the existence of 12 transmembrane helices and many retained amino acid residues and major structures. 15 Due to the understanding of the transport process for the lack of glucose in vivo It is important to balance the diseases printed by the Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperatives, such as diabetes or Fanconi-Bickel syndrome, so the transport of glucose in mammals is the focus of many studies. Eight glucose transporters have been identified so far [GLUT1 to GLUT5, GLUT8, GLUT9/SLC2A9, GLUT9 (GenBank Accession Number 20 ΥΠ 803)] 'It contributes to the promotion of glucose uptake. The important tasks of these transporters include glucose uptake into various tissues, storage in the liver, insulin Dependently, it is taken into the muscle cells and fat cells, and is measured by the glucosinolates of the spleen 10,000 cells. GLUT1 transmits glucose into the red blood cells and crosses the brain barrier, but the paper scale applies to the Chinese national standard (CNS) ) A4 size (210x297 public «) 1323281 A7

Also expressed in many other organizations, 'flying GLUT4 is limited to islet tissue, mainly muscle and fat. In these Tenguin-dependent tissues, controlling the (4) T4 transporter to direct intracellular or primary membrane intervals constitutes an important mechanism for regulating glucose uptake. Under the insulin membrane 5, intracellular GLUT4 is redistributed to the plasma membrane to promote the uptake of sugar. GLUT1 also exhibits its distribution in cells in these simian-dependent tissues and is also affected by insulin, to a lesser extent. In addition, the relative efficiency of GLim or GLUT4 catalyzing sugar transport is determined not only by the extent to which each transporter directs to the cell surface, but also by its kinetics. The study of the roles and detailed properties of individual glucose transporter isoforms in these insulin-dependent tissues becomes a complex task due to the fact that different glucose transporters of the same type will behave together, and glucose will be rapidly metabolized. Heterologous expression systems, such as Xenopus egg cells, tissue culture cells, insect cells, and yeast cells, have been used to address these issues. However, these systems present some difficulties: heterogeneously expressed transporters are too weak, glucose transporters inherent in these systems, intracellular retention of many transporters, or the production of virtually inactive transporters. In addition to the heterologous and functional Glut4 glucose transporters, it is not known that organisms can express additional hexose transporters, particularly the intrinsic hexose transporter. This has led to a number of disadvantages in the search for compounds that modify the transport properties of the Glut4 protein. Since Glut4 is known to play an important role in reducing blood glucose levels in combination with insulin and other factors, the use of these compounds as ingredients in pharmaceutical preparations will be printed in the Ministry of Economic Affairs. This paper scale applies to the national standard for solids (CNS>A4 specification (210 X 297 public « ) 1323281 A7 B7 V. INSTRUCTIONS (Ο When interested. Creatures that express the function of Glut4 transporter protein can be used to find direct effects on transport The compound of the compound does not occur (4) the side effects of the factor transfer. Therefore, the side effects of the compounds are less obvious. In addition, if the yeast strain can be used, it will be more advantageous in the treatment and supply of the village material. It is a strain of Saccharomyces cerevisiae that cannot grow on a substrate with six carbon sugar as the sole carbon source, but after it exhibits the QLUT4 gene, it resumes its ability to grow on a substrate with a hexose carbon as a carbon source. Such strains, for example, can be produced by mutation or deletion of the relevant gene body sequence. The six-carbon sugar system means _, such as glucose, having 6 carbon atoms. Galactose or mannose, and ketose having one carbon atom, such as sugar or sorbose. The present invention further relates to a Saccharomyces cerevisiae strain containing the GLUT4 gene as described above. 15 Among Saccharomyces cerevisiae, 17 are known. The carbon sugar transporter and the other Ministry of Economic Affairs Intellectual Property Bureau employees cooperate to print three maltose transporters, and if they are strong enough, they can transport the six carbon sugar into the yeast. One strain is known to be able to ingest six All transporters of carbohydrates were removed by deletion. Therefore, this strain contains only two genes, MPH2 and MPH3, which are homologous to the maltose transporter. The MPH2 20 and MPH3 genes are inhibited by the presence of glucose in the medium. The production and identification of yeast plants are described in Wieczorke et al., FEBS Lett. 464, 123-128 (1999). This strain cannot grow in a matrix with glucose as the sole carbon source. Functionally, Glutl (hxt) The appropriate vector for fgyl-Ι strain can be selected from this strain. However, if it is to be carried with this paper, the Chinese National Standard (CNS) A4 specification (210 X 297 public) B7 (4) The GLUT4 base printed by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs, under the yeast promoter (4), is transformed into the yeast record hxt fgyM, and only a small amount of glucose is transported. The function of the nucleus requires the yeast to adapt to Glut4 for a significant amount of glucose transport. These yeasts can be used as glucose-only carbon by using the single-glucose-transfer GUU4. The substrate of the source is isolated. For this purpose, the GLUT4-based yeast strain fgyM with the yeast promoter (IV) is transformed. The yeast cells which have been transformed according to this method are plated on a medium containing glucose as a carbon-only source, and then cultured. For example, after 3 days of TC culture, the growth of individual colonies can be observed, and one of the colonies is isolated. If the yeast body of the colony is removed, no occurrence occurs on the medium with glucose as the sole carbon source. If the yeast plastid with the GLUT4 gene under the functional control of the yeast promoter is reshaped into a strain that no longer contains the vector plastid, the strain 15 will be re-used as the sole source of glucose. The ability to grow on the medium. The production of Saccharomyces cerevisiae strains that may take up glucose using the Glut4 transporter will be described in detail in the examples. This strain does not exhibit the yeast hexasaccharide transporter' and can utilize the Glut4 transporter gene, for example The gene that has been transformed into this strain, the six-carbon sugar, especially glucose, is taken into the cell. The yeast strain with this characteristic has been internationally recognized according to the Budapest Treaty for the purpose of the patent procedure. , deposited at the German Strain Conservation Center in Braunschweig, Germany (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), registration number For DSM 14035, DSM 14036 or DSM 14037 (Table • 6 - This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1323281 A7 B7 V. Inventive Note (5) 1) A preferred embodiment of the yeast strain is to express the GLUT4 gene under the functional control of the yeast-expressing promoter. Those skilled in the art will be familiar with the appropriate yeast-expressing promoter, an example of which is the 5 S0D1 promoter (superoxide). Dismutase, ADH promoter (ethanol deaminase), acid phosphatase promoter, HXT2 promoter (glucose transporter 2), HXT7 promoter (glucose transporter 7), GAL2 promoter (galactose transporter) Etc. For the purpose of performance, a part of the yeast vector consisting of a yeast-expressing promoter and a GLUT4 gene construct system. Table 10 now 'this yeast vector can be self-replicating particles independent of yeast genome' or stably incorporated In yeast matrices, in principle, suitable yeast vectors are all polynucleotide sequences that can be propagated in yeast. Particularly useful yeast vectors are artificially dyed for yeast plastids or yeast. In particular, the yeast vector contains a replication source (2β, ars) for initiation of replication, and a selection marker usually consisting of a nutrient deficiency marker or an antibiotic resistance gene. The yeast vector known to those skilled in the art is For example, BM272, pCS19, pEMBCYe23, pFL26, pG6, pNN414, pTV3, etc. Principles Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed on 'any species of GLUT4 gene can be expressed. Better from human, small The GLUT4 gene of mouse or rat, the polynucleotide 20 and amino acid sequence of Glut4, can be obtained from the following gene bank registration data: M20747 (cDNA; human), EMBL: D28561 (cDNA; rat), EMBL: M23382 ( cDNA; mouse), Swissprot: P14672 (protein; human), Swissprot: P19357 (protein; rat) and Swissprot: P14142 (protein; mouse). The GLUT4 gene is particularly preferred for the National Standard for Sleeping (CNS) A4 specification (210 X 297 mm). 1323281 A7 B7 5. Inventive Note (6) Expressed with the vector YEp4H7-HsGlut4 (SEQ ID No. 9) . The GLUT4 gene of this vector is derived from humans. Those skilled in the art will be familiar with the production of a yeast vector containing the GLUT4 gene expressed in cells. The generation of such vectors is described in the examples. The yeast-containing gene that expresses the gene is transformed into the yeast that will express the gene. A method suitable for this purpose is, for example, electroporation using a vector DNA or a competent cell culture method. Transformation is a technique known to those skilled in the art for introducing foreign DNA, particularly plastids or vectors, into microorganisms such as yeast or bacteria. Detailed experimental procedures, such as yeast transformation, yeast vector, screening of mutant strains of yeast, or performance of proteins in 10 yeast, are described in "Methods in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual; Adams". Alison (Edt.);

Cold Spring Harbor Laboratory; ISBN: 0_87969-508_0" Hand Printed by the Intellectual Property Intelligence Bureau's Staff Consumer Cooperative. Evidence that the GLUT4 gene has been expressed in yeast according to the present invention can be provided by Northern blotting, Western blotting, glucose uptake studies, and glucose conversion studies or other methods. The Northern Ink Point method involves applying an RNA that is isolated from the organism to the support (eg, nitrocellulose). After immobilization, the support that now contains the biological RNA, and the GLUT4 polynucleotide sequence are Radiolabeled or fluorescently labeled DNa - 20 cultures. The performance of GLUT4-mRNA according to the invention in yeast is evidenced by the appearance of black bands. In contrast, no black bands were detected in rNA of yeast which was identical but not transformed with the GLUT4 expression vector. The Western blot method involves applying the protein extract of the organism to be applied to the membrane support (for example, nitrocellulose), and the paper scale to be expressed by the antibody is applicable to the Chinese National Standard CCNS) A4 specification (210x297 public 5). 1323281 A7 B7 V. INSTRUCTIONS (7) Protein.

Antibodies to the Glut4 protein are available, for example, from Alpha Diagnostic International, Inc., 5415 Lost Lane, San Antonio, TX 78238 USA. The analytical system required to detect binding antibodies is also available from this supplier. The expressed Glut4 protein line was tested against the same yeast strain without the Glut4 protein. For the glucose uptake study, the test organism is supplied with radiolabeled glucose as the sole carbon source. In contrast to control strains that are identical but do not contain the Glut4 transporter, yeast with Glut4 as the sole glucose transporter transports radiolabeled glucose into the interior of the cells. Glucose conversion can be tested on a nutrient medium containing glucose as the sole carbon source. In contrast to the control group, which is identical but does not contain the Glut4 transporter, the yeast strain with the Glut4 transporter as the sole glucose transporter can be grown on a nutrient medium with glucose as the sole carbon source. Those skilled in the art are familiar with the methods just mentioned. For a detailed description, see, for example, "Current Protocols in Molecular Biology; Edited by: FM, Ausubel, R. Brent, RE Kingston, DM Moore, JG Seidman, JA Smith, K, Struhl; 2000 by John Wiley & Department of Economics Intellectual Property Bureau employee consumption cooperative printing

Sons published (currently in the update). The present invention is preferably a Saccharomyces cerevisiae strain which exhibits the 20 Glut4 gene derived from human, mouse or rat. The present invention is particularly preferably a Saccharomyces cerevisiae strain which exhibits a polynucleotide sequence comprising a coding region of the human Glut4 gene. In a further preferred embodiment, the invention resides in, for example, the German Culture Collection Center of the German city of Braunschweig, registered -9- This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297) - - 'Inventive Note (8) The Department of Economic Intelligence's Intellectual Property Office staff eliminates the number of DSM 14038, DSM 14039 or DSM 14〇4〇 printed by the Department of Intellectual Property. These strains are listed in the table! This table provides information on yeast strains, plastids, and growth conditions of these yeasts used for plastid transformation. 5 The invention also relates to the production of a strain of Saccharomyces cerevisiae according to the invention, which is obtained by the following procedure: a) providing a yeast which is no longer grown on a substrate having hexose as the sole carbon source; b) The plastids of the GLUT4 gene under yeast functional control in yeast are transformed into yeast a) and c) the strain obtained according to b) transformation is plated on a medium containing six carbon sugar as the sole carbon source. d) Select a strain that is cultured on this medium according to c) and that uses the gamma gene to support the uptake of hexose. The invention also relates to the growth of growth of such strains. In order to provide the yeast according to the present invention, the first step will no longer be grown on a substrate which is a carbon-only source, but in its performance of the gamma gene, it is restored to a carbon source of six carbon sugars. The ability to grow on the substrate is off: the yeast strain is isolated. This can be done by mutating or deleting the phase sequence encoding the six carbon sugar transporter. After the yeast is provided, the yeast is further grown. The growth line is carried out in a medium using a microbiological standard method. A suitable medium for growing the yeast is, for example, a nutrient base, particularly YPD medium (yeast extract/gland/dextrose) or a selective medium. In these culture medium, the yeast cells are grown, centrifuged and separated from the culture medium, and then, according to the prescription of the country of the country (CNS) A4 specification (21〇X 297 public Chu) 10 15 2〇 binding 13223281 A7 B7 V. Invention For the purpose of the method (9), it is suspended in an aqueous medium containing, for example, a buffer substance, a salt or other additives to form an aqueous suspension. Those skilled in the art can refer to the above mentioned "1^丨110(15丨11丫6331〇6116^5,1997: A Cold Spring Harbor Laboratory Course Manual; Adams 5 Alison (Edt.); Cold Spring harbor Laboratory Information on the growth of yeast can be found in ISBN: 0-87969-508-0. In the preferred embodiment of the above-described yeast strain, the GLUT4 gene which can be expressed in yeast under the functional control of the promoter is used. In the case of transformation, reference may be made to the above-mentioned "Methods in.; 10 Yeast Genetics jo. The GLUT4 gene line specially used for transformation to produce such yeast strains is obtained from human, mouse or rat. GLUT4 gene. In addition, a GLUT4 gene line for transformation is particularly present in the polynucleotide sequence as shown in SEQ ID No. 9 or SEQ ID No. 10. SEQ ID No. 9 discloses 15 yeast vector Yep4H7-HsGLUT4 Polynucleotide sequence. This vector contains a polynucleotide sequence functionally controlled by the HXT7 promoter, which encodes the amino acid sequence of the human GLUT4 gene. SEQ ID No. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative 1〇 contains The polynucleotide sequence of H2rg4g2. The yeast vector H2rg4g2 carries the rat GLUT4 20 gene functionally controlled by the HXT2 promoter. The GLUT4 gene is functionally controlled by the promoter and is transcribed to a Glut4 protein by the promoter. For the disclosure of the GLUT4 sequence and the method used, reference may be made to the literature already mentioned above. The invention further relates to the use of the following procedure steps for increasing or decreasing the 11th paper size applicable to the Chinese National Standard (CNS) A4 specification. (210 X297 mm) V. Description of invention (1()) 10 15 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperation Du printing 20 : Γ: = protein transport hexasaccharide amount of compound forging method: ): for The Saccharomyces cerevisiae b) according to the present invention exhibits a hexose carbon uptake amount; c) provides a compound; d) ^ according to a) the provided yeast is contacted according to the provided substance, e) d) After the contact, take the amount of the six-carbon sugar in the yeast strain 'Κ Ϊ 藉 0 by comparing d) to the amount of the six-carbon sugar in the liquid, that is, the amount measured according to b) and e) , Zhong Ding increases or decreases the use of G a hexaose amount of a compound transported by the lut4 protein f. In order to provide the yeast according to the invention, the first step will no longer be grown on a substrate having a hexose-carbon source as the only carbon source, but after expressing the gamma "gene" Then, it restores its ability to grow on a substrate with six carbon sugar as the only carbon source. The strain of this strain contains a material that is functionally controlled by the yeast promoter GLUT4 gene (4) to be transformed. The production of such yeast strains is described in the Examples. Further, it is necessary to grow the yeast after providing the yeast. The growth standard (4) biological standard method is carried out in an appropriate medium. A suitable medium for growing the yeast is, for example, a complete medium 'particularly a YPD medium (yeast extract/gland/dextrose medium) or a selective medium or the like. In these mediums, the yeast cells are grown, grown, centrifuged to separate them from the medium, and then suspended in a medium containing, for example, buffer substances, salts or other additives according to the purpose of the method used. National Standard (CNS) A4 Specification (210 x 297 public) Order 1332281 A7 B7 V. INSTRUCTIONS (11) In the aqueous medium of the additive to form an aqueous suspension, those skilled in the art can Information on yeast growth is found in "Methods in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN: 0-5 87969-508-0". The hexose uptake of the yeast strain provided as described above can be determined using a radiolabeled glucose uptake study. To achieve this, a specific amount of yeast cells, for example, 60 mg wet weight per ml, is suspended in, for example, 100 μl of buffer, and a quantitative amount of 14C- or 3Η·labeled 10 glucose is used as the sole carbon source. Handle it. Cell culture is performed to sample a specific amount of cells at a specific time. LSC (liquid scintillation counting) is used to help determine glucose uptake. However, the hexose intake of the yeast strain as provided above can also be determined by a growth test on a medium having glucose as the sole carbon source. To achieve this, after the addition of the compound, the growth rate of the strain is determined, for example, by periodically measuring the optical density of the culture solution at 600 nm. This value is compared with the growth rate of the control strain (e.g., wild type yeast strain). The Ministry of Economic Affairs' Intellectual Property Bureau's Bayer Consumer Cooperative Printed Compounds are supplied by chemical synthesis or by separation from organisms. Chemical synthesis can also be automated. The 2 oxime compound synthesized or isolated can be dissolved in a suitable solvent. Suitable solvents are, in particular, 'containing a specific amount of an organic solvent, for example, DMS0 (dimethyl hydrazine), a winter aqueous solution. The contact of the yeast strain with the compound used to identify or increase the amount of the hexose to be transported by the Glut4 protein is specifically accomplished in an automated laboratory system provided for this purpose. These systems can be made up of special grades -13 - paper size (+ (210x297 mm) B7 B7 10 15 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Print 20 I, Invention Instructions (12) Prepare shelter room, micro culture A dish, a microcentrifuge tube or a laboratory glassware. Automated laboratory systems are designed to achieve high productivity. Therefore, the procedure as described immediately by means of an automated laboratory system is also referred to as HTS (High Productivity Screening). After the yeast is brought into contact with the compound, the amount of hexose (especially glucose) which is carried into the cells by the yeast cells under these conditions is measured. To this end, the same procedure as described for determining the glucose uptake of the yeast strain that is not in contact with the compound can be used. The identification of a compound which increases or decreases the amount of hexose to be transported by the Glut4 protein is carried out by using the amount of the six carbonaceous amount in the strain before and after the contact of the comparative strain with the compound. The invention further relates to an agent for the treatment of diabetes and obesity, comprising a compound which has been identified and, if appropriate, further developed using the above-described method using the Glut4 gene, and an adjuvant for formulating the agent. Further development of this identified compound means that, firstly, the specificity of the target protein (in this case Glut4) is improved; secondly, the availability in animal or human organisms is increased; and third, any There has been a decrease in side effects that are not desired. To this end, those skilled in the art can use a range of methods including, for example, but not limited to, the use of pharmacological animal models such as diabetic or obese mice, in vitro biochemical assays, and the actual compound and Glut4 protein. The use of structural patterns. The adjuvant for the formulation of the medicament allows the active substance to be adjusted for the application, the distribution of the active ingredient to the intended application, and the development of the active substance. The adjuvant J is a filler, a binder, a disintegrating agent or a glidant such as lactose, cane 1323281 A7 B7 5. Inventive Note (!3) Mannitol, Sorbitol, Cellulose, Starch, Phosphoric Acid Dicalcium, polyglycols, alginate, polyvinylpyrrolidone, carboxymethylcellulose, talc or cerium oxide. Diabetes is caused by a lack of insulin or a decrease in insulin action, and there is a sign that glucose is secreted together with urine and at the same time has an abnormal increase in blood glucose (hyperglycemia). The lack or reduction of insulin action will cause somatic cells to be ingested into the blood by not fully absorbing and converting glucose. In adipose tissue, hormones that antagonize insulin have an effect of increasing fat breakdown, so that the content of free fatty acids in the blood must increase. Obesity is caused by excessive calorie intake, resulting in an imbalance of energy, resulting in an abnormal increase in body weight and damage to health. The invention further relates to the use of a compound which has been identified and, if appropriate, further developed by the method of using Glut4 protein, for the preparation of a medicament for the treatment of urinary uremia and obesity. These agents are in the form of dosages of pharmacologically active substances for the treatment of diseases or bodily functions in humans and animals. Examples of dosage forms known for oral treatment are powders, granules, ingots, Ministry of Economic Affairs, Intellectual Property Bureau, Bayer Consumer Cooperatives, printing agents, pills, tinctures, dragees, capsules, liquid extracts, elixirs and syrups. Examples of dosage forms for external administration are aerosols, sprays, 20 gels, ointments or powders. Injectable or infused solutions are administered parenterally, in the form of vials, vials or prefilled syringes. These and other forms of pharmaceutical agents are known to those skilled in the pharmaceutical arts. The invention further relates to an assay for the use of the following procedure for increasing or decreasing the amount of a six-carbon sugar transported by Glutl protein: -15- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) B7 V. INSTRUCTIONS (14) a) Provide the ability to grow on a substrate with six carbon sugar as the sole carbon source, but after it exhibits the GLUT1 gene, restore its ability to grow on a substrate with six carbon sugar as the sole source. a Saccharomyces cerevisiae strain containing a factor capable of functioning in yeast under the functional control of a promoter; base b) determining the hexose intake of the strain according to a); c) providing a compound; d) The yeast strain provided according to a) and the human touch provided according to c); : 物接 10 e) determined after the contact according to d), ingesting the hexose sugar in the yeast strain "W « 0 Compounds which increase or decrease the amount of hexose to be transported by Glutl protein are identified by comparing the amount of hexose carbon in the ingested strain before and after exposure according to d). In order to provide the yeast according to the present invention, the first step: 丨 delete no longer forms a six-breaking sugar transporter, and the result == the sugar grows on the substrate of the only-carbon source, but after it exhibits GW due to ^, it is restored The 6-carbon broth was used to ferment 1 the growth factor of the substrate and the parent strain was detached. These strains are deposited in Germany with two storage numbers DSM_, deleted or = 'known to have 17 six-carbon sugar transporters and another disaccharide transporter can transport six carbon sugar into yeast. All transporters are removed by the deletion method-16-

External bacteria 1323281 A7 B7 V. Description of invention (15) Except. The production and timing of this yeast strain is described in wiecz〇rke spit FEBS Lett. 464, 123-128 (1999). This strain could not be grown in a matrix that was printed as the sole carbon source by the Consumers' Cooperatives of the Sixth Sugar Ministry of Economics. If the plastid vector carrying the GLUT1 gene under the control of the yeast promoter is transformed into these yeast strains, grape 5 sugar transport still does not occur. The functional performance of GluU requires further adaptation of this yeast strain to allow glucose transport using Glutl. These yeast strains which take glucose into the cells using a single glucose transporter Glutl can be isolated from the matrix in which glucose is the sole source of carbon. For this purpose, yeast strains which no longer exhibit intact hexose transporter proteins were transformed with a yeast vector carrying the GLUT1 gene 10 under the functional control of the yeast promoter. The yeast cells which have been transformed according to this method are plated on a medium containing glucose as the sole carbon source, and then cultured. After incubation at, for example, 30 ° C for several days, the growth of individual colonies can be observed, and one of the colonies is isolated. If the yeast plastids of this colony were removed, no growth occurred on the medium with 15 glucose as the sole carbon source. Now if the yeast plastid with the GLUT1 gene under the functional control of the yeast promoter is transformed into a strain that no longer contains the vector plastid, the strain will be re-extracted on the medium with glucose as the sole carbon source. The ability to grow. In order to transform a yeast strain, a GLUT1 gene derived from human, mouse or 20 rat can be particularly used. The polynucleotide and amino acid sequences of Glutl are disclosed in the following numbers for the indicated libraries: EMBL: M20653 (cDNA; human), EMBL: M13979 (cDNA; rat), EMBL: M23384 (cDNA; mouse), Swissprot: P11166 (protein; human), Swissprot: P11167 (protein; rat) and Swissprot: P17809 (egg-17) This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public policy) 1323281 A7 B7 V. Description of the invention (16) White matter '· mice). The production of such yeast strains is described in the examples. After the yeast is provided, it is further required to grow the yeast. The growth line is carried out in a suitable medium using a microbiological standard method. A suitable medium for growing yeast is, for example, a complete medium 'particularly YPD medium (yeast extract/gland/dextrose medium) or selective medium, etc.) in which yeast cells are grown, grown, and centrifuged. It is separated from the culture medium and then suspended in an aqueous medium containing, for example, a buffer substance, a salt or other additives for the purpose of the method used to form an aqueous suspension. Familiarity 10 This technique can be described in "Methods in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN: 0-87969-508-0 Let us find information to make yeast grow. In a preferred embodiment of this method, a Saccharomyces cerevisiae strain comprising a GLUT1 gene under the functional control of a promoter which is expressed in yeast 15 is provided. These strains suitable for this method are deposited in the preservation of German strains.

The registration number is DSM 14033, DSM 14026 or DSM Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed 14033. The GLUT1 gene consisting of a plastid is disclosed in SEQ ID No. 11 or 20 SEQ ID No. 12. SEQ ID No. 11 contains yeast vector Υβρ4Η7-

HsGlutl. This vector contains the polynucleotide sequence of the human GLUT1 gene functionally controlled by the HXT7 promoter. SEQ ID No. 12 comprises the polynucleotide sequence of the yeast vector H2rglg2. This plastid contains the GLUT1 gene from the rat that is functionally controlled by the HXT2 promoter. -18- This paper scale applies to China National Standard (CNS) A4 specification (2丨〇 X 297 public « ) A7 B7

1323281 V. INSTRUCTIONS (17) The hexose uptake of a yeast strain provided as described above can be determined using a radiolabeled glucose uptake study. To achieve this, a specific amount of yeast cells, for example, 60 mg wet weight, is suspended in, for example, 100 μl of buffer, and quantified or 3 = = glucose is used as the only carbon source for reduction. . Cell culture was performed and a specific amount of cells was taken at & time. a LSC (Liquid Scintillation Count) helps determine glucose uptake. The k-series of the σ substance is formed by a chemical synthesis method or a method of separating from the biological Zhao. Chemical synthesis can also be automated. The compound synthesized or isolated can be dissolved in a suitable solvent. A suitable solvent is, in particular, an aqueous solution containing a specific amount of an organic solvent such as DMSO (dimethyl sulfoxide). The contact of the yeast strains with the compounds used to identify or increase the amount of hexoses that utilize Glutl protein transport by the Ministry of Economics, the Ministry of Finance, and the Ministry of Finance, is specifically done in an automated laboratory system for this purpose. These systems may consist of special rooms, shelters, microcentrifuge tubes or laboratory glassware. Automated laboratory systems are typically designed to achieve high productivity. Therefore, the procedure described by means of an automated laboratory system as just described is also referred to as HTS (High Productivity Screening). After the yeast was brought into contact with the compound, the amount of hexose (especially glucose) which was carried into the cells by the yeast 20 cells under these conditions was measured. To this end, the same procedure as described for determining the glucose uptake of the yeast strain that is not in contact with the compound can be used. The bell of the compound which increases or decreases the amount of the hexose to be transported by the Glutl protein is carried out by using the comparative strain before and after the contact with the compound, -19-1323281 A7 B7, and the invention (18). Further, the present invention relates to an agent for treating diabetes and obesity comprising a compound which has been identified and, if appropriate, further developed using the above-described procedure using the Glutl gene, and an adjuvant for formulating the agent. This further development of the identified compound means that, firstly, the specificity of the target protein (in this case Glutl) is improved; secondly, the availability in animal or human organisms is increased; and thirdly, Any side effects that are already present and not desired are reduced. To this end, those skilled in the art can use a range of methods including, for example, but not limited to, pharmacological animal models such as the use of diabetic 10 rats or obese mice, the use of in vitro biochemical assays, and the actual compound and Glutl protein. The use of structural patterns. The adjuvant for the formulation of the drug can be used to adjust the active substance for the application, the distribution of the active ingredient to the intended application, and development. Examples of such adjuvants are fillers, binders, disintegrants or glidants such as lactose, sucrose, mannose 15 alcohol, sorbitol, cellulose, starch, dicalcium phosphate, polyglycols, alginic acid Salt, polyvinylpyrrolidone, carboxymethylcellulose, talc or dioxide. Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing Diabetes, is a chronic metabolic condition caused by a lack of skin insulin or a decrease in insulin's effect. Glucose is secreted together with urine and has blood. 20 Glucose levels are abnormally increased (hyperglycemia) ) signs. The lack or reduction of the effect of 姨 素 ’ will cause the somatic cells to be ingested into the blood by not fully absorbing and converting glucose. In adipose tissue, hormones that antagonize insulin have an effect of increasing fat breakdown, which inevitably increases the content of free fatty acids in the blood. -20- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1323281 B7 ----------___ V. Description of invention (19) Obesity is due to excessive calorie intake 'Making energy imbalance, leading to abnormal weight gain, and causing damage to Ji Kang. The invention further relates to the use of a compound which has been identified and, if appropriate, further developed by the method of using Glutl protein, for the preparation of a medicament for the treatment of urinary dysfunction and obesity. These agents are in the form of dosages of pharmacologically active substances for the treatment of diseases or bodily functions in humans and animals. Examples of dosage forms known for oral treatment are powders, granules, granules, pills, elixirs, dragees, capsules, liquid extracts, elixirs and syrups. Examples of dosage forms for external administration are aerosols, sprays, 10 gels, ointments or powders. Injectable or infused solutions are administered parenterally, in the form of vials, vials or prefilled syringes. These and other forms of pharmaceutical agents are known to those skilled in the pharmaceutical arts. Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives, In addition, the present invention relates to the polynucleotide sequence of SEQ ID No. 13 and the polynucleotide sequence of SEQ ID No. 14. SEQ ID No. 13 and 15 SEQ ID No. 14 Polynucleotide sequence Mutant of the gamma rat Glutl gene, which results in the substitution of the individual amino acid of the protein in question e SEQ ID No. 13 Polynuclear acid sequence coding a Glutl protein in which the amino acid chain position 69 is substituted with methionine; the SEQ ID No. 14 polynucleotide sequence encodes an amino acid chain position 70 alanine substituted with methionine 20 Glutl protein. This two-protein mutant has been shut down even by the deletion of the six-carbon sugar transporter, but glucose uptake is not maintained by the wild-type (10) "protein-maintained strains'. Such mutants can be obtained, for example, by selection mutations or inhibitor mutations, or by exogenous mutation formation. -21 * This paper size is used in the general standard® National Standard CCNS>A4 specification (2丨Ox297·^^·1323281 A7 B7 5. Inventive Note (20) The present invention further relates to SEQ ID No. 13 or SEQ ID No. 14 The G丨utl protein encoded by the polynuclear acid sequence. The present invention also relates to a yeast strain comprising the polynucleotide sequence of SEQ id No. 13 and the polynucleotide sequence of SEQ ID No. 14. These yeast strains 5 are DSM 14026 and DSM 14027. The deposit number is deposited in the German Culture Collection Center. To produce these strains, the yeast vector corresponding to SEq ID N〇13 or SEQ ID No. 14 is transformed into a matrix that cannot be used as the sole carbon source with hexose Growth on the 'only when the GLUT1 gene is expressed in this strain' is finally restored to the yeast strain with a growth capacity of 10 on a substrate with six carbon sugar as the sole carbon source. Then, after the transformation, the cells are plated to glucose. In the medium of the sole carbon source, the colonies grown in this medium are isolated. The yeast strain transformed in this way is suitable as an example of a method for identifying a compound which increases or decreases the amount of hexoses transported by Glutl protein. 15 Abbreviation Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed HXT Hexasaccharide Transporter ORF Open Translation Architecture PCR Polymerase Chain Reaction Example 20 Growth of Yeast Strains The yeast strains described herein were obtained from strain CEN.PK2-lC ( M4ra leu2-3, 112 ura3-52 trpl-289 his3-Al MAL2-8C SUC2). The production of a yeast strain with a deletion of the six-breaking sugar transporter gene (good) is described in

Wieczorke et al., FEBS Lett. 464, 123-128 (1999): EBY. -22- This paper scale applies to the Chinese National Standard (CNS) A4 specification (21〇X 297 public S) 1323281 A7 B7 V. Description of invention ( 21) 18ga (ΜΑΤα Δ hxtl-17 Δ gal2 AagtlAstU leu2~3, 112 ura3-52 trpl-289 his3-Al MAL2-8C SUC2), EBY.VW4000 {MATa Δ hxtJ-17 Δ gal2 Δ agtl Δ mph2 Δ mph3 Δ Stll leu2-3, 112 ura3-52 trp 289 his3-Al MAL2-8C SUC2). The culture medium is based on 1% yeast extract and 2% gland (YP), while the basic medium consists of 0.67% Difco yeast nitrogen base (YNB) without amino acid, and contains nutrient requirements. Additives and different carbon sources. At 30 ° C, the yeast cells were subjected to aerobic growth in a stationary oscillator or a canola culture. The optical density (OD6QQ) of 600 nm was measured to monitor the growth of the cells. Glucose uptake assay Glucose transfer was measured by uptake of D-[U-14C]-glucose (Amersham) and kinetic parameters were determined by Eadie-Hofstee plot. The cells were centrifuged, washed with phosphate buffer, and resuspended in phosphate buffer at a concentration of 15 mg per milliliter (fresh weight). Glucose uptake was determined at a glucose concentration between 0.2 and 100 mM and a specific activity of the matrix between 0.1 and 55.5 kBq/z mol1. The cells and glucose solution were printed at 30 °C for 5 minutes at the Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative. Glucose was treated with cells treated with radioactive glucose. After 5 seconds of incubation, 10 ml of ice-cold stop buffer (0.1 20 M KiP〇4, pH 6.5, 500 mM glucose) was added and the cells were quickly filtered onto a glass fiber device (0=24 mm, Whatman). The filter was immediately washed three times with ice-cold buffer and the incorporated radioactivity was measured with a liquid scintillation counter. Determination of the inhibitory effect of cytochalasin B (final concentration 20 μM in ethanol) 'Insist the cells in the presence of a solvent or only solvent for 15 minutes -23- This paper size applies to the National Standard (CNS) A4 specification (210 X 297 public « ) 1323281 A7 B7 V. Inventive Note (22), a 15-second ingestion test was performed with 50 mM or 100 mM radioactive glucose. Construction of H2rg4g2 (SEQ ID No. 10) and H2rglg2 (SEQ ID No. 12) 5 H2rg4g2 and H2rglg2 lines comprise the GLUT4 gene (in SEQ ID No. 10) or the GLUT1 gene derived from rat (in SEQ ID No. . 12) DNA construct of the functionally linked HXT2 promoter (promoter of yeast glucose transporter 2). The plastids GLUT1-pTV3 and 01^丁4-pTV3 (Kasahara and Kasahara, Biochem J. 315, 177-182 10 (1996); Kasahara and Kasahara, Biochim. Biophys. Acta 857, 146-154 (1997)) 0.5 The kb promoter fragment was replaced by a 0.5 kb DNA fragment (Genbank: P23585) containing the yeast/WT2 promoter from -452 bp to +9 bp, respectively. YEp4H7-HsGLUT 1 (SEQ ID No. 11) and YEp4H7- 15 HsGLUT4 (SEQ ID No. 9) Department of Construction Economics Intellectual Property Office Staff Cooperatives printed YEp4H7-HsGLUTl and YEp4H7-HsGLUT4 as plastids, of which HXT7 promoter (Promoter of HXT7 gene) The promoter fragment at position -392 to -1 is functionally linked to the human GLUT1 gene (in SEQ ID No. 11) or the GLUT4 gene (in SEQ ID No. 9). Since the intact 20 HXT7 promoter is inhibited by glucose, a promoter fragment is used. The 0.4 kb promoter fragment from p426MET25 (Mumberg et al., Nucleic Acids Res. 22, 5767-5768 (1994)) is included

Position-392 to -1/ΟΤ7 promoter fragment substitution, using the ΗΧΤ7 gene (Genbank: Ρ39004) as a template, using the primer P426H7-1 (SEQ-24- This paper scale applies the Chinese National Standard (CNS) A4 specification ( 210 X 297 ng β ) 1323281 A7 B7 V. Inventive Note (23) ID No. 1) and P426H7-2 (SEQ ID No. 2) were amplified by PCR to obtain plastid YEp4H7 (SEQ ID No. 15). . HSG1-F7/T2-HSG1 (SEQ ID No. 3.4) and HSG4-F7/T2-HSG4 (SEQ ID No. 5.6) were used as primers for Glutl and Glut4, respectively, to human 5 GLUT1 (EMBL: M20653) and human GLUT4. c DNA (Genbank: M20747) was used as a template, and human GLUT1 and GLUT4 ORFs (open translation architecture) were amplified by PCR for 10 cycles. The PCR product used primer T71-ORF (SEQ ID No. 7) and T2. -HSG1 (SEQ ID No. 4) or T2-HSG4 (SEQ ID No. 6) was subjected to 10 10 cycles of reamplification. The final PCR product, upstream or downstream of the GLUT ORF sequence, contains sequences homologous to the EOp4H7/OT7 promoter region or the Crc/terminator region (iso-cytochrome cl). This was transformed into yeast strain EBY.F4-1 with the linearized YEp4H7: oRI, and then homologously recombined in yeast, in 2% strength wheat 15 bud sugar medium. The urinary mouth bites the original trophic growth to select the transformed product. Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed GLUT1 and GLUT4 from rats in the absence of hexose-carbon transport yeast strains, yeast multi-copy performance, 艎GLUTl-pTV3e and GLUT4- 20 pTV3e with galactose-induced And the glucose transporter genes GLUT1 and GLUT4 from rats under the control of a glucose-inhibited yeast promoter. In this two constructs, the promoter was replaced by a glucose-induced yeast/«T2 promoter. These vectors were transformed into the yeast strain EBY.18ga("/j;ci). 'This strain cannot take any six-carbon prayer, so -25- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210x297 mm). 1323281 A7 B7 V. INSTRUCTIONS (24) It is not possible to use ΐ glucosamine or other hexoses. The cells were plated on a sufficient medium to cover the carbon source. Use the impression of Lakida '*tryptophan's maltose, but with different glucose concentrate plates: raise: 5 mM, 100 mM without the same basic culture sense 10 mM '50 for more than a week, the transgenic strain is not Can not be on. Even growing at 3 °C. This proof - does not support glucose uptake in the parent strain with Glut4 grapes ===. Glucose uptake through the Glutl transporter in normal fermented (four) mother cells =: suppress:, "up*(10), this fiber obviously:::== gluti^〇LUT4#w^#^i〇15 quasi- Carbon source, *YNB medium on the aquaculture dish. After the UV-fired shot with the dose of ==, in the case of the GLUT4 transforming strain printed on the GLUT4 employee No inhibitors were observed, and GLUT1 transformants had several inhibitors that could grow on glucose. The growth of the GLUT1 inhibitors on the selective YP maltose medium grew 15 generations. Above 20; all cells of the plastid can not regenerate longer than glucose = carbon source ===. Therefore, it can be proved that the growth of glucose as the sole carbon source is dependent on the original wild-type H2rglg2 plastid. This latter, in which one of the several yeast strains was re-obtained on glucose, confirmed that the strain contained a functional GLI in its genome. Ti showed the paper size for the National Standard (CNS) A4 specification (210). X 297 mm) 1323281 A7 B7 A mutation indicating the inhibition of (25). The mutated double gene was named /g)^W (representing "functional expression of GLUT1 in yeast"), and the strain was named EBY.S7 〇 separated from other inhibitor mutants The H2rglg2 plastid was amplified in B. coli and transformed into the original yeast strain EBY.18ga (J/2j:i) lacking glucose transport. Several of these qualities were allowed to grow on synthetic media with 1 mM glucose as the sole carbon source. Thus, their GLUT1 sequences contain mutations that convert the corresponding GLUT1 protein in yeast into a successful glucose transporter. For example, the mutants comprise a proline acid substituted with methionine at position 69 of the amine acid chain (SEQ ID No. 13) or a propylamine at position 70 of the amino acid chain. Acid substitution (SEQ ID No. 14). As described above, the mutant of SEQ ID No. 13 was found during screening of the mutant strain. The mutant of SEQ ID No. 14 is obtained as described above by using in vitro mutation formation. The principle of in vitro mutation is described by Boles and Miosga (1995) (Boles and Miosga,

Curr Genet. 28, 197-198 (1995)). In the first PCR reaction, the plastid YEpH2-rGLUTl (20 Ng) was used as the DNA template, and the EDeqxt2 (SEQ ID No. 16) and glutmet2 (SEQ ID No. 17) were printed by the Intellectual Property Office of the Ministry of Economic Affairs. ) (each is 100 picomoles (pmol)) (PCR conditions: 95 ° C for 45 seconds, 50 ° C for 30 seconds, 20 72 C for 2 minutes '25 cycles ' taq polymerase). The primer glutmet2 contains a sequence in which the normal GLUT1 gene is modified and the alanine of the GLUT1 amino acid position 70 in the rat is replaced by methionine. The resulting pCR fragment was purified by agarose gel electrophoresis followed by gel extraction. In the second PCR reaction, the purified PCR fragment (20 Ng) and -27- paper scale are applicable to the Chinese National Standard (CNS) A4 specification (2丨〇χ 297 gong ^ ) 1323281 at B7 V. Invention Description (26) plastid GLUT1-pTV3 (Kasahara and Kasahafa, Biochem J. 315, 177-182 (1996)) as a DNA template (50 ng), and primers seqhxt2 and seq2gal2 (SEQ ID No. 18) (each 100 picomoles) (PCR conditions: 95 ° C for 45 seconds ' 54 ° C for 30 seconds, 72 ° C for 2 minutes, 20 cycles, taq 5 polymerase). Since the primer seqhxt2 binds only to the fragment of the first PCR reaction', only the DNA sequence which causes the alanine of the amino acid position 70 to be replaced by methionine is amplified in this second PCR reaction. The resulting PCR fragment having the mutant GLUT1 gene was subjected to agarose gel electrophoresis, followed by gel extraction, and exchanged with the wild-type GLUT1 gene in the plastid YEpH2-rGLUT10. This plastid (SEq ID N〇. 14) was transformed into a yeast strain EBY.18ga ("Ax" lacking glucose transport, which was grown on a synthetic medium with glucose as the sole carbon source. Ingestion via Glut4 transporter Glucose Saccharomyces cerevisiae strain EBY.S7 (zi/ixi/g3; plant/) apparently contains the gene 鳢 mutation, viz 15 /g;; plant /, which makes Glutl functional in yeast to support glucose through the protoplast The membrane is taken up into the cells. After the strain EBY.S7 (z3/^允少) is transformed by H2rg4g2, the Ministry of Economic Affairs Intellectual Property Office employee consumption cooperative can print on the medium with glucose as the sole carbon source. Inhibitor colonies were isolated. 20 Nine of these GLUT4 inhibitor mutants were grown on non-selective γρ maltose media for more than 15 generations. All cells that lost their plastids could not regenerate longer than 1 mM glucose medium. Therefore, it was confirmed that the earlier growth was GLUT4-dependent. The H2rg4g2 plastid was further isolated from the nine inhibitory strains and amplified in E. coli and -28- This paper scale was applied to the Chinese National Standard (CNS) A4. (21〇χ 297 public policy) 1323281 A7 B7 V. INSTRUCTIONS (27) Transformed back to the yeast strain EBYS7, which originally lacked glucose transport. This kind of quality was found in the absence of 10 glucose as the sole carbon source. On synthetic medium, this proved to be free of the form of the "activated" GLUT4 mutant. The original wild-type H2rg4g2 plastid was re-transformed into the 5 nin-inhibitor strains without plastids, relative to the transport containing no Giut4. The control vector of the protein gene, all of which are capable of growing on glucose. The corresponding mutant of this strain is named 〆·You (χ=19). The mutated dual gene fgy4-X induces the GLUT4 gene. Functional manifestations in strains. Thus the object of the invention is achieved. A review of the yeast strain 10 used in accordance with the invention is provided in the attached table. The table lists a review of the yeast strains used according to the invention, including genotypes, needs The growth conditions and the registration number of the German strain storage center. Printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative

1323281 & 扦添私*.:-^呤财^»^0.67. /0«砟唪|»轚3二8}; 16.2. Surgery 4 - God axe to wear: 6 Hong Kong itching 044 35.4, celluloid 0.19 32). Poor «Mo 0.25 015. 轫 letter ^ 0.44 32). 3: a^1f$i*.JiJl cold 1S&2% 3 :»ιί Itchy cold extraction 0.2 » 2% Λ3 (extraction * quenching; 1Li»t*珈"晬). iS Mb 〇CO CO σ> 〇1 a CO ΙΟ thin m 00 -< s < L· s < S < zm GO < ι m CD -< _1 s < ig I m W < ω m CD <&> S < s » S 7< ·* 〇> S < ^Λ. S 0Β Μ •. * Solution MATa 0hxtU170gal20agt1 ^sU1 fgy4~ 7 fgy4.1 丨eu2-3,112 ura3- 52 trp1.289 his3-01 MAL2-SF SUC2 =r厶$ itl -Λ b. |δ·|: 卜ίδ^ 苕& K:S §f III *厶fe if! Qiao: ώ-03 N| 〇 ** tv δΙ& §f ill 1^1 _ c>** t^. f •δ fe· K;5 δ wear• MA Ta dhxt 1-17 0gai2 Aagt 1 Jsf/ϊ fgy4-4 fgy4-1 Ieu2- 3t 112 ura3-52 ίφ1·289 his3-01 MAL2-(F SUC2 MATa 0hxt1-170gal2 0agt1 AstH fgy4· 1 fgy4-1 Ieu2-3t112 ura3.52trp1.289 his3-A1 MAL2^ SUC2 III S Co 111 _ It »3 1 MATa^ix"-i7Z^a/2i3ap"4s""jgy" 1 ieu2-3J 12 ura3-52 ΐφΐ^β,θ his3^1 MAL2-Sr SUC2 MATa 0hxt1-17 Agai2 0agt1 Δεϋ1 fgy1-11eu2-3J 12 ura3-52 ϊφ1^2Β,9 his3^1 i MAi2-ST SUC2 buckle s· δΐ&?> a sr g- Ml buckle s< δ|! 55^ f| it CO ^ |i Ml to s* δΙ ! -f| 13⁄4 Η :3⁄4 ·..·;*/ S 1 mm :'.f 譲I 1 残番淳洱P • ro 8? st 幸 幸 ** ro C»5S 漭 彝 彝 筅 筅 筅 筅 筅 铕莩 铕莩 铕莩 铕莩 铕莩 铕莩 铕莩 铕莩 3 3 3 3 3 3 ^55» 爵嶙;铱@典漭锅•痛·. 涔A Ja.' 85 尹Φ啭瑢μμ' Linggang 瘁A 呦》钧铱钧铱 $ SL." 迕令笋 W W 瘁啪*! 珈 放 放 • • • • • • • • • • • 择 择 择 择 择 择 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ - - - - - - - - - - - - - - - - - - - - - - - - - - » a 瑢琴* ro 荦 荦 at 令 令 令 令 令 令 令 令 令 令 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; | Μ Μ m I.4 1 Let m < ^ m Arp «| p > C Μ 典 ? Completion cg>>E l! 捋i If 4 j • I ll deduction i §p έ§ < —m go 5 --§ < ΓΠ 翁 Ω J3 Η 5< 〇CO S: ί :玛 iS ;〇.;. :藏ate Λ Λ ;···· :W DSM 14040 S 2 s iD •AA i 5 g CO ο (/) S: § a S αι SS 4^ s § σ ω sas ro民2 ro民«ag IS σ CD sp ®#>

•30·

Claims (1)

Patent Application No. 91112263 ROC Patent Appln. No.91112263 Text of the amended patent application - Annex (II) - Encl.nn (August 25, 1998, Submitted > (Submitted on August 25, 2009) 1. A strain of Saccharomyces cerevisiae, deposited in the German Collection of Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) 'Accession number DSM 14035, DSM 14036 or DSM 14037 (stored in the Food Industry Development Institute, sent to the Ministry of Economics The Property Bureau staff consumption cooperative prints 1323281 5 The deposit number is CCRC 920040, CCRC 920041 or CCRC 920042) ° 2. The method of producing the Saccharomyces cerevisiae strain as described in the scope of the patent application 'includes: a) provides yeast; 〇b) using the mutation or deletion of the relevant gene sequence of the HXT gene to eliminate the function of all six carbon sugar transporters of yeast derived from a); c) the separation cannot be grown on a substrate with six carbon sugar as the sole carbon source The strain 'but if the Glut4 gene is expressed in this strain, its growth ability on a substrate with six carbon sugar as the sole carbon source can be restored. 15 3. The Saccharomyces cerevisiae strain according to claim 1, which comprises the GLUT4 gene. 4. The Saccharomyces cerevisiae strain, as claimed in item 3 of the patent application, is deposited with the German Species Preservation Center under the accession number DSM 14038 (registered at the Food Industry Development Institute under the accession number CCRC 920043). 2〇5. A method for producing a yeast strain such as a patent model gj帛3 or 4, comprising: a) providing yeast; b) using a mutation or deletion of a related gene sequence of the HXT gene. The function of all six carbon sugar transporters in yeast; this paper scale applies to the standard of the Chinese national standard J:\menu\Pending-91\91227(CLAIMS)-connected 4.doc 1323281 A8 B8 C8 D8_ VI. Patent scope C) will contain the quality of the GLUT4 gene that can be expressed in yeast under the functional control of the promoter. Body, shaped into yeast b); 'd) the bacteria obtained according to c) transformation; ^ plate and ^ on the medium containing six carbon sugar as the sole carbon source; 5 e) separation according to d) for plate culture And the strain grown on the medium. 6. The method of claim 5, wherein the GLUT4 gene obtained from human, mouse or rat is used for transformation. 7. The method of claim 5, wherein the method of transforming is carried out using a vector having the polynucleotide sequence of SEQ ID Ν · 9 or 10. Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives 2 This paper scale applies to China National Standard (CNS) A4 specification (210x297 mm)